key: cord- -qz gtd authors: greatorex, jane s.; digard, paul; curran, martin d.; moynihan, robert; wensley, harrison; wreghitt, tim; varsani, harsha; garcia, fayna; enstone, joanne; nguyen-van-tam, jonathan s. title: survival of influenza a(h n ) on materials found in households: implications for infection control date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qz gtd background: the majority of influenza transmission occurs in homes, schools and workplaces, where many frequently touched communal items are situated. however the importance of transmission via fomites is unclear since few data exist on the survival of virus on commonly touched surfaces. we therefore measured the viability over time of two h n influenza strains applied to a variety of materials commonly found in households and workplaces. methodology and principal findings: influenza a/puertorico/ / (pr ) or a/cambridge/aho / (pandemic h n ) viruses were inoculated onto a wide range of surfaces used in home and work environments, then sampled at set times following incubation at stabilised temperature and humidity. virus genome was measured by rt-pcr; plaque assay (for pr ) or fluorescent focus formation (for pandemic h n ) was used to assess the survival of viable virus. conclusions/significance: the genome of either virus could be detected on most surfaces h after application with relatively little drop in copy number, with the exception of unsealed wood surfaces. in contrast, virus viability dropped much more rapidly. live virus was recovered from most surfaces tested four hours after application and from some non-porous materials after nine hours, but had fallen below the level of detection from all surfaces at h. we conclude that influenza a transmission via fomites is possible but unlikely to occur for long periods after surface contamination (unless re-inoculation occurs). in situations involving a high probability of influenza transmission, our data suggest a hierarchy of priorities for surface decontamination in the multi-surface environments of home and hospitals. influenza transmission is well documented in households and other residential settings [ ] [ ] [ ] [ ] . yet the underlying mechanisms of transmission remain poorly understood and hotly debated [ , ] . although transmission by aerosols (particles typically , mm in diameter), larger droplets and contact transmission (direct and via fomites) probably all play some role, the relative importance of each is uncertain, which has led to difficulties regarding the provision of evidence-based infection control advice for both pandemic and seasonal influenza [ ] . if virus can survive for meaningful periods on surfaces and objects, or alternatively, if surfaces are frequently re-inoculated (e.g. by toddlers), then it is feasible that transmission via fomites might occur. the potential for transmission of influenza by indirect contact (i.e. via fomites) is linked to the ability of virus to survive in transmissible titres on commonly touched surfaces; however few data exist on this subject. parker et al ( ) demonstrated improved survival of influenza viruses in the presence of human mucus [ ] ; and in , buckland demonstrated experimentally that influenza virus was inactivated relatively quickly on glass, probably through desiccation [ ] . in , widely cited work by bean et al showed that both influenza a and b, directly applied to stainless steel surfaces or hard plastic, could survive for - hours, and be transferred, from there to hands, for hours; survival was much shorter on porous materials such as paper and cotton ( - hours) , with transferability to hands for only minutes [ ] . in contrast, thomas et al, recently demonstrated survival of human seasonal a (h n ) and a (h n ) on swiss banknotes for up to three days, increasing to up to eight days when applied with nasopharyngeal secretions from children ( days if applied at very high concentration). although viable virus was recovered at each of these time points, it was noted that virus load declined sharply after the first few days; no other materials were tested [ ] . other studies have detected influenza virus on fomites in homes and health and childcare facilities, using rt-pcr to establish the presence of the viral genome [ ] [ ] [ ] . however, data obtained using this technique (even quantitatively) do not distinguish adequately between viable and non-viable virus and are therefore problematic to interpret in the context of practical infection control guidance. in another recent study, virus was detected by pcr on commonly touched household surfaces, but only one sample proved culture positive [ ] . however, the time from deposition to recovery was not known, nor the extent of any cleaning undertaken. we evaluate the survival of influenza a (h n ) viruses deliberately applied to a range of commonly touched household and workplace surfaces, using rt-pcr for genome detection and culture methods to determine viability. we conclude that rt-pcr is only useful to demonstrate the absence of virus and that on most surfaces, virus viability drops rapidly. nevertheless, on certain non-porous surfaces, viable virus persists for several hours, rendering fomite transmission possible without re-inoculation. to test the surface survival of influenza virus, we used a variety of materials commonly encountered in the home and workplace, including a hospital setting ( table ) ; choice of surfaces to be tested was discussed with the department of health, england to ensure relevance to public health policy. these included fibrous materials such as the ubiquitous j-clothh (associated brands) widely used for cleaning, a silver impregnated fabric with known bacteriostatic properties (toray textiles europe ltd.) of the type sometimes encountered in hospital staff clothing to combat nosocomial bacterial infections, as well as fabric from a child's soft toy. the latter fabric was made of non-absorbent polyester and, although a porous item overall, individual fibres might perform as a nonporous surface. a variety of non-porous plastic surfaces representing objects highly likely to be touched by multiple individuals such as light switch, telephone and keyboard plastics were also tested, as well as porous and non-porous 'background' materials such as various wood surfaces, glass, perspex/plexiglass (poly (methyl methacrylate) -a thermoplastic often used as a light or shatterresistant alternative to glass) and metals. as a control surface, we used standard laboratory polystyrene culture dishes. as viruses, we used two human h n strains: the laboratory adapted a/puerto rico/ / (pr ) strain because of ready availability and robust, convenient assay systems with a wide dynamic range, and an isolate of the current pandemic virus a/cambridge/ah / (ah ), as a low passage history representative of a virus likely to be encountered in the current environment. the source and disinfection method used to clean the various surfaces before testing are listed in table . human influenza a virus pr (cambridge lineage) was grown in embryonated hens' eggs and harvested at a titre of pfu/ ml. for inoculation of the surfaces, the virus was diluted : in % bsa and serum free media (dulbecco modified eagles medium, dmem, gibco, uk). this represented a viral titre approximating . tcid /ml, just above the upper end of titres reported for human shedding [ , ] . preliminary experiments established that virus survival was improved by the addition of extra protein to the suspension. we tested . % or % bsa as well as four preparations of artificial mucus produced from pig stomach mucosa (nbs biologicals), pig stomach mucin types ii or iii or bovine sub maxillary glands mucin, type i-s (all from sigma aldrich). % bsa had the largest effect on titre and duration of survival, followed by the bovine mucin (data not shown). in the interests of simplicity and reproducibility, % bsa was therefore used in all subsequent experiments. to test a pandemic influenza a (h n ) virus strain on selected surfaces, a clinical isolate designated influenza a/cambridge/aho / (ah ) was passaged once in mdck cells and then grown in caco- cells (colorectal adenocarcinoma cells, atcc htb- tm ). the virus is a recent isolate from an received sterile in packaging from manufacturer. fumigation in a cliii room using a laycock fumigator (tolbest ltd). doi: . /journal.pone. .t immunocompetent patient who was hospitalised briefly at the start of their illness, but recovered. this virus does not form discrete plaques in mdck cells and could therefore not be titred by this method. instead, virus stocks were quantified by qpcr for segment [ ] . although this method scores viable and non-viable virus particles alike, preparations of wild type influenza a viruses generally have similar particle:pfu ratios and quantitative comparison of rt-pcr and other titration methods have shown good agreement [ , ] . the ah stock contained . genome copies/ml and was used at a : dilution as for pr . for comparison, the pr stock had a genome titre of . genome copies/ml. mouse monoclonal aa h (abcam) was used to detect influenza np by immunofluorescence. surfaces were cut into cm pieces and sterilised by a variety of means depending on the surface to be tested (e.g. autoclaving, fumigation etc). sterile surfaces were glued into sterile -well tissue culture dishes using cyanoacrylate adhesive (henkel, uk). preliminary experiments (data not shown) demonstrated that dried adhesive alone was non-inhibitory to influenza virus. under the same conditions of temperature and humidity (ranges - uc and - % respectively), ml volumes of virus were applied to six samples of each surface at the same time. sampling was conducted immediately -time zero -to demonstrate recoverability. a cotton swab was moistened by dipping in ml of virus transport medium (vtm, remel, uk) and then wiped carefully in different directions for minute across the top of the surface. keeping everything on ice, the swab was placed into the tube containing the residual ( ml) volume of vtm and vortexed for minute. after this, the sample was split directly into eppendorf tubes and stored on dry ice prior to freezing at uc. the remaining samples in the plate were kept in a plastic, lidded box at constant temperature and humidity. at , , , and hrs, further samples were taken and stored. after initial experiments it was clear that the virus did not survive in detectable amounts for more than hrs, therefore for the majority of the experiments only the first time points ( , , , and hrs) were taken. initial experiments with pr virus also showed that loss of virus on the swab was not a major factor, with recovery of virus at time zero from polypropylene surfaces approaching % of initial titre (data not shown). the qrt-pcr assay used has been described previously [ ] . in brief, primers and probes to the matrix gene of influenza a were used to detect the presence of the virus on the surfaces. samples from all time points were stored and then extracted. virus genome was amplified to check that the quantity of virus deposited on the different surfaces was consistent and to determine whether any of the surfaces affected the genome over time. plaque assays were performed as previously described in mdck cells using avicell overlays [ , ] , in duplicate or where possible in triplicate. to detect ah virus by fluorescent focus assay, infectious material from swabs was first allowed to amplify by inoculation into mdck cells and incubation for h. supernatant virus was then diluted : in serum free dmem and ml used to inoculate . mdck cells in a well tissue culture plate. after virus absorption, the cells were overlaid with ml serum free dmem media containing mg/ml worthington's trypsin and . % bsa and incubated overnight at uc. the following day they were fixed with % formaldehyde in pbs, permeabilised by the addition of . % triton in pbs for minutes at rt and fluorescently stained with anti-np monoclonal antibody and counterstained for dna with , -diamino- -phenylindole (dapi) as previously described [ ] . cells were examined blind by two people and scored semi quantitatively for the presence of infected cells using a standardised schema ( no fluorescence seen; +/ some fluorescence seen (, % cells infected); + - % cells infected; ++ . % cells infected). the literature indicates immunofluorescence to be at least as sensitive in general as plaque assay [ , , ] . confirming this, tests using serial dilutions of known quantities of pr virus, our method reliably detected pfu of virus in the original sample prior to amplification and % of the time detected pfu (data not shown). to test the surface survival of the virus genome, replicate samples of the various materials were inoculated with ml samples containing pfu of virus and incubated for defined periods of time before sample recovery was attempted by swabbing. it was noted that the liquid was absorbed by the wooden surfaces within minutes whereas a droplet could be seen on non-porous surfaces for considerably longer, although in all cases, surfaces had dried by hours. material eluted from the swabs was then titred for virus genome by quantitative rt-pcr. for both pr (fig. a , table ) and ah (table ) viruses the results were unambiguous. on most surfaces, the viral genome persisted well, with only around a - fold drop from the initially recoverable titre after h. the exceptions were unsealed wood surfaces, where both viruses lost genome titre rapidly and on pine surfaces in particular, became undetectable after a few hours. thus in general, viral rna survives well for at least h and few surfaces had any significant 'contact effect' in immediately reducing genome titre. when pr surface viability was assessed by plaque assay, virus inoculated onto a control surface of a tissue culture dish could be recovered efficiently at t , but thereafter infectivity fell away rapidly with no live virus recovered at h (table ). fitting the data to a one-phase exponential decay model (fig. b ) estimated the t / of the virus under these conditions to be around . h. a similar pattern of rapid loss of infectivity was seen when the household surface samples were tested, with the difference that greater initial losses of infectivity ranging between -fold (telephone handset) to nearly -fold (unsealed pine) were seen (table ). nevertheless, viable virus was recovered at h (but not later) from the silver-impregnated cloth, soft toy fabric and in trace quantities, from light switch material. the only material (other than the control tissue culture dish) for which even low amounts of viable virus could be detected at h was stainless steel. thus despite the persistence of the viral genome on a wide variety of household surfaces, pr infectivity decayed sharply, with evidence of significant contact effects from some materials; most notably unsealed pine, but also a wide variety of other porous and nonporous surfaces. to test whether these findings could be extrapolated to a currently circulating virus, we next tested the survival of ah virus, a pandemic isolate, on a subset of the materials. unlike pr , as a recent clinical isolate this virus does not grow to high titres in the laboratory and nor was a workable plaque assay available. we therefore used a fluorescent focus assay in which live virus is detected by immunofluorescent detection of the viral nucleoprotein in infected cells. to boost the sensitivity with which viable virus could be detected, infectious virus present in the swabs was first amplified by growth in mdck cells before subsequent assay. the assay therefore provides a highly sensitive but semi quantitative measure of virus infectivity, ideally suited to working with low titre samples [ , ] . by this measure, the ah virus persisted for at least h on the control tissue culture dish material, although titres were evidently lower at and h ( table ) . consistent with the results obtained with pr virus, all household surfaces tested showed lower persistence of infectious virus, with none providing recoverable titre at h and the majority failing to produce live material at h. once again the pine surface showed very rapid inactivation of viability, with no infectivity recovered at h. thus both an historic virus isolate and an example of the recent pandemic strain fail to survive in high titres for long periods of time on a variety of household surfaces, but with significant survival over shorter time spans on certain materials. prior to the influenza a(h n ) pandemic of - , few data were available with regard to virus survival on different household surfaces. with a few notable exceptions [ , ] , the majority of studies had been carried out based on rt-pcr to detect the presence of the genome [ ] [ ] [ ] ; these shed no light on the presence or absence of viable virus. in this study we sought to provide contemporary data about virus survival on a wider range of materials found in or on household surfaces than previously described in the literature; these exemplars were chosen after discussion with uk pandemic policy makers. however, one limitation is that our study was confined to h n influenza a viruses (pr and the pandemic virus) due to resource issues. however, we know of no evidence to suggest there are substantial differences in survival between human influenza viruses. moreover, when we compared the survival of pr virus with two seasonal isolates of influenza a (a/solomon islands/ / / (h n ) and influenza a/brisbane/ / / (h n ), obtained from professor alan hay of the national institute of medical research, mill hill), we saw no significant differences, with all three viruses losing plaque titre on a plastic surface with a t / of around minutes (data not shown). we therefore think it is reasonable to generalise from the findings here to other human strains of influenza a. further studies, especially of influenza b are warranted however. we applied concentrations of virus (, tcid ), which were within the range of those reported in the respiratory secretions of naturally infected individuals [ , ] . in addition, we suspended virus in % bovine serum albumin (bsa), reflecting our (unpublished) finding that bsa improved virus survival, and similar findings from thomas et al [ ] using mucus obtained from children. our experiments were conducted within a narrow range of humidity and temperature conditions consistent with normal human indoor living conditions in temperate zones, and all survival assays were performed in duplicate and where possible triplicate. we used plaque assay techniques and immunofluorescence techniques for pr and pandemic viruses, respectively. the differing methodologies used to detect the two strains of h n virus (lower titre inoculum of the pandemic ah virus but higher sensitivity detection method) make it difficult to directly compare the survival of the two strains, but we see little to suggest any major difference. our data on the survival of the laboratory adapted pr virus indicated that viable virus was no longer recoverable in detectable amounts from of ( %) surfaces four hours after deposition; however, contrary to the findings of bean et al., non-porous surfaces were not consistently more conducive to virus survival than porous ones [ ] . nevertheless, no test surfaces supported detectable virus survival beyond nine hours. broadly similar outcomes in which infectivity tended to be lost after - hours were obtained with the recent pandemic isolate ah . overall, our results indicate that influenza virus does not remain viable in large quantities on most surfaces in indoor domestic conditions for more than a few hours. our data are consistent with recent findings from a study of environmental deposition of pandemic h n virus in the homes of infected patients, involving our laboratory, when almost % of tested surfaces yielded viable virus [ ] . however, in this and similar studies in community settings where environmental samples are taken relatively infrequently and the infectious source remains present, it is not possible to establish the time elapsed since virus deposition [ , ] . with regard to the testing of specific materials, we examined survival on a range of porous items: a children's soft toy, a silver impregnated fabric with known bacteriostatic properties of the type sometimes encountered in hospital staff clothing to combat nosocomial bacterial infections, and a branded cleaning cloth (j clothh, associated brands). we hypothesised that the inclusion of an antimicrobial agent, microbanh (microban international ltd) in the j cloth might inhibit viral growth. microbanh is based on triclosan and has been demonstrated to have anti-bacterial and anti-fungal activity; it has not however, been demonstrated or claimed to be anti-viral. notwithstanding, in our laboratory setting, some constituent or quality of the j clothh appeared to limit virus survival to under hours. the result for the silver impregnated fabric also deserves further comment. whilst silver has been demonstrated to have bacteriostatic properties, it has not been documented to show antiviral activity. our data would tend to suggest that it is not significantly inhibitory to influenza a. surfaces that allowed pr virus to survive longest (between four and nine hours) included light switch material (polyvinyl chloride) and a computer keyboard. interestingly these are likely to be the materials from which the most frequently touched communal household objects are made. both pr and pandemic viruses survived less than four hours on all of the wood surfaces tested. this may have been due to a number of factors including porosity of the surface, oils in the wood or a potentially virucidal 'contact effect' of varnish finishes. pine oil in particular has been demonstrated to have virucidal activity against respiratory viruses [ ] . our findings suggest they are not hospitable environments for enveloped viruses. as observed in other studies, we found that stainless steel supported the viability of influenza viruses longer than other tested metals. metals have been demonstrated to have low levels of anti viral activity [ ] [ ] [ ] ; and stainless steel has previously been demonstrated to support influenza virus viability for longer than that of copper [ ] . confirmation of these results raises questions about the use of stainless steel in healthcare and daycare settings in particular. in conclusion, testing two h n strains of influenza a (one of which was a pandemic virus) demonstrates that in an environment that is consistent with indoor domestic settings in temperate zones, virus deposited onto the touched environment is likely to survive up to a few hours, though rarely more than nine hours, on the vast majority of surfaces. metallic and non-metallic non-porous materials pose the greatest risk and should be targeted for frequent cleaning if situated in close proximity to patients infected with influenza virus; fortunately the latter are also more conducive to surface cleaning with a wide variety of simple cleaning agents [ ] . whilst our data suggest that the risk of virus transmission might last several hours after deposition, we generated very little data suggesting that appreciable amounts of virus survived much beyond nine hours. this probably means that frequently touched environments such as classrooms, offices and living rooms, which are then left unoccupied overnight, will not contain much viable virus on surfaces by the next morning. nevertheless, the data still support frequent cleaning of commonly touched items and surfaces throughout the working day, particularly when symptomatic persons are present, for example in physician waiting rooms. in terms of cleaning regimens, one critically important consideration is that survival of virus in high titres for prolonged periods is not necessary for fomite transmission if surfaces are frequently re-inoculated (e.g. by toddlers). however the contribution of such indirect transmission relative to respiratory droplets directly from one person to another or relative to aerosol transmission remains unknown. estimating household and community transmission parameters for influenza statistical procedures for estimating the community probability of illness in family studies: rhinovirus and influenza simulation studies of influenza epidemics: assessment of parameter estimation and sensitivity using data on social contacts to estimate age-specific transmission parameters for respiratory-spread infectious agents review of aerosol transmission of influenza a virus transmission of influenza a in human beings influenza and related infection control issues resistance of the melbourne strain of influenza virus to desiccation loss of infectivity on drying various viruses survival of influenza-viruses on environmental surfaces survival of influenza virus on banknotes effectiveness of common household cleaning agents in reducing the viability of human influenza a(h n ) biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity genome packaging in influenza a virus virus shedding and environmental deposition of novel a(h n ) pandemic influenza virus: interim findings mutational analysis of cis-acting rna signals in segment of influenza a virus interaction of the influenza virus nucleoprotein with the cellular crm -mediated nuclear export pathway comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay and virus isolation for detection of respiratory viruses in nasopharyngeal secretions enhanced detection of infectious airborne influenza virus the occurrence of influenza a virus on household and day center fomites respiratory viral rna on toys in pediatric office waiting rooms occurrence of bacteria and viruses on elementary classroom surfaces and the potential role of classroom hygiene in the spread of infectious diseases influenza virus contamination of common household surfaces during the influenza a (h n ) pandemic in bangkok, thailand: implications for contact transmission the antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for sars coronavirus neutralizing viruses in suspensions by copper oxide-based filters inactivation and morphological changes of avian influenza virus by copper ions -oximinocarboxylates of certain metals and their antiviral activity inactivation of influenza a virus on copper versus stainless steel surfaces we thank penny powell, roberto vivancos and darren sexton at the university of east anglia for their contributions to early discussions; helen wise and amanda stuart for help with the virus work. some materials were kindly supplied free of charge by brushwood ltd uk (kitchen work surfaces) and toray textiles europe ltd (silver-containing fabric); the remaining items were sourced commercially or as shown in table . key: cord- -qrt zmz authors: miyakawa, kei; matsunaga, satoko; yamaoka, yutaro; dairaku, mina; fukano, kento; kimura, hirokazu; chimuro, tomoyuki; nishitsuji, hironori; watashi, koichi; shimotohno, kunitada; wakita, takaji; ryo, akihide title: development of a cell-based assay to identify hepatitis b virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: qrt zmz sodium taurocholate cotransporting polypeptide (ntcp) is a major entry receptor of hepatitis b virus (hbv) and one of the most attractive targets for anti-hbv drugs. we developed a cell-mediated drug screening method to monitor ntcp expression on the cell surface by generating a hepg cell line with tetracycline-inducible expression of ntcp and a monoclonal antibody that specifically detects cell-surface ntcp. using this system, we screened a small molecule library for compounds that protected against hbv infection by targeting ntcp. we found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface ntcp with an ic( ) of ~ μm. we also found that glabridin could attenuate the inhibitory effect of taurocholate on type i interferon signaling by depleting the level of cell-surface ntcp. these results demonstrate that our screening system could be a powerful tool for discovering drugs targeting hbv entry. hepatitis b virus (hbv) is the causative agent of chronic hepatitis b (chb), which can lead to liver cirrhosis and hepatocellular carcinoma. the world health organization reported that over million people worldwide have chronic hbv [ ] . despite the effectiveness of the hbv vaccine, worldwide prevalence of the disease remains high, and chb is a major global health problem. current therapeutic regimens for chb include pegylated interferon (ifn) and nucleoside/ nucleotide analogues. both treatments aim to prevent progression of the disease to liver failure, cirrhosis, and hepatocellular carcinoma. however, these treatments have limited effectiveness for hbv clearance [ ] . in fact, pegylated ifn maintains viral suppression only in approximately % of patients [ ] . nucleoside and nucleotide analogues inhibit hbv replication by targeting viral dna polymerase, but long-term treatment is required to achieve clinical benefits. for example, a -month course of lamivudine achieves clearance of hepatitis b e antigen (hbeag) in approximately % of patients with chb [ ] . moreover, long-term treatment can be associated with a higher risk of side effects and emergence of drug resistant viruses, resulting in treatment failure and disease progression. therefore, it is vital to research paper www.oncotarget.com develop new types of antiviral drugs for hepatitis b treatment. in the hbv life cycle, the hepatitis b surface antigen (hbsag) initially attaches to heparan sulfate proteoglycans on the host cell surface [ ] . this attachment seems to be relatively low affinity and reversible, but is essential for the subsequent more specific interaction between hbs and the hepatocyte-specific bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) [ ] . although various membrane proteins have been reported to be hbv entry receptors, accumulating evidence now suggests that ntcp is an essential receptor for hbv infection [ ] . discovery of this receptor has dramatically increased our understanding of the molecular basis of hbv entry. viral entry is currently one of the most important targets in the search for new drugs to treat viral infections and identification of ntcp has kindled interest in exploring compounds that inhibit hbv entry. ntcp is exclusively expressed at the basolateral membrane of hepatocytes [ ] [ ] [ ] , and is involved in reuptake of conjugated bile acids from the bloodstream into hepatocytes [ ] . ntcp is one of the factors that highly restrict host tropism of hbv to hepatocytes. the specific interaction of the pres domain of hbsag with ntcp triggers hbv attachment and initiates entry into hepatocytes. the synthetic peptide drug myrcludex b exhibits significant anti-hbv activity by competitively inhibiting this interaction, both in vitro and in vivo, and is currently undergoing a phase ii clinical trial for chronically hbv-infected patients [ , ] . physiological substrates of ntcp such as taurocholic and glycocholic acids can also inhibit hbv infection, suggesting the competitive binding of bile acids and pres to ntcp [ ] . interestingly, pres -ntcp binding could trigger type i ifn signaling, whereas bile acid treatment does not [ ] . bile acid intake via ntcp was found to inhibit the ifn pathway in the physiologically relevant range of concentrations [ ] . these observations support the possibility that ntcp also takes part in the ifn-mediated innate antiviral response. because high expression often causes cell cycle inhibition, there are very few conventional hepatocellular carcinoma cell lines with ectopic expression of high levels of ntcp. moreover, there are no commercially available monoclonal antibodies (mabs) specifically recognizing cell-surface ntcp due to the difficulty of producing full-length ntcp protein with native structure. in the current study, we generated hbv-susceptible hepg cells with a tetracycline-inducible ntcp gene (intcp cells) without affecting cell growth. we also generated a high quality mab (clone a ) to efficiently detect cell-surface ntcp. using these tools, we identified the compound glabridin, which significantly inhibits hbv infection through the downregulation of ntcp from the cell surface. endogenous ntcp expression is rarely detectable in hepatoma cell lines such as hepg and huh , and these cells are not susceptible to hbv infection. therefore, we generated the intcp cell line, a hepg based cell line harboring a tetracycline-inducible human ntcp gene ( figure a) . treatment of the intcp cell line with the tetracycline analogue doxycycline (dox) caused expression of ntcp in a dox-dependent manner, and ntcp expression was -to -fold higher than endogenous expression in differentiated heparg cells and primary hepatocytes, as revealed by western blot and quantitative reverse transcription-pcr ( figure b- d) . to determine whether dox-induced ntcp protein localized to the plasma membrane, we performed a pres peptide binding assay. we found that the pres peptide detected dox-treated intcp cells but not untreated cells, indicating the presence of ntcp on the cell surface ( figure e ). consistent with a previous report [ ] , a western blot of ntcp showed major bands of - kd that were shifted to a single band of - kd after treatment with peptide n-glycosidase (pngase), implying that ntcp was modified by n-glycosylation (supplementary figure a) . although ntcp expression has been reported to affect cell proliferation [ ] , intcp cells showed no differences in cell cycle progression or cell expansion with or without dox treatment ( figure f, g) . we subsequently examined the susceptibility of intcp cells to hbv infection. at an moi of geq/cell, intcp cells showed high susceptibility to hbv infection (~ % infected) without dmso treatment ( figure h , supplementary figure b ). this infection was significantly inhibited by pres peptide treatment ( figure h ), which indicates that hbv infection was ntcp-mediated. taken together, these results show that dox-induced ntcp proteins are exposed on the cell surface and functionally interact with pres . although the above results suggest that ntcp proteins localize on the cell surface, this could not be directly demonstrated due to the lack of suitable antibodies for flow cytometry or immunofluorescence microscopy analysis. therefore, we generated a monoclonal antibody (mab) for this purpose. because recombinant ntcp protein tends to form insoluble aggregates, we utilized the wheat germ cell-free system, which has been shown to have advantages for the production of membrane proteins [ , ] . the synthesized ntcp proteins were purified and used to immunize mice, and more than hybridoma clones were established (figure a ). using a www.oncotarget.com flow cytometer-based screening assay with dox-treated and untreated intcp cells, we identified a hybridoma clone producing anti-ntcp mab, clone a ( figure b ). the a mab could recognize endogenous ntcp in differentiated heparg cells (supplementary figure ) . we performed immunofluorescence microscopy using the a mab in various cell lines and cell-surface ntcp was clearly observed ( figure c , d). because the three-dimensional organoid culture recapitulates cell-cell interactions and recent studies have indicated some advantages of hepatoma organoids in hepatitis virus infection [ ] , we investigated the localization of ntcp in hepatoma organoids. we embedded intcp cells in hydrogel before performing immunofluorescence microscopy with the a mab to visualize ntcp. ntcp localization was widespread in the membrane of internal cells as well as on the surface of the organoid ( figure e ). epitope mapping using recombinant ntcp mutants revealed that the a mab recognizes amino acids - of ntcp ( figure f ). to test whether the a antibody can inhibit hbv infection, we pretreated intcp cells and primary human hepatocytes with a mab and subsequently infected cells with wild type hbv and hbv encoding a luciferase reporter gene (hbv-nl) [ ] . the a mab failed to inhibit hbv infection ( figure g , h), suggesting that the interaction between a mab and ntcp neither blocks hbv-host cell interaction nor causes downregulation of ntcp from the cell surface. because the a mab binds ntcp but does not interfere with its localization and function, it can be used to screen for antiviral compounds that modulate the level of cell-surface ntcp. briefly, intcp cells were treated with different low-molecular-weight chemical compounds from a library derived from natural plant extracts for hours and we subsequently quantified cell viability and the amount of cell-surface ntcp using the a mab ( figure a ). we identified two compounds, geraldol and glabridin, that could decrease the amount of cell-surface ntcp without observable cytotoxicity ( figure b ). flow cytometry analysis using the a mab demonstrated that both geraldol and glabridin decrease the levels of cell-surface ntcp in a dose-dependent fashion ( figure c ), but further analysis revealed that geraldol negatively regulates the tetracycline-responsive promoter activity ( figure d ). microscale thermophoresis analysis of the biomolecular interaction [ ] revealed that glabridin could weakly but directly interact with ntcp ( figure e ). therefore, we focused on glabridin for further functional analyses. flow cytometry analysis showed that glabridin downregulated the amount of cell-surface ntcp in hepg -hntcp-c [ ] and heparg cells with ic values of µm and µm, respectively ( figure a ). notably, treatment with µm glabridin had no effect on the level of ntcp mrna (supplementary figure a) , suggesting that glabridin affects ntcp protein rather than ntcp gene expression. consistently, treatment with glabridin rapidly (within three hours) modulated the membrane localization of ntcp, but not that of the transferrin receptor ( figure b ). similar results were obtained with cell-surface biotinylation analysis in which only cell-surface proteins were purified and detected by western blotting (supplementary figure b ). immunofluorescence microscopy using the a mab showed that in glabridin-treated cells, ntcp mainly accumulated in the cytoplasm ( figure c ). to test the role of endocytosis in ntcp localization, we treated cells concurrently with glabridin and inhibitors of clathrinmediated or caveolar endocytosis. we found that genistein, a specific inhibitor of caveolar endocytosis, completely disrupted ntcp internalization ( figure d ), suggesting that glabridin causes the internalization of ntcp through caveolar endocytosis. because internalized membrane proteins are typically trafficked to degradation or recycling pathways, we next performed a cycloheximide chase assay. interestingly, ntcp protein levels were relatively stable in control cells, but the half-life of ntcp was significantly shorter in glabridin-treated cells ( figure e ). this suggests that glabridin reduces the amount of ntcp on the cell surface by promoting its endocytosis and subsequent intracellular degradation. previous reports have indicated that glabridin induces apoptosis in certain cancer cells [ ] , but we observed no negative effect on cell viability under our experimental conditions ( figure f ). we next assessed the impact of glabridin on hbv infection in hepatocytes. time-of-addition experiments demonstrated that µm glabridin inhibited hbv infection when added early in infection but became less effective when added later ( figure a ), suggesting that it acts at an early stage of the viral life cycle. indeed, when glabridin was added to hbv-producing cells, it did not affect the amounts of core-associated viral dna and/or rna (supplementary figure ) . consistent with these observations, intcp cells treated with glabridin three hours prior to infection showed a significant reduction in the percentage of infected cells and the amount of hbsag secretion ( figure b , c). we performed a parallel analysis with differentiated heparg cells and confirmed that glabridin blocked hbv infection and downregulated endogenous ntcp with an ic value of µm ( figure d ). these results suggest that glabridin inhibits hbv infection by removing ntcp from the cell surface. ntcp is a transporter for bile acid uptake. we investigated the effect of glabridin on ntcp-mediated www.oncotarget.com anti-ntcp mab generation. using a wheat germ cell-free protein production system, large amounts of ntcp were synthesized with high solubility. following mouse immunization, we established over hybridoma clones. (b) selection of a clones producing anti-ntcp mab. culture supernatants of hybridoma clones were used as primary antibodies for flow cytometry analysis of dox-treated (red line) or -untreated intcp cells (blue line). (c-e) immunofluorescence staining of cell-surface ntcp by a mab on intcp cells (c), hepg -hntcp-c cells (d), and intcp-derived spheroid (e). scale bars: µm. (f) epitope mapping of a mab. recombinant wild-type or partially truncated ntcp proteins tagged with his were generated using wheat germ extracts and subjected to western blotting using anti-his or a antibodies. the predicted epitope of a mab is shown in pink. (g, h) a mab fails to inhibit hbv infection. intcp cells (g) and primary human hepatocytes (h) were infected with hbv or its reporter virus (hbv-nl) respectively, in the presence of a mab. anti-hbs mab (clone a , which recognizes the pres domain) was used as a control. viral infectivity was determined by intracellular hbcag staining (g) or nanoluc activity (h) of infected cells. uptake of taurocholate (tca) in a sodium-containing condition and found that glabridin reduced tca uptake in a dose-and time-dependent manner ( figure a ). a previous study has shown that ntcp-mediated bile acid transport affects the expression of ifn stimulatory genes (isgs) in primary human hepatocytes [ ] , so we investigated whether glabridin counteracts this function of bile acid. primary human hepatocytes were treated with type i ifn and tca in the presence or absence of glabridin. treatment with ifn upregulated various isg proteins including mx and bst , which are known to inhibit hbv replication [ ] [ ] [ ] [ ] , while concurrent treatment with tca reduced this effect ( figure b ), in accordance with previous observations [ ] . interestingly, the addition of µm glabridin partially cancelled the effect of tca in isg expression ( figure b ). these findings suggest that glabridin enhances the innate immune response by suppressing bile acid uptake in hepatocytes through the downregulation of cell-surface ntcp. in this study, we generated intcp cells, which have high ntcp expression and high susceptibility to hbv infection, and also developed a monoclonal antibody (mab) that recognizes cell-surface ntcp. using these tools, we identified glabridin as a compound that inhibits hbv infection by downregulating levels of its entry receptor, ntcp. although primary hepatocytes express ntcp at low levels for the uptake of bile acids, endogenous ntcp in hepatocellular carcinoma cell lines is not sufficient to achieve successful infection with hbv in vitro. hepatocellular carcinoma cell lines stably expressing ntcp have been created, and exhibited increased susceptibility to hbv infection [ ] , but it has been shown that continuous ntcp expression can induce cell cycle arrest at the g /g phase [ ] , indicating that ectopic ntcp expression may be unfavorable for the long-term culture. to overcome these issues, we created the intcp cell line, featuring inducible ntcp expression, using the third-generation tetracyclineinducible gene expression system in hepg cells. upon transient treatment with tetracycline derivatives, this cell line exhibits high expression of cell-surface ntcp and becomes highly susceptible to hbv infection without any observable cell cycle arrest. this unique feature of our newly developed cell line makes it a useful tool for further biological analysis of ntcp in hepatic cells. we created a new mab recognizing cell-surface ntcp by using a wheat germ cell-free protein production system to synthesize full-length recombinant ntcp protein, which was then used to immunize mice. generally, the quality of an antibody is determined largely by the antigen used for immunization. preparation of immunogens derived from highly insoluble membrane proteins is challenging, and ntcp tends to be insoluble in conventional cell expression systems due to its multiple transmembrane domains. there are several methods for preparing antigens derived from insoluble proteins, including use of synthetic peptides containing the predicted immunogenic epitope of extracellular domains [ ] , but synthetic peptides are structurally linear and do not recapitulate the native structural features of membrane proteins. by contrast, the wheat germ cell-free protein production system utilizes a eukaryotic translation system to synthesize structurally intact and biologically active proteins similar to those expressed in mammalian cells [ , ] . this approach enabled us to create a mab capable of detecting a spatial antigen within the region of ntcp exposed on the cell surface. our work demonstrates the advantage of the cell-free system in the production of proteins with multiple transmembrane domains [ , ] . the three-dimensional structure of ntcp protein has not been well characterized and the number of transmembrane domains is controversial at present. several groups have predicted that ntcp has - transmembrane domains and that the c-terminus of ntcp is located in the cytoplasm [ , ] . we found that our a mab recognizes amino acids - of ntcp on intact cells without membrane permeabilization, implying that this epitope is possibly exposed to the extracellular space. more precise structural biological studies should be carried out to elucidate the topology of the ntcp protein. immunofluorescence microscopy experiments using our a antibody revealed that ntcp localized on the plasma membrane, frequently at cell-cell contact sites. this localization was more obvious in a hepatocyte organoid culture system, suggesting that the localization of ntcp may depend on cell polarity. because the threedimensional culture system recapitulates cell polarity, it is useful for analyzing the function of ntcp in sodium taurocholate transport as well as hbv infection. recent studies demonstrated that the three-dimensional culture system is suitable for analyzing polarized hbv transmission [ , ] . it is not well established whether ntcp plays a role in viral transmission in polarized cells, and our newly developed a mab may be useful for pursuing this intriguing question. several compounds have been shown to target ntcp. recent studies demonstrated that antagonists of retinoic acid receptor ro - [ ] or the cytokine interleukin- [ ] could reduce ntcp expression. a previous report also indicated that the green tea extract epigallocatechin- -gallate (egcg) also inhibited hbv entry into primary human hepatocytes [ ] . interestingly, egcg induced endocytosis of ntcp from the plasma membrane followed by protein degradation. in the current study, we found that glabridin, a compound from licorice extract, also causes ntcp receptor downregulation. although the precise mechanisms are unclear, our findings intcp cells were treated with glabridin ( , , and µm) for three or hours. after incubation with [ h]-taurocholate (tca) for minutes, cells were washed and intracellular radioactivity was quantified. cyclosporin a (csa, µm) was used as a positive control in this assay. * p < . , ** p < . , two-tailed unpaired t-test. (b) glabridin counteracts bile acids to promote innate immune signaling. primary human hepatocytes were sequentially treated with ifn-β ( u/ml), tca, and glabridin ( µm), as shown in left panel. the expression of representative isgs, including mx and bst , was quantified using qpcr. ns, not significant; ** p < . , two-tailed unpaired t-test. www.oncotarget.com suggest that glabridin directly binds ntcp and causes its internalization by caveolar endocytosis. by estimating protein turnover using a cycloheximide chase assay, we were able to infer that glabridin promotes the intracellular degradation of ntcp. interestingly, glabridin is orally bioavailable and is known to rapidly accumulate in the liver [ ] [ ] [ ] . since ic value of glabridin for inhibiting of hbv entry is relatively high (~ µm), this compound might have little translational benefit in hbv research. however, the data obtained from our study demonstrates the potential scope of our current methodology in drug discovery for hbv therapeutics. it is possible that the internalization of ntcp could inhibit its transporting activity thereby causing unfavorable effects on hepatocytes. however, people with i t or s f mutations in the ntcp gene show decreased surface expression and transporting activity of ntcp, but there have been no reports of serious diseases resulting from these mutations to date [ , ] . the frequent occurrence of physiological downregulation of ntcp is also notable. this downregulation is typically controlled by transcription factors, such as hnf α, under cholestasis conditions [ ] . furthermore, recent papers have shown that ntcp is the main transporter for conjugated bile acids into the liver, but other auxiliary transporters, such as oatp, may compensate when ntcp is absent [ , ] . although these reports hint that transient downregulation of ntcp might not induce immediate adverse effects on the liver, the effect should be further investigated in in vivo models to determine whether ntcp inhibition is a reasonable anti-hbv drug target. in conclusion, we identified as glabridin as a natural compound capable of inhibiting hbv infection by impairing viral entry into host cells, with an additional effect of enhancing the antiviral immune response. furthermore, our data demonstrate that our newly developed cell line and antibody will serve as powerful tools for drug discovery targeting hbv entry and for exploring molecular mechanisms underlying hbv spread. to generate intcp cells, a hepg tet-on advanced cell (clontech) parental cell line was transduced with a retroviral vector encoding the ntcp gene fused to a tetracycline-responsive element, then was selected with puromycin ( µg/ml), and cultured with dmem (wako) supplemented with % fbs. unless otherwise indicated, intcp cells were treated with doxycycline (sigma-aldrich) for hours before experiments. the intcp spheroid was made using the -d life dextran-cd hydrogel sg kit (cellendes) and cultured for seven days on a chamber slide (thermo fisher scientific). primary human hepatocytes (pxb-cells) were purchased from phoenixbio. heparg cells were purchased from biopredic international and differentiated according to the manufacturer's instructions. the chemical compound library from natural plant extracts was obtained from tokiwa phytochemical. nocodazole, cyclosporin a, genistein, and pitstop were purchased from sigma-aldrich. glabridin, staurosporine, and ifn-β were obtained from wako. a protemist xe robotic protein synthesizer (cellfree sciences) was used for the generation of full length ntcp and its truncated derivatives as previously described [ , ] . immunization of balb/c mice with recombinant ntcp and generation of hybridoma cells producing anti-ntcp antibody were performed as previously described [ ] . purification of antibodies in the culture supernatant of the hybridoma clones was performed by centrifugation at , rpm for minutes and elution with acrosep hyper df columns (pall). samples were then concentrated using amicon ultra filters (merck millipore). immunoglobulin characterization was carried out using the isostrip mouse monoclonal antibody isotyping kit (roche). cells were fixed with % paraformaldehyde, blocked with % normal goat serum (thermo fisher scientific), and stained with either anti-ntcp mab (clone a ) or anti-hbcag polyclonal antibody (dako) and alexa fluor-conjugated secondary antibodies (thermo fisher scientific). alexa fluor -conjugated phalloidin (thermo fisher scientific) was used for f-actin staining. for intracellular staining, cells were permeabilized with . % triton x- before blocking. for the pres -peptide binding assay, intcp cells pretreated with µg/ml dox for hours were incubated with nm fitc-conjugated pres peptide (the first amino acid residues of small hbs domain fused with n-terminal myristoyl group and c-terminal fitc) at ° c for two hours. cells were then washed with pbs and fixed with % paraformaldehyde. microscopic imaging was performed with an fv -d confocal laser scanning microscope (olympus) or bz- fluorescence microscope (keyence). cells were detached with mm edta in pbs and fixed with % formaldehyde before incubation with either anti-ntcp ( a ) or anti-transferrin receptor (genetex) antibodies at ° c. cells were then stained with pe-conjugated secondary antibody and analyzed using a facscanto ii instrument (bd biosciences). for the drug screen, cells were treated with candidate compounds www.oncotarget.com ( µm) hours before analysis. data were analyzed with flowjo software (treestar). wild-type hbv was derived from the supernatants of hepg . . cells, which were stably transfected with a complete hbv genome. hbv reporter viruses (hbv-nl) were produced by transient transfection of hepg cells with puc . -hbv/nl and puc-hbv-d, as previously described [ ] . the collected supernatants were filtered through a . -μm filter (merck millipore), and concentrated approximately times using a peg virus precipitation kit (biovision). cells were infected with wild-type hbv at a concentration of , genome equivalents per cell in the presence of % peg for hours. alternatively, cells in a -well plate were inoculated with µl of hbv-nl in the presence of % peg for hours. hbv-infected cells were cultured in fresh medium for an additional - days and their infectivity was determined by intracellular hbcag staining or extracellular hbsag quantification, as previously described [ ] . the infectivity of hbv-nl was quantified using the nano-glo luciferase system (promega), according to the manufacturer's instructions. samples in sds loading buffer were loaded onto % polyacrylamide gels, electrophoresed, and blotted onto pvdf membranes (merck millipore), as previously described [ ] . membranes were probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (ge healthcare). for protein degradation analysis, cycloheximide ( µg/ml) was added - hours before harvesting cells. the primary antibodies used were as follows: anti-ntcp, anti-vinculin, anti-α-tubulin (sigma-aldrich) and anti-his (genetex). detected proteins were visualized using a fluorchem digital imaging system (alpha innotech). band analysis was performed with imagej software (national institutes of health). messenger rna extraction and subsequent cdna synthesis was performed using trizol reagent (thermo fisher scientific) and revertra ace (toyobo), respectively, according to each manufacturer's instructions. gene expression was then analyzed by qpcr using sybr premix ex taq ii (takara) and a cfx real-time pcr detection system (bio-rad). the primer pairs used were ′-ggacttcgagcaagagatgg- ′ and ′-agcactgtgttggcgtacag- ′ for actb; ′-atggaggcccacaacg cgtctgccc- ′ and ′-cagaaggtggagcaggtggtcatcac- ′ for ntcp; ′-ggctgtttaccagactccgaca- ′ and ′-cacaaa gcctggcagctctcta- ′ for mx ; and ′-tctcctgcaaca agagctgacc- ′ and '-tctctgcatccagggaagccat- ' for bst . recombinant ntcp-gfp and gfp proteins were incubated with different concentrations of compounds in mm potassium phosphate buffer (ph . ) containing mm nacl, . % bsa, and . % ddm (n-dodecylβ-d-maltopyranoside) for one hour at room temperature. samples were loaded on monolith nt. standard treated capillaries (nano temper technologies) and analyzed with a monolith nt. blue red microscale thermophoresis instrument. cell cycle analysis was performed with a tali image-based cytometer (thermo fisher scientific). cell viability was measured with cell counting kit- (dojindo) or celltiter-glo assay (promega) according to the manufacturer's instructions. cells were treated with [ h]-taurocholic acid (tca) in a sodium-free or sodium-containing buffer at ºc for minutes to allow tca uptake into cells. cells were washed and intracellular radioactivity was measured using a liquid scintillation counter. all graphs represent means and standard deviations. the statistical significance of differences between two groups was tested using a two-tailed unpaired t test with prism software (graphpad). km designed and performed research, analyzed data, and wrote the manuscript; sm, yy, md, and kf performed research and analyzed data; hk and tc analyzed data; hn, kw, ks, and tw contributed reagents and analyzed data; and ar designed and supervised the research, analyzed the data, and wrote the manuscript. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity virologic monitoring of hepatitis b virus therapy in clinical trials and practice: recommendations for a standardized approach pegylated interferon alfa- b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial nucleoside analogues for chronic hepatitis b: antiviral efficacy and viral resistance entry of hepatitis b and hepatitis d virus into hepatocytes: basic insights and clinical implications sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for speciesspecific entry into hepatocytes characterization of cloned rat liver na(+)-bile acid cotransporter using peptide and fusion protein antibodies in situ localization of the hepatocytic na+/ taurocholate cotransporting polypeptide in rat liver chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas sinusoidal (basolateral) bile salt uptake systems of hepatocytes first-in-human application of the novel hepatitis b and hepatitis d virus entry inhibitor myrcludex b treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study kinetics of the bile acid transporter and hepatitis b virus receptor na+/taurocholate cotransporting polypeptide (ntcp) in hepatocytes solute carrier ntcp regulates innate antiviral immune responses targeting hepatitis c virus infection of hepatocytes bile acids modulate the interferon signalling pathway down-regulation of ntcp expression by cyclin d in hepatitis b virus-related hepatocellular www.oncotarget.com carcinoma has clinical significance production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system a cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters single particle imaging of polarized hepatoma organoids upon hepatitis c virus infection reveals an ordered and sequential entry process novel reporter system to monitor early stages of the hepatitis b virus life cycle molecular interaction studies using microscale thermophoresis evaluation and identification of hepatitis b virus entry inhibitors using hepg cells overexpressing a membrane transporter ntcp glabridin mediate caspases activation and induces apoptosis through jnk / and p mapk pathway in human promyelocytic leukemia cells mxa inhibits hepatitis b virus replication by interaction with hepatitis b core antigen the interferon-inducible protein tetherin inhibits hepatitis b virus virion secretion identification of bst- /tetherin-induced hepatitis b virus restriction and hepatocyte-specific bst- inactivation molecular dissection of hbv evasion from restriction factor tetherin: a new perspective for antiviral cell therapy cell culture models for the investigation of hepatitis b and d virus infection generation and identification of peptide-based monoclonal antibodies against vacuolar proton pyrophosphatase of toxoplasma gondii advances in genome-wide protein expression using the wheat germ cell-free system cell-free expression systems for eukaryotic protein production organization of the membrane domain of the human liver sodium/bile acid cotransporter cloning and functional characterization of human sodium-dependent organic anion transporter (slc a ) concise review: organoids are a powerful tool for the study of liver disease and personalized treatment design in humans and animals cultivation of hepg . . on cytodex- : higher yield of hepatitis b virus and less subviral particles compared to conventional culture methods dysregulation of retinoic acid receptor diminishes hepatocyte permissiveness to hepatitis b virus infection through modulation of sodium taurocholate cotransporting polypeptide (ntcp) expression interleukin inhibits hbv entry through ntcp down regulation epigallocatechin- -gallate inhibits entry of hepatitis b virus into hepatocytes determination of glabridin in human plasma by solidphase extraction and lc-ms/ms role of p-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of glycyrrhiza glabra role of p-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of glycyrrhiza glabra ethnicity-dependent polymorphism in na+-taurocholate cotransporting polypeptide (slc a ) reveals a domain critical for bile acid substrate recognition genetic polymorphisms in na+-taurocholate co-transporting polypeptide (ntcp) and ileal apical sodium-dependent bile acid transporter (asbt) and ethnic comparisons of functional variants of ntcp among asian populations hepatocyte nuclear factor- alpha is a central transactivator of the mouse ntcp gene sodium taurocholate cotransporting polypeptide (slc a ) deficiency: conjugated hypercholanemia without a clear clinical phenotype hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice wheat germ cell-free system-based production of hemagglutininneuraminidase glycoprotein of human parainfluenza virus type for generation and characterization of monoclonal antibody development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen www.oncotarget.com we thank naohito nozaki for antibody production and haruka sato, kyoko ohnishi, and sho nonoyama for their technical assistance. the authors declare that they have no competing interests. this work was supported in part by an amed grant-in-aid for the program on the innovative development and the application of new drugs for hepatitis b (jp fk to ar and jp fk to km), the creation of innovation centers for advanced interdisciplinary research areas program (to ar), and by a gsk japan research grant (to km). key: cord- - chba ru authors: schmidt, michael g.; banks, andrea l.; salgado, cassandra d. title: role of the microbial burden in the acquisition and control of healthcare associated infections: the utility of solid copper surfaces date: - - journal: use of biocidal surfaces for reduction of healthcare acquired infections doi: . / - - - - _ sha: doc_id: cord_uid: chba ru for more than a century, healthcare has been challenged to keep environmental surfaces clean to control microbes and improve patient outcomes. however despite an annual cost exceeding ten billion dollars cleaning with disinfection has done little to reduce the incidence of healthcare-associated infections (hai). this chapter will review the scientific evidence delineating the role that the environment and healthcare workers play in the acquisition and movement of the microbes implicated in hai and how through controlling the microbial burden of the built clinical environment it is possible to mitigate the rate of hai acquisition. specifically evidence demonstrating the effectiveness of solid copper surfaces for its ability to continuously limit the concentration of bacteria found on surfaces and objects within the built environment will be reviewed in concert with a discussion of how through the mitigation of the environmental burden copper surfaces are able to concomitantly reduce the incidence of hai. insights provided by this chapter are intended to facilitate an understanding and importance of the need to use a comprehensive or systems based approach to fight healthcare associated infections. the microbes implicated in hai and how through controlling the microbial burden of the built clinical environment it is possible to mitigate the rate of hai acquisition. specifically evidence demonstrating the effectiveness of solid copper surfaces for its ability to continuously limit the concentration of bacteria found on surfaces and objects within the built environment will be reviewed in concert with a discussion of how through the mitigation of the environmental burden copper surfaces are able to concomitantly reduce the incidence of hai. insights provided by this chapter are intended to facilitate an understanding and importance of the need to use a comprehensive or systems based approach to fight healthcare associated infections. hospital associated infections (hai) continue to be a common and significant complication of hospitalization, leading to increased morbidity and mortality. it was estimated that in , there were approximately . million healthcareassociated infections, which resulted in approximately , deaths [ ] . a more recent meta-analysis of the costs and financial impact of hai on the us healthcare system reported that the total annual costs for the five major infections (central line-associated bloodstream infections (clabsi), ventilator-associated pneumonia (vap), surgical site infections (ssi), clostridium difficile infection (cdi), and catheter-associated urinary tract infections (cauti)) were $ . billion ( % confidence interval (ci) $ . to $ . billion) [ ] . there has been an unprecedented movement for healthcare facilities to improve patient safety and certainly prevention of hai represents a major portion of that effort. the process by which a patient acquires an infection while hospitalized is complex. this has been elegantly illustrated and described by dr. weinstein ( fig. . ) , highlighting the role of the patient's endogenous flora, exposure to exogenous flora, as well as the influence of devices and pressure from antibiotic use [ ] . recent development and implementation of strategies to prevent hai have included such efforts as antimicrobial stewardship, interrupting transmission of epidemiologically important organisms, and infection specific prevention bundles; however, there is renewed interest in defining the role of environmental contamination in transmission of nosocomial pathogens and development of hai. [ ] ). the complexity and dynamic nature of the microbial pressure being introduced into the built clinical environment is dependent on stochastic nature inherent to healthcare the purpose of this chapter is to review the role of the environment of care as it pertains to microbial contamination and risk of hai to patients as well as describe the novel use and efficacy of antimicrobial copper surfaces in mitigating this risk. we will discuss problematic pathogens in healthcare, their ability to contaminate and persist in the environment, their ability to contaminate the healthcare provider, and ultimately their ability to directly or indirectly result in colonization and infection in the patient. we will briefly review the traditional measures utilized to reduce the microbial burden associated with the healthcare environment but focus our discussion on the use of continuously active antimicrobial solid copper for this purpose. given that, we describe the proposed mechanism of action for copper's antimicrobial property, its activity against pathogens commonly found in healthcare, as well as the clinical efficacy of placing solid copper surfaces into the patient care environment. the majority of healthcare associated infections are thought to occur via transmission from the patient's own endogenous flora. however, there is increasing evidence that there exists significant transmission of microbes from healthcare personnel and the hospital environment to vulnerable patients. a study published in estimated that the causative source of an hai in the intensive care unit (icu) was the patients' endogenous flora - % of the time and antibiotic driven changes in flora - % of the time. cross-infection via the hands of personnel accounted for - % of cases and other sources, including contamination from the environment, accounted for the remaining % [ ] . it has been established that the inanimate hospital environment can become contaminated with nosocomial pathogens after exposure to colonized patients [ ] . this environment includes surfaces within the hospital room (bedrails, bedside tables, etc.) and medical equipment. a review of the available literature in concluded that personal and environmental hygiene reduced the spread of infections [ ] . more recent literature has provided additional evidence that contaminated hospital surfaces are a source of transmission of nosocomial pathogens [ ] . otter and colleagues delineated the continuous, omni-directional and complex nature of how microbes can easily move between infected or colonized patients, healthcare workers, and objects resident in the built environment (chap. and ref [ ] ) ( fig. . ). microbes have an innate ability to contaminate and potentially establish residence on any surface. surfaces with frequent hand contact and in close proximity to the patient are often colonized with nosocomial pathogens, and most of these pathogens can remain viable on these inanimate surfaces for weeks to months (chap. and ref [ ] ). further, the distribution and dispersal of the microbes from healthcare workers, visitors and patients can contribute to the resident microbial flora of the built environment. humans shed a minimum of ten million of their million skin cells per day. routine activities such as walking can result in the loss of approximately skin particles per minute with a complete layer of skin cells being lost and replaced from healthy individuals on average approximately every days [ ] . the displaced skin cells are covered with the endogenous flora of the individual. not all individuals shed skin equally. in one study of microbial dispersal by skin in a hospital ward, noble defined a 'staph aureus disperser' as a patient who contributed greater than six s. aureus per cubic meter of air [ ] . given that the mean concentration of bacteria within the ward was the equivalent of viable bacteria per cubic meter, the concentration of s. aureus observed was thought to represent % of the total flora [ ] . over the years the number has been revised to suggest that an individual is a 'staph aureus disperser' when they are able to disseminate more than four viable particles per microbe per cubic meter of air [ ] . causality, or the linkage of an environmental isolate to that organism responsible for disease in individuals has been demonstrated as early as when deforest and kerr reported cases of eczema which occurred amongst nurses that were caused by streptococci that were shed [ ] . with the advent of molecular techniques, such as pulsed field gel electrophoresis (pfge) and whole genome sequencing, the ability to demonstrate casualty has now become much more straightforward but is still nevertheless time intensive and cost prohibitive. once established within the built environment the microbe must then be able to resist the perturbations introduced as a consequence of cleaning and other infection control measures. to that end, some pathogens have become resistant to certain factors must be met for a microbe to transition from its role as an inhabitant of the surfaces associated with the built clinical environment to pathogen that can be transmitted to a patient or healthcare worker. first, the pathogen must be able to survive on the objects and surfaces within the environment for a sufficiently long period of time while retaining its ability to be virulent or its ability to colonize a susceptible host after its subsequent liberation from the surface and resulting transmission/establishment. second, contamination of the environment by a particular pathogen must be sufficiently frequent to account for its loss from the object or surfaces as a consequence of routine cleaning, desiccation, or starvation. third, the agent must be present at a concentration sufficient to establish itself upon encountering the new host or location. certain nosocomial pathogens, such as norovirus, have incredibly small infectious doses with a median dose of viruses [ ] while the environmental dose of the causative agent of the majority of cauti, escherichia coli, is not as evident. the infectious disease society of america (idsa) has classified that in the absence of symptoms a concentration of colony forming units (cfu) per ml coupled where bacterial species is present in the urine of a catheterized patient that the individual has an asymptomatic catheter associated bacteriuria (ca-asb) [ ] . a cauti is defined as the "presence of symptoms or signs compatible with urinary tract infections (uti) with no other identified source of infection along with cfu/ml of bacterial species" from a catheterized or previously catheterized ( h) urine sample [ ] . however the guidelines are silent as to the origin and/or concentration of the microbe(s) required to establish the ca-asb or cauti. the concept of infectious dose from the environment as it pertains to nosocomial infection has not been rigorously studied for the majority of the hai. further study is warranted. hospitals have put into place measures in an attempt to decrease the contamination or likelihood of colonization of healthcare workers with infectious pathogens. focus has been placed on increased hand hygiene, contact precautions, and enhanced environmental cleaning. in pittet and colleagues presented an evidence-based model arguing for improved hand hygiene practices during patient care as being the most important method for preventing hai and spread of antimicrobial resistant pathogens [ ] . in their model, five steps are required for the transmission of pathogens within the clinical care setting. collectively, the model considers the microbes and their transmission from objects, healthcare workers, and patients to the next individual or object. the first step requires that the microbe be present or resident on the patients'/healthcare workers' skin or immediate environment. the concentration of the nosocomial pathogen can vary from as few as to over cfu per cm . subsequently, the microbe must be transferred to the healthcare worker. simple acts such as lifting a patient, obtaining a blood pressure, pulse, or assessing a temperature can easily result in the transfer of between and , cfu of a common gramnegative pathogen klebsiella spp. [ ] . in fact these authors learned that % of the staff of an intensive care unit were found to have klebsiella contaminating their hands when screened and that the serotypes were related to those isolated from infected or colonized patients within the icu on the same day [ ] . further advancing the importance of hand hygiene was a study that found healthcare workers were as likely to contaminate their hands or gloves from commonly-touched environmental surfaces as from direct contact with colonized patients [ ] . the third aspect of the model is dependent upon the biology of the microbe. some microbes can survive for longer periods of time on hands than others. epidemic and non-epidemic strains of e. coli and klebsiella spp. were found to have significantly different survival times [ ] supporting the argument that bacterial properties other than the survival of a typed strain under defined conditions may contribute to the ability of a microbe to be easily transmitted and retained within healthcare setting. in other studies workers found that bacterial colonization of the hands of healthcare workers progressively increased with time [ , ] . in these two studies they found that the concentration of commensal and pathogenic flora increased as a consequence of patient care. additionally, the authors reported that the dynamics of hand contamination were independent of whether or not the healthcare worker was working while gloved or ungloved [ , ] . such an establishment of causality in the development of hai, and an intrinsic ability to survive on the hands of the healthcare workers, provides strong support for a role for hand hygiene for limiting the incidence and controlling the spread of hai. the fourth and fifth aspects of the model advanced by pittet and colleagues addresses the issue of defective and/or absent hand cleansing and how it can lead to the cross transmission of the microbes [ ] . here they have raised the issue of the need to microbiologically validate proper hand cleansing in order to control the spread of microbes regardless of their source. in citing a study by sala and colleagues, they describe how an outbreak of norovirus was traced to an infected food handler within a hospital cafeteria. here the implicated foodstuffs consumed during the outbreak were handmade by the infected worker [ ] . independently, it has been shown that norovirus contaminated fingers can sequentially transfer this virus to up to seven surfaces [ ] . sequential transfer is not only confined to human to surface transfer. in the same study, the virus was found to move from contaminated cleaning cloths to clean hands and surfaces [ ] . recently, snitkin and colleagues used whole genome sequencing to track an outbreak of carbapenem-resistant klebsiella pneumonia that occurred at the u.s. national institutes of health clinical center where they learned that despite early implementation of infection control procedures, including aggressive hand hygiene controls, the microbe persisted in the environment [ ] . consequently, the built environment can serve as a reservoir from which clean hands can serve as a source of hai. when a patient is known to be colonized or infected with a transmittable pathogen, dedicated equipment (i.e. stethoscopes) should be used when possible along with other personal protective equipment such as gowns, gloves and masks. frequently touched hospital surfaces and medical equipment, such as doorknobs, bed rails, faucet handles, and intravenous (iv) poles, have been identified as reservoirs of pathogenic microbes [ , ] . in addition to medical equipment and healthy or intact skin, there have been reports of the transfer of bacteria to the gloves and gowns of healthcare workers after patient contact [ , , , ] . specifically, morgan and colleagues reported that the transfer of multi-drug resistant bacteria (mdr) to the gowns and gloves of healthcare workers occurred after routine contact, and that this was found to increase as environmental contamination increased [ ] . the intent of the study was to evaluate the differential rate of contamination by a mdr variant of acinetobacter baumannii compared with other mdr bacteria while attempting to understand the importance of environmental contamination in the transfer of mdr bacteria to personal protective equipment (ppe, (gowns and gloves)) of healthcare workers. here the microbe most frequently recovered was the extremely recalcitrant multidrug resistant variant of a. baumannii. most striking however, were the conclusions that resulted from the modeling of their data. here a positive environmental culture was found to be the strongest risk factor associated with the contamination of the clothing of the healthcare worker by mdr bacteria (odds ratio (or) . ; % ci . - . ) [ ] . other independent variables, such as presence in the patient's room for greater than min (or . ; p ¼ . ), performing a physical examination (or . ; p ¼ . ) or contact with a ventilator (or . ; p ¼ . ) were similarly significant in raising the likelihood or risk of transfer of mdr bacteria but at rates lower than the rate observed for a positive environmental culture [ ] . intuition would suggest transfer was greater when interacting with a patient. however, the higher risk associated with a positive environmental culture serves to reinforce the importance that the microbial burden of the built clinical environment represents to the set of circumstances required for colonization and infection of patients while hospitalized. [ ] . in another study where the environments of patients colonized or infected with vre were evaluated upwards of % of the environmental samples collected were found to harbor vre [ ] . the samples included patient gowns, medical equipment used for care, as well as environmental surfaces [ ] . controlling the spread of vre to subsequent room occupants is challenging in that this microbe can be resistant to the disinfectants used for routine and terminal cleaning; even the use of bleach-based products have been reported to fail in their ability to eradicate the microbe from surfaces [ , ] (see also chap. ). independent of cleaning, the issue of transference of pathogens from the environment to subsequent occupants can be inferred from studies demonstrating the long-term survival of the microbes on surfaces within the built environment. mrsa and other nosocomial pathogens, including vre and c. difficile can survive for months on dry surfaces (chap. and ref [ ] ). mrsa has been documented for its ability to survive within hospital dust for up to a year [ ] . further, frequently touched hospital surfaces, such as doorknobs, have been implicated as reservoirs from which pathogens can be routinely recovered and thus transferred [ ] . mrsa, like vre, is ubiquitous in the hospital environment, especially in the vicinity of patients known to be colonized or infected [ , ] . the chief method of spread is poor compliance with infection control measures, such as hand hygiene, by healthcare workers. several studies have described endemic and epidemic contamination of the environment with mrsa. a recent review by dancer and colleagues found that the site contamination mean for common objects in the patient's room with mrsa was %, with high percentages found for such surfaces as overbed tables ( %), bed rails ( %), and other furniture ( %) [ ] . the risk of acquiring mrsa or vre by a patient being admitted into a room that was previously occupied by a patient known to harbor mrsa or vre was described by huang and colleagues [ ] . the added risk of acquisition of mrsa to the , 'eligible' patients examined by their study was found to increase by an adjusted odds ratio of . (p ¼ . ). specifically, amongst the patients whose prior room occupant was mrsa positive (n ¼ , ), . % of this cohort acquired mrsa, while only . % of the patients who occupied a room previously housing a mrsa negative patient (n ¼ ) acquired mrsa. a similar risk profile of acquisition of the drug resistant microbe was similarly observed with vre. here . % of patients who occupied a room that previously housed a vre positive patient (n ¼ , ) developed vre while the infection rate in patients housed in rooms previously occupied by a vre negative patient (n ¼ , ) had an attack rate of . % (adjusted odds ratio of . ; p ¼ . ). the authors concluded that acquisition from previous occupants accounted for % increased odds of transmission of mrsa and vre strongly suggesting a role for environmental contamination, despite room cleaning methods that exceeded the national standard [ ] . a review of the topic of the risk of nosocomial pathogen acquisition from prior room occupants was recently published [ ] . here otter and colleagues reviewed the increased risk associated with other mdr microbes. again the trend was the same. patients who occupied rooms where the former patient was infected or colonized with pseudomonas aeruginosa or a. baumannii [ ] , or c. difficile [ ] resulted in a similar increase risk of acquiring the previous occupants pathogen. a study in showed that a prior room occupant with a cdi was a significant risk factor for cdi acquisition by the subsequent occupant [ ] . this spore-forming anaerobic bacterium can survive for many months on hospital surfaces and is recalcitrant to usual cleaning methods [ ] . studies have shown very high environmental surface contamination rates, particularly in areas within close proximity to the patient. in a trial conducted in france, approximately % of healthcare workers who were caring for patients with a cdi were found to have c. difficile spores associated with their hands [ ] . the authors concluded that contamination of the hands was positively associated with exposure to fecal soiling and lack of glove use. several gram-negative nosocomial pathogens, such as p. aeruginosa and a. baumannii, increasingly associated with multi-drug resistance, have similarly been recovered from high touch surfaces such as beds, tables, and infusion pumps [ ] . outbreaks, thought to have occurred because of patient to patient spread of mdr gram negatives, can be devastating to patients and hospitals, resulting in high numbers of cases and high morbidity and mortality. responses have included robust and aggressive approaches towards infection control often including enhanced environmental cleaning and in extreme cases closure of the affected unit or substantial areas of the hospital [ , , ] . fortunately, the majority of the clinically relevant gram-negative microbes associated with the built clinical environment are not viable after drying. half-lives routinely encountered are h or less [ ] . an emerging nosocomial fungal pathogen, which has become a common cause of central line associated bacteremia in healthcare, is candida albicans. there are fewer studies documenting the extent of environmental contamination with fungi; however, c. albicans has been shown to be able to survive anywhere from days to up to months on inanimate surfaces [ ] . the majority of candida infections are likely from endogenous sources. however, through molecular typing, evidence of transmission via environmental sources has been suggested; identical strain types were recovered from patients infected with candida and from hospital surfaces from the rooms of the affected patients [ ] . there are several classes of pathogenic viruses that can be found on hospital surfaces. respiratory viruses such as influenza, coronavirus, and rhinovirus can persist on surfaces for a few days [ ] . viable influenza virus can be transferred from surface to skin, leading to the potential transfer to patients [ ] . gastrointestinal tract viruses, such as rotavirus and astrovirus, can persist for around months [ ] . rotavirus is a well-known cause of gastrointestinal illness outbreaks, especially in day care centers where it is spread through contamination of toys [ ] . norovirus has been shown in several studies to be consistently transferred to frequently touched sites in a hospital, such as door handles and telephones [ ] . closure of units and deep environmental cleaning similar in scope, time and expense seen with mdr-gram negative outbreaks are often needed to control norovirus outbreaks in hospitals. in summary, since the seminal paper by weinstein in , substantial evidence implicating the environment as a continuous source of risk for the acquisition of hai has accumulated to such an extent that there now exists significant interest in learning how to manage and provide best-practice applications for infection control for hospitals [ , , , ] . evident from the previous discussion, microbes have an intrinsic ability to survive and ultimately colonize common touch surfaces where acquisition and transport from surfaces to humans is common. healthcare workers have the potential to transfer these microbiological contaminants not only from patient to patient but amongst themselves and back to surfaces, refreshing or adding to the complexity of the microbial reservoir involved in transmission. there have been many studies looking at the control of contamination of common hospital touch surfaces both from hand to surface contact and vice versa. investigators have shown that the gloves of nurses frequently collected viable mrsa after touching inanimate objects near colonized patients [ ] . in concert with aggressive hand hygiene campaigns recent hygiene guidelines specifically recommend that particular attention be paid to the disinfection of patient-care surfaces, especially surfaces designated "high touch objects" (htos) as a target of infection prevention and control [ ] . the guidelines note that such objects could potentially contribute to secondary transmission by contaminating hands of healthcare workers (hcws) or by contacting medical equipment that subsequently contacts patients [ , , , , , , ] . routine or daily cleaning coupled with cleaning immediately after patient discharge (terminal cleaning) of the surfaces and objects within the room with subsequent application of a hospital grade disinfectant has been an accepted method for controlling and limiting the spread of infectious agents [ ] . a concentration of between . and aerobic cfu per square centimeter has been proposed as the benchmark where bacterial levels below this value are considered to represent a minimum of risk while concentrations greater are suggestive of an increased risk of hai acquisition [ , ] . no touch solutions for the disinfection of at-risk environments within healthcare settings are quickly gaining acceptance as technologies that have been found to be an effective and comprehensive addition to systems-based solutions for infection control. the technologies have been studied in concert with aggressive hand hygiene campaigns, appropriate routine and terminal cleaning of patient care environments, and an active surveillance and isolation protocol for patients entering care who are already colonized with vre, mrsa, c. difficile or other multi-drug resistant microbes such as klebsiella pneumoniae carbapenemase (kpc). as a consequence of this, one is left to wonder whether or not the antimicrobial effectiveness is providing an additive effect or whether the antimicrobial effectiveness of these 'no-touch technologies' are acting synergistically. as the name suggests, no-touch technologies do not come in direct contact with colonized, contaminated or soiled surfaces. rather, they distribute their microbiocidal activity through the atmosphere by either delivering a lethal concentration of electromagnetic energy in the ultraviolet spectrum or by the real-time distribution of reactive oxygen species, such as hydrogen peroxide, singlet oxygen, hydroxyl radical or oxyanions. in general both systems have been found to effectively reduce the concentration of microbes by at least logs [ ] . both systems have their limitations (see chap. ). each requires skilled labor to place the equipment and commence the disinfection cycle in the location subjected to disinfection. the disinfection reach of ultraviolet light is subject to the effects of shadowing and 'cornering'. this typically requires that the equipment be placed in the center of the room to insure uninform distribution of the lethal ultraviolet energy. additionally, the room must be vacant and any associated ultraviolet energy need be prevented from leaking into areas occupied by people as the ultraviolet (uv) light energy can damage eyesight and result in skin burns. the energy can also shorten the life of equipment in the room as routine exposure to uv light can accelerate decay by increasing the brittleness of many of the plastics used in the fabrication of healthcare associated equipment. the use of an automated uv-c light emitting system for the inactivation of vre, c. difficile and species of acinetobacter has been found to be effective in debulking the built environment of these pathogens. in one study, employing an automated emitter in two hospitals, the concentrations of bacteria were reduced for all of the environmental sites tested and occurred regardless of whether the sampled location was in direct or indirect line of sight of the uv source [ ] . further, the extent of the reduction to the microbial burden was found to be significant for vre and c. difficile but not acinetobacter spp. [ ] . however, the data were sufficiently compelling to lead the authors to conclude that the use of an automated uv-c no-touch disinfection device can lead to a decrease in the bioburden of important nosocomial pathogens in 'real-world' active clinical environments [ ] . another multi-hospital intervention used a pulsed xenon based uv delivery mechanism in concert with screening and hand hygiene education, together, the three were able to significantly reduce ( %, p ¼ . ) the incidence of hospital associated mrsa infections in the study population [ ] . given that this was a bundled intervention the contribution of the individual components of the bundle cannot be discerned. however, the data do reinforce the common belief that any effective infection control program requires a systematic approach in order to be effective. as early as vapor phase hydrogen peroxide (hpv) has been advocated as an effective surface decontaminant and sterilant [ ] . in the intervening years a number of devices have been developed to deploy this disinfectant/sterilant as a vapor into the built clinical environment. in one study conducted by passaretti and others, an evaluation of the environmental and clinical impact of this no-touch technology was assessed [ ] . in a month prospective cohort intervention trial involving high risk units from a bed tertiary care hospital, they learned that patients admitted to rooms decontaminated using hpv were % less likely (p < . ) to acquire any multidrug resistant microbe and % less likely to acquire vre (p < . ) after adjusting for other factors [ ] . again, the complexity inherent to the transmission and distribution of microbes within the built environment, coupled with the stochastic nature of care, well illustrates that the risk of acquiring c. difficile, mrsa, and multidrug-resistant gram-negative rods were reduced, but failed to reach significance. however, in spite of the failure to reach significance the effectiveness of this no-touch infection control solution was able to significantly alter the proportion of rooms environmentally contaminated with mdrs. here the concentration of mdrs in the hpv treated units were significantly reduced (relative risk, . , p ¼ . ), but not on non-hpv treated units leading the authors to conclude that the use of hpv can reduce the risk of acquiring mdrs compared with standard cleaning protocols [ ] . in spite of the success demonstrated here and in other studies [ , ] vaporphase disinfection of the built environment has limitations in that the ventilation to the room must be controlled/and or limited for the duration of the disinfection cycle. this time can vary depending upon the concentration of peroxide or disinfecting gas used. these two technologies, hpv and uv, have been found to be effective for the disinfection of inanimate objects and surfaces. however, neither technology is intended as a substitute for cleaning or for the removal of soil from the resident objects and surfaces within the built patient care environment (see also chap. ). an appropriately trained environmental service team must accomplish cleaning, with subsequent disinfection of the built environment. recently, we have begun to witness the incorporation of another 'no-touch' technology. however, unlike uv and vapor phase oxygen radicals (h o ) that distribute their antimicrobial activity through the atmosphere, this technology requires the microbe come in contact or be in close proximity with the material in order to facilitate its antimicrobial activity. in contrast to uv and hvp, once placed, this no-touch system simply requires that the fugitive microbe come in contact with the surface in order to effect disinfection. thus, the inactivation or killing of the microbe does not require any user intervention once deployed. one such example of this type of no-touch technology is solid antimicrobial copper. the resident microbial burden associated with the built environment is continuously reduced through the strategic placement of solid copper surfaces onto critical high touch surfaces within the patient care setting [ ] . copper has been used by humans for millennia, first as tools and then as a measure to fight the spread of infectious agents. metallic copper intrinsically displays a strong antibacterial activity in aquatic systems [ , ] as well as on dry surfaces [ , , , , ] . in the united states environmental protection agency (epa) registered five families of copper-containing alloys as antimicrobial, establishing that products manufactured from one of these registered alloys can make public health claims wherein the label indication states that the alloys kill greater than . % of bacteria within h of exposure [ ] . it is anticipated that the solid antimicrobial copper surfaces will remain microbiocidal for the life of the product (> years). a variety of controlled studies have looked at the antimicrobial activity of copper surfaces against specific human pathogens [ , , , , , , ] . in fact solid copper surfaces have been found to be microbicidal to well over bacteria, fungi and viruses. of the microbes listed in table . , five were evaluated in the studies used to grant the public health registration by the united states epa. the public health claims granted illustrate the robust nature of the antimicrobial activity. alloys granted registration contain greater than % metallic copper and were found to continuously kill greater than . % of gram-negative and grampositive bacteria within h of exposure even after repeated contamination illustrating how solid copper surfaces will inhibit the buildup of microorganisms between routine cleaning and sanitizing steps. the public health claims attributed to solid copper have been evaluated to limit the bacterial burden found on commonly touched surfaces and objects in active healthcare environments. in a recent hospital trial bacterial reductions up to one third were recorded using copper alloys in place of plastic or aluminum surfaces on light switches, door knobs and push plates [ ] . casey and others [ ] observed a median microbial reduction of between and % (log . - . ) on copper surfaced push plates, faucet handles, and toilet seats while schmidt and colleagues demonstrated significantly lower bacterial burdens on six htos, averaging an % (log . ) reduction for all of the objects over the course of a month multi-center trial [ ] . current cleaning methods can effectively remove pathogens from surfaces but studies have shown that more than half of the trial surfaces were not adequately terminally cleaned, and became re-contaminated within minutes [ , ] . the rails of hospital beds, as a consequence of coincident interactions with patients, hcws, and visitors are one of the most frequently touched items found in the built patient care environment. schmidt and colleagues found when they quantitatively assessed the bacterial burden present on bed rails that, through the surfacing of the rail with metallic copper, the concentration of bacteria resident on this frequently touched surface was continuously at or below the threshold representing a risk of transfer regardless of whether or not the surface was measured before or after routine cleaning [ ] . further, the environmental monitoring of bed frames has consistently shown that the rails of hospital beds typically exceed a suggested threshold of risk more than any other object in the patient's room [ , , , , ] . it was evident that bed rails covered with solid copper are able to augment cleaning and thereby continuously support the control of the concentration of associated aerobic bacteria. this observation was consistently maintained in spite of the kinetic nature of care present in the environment of the icu. lower risk concentrations, less than . cfu/cm , were associated with over % of the sampled beds [ ] . further, mrsa and vre were absent from all but of the , copper objects sampled arguing that the risk mitigation provided by copper surfaces might be greater than the average concentrations reported suggest [ ] . weber and rutala [ ] in their commentary of the evaluation of no-touch copper conducted by karpanen and colleagues argued that it was impractical or impossible to coat each of the environmental surfaces with copper [ ] . however, the data provided by schmidt and colleagues suggest that the strategic placement of solid copper surfaces in high touch areas is key, and offers a novel strategy to limit the bacterial burden on a continuous basis [ ] . copper-alloyed surfaces offer a continuous way to limit and/or control the environmental burden. hospital and environmental services need not perform additional steps, follow complex treatment algorithms, obtain "buy-in" from other providers or require additional training or oversight. the other 'no touch' methods presently in wide scale use for room disinfection rely on discontinuous modalities of application in order to reduce the environmental bacterial burden [ ] . hydrogen peroxide vapor is introduced as a gas into a sealed room. ultraviolet light achieves its effectiveness through the transient transmission of germicidal radiation within an unoccupied room. consequently, like the epa registered disinfectants regularly used to disinfect patient rooms subsequent to cleaning, both uv and hpv will likely suffer from the same limitations of the rapid restoration of the bacterial burden intrinsic to high touch objects. in addressing the question of whether or not the strategic placement of copper might ameliorate the rate with which hai are acquired, salgado and colleagues [ ] found from the conduct of a multi-center trial that the limited placement of copper as described by schmidt and colleagues [ ] resulted in a significant reduction to the hai rate and/or mrsa or vre colonization rate in medical intensive care rooms (icu). the collective rate for hai infection or mrsa/vre colonization was found to be significantly lower by % ( . %) in the copper arm of the study when compared against the ( . %) rate observed in the control rooms (p ¼ . ). when the data were considered separately for hai alone, the rate of infection was significantly reduced ( %) from . to . % (p ¼ . ). more importantly, these investigators were able to demonstrate that burden and infection were directly linked. in the analysis of the quartile distribution of hais stratified by microbial burden measured in the icu rooms during the patient's stay they learned that there was a significant association between burden and hai risk (p ¼ . ), with % of hai occurring among patients cared for in a room with a burden of more than cfu (fig. . ) [ ] . the mechanism of action associated with the antimicrobial properties of solid copper surfaces is multifaceted (chap. ). upon coming in contact with the metallic copper surfaces of objects, the electron potential of the microbe in concert with copper facilitates a cascade of irreversible events leading to the rapid death of the bacterium. given the inherent ability of solid metallic copper and its alloys containing greater than % copper for the conduction of electricity, the electrons resident in the membrane of the bacterium that are sufficiently close to the metallic surface coupled with the high flux required by living cells result in the rapid collapse of the proton motive force of the microbe. the subsequent dissipation of the proton motive force (pmf) has been observed through the use of dyes that measure the membrane potential. warnes and others have reported on this observation on numerous occasions for both gram positive and gram negative bacteria [ ] [ ] [ ] ] . subsequent to the collapse of the membrane potential a concomitant production of free radicals immediately develops within the cytoplasm of the bacterium. the free radicals facilitate the peroxidation of the membrane, bleaching of cellular proteins and the cleavage and subsequent complete destruction of the nucleic acids resident in the cytoplasm of the effected microbes. additionally upon peroxidation of the membrane there is a loss of membrane integrity resulting in the subsequent leakage of the cytoplasm from the cell and diffusion mediated transport of copper ions into the cytoplasm. the copper ions then act in concert with the free radicals resulting in a fenton reaction that leads to further irreparable damage to the cell [ ] . the entire process occurs quickly resulting in the collapse of a population within minutes. thus, the likelihood that the population will develop resistance to this multifaceted mechanism of death is unlikely. there have been reports in the literature of bacteria being isolated from copper coins but upon challenging the 'resistant' isolates they were found to be uniformly sensitive to metallic copper [ ] . a likely explanation for their recovery from the surface is likely a consequence of a failure to sufficiently collapse the pmf of the entire community as either a function of proximity of the surviving microbes to the metallic surface or the absence of sufficient electron flux through the membrane to initiate the cascade required for death. in the study conducted by salgado and colleagues, six highly touched objects within the icus were selected based from a limited survey where the contact surfaces being the most highly contaminated were identified. the six items were then fabricated from a variety of antimicrobial copper alloys, where the criteria for alloy selection were reflective of the ability of the antimicrobial alloys to be readily fabricated into that particular component ( fig. . ) . properties of strength and durability were operationally defined such that the resulting component would be able to withstand the rigors placed on the finished goods within the built environment of an active clinical setting and for the ability of the materials to withstand standard hospital cleaners, including sodium hypochlorite. additionally, the surface finish was to provide consistent wear and aesthetics over the lifespan of the product. all of the copper alloys used for component fabrication were made from solid alloys registered with the epa [ ] . subsequent to the published report, manufacturers have introduced numerous products fabricated from epa registered solid copper that meet or exceed the criteria used by the referenced authors [ , , ] . from a design standpoint, it is important to note that these results were 'additive' to other infection-control implementations already in place. single patient rooms, hand-washing sinks, hand sanitizing alcohol dispensers, contact precautions required of mrsa and vre carriers/infected patient(s), and an active hand hygiene staff education program were already in place in the units of the hospitals studied. should these conclusions expand to other areas of the hospital, then employing inherently antimicrobial surfaces could represent a significant enhancement to mitigating infectious bacteria within hospitals. for example, by instituting a 'best practices' approach that implemented cleaning and hand hygiene designs and protocols, the california's healthcare-associated infection prevention initiative showed a reduction of hai by . %. with many of these best practices already in place, the initial findings from the clinical trials are showing an additional double-digit reduction in infections. although the relative infection rate in the medical icus where the clinical effectiveness of antimicrobial copper surfaces were evaluated is generally higher than hospitals at large, patients in icus are typically not mobile, and their interaction with the built environment is very limited. consequently, items where antimicrobial copper alloys might have been easily incorporated, e.g. grab bars, sinks, faucets, paper dispensers, shelves and towel racks were not present. the further evaluation of antimicrobial copper surfaces is warranted beyond the medical icu to include, but not be limited to, the effect of inherently antimicrobial materials in fig. . epa registered antimicrobial cooper alloys used in the fabrication or surfacing of high touch items. items were fabricated from a variety of epa registered antimicrobial copper alloys as listed. the criterion used to select an alloy was reflective of the ability of the antimicrobial alloy to be readily fabricated into that particular component and withstand the rigors of healthcare general wards where patients have greater interaction with other objects in the built environment. similarly, investigations should also be conducted in emergency and recovery rooms, in hospital rehabilitation units, pediatric and neonatal units, dialysis centers, burn units, transplant units and cancer centers with immunecompromised patients. at issue is the central theme that antimicrobial copper surfaces continuously and passively limit the concentration of bacteria within the built environment. salgado and colleagues were able to demonstrate that infections were correlated with burden. thus, other healthcare environments that may arguably receive less day-to-day hygienic oversight than hospital patient rooms, such as visiting area, long-term care facilities, long-term rehab centers, outpatient clinics and elder care facilities should also be investigated as they too may directly benefit from the antimicrobial activity of copper. the study of pathogen transmission in the hospital and the impact of colonization and infection with nosocomial organisms have established the epidemiologic importance of the environmental microbial burden associated with the built clinical environment. these studies have outlined the complexity of this concept and have led to robust recommendations for infection prevention that have undoubtedly prevented undo morbidity and mortality. however, with renewed interest and study the risk contribution provided by the built environment towards patient care warrants a better understanding of the dynamics of colonization and infection. through our discussion here we hope that we have been able to identify potential avenues for improvement with adjunctive use of newer technologies. these include the use of uv light and hpv disinfection, and the potential value of the use of solid antimicrobial copper surfaces. through a multifaceted and continuously active mechanism of action, solid copper surfaces placed in key locations within the patient room can significantly reduce the overall microbial burden; have demonstrated their ability to continuously maintain this concentration at a level representing a minimal risk for hai acquisition and most importantly, have translated meaningful benefit to patients by their association with significant reduction of hai. further study to identify the optimal amount of copper surfaces needed as well as the optimal placement in rooms and areas within healthcare facilities is necessary to fully understand the potential impact. what is the evidence for a causal link between hygiene and infections? sub-lethal effects of several metallic salts organic compound combinations upon the heterotrophic microflora of a natural water decontamination of targeted pathogens from patient rooms using an automated ultraviolet-cemitting device intrinsic bacterial burden associated with intensive care unit hospital beds: effects of disinfection on population recovery and mitigation of potential infection risk environmental contamination during a carbapenem-resistant acinetobacter baumannii outbreak in an intensive care unit quantifying bacterial transfer from patients to staff during burns dressing and bed changes: implications for infection control effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces acquisition of nosocomial pathogens on hands after contact with environmental surfaces near hospitalized patients shedding of staphylococcus auerus by human carriers environmental contamination due to methicillin-resistant staphylococcus aureus (mrsa) fighting nosocomial infections with biocidal non-intrusive hard and soft surfaces environmental contamination makes an important contribution to hospital infection vancomycin-resistant enterococcus. detection, epidemiology, and control measures impact of hydrogen peroxide vapor room decontamination on clostridium difficile environmental contamination and transmission in a healthcare setting guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/ shea/apic/idsa hand hygiene task force environmental contamination due to methicillin-resistant staphylococcus aureus: possible infection control implications evaluating hygienic cleaning in health care settings: what you do not know can harm your patients effect of single-versus doublegloving on virus transfer to health care workers' skin and clothing during removal of personal protective equipment hands as route of transmission for klebsiella species role of copper in reducing hospital environment contamination impact of environmental decontamination using hydrogen peroxide vapour on the incidence of clostridium difficile infection in one hospital trust how do we assess hospital cleaning? a proposal for microbiological standards for surface hygiene in hospitals importance of the environment in meticillin-resistant staphylococcus aureus acquisition: the case for hospital cleaning the role of environmental cleaning in the control of hospital-acquired infection carbapenem-resistant acinetobacter and role of curtains in an outbreak in intensive care units a case of eczema as a source of a streptococcal epidemic investigation and management of an outbreak of multidrugcarbapenem-resistant acinetobacter baumannii in cambridge antimicrobial activity of copper surfaces against suspensions of salmonella enterica and campylobacter jejuni in: block ss (ed) disinfection, sterilization, and preservation survival on skin and surfaces of epidemic and non-epidemic strains of enterobacteria from neonatal special care units nosocomial infection with microsphere beds metallic copper as an antimicrobial surface transfer of staphylococcus aureus via nurses' uniforms comparison of the microbiological efficacy of hydrogen peroxide vapor and ultraviolet light processes for room decontamination a infectious diseases society of america ( ) diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: international clinical practice guidelines from the infectious diseases society of america contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? risk of acquiring antibiotic-resistant bacteria from prior room occupants acute copper and cupric ion toxicity in an estuarine microbial community the antimicrobial efficacy of copper alloy furnishing in the clinical environment: a crossover study vapor-phase hydrogen peroxide as a surface decontaminant and sterilant estimating health care-associated infections and deaths in u.s. hospitals how long do nosocomial pathogens persist on inanimate surfaces? a systematic review contamination of healthcare workers' hands with clostridium difficile spores after caring for patients with c. difficile infection use of audit tools to evaluate the efficacy of cleaning systems in hospitals cross-transmission of multidrug-resistant acinetobacter baumannii clonal strains causing episodes of sepsis in a trauma intensive care unit role of environmental contamination as a risk factor for acquisition of vancomycin-resistant enterococci in patients treated in a medical intensive care unit the antimicrobial activity of copper and copper alloys against nosocomial pathogens and mycobacterium tuberculosis isolated from healthcare facilities in the western cape: an in-vitro study survival of bacteria on metallic copper surfaces in a hospital trial transfer of multidrug-resistant bacteria to healthcare workers' gloves and gowns after patient contact increases with environmental contamination finding a benchmark for monitoring hospital cleanliness dispersal of skin microorganisms the dispersal of staphylococci in hospital wards inactivation of influenza a virus on copper versus stainless steel surfaces potential use of copper surfaces to reduce survival of epidemic meticillin-resistant staphylococcus aureus in the healthcare environment risk of acquiring multidrug-resistant gram-negative bacilli from prior room occupants in the intensive care unit contamination of room door handles by methicillinsensitive/methicillin-resistant staphylococcus aureus the role played by contaminated surfaces in the transmission of nosocomial pathogens evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings an evaluation of environmental decontamination with hydrogen peroxide vapor for reducing the risk of patient acquisition of multidrug-resistant organisms dynamics of bacterial hand contamination during routine neonatal care world alliance for patient safety who global patient safety challenge ( ) evidence-based model for hand transmission during patient care and the role of improved practices bacterial contamination of the hands of hospital staff during routine patient care mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces undetected vancomycinresistant enterococcus stool colonization in a veterans affairs hospital using a clostridium difficile-focused surveillance strategy influence of plumbing materials on biofilm formation and growth of legionella pneumophila in potable water systems influence of temperature and plumbing material selection on biofilm formation and growth of legionella pneumophila in a model potable water system containing complex microbial flora uses of inorganic hypochlorite (bleach) in health-care facilities guideline for disinfection and sterilization in healthcare facilities an outbreak of food poisoning due to a genogroup i norovirus copper surfaces reduced the rate of healthcare-acquired infections in the intensive care unit isolation and characterization of bacteria resistant to metallic copper surfaces chemical disinfection to interrupt transfer of rhinovirus type from environmental surfaces to hands institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission copper continuously limits the concentration of bacteria resident on bed rails within the icu sustained reduction of microbial burden on common hospital surfaces through introduction of copper characterization and control of the microbial community affiliated with copper or aluminum heat exchangers of hvac systems copper continuously limits the concentration of bacteria resident on bed rails within the intensive care unit guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) inactivation and sub-lethal injury of salmonella typhi, salmonella typhimurium and vibrio cholerae in copper water storage vessels traditional copper water storage vessels and sub-lethal injury of salmonella enterica serovar typhi and vibrio cholerae evaluation of hospital room assignment and acquisition of clostridium difficile infection impact of a multi-hospital intervention utilising screening, hand hygiene education and pulsed xenon ultraviolet (px-uv) on the rate of hospital associated meticillin resistant staphylococcus aureus infection tracking a hospital outbreak of carbapenem-resistant klebsiella pneumoniae with whole-genome sequencing antimicrobial activity of copper surfaces against carbapenemase-producing contemporary gram-negative clinical isolates contamination of hands with methicillin-resistant staphylococcus aureus after contact with environmental surfaces and after contact with the skin of colonized patients norwalk virus: how infectious is it epa registers copper-containing alloy products nosocomial candida glabrata colonization: an epidemiologic study better environmental survival of outbreak vs. sporadic mrsa isolates prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray mechanism of copper surface toxicity in escherichia coli o :h and salmonella involves immediate membrane depolarization followed by slower rate of dna destruction which differs from that observed for gram-positive bacteria biocidal efficacy of copper alloys against pathogenic enterococci involves degradation of genomic and plasmid dnas horizontal transfer of antibiotic resistance genes on abiotic touch surfaces: implications for public health inactivation of norovirus on dry copper alloy surfaces mechanism of copper surface toxicity in vancomycinresistant enterococci following wet or dry surface contact potential for preventing spread of fungi in air-conditioning systems constructed using copper instead of aluminium survival of clostridium difficile on copper and steel: futuristic options for hospital hygiene potential action of copper surfaces on meticillin-resistant staphylococcus aureus commentary: self-disinfecting surfaces role of hospital surfaces in the transmission of emerging health care-associated pathogens: norovirus, clostridium difficile, and acinetobacter species epidemiology and control of nosocomial infections in adult intensive care units antimicrobial efficacy of copper surfaces against spores and vegetative cells of clostridium difficile: the germination theory are hygiene standards useful in assessing infection risk? the survival of escherichia coli o on a range of metal surfaces survival of listeria monocytogenes scott a on metal surfaces: implications for cross-contamination health care-associated infections: a meta-analysis of costs and financial impact on the us health care system key: cord- -ycg waay authors: peng, xiaolei; rajeeva, bharath bangalore; teal, daniel; zheng, yuebing title: plasmofluidics for biosensing and medical diagnostics date: - - journal: nanotechnology characterization tools for biosensing and medical diagnosis doi: . / - - - - _ sha: doc_id: cord_uid: ycg waay plasmofluidics, an extension of optofluidics into the nanoscale regime, merges plasmonics and micro-/nanofluidics for highly integrated and multifunctional lab on a chip. in this chapter, we focus on the applications of plasmofluidics in the versatile manipulation and sensing of biological cell, organelles, molecules, and nanoparticles, which underpin advanced biomedical diagnostics. infectious diseases, which are caused by bacterial (e.g., pneumonia), mycobacterial (e.g., tuberculosis), viral (e.g., hiv), fungal (e.g., candidiasis), and parasitic pathogens (e.g., malaria), have considerable impact on global economy, health, and security [ , ] . for instance, recent years have witnessed large outbreaks of ebola in africa and zika in central and south america, and there have been serious concerns and heated debates on public health at the global scope. cancers, which are characterized by the uncontrolled growth and spread of abnormal cells and induced by various factors such as tobacco, infectious organisms, genetic mutations, and immune conditions, are the leading cause of death for much of the us population [ ] . the threats from infectious diseases and cancers become increasingly prevalent due to factors such as changing trends in human and animal migration, increasing urbanization, environmental deterioration, and climate change. early disease diagnosis and effective treatment have become critical for the clinical regulation and management of infectious diseases and cancers. convectional disease diagnostics has relied on techniques such as optical microscopy, culture, immunoassays, and nucleic acid amplification [ ] . a standard process for disease diagnostics includes the following steps: (a) collection and transport of biological samples such as blood, urine, and tissue swabs from the point of care; (b) analysis of the samples by experienced staffs in a centralized laboratory; and (c) notification of the results to the clinicians and patients. due to highly frequent access to energy resources like electricity, time-consuming procedures, and need for well-trained personnel, the conventional clinical methods prelude the rapid disease detection and response at the primary care. their drawbacks are prominently revealed in resource-limited and underdeveloped areas. point-of-care (poc) devices, which enable on-site test and follow-up action, are promising to improve the diagnosis and management of infectious diseases and cancers in various clinical settings. these include areas where healthcare infrastructure is limited and high-quality timely medical care is inaccessible [ , ] . with the advancements of nanotechnologies and micro-/nanofluidic technologies, many innovative poc biosensors with optical, electrical, and mechanical interrogations have been developed. however, two major challenges, matrix effect and system integration, have prevented the further developments and uses of poc devices [ ] . most biosensors can have excellent performance with pristine samples. however, they still need to be strictly evaluated with clinical samples due to the matrix effect. generally, the matrix of human fluids becomes more complex in infected states, which could lead to the clogging of microfluidic channels and the decrease of the transduction signals. transformation of poc devices from proof-of-concept and benchtop to bedside care and point-of-care settings requires the system integration. however, to integrate and package various modules for sample preparation and signal detection into a fully automated and user-friendly platform remains challenging. plasmofluidics, which seeks to synergize plasmonics and micro-/nanofluidics for lab on a chip, is promising for advancing the next-generation poc devices that feature high compactness, high integration, and multiple functions as well as the frontiers of biomedical research [ ] . with their capability of controlling light at the nanoscale beyond the diffraction limit, surface plasmons such as surface plasmon polaritons (spps) and localized surface plasmon resonances (lsprs) [ ] are effective at optically manipulating, sensing, and analyzing biological cells and molecules [ ] [ ] [ ] . micro-/nanofluidics exploit rich fluidic behaviors at the micro-/nanoscale to enable low-load, high-throughput, cost-effective, and precise delivery of analyte samples. therefore, plasmofluidic platforms can serve the purpose of processing, sensing, and analyzing biological objects in clinical samples, paving the way toward affordable and portable healthcare devices for their uses in primary care and resource-constrained settings. there have been several successful review articles on plasmonic biosensing and medical diagnostics [ , , ] . however, a comprehensive review article focused on the emerging field of plasmofluidic sensing is not available yet. to fill the blank, our chapter covers classification, working principles, design strategies, and applications of the state-of-the-art plasmofluidic systems for sensing and medical diagnostics. experimental methodology biosensing and medical diagnostics often rely on the capability of manipulating biological particles and molecules in fluidic environments. three prominent techniques that exploit plasmon-enhanced optical near fields to manipulate particles and molecules in fluids have been developed: (i) spp-based plasmonic tweezers, (ii) lspr-based plasmonic tweezers, and (iii) plasmonic tweezers based on selfinduced back-action (siba). both top-down fabrication techniques such as electron beam lithography and focused ion beam lithography and bottom-up fabrication techniques such as directed assembly and self-assembly have been applied to engineer plasmonic structures for targeted tweezing platforms [ , ] . spps can propagate along the metal-fluid interface with evanescent characteristics perpendicular to the interface. accordingly, spp-based tweezers have been used to transport and assemble microscale particles [ ] [ ] [ ] . a patterned spp landscape was constructed from an array of micrometer-sized gold disks on a glass substrate to achieve parallel trapping of individual particles, as shown in fig. . a [ ] . the simulated trapping potential in fig. . b shows that the trapped particle on the gold disk is stabilized in a forward position along the spp propagation direction. due to the localized electromagnetic fields associated with the excitation of lsprs, lspr-based tweezers are often applied to trap single or multiple particles near the plasmonic structures. plasmonic nanoantennas such as nanodot pairs [ ] , diabolo structures [ ] , and bowtie structures [ ] were commonly used to induce strong near-field confinement and enhancement in the nanoscale dielectric gaps known as "hot spots," which provide stable trapping of nanoparticles at low optical power. trapping stiffness was enhanced by two orders of magnitude with a metal nanodot pair in a conventional optical tweezers setup, as shown in fig. . c-d [ ] . despite the localized nature of lsprs, recent research efforts have achieved long-range transportation of particles by using arrays of plasmonic c-shaped engravings [ ] and gold nanoislands with a network of "hot spots" [ ] . lspr-based tweezers exhibit limits in manipulating particles that are smaller than nm due to the high-power requirement and significant thermal effects. sibabased plasmonic tweezers were developed to reduce the required power intensity by more than an order of magnitude [ ] . as illustrated in fig. . e, a nanoaperture redshifts in its resonance wavelength when the trapped particle has a larger refractive index than solvent, enhancing the laser beam transmission through the nanoaperture. when the particle tends to escape from the aperture, a drop in the light transmission generates a restoring force and pulls the particle back to the equilibrium position. in other words, when the particle is moving away from the optical potential well, the siba force will deepen the potential well and maintain the trapping state, as illustrated in fig. . f-h. along with the enhanced electromagnetic fields, surface plasmons can induce strong light absorption and photothermal effects [ ] . the competing factors between light absorption and heat dissipation determine the temperature increase and spatial temperature distribution. for a spherical gold nanoparticle immersed in water and illuminated by a laser of lspr wavelength, the temperature increase is uniform in the particle and inversely proportional to the distance outside the particle, resulting in a thermal gradient built up at the metal-water interface [ ] . for metal nanoparticle arrays as illustrated in fig. . a, two heating regimes exist depending on the particle size, particle number, and geometry of the array. these are temperature confinement regime and temperature delocalization regime, as shown in fig. . b [ ] . to reach the temperature confinement regime, both large spacing between nanoparticles and small illumination area are preferred. the plasmon-assisted heating was exploited to control fluid motion at the microscale and nanoscale [ ] . in typical plasmofluidic systems where the characteristic size of the plasmonic structure is very small (~ nm), thermal diffusion of the fluid dominates in heat dissipation and thermal convection plays a minor role. as shown in fig. . c-d, the thermal convection velocity at small plasmonic structures is limited to - nm/s. in this case, the thermal convection can barely contribute to the motion of the particles in the fluidic environment. the thermal convection field can be further damped by more than one order of magnitude when the height of the fluid chamber is reduced to $ μm, as illustrated in fig. . e. optofluidic biosensing platforms based on surface plasmons have advantages of being highly integrated, miniaturized, and sensitive. spp-based sensors typically interrogate the change in resonance angle in attenuated total internal reflection to detect target specimens and to extract binding kinetics, as shown in fig. . a [ , ] . the specimen-binding events change the dielectric constant over the metal layer. several approaches have been applied to excite spps, including prism coupling [ , ] , grating coupling [ ] , and waveguide coupling [ ] . lspr-based sensors utilize the strong electromagnetic response of metal nanoparticles to refractive-index changes in their surroundings [ , , , ] . various nanostructured metal arrays such as nanohole arrays [ , ] , nanowell arrays [ ] , nanocross arrays [ ] , nanocube arrays [ ] , nanomouth arrays [ ] , nanomushroom arrays [ ] , nanodisk arrays [ , ] , and nanobowtie arrays [ ] have been designed for the refractive-index sensing with high figure of merit (fom). various techniques such as focused ion beam lithography, electron beam lithography, soft lithography, and nanosphere lithography have been employed to fabricate the large-scale arrays with good repeatability [ , [ ] [ ] [ ] . of particular interest are metal nanohole arrays integrated with complex fluidic structures for versatile and parallel sensing [ , ] . on-chip nanohole array-based sensors feature simple optical instrument and high synergy with microfluidic schemes. in the flow-over mode ( fig. . b), only the upper metal surfaces of the nanohole arrays are exploited like those in the convectional spp-based sensors. through scaling analysis and numerical simulation, escobedo's group has found that the flow-through mode features~ -fold enhancement of time response over the flow-over mode in typical biosensing applications [ ] . further experiments showed that the flow-through mode enabled active delivery, concentration, and sensing of analytes, leading to one order of magnitude increase in sensing efficiency and two orders of magnitude improvement in lod [ ] . in general, a lspr biosensor consists of the following components: (i) a recognition element in conjunction with the plasmonic substrate, (ii) a transducer to convert the interaction between target and recognition element to optical or electrochemical output signals, and (iii) a system to interrogate the signals. spectroscopic measurements are the most common approach for the signal analysis, which include (i) transmission spectroscopy ( [ ] . surface plasmon resonance imaging (spri) has been developed to perform large-area measurements at high resolution [ ] . it is a label-free method of visualizing binding activities across an arrayed biochip via a video ccd camera. surface-enhanced raman spectroscopy (sers), which often arises from the strong electromagnetic field enhancement proximate to the metal surface, has proved as a powerful tool for label-free analysis of molecules. sers interrogates raman shifts originating from molecular vibrational energy levels [ ] [ ] [ ] . metal colloids, colloid aggregations in solutions, and metal nanoparticle arrays on substrates are commonly used for sers-active sensors integrated with microfluidics [ , ] . there are two major microfluidic approaches toward sers-active plasmofluidic sensors: (i) mixing the sample with sers-active colloids in a microfluidic channel [ ] . such plasmofluidic sensors have the advantages of measuring biological particles and molecules in their native watery environments. however, since the molecular adsorption or binding to the sersactive surfaces relies on the diffusion of the analytes in solutions, the raman signal intensities will be weaker than those of sers based on dried samples on solid-state raman substrates. various active and passive concentration techniques have been developed to improve the sers lod in fluidic systems [ ] . recently, we have developed a technique that uses plasmon-enhanced thermophoresis for reversible and dynamic assembly of plasmonic nanoparticles for in situ sers analysis of molecules, as shown in fig. . a [ ] . once a temperature gradient is created on a plasmonic substrate that is illuminated with a low-power laser beam, the positive-charged nanoparticles will migrate to the hot region under a thermally induced local electrical field ( fig. . b) and form nanoparticle assemblies at the laser spot ( fig. . c ). as shown in fig. . d, the dynamic assemblies feature a high particle density and small interparticle distance, which are suitable for the excitation of multiple electromagnetic "hot spots" for sers with enhanced sensitivity. the metal surface is functionalized with recognition elements for selective detection. once biological particles or molecules are captured by the metal surface, the spp mode will be modified, and a signature in the reflected light will be probed by a detector for analysis (reproduced with permission [ ] . copyright american chemical society). (b) schematic depiction of an on-chip lspr sensing platform based on metal nanohole arrays. the fluid can be transported over or through the nanoholes. the platform also includes external components such as a light source, a detector, and a fluidic actuator (reproduced with permission [ ] . copyright rsc publishing). schematics of the instrumental setups for lspr sensing that perform optical (e) transmission, (f) reflection, and (g) scattering measurements. aunt assembly manipulations of biological particles and molecules such as trapping, immobilization, and pre-concentration are becoming a critical component of biosensing and analysis in fluidic environments. plasmonic tweezers have been employed in manipulating various bio-specimens such as cells [ , ] , dnas [ , ] , and proteins [ ] [ ] [ ] at high spatial resolution and low optical power. using simple optics to create the trapping force, plasmonic tweezers can be readily incorporated into microfluidic systems to design novel plasmofluidic chips with functionalities such as single-particle trapping [ , ] , parallel trapping [ ] , co-trapping [ ] , and kinetic detection of biological objects [ , ] . one has also recognized multiple issues of current plasmofluidic systems and proposed new solutions. first, due to the near-field nature of surface plasmons and weak thermoplasmonic convection, only particles that diffuse to the close proximity to the plasmonic structures can be captured [ ] . second, isolated plasmonic nanostructures are commonly used to avoid collective heating and particle agglomeration, preventing long-distance transportation and dynamic manipulations. third, in contrast to conventional optical tweezers that are capable of three-dimensional ( d) manipulations, plasmonic tweezers are often limited to two-dimensional ( d) trapping of objects at the plasmonic structures. new solutions to these issues in the plasmofluidic systems include the adoption of optical components with a higher level of spatial control [ ] and the exploitation of loss-induced heating at the plasmonic structures [ ] . the capability of trapping fragile biological objects such as cells, dnas, and proteins in fluids is of significant importance in cellular and molecular biotechnology. e. coli cells were trapped in parallel with near-infrared lsprs on the arrays of plasmonic nanoantennas in a kretschmann optical setup [ ] . the cells were stably aligned along the antennas' long axis, and their continuous growth and division were kept over h. there was no difference in the average division time between the optically trapped cells and those outside the illuminated region. in contrast, optical tweezers have challenges in trapping the e. coli cells for their low refraction index ( . for visible light) and small dimensions (  . μm at infant stage). we invented opto-thermophoretic tweezers that can achieve light-directed versatile manipulations of biological cells at an optical power - times lower than that of optical tweezers, as illustrated in fig. . a [ ] . by harnessing the permittivity gradient in the electric double layer of the charged surface of the cell membrane, we succeeded at the low-power cell trapping with a plasmon-enhanced temperature gradient field. arbitrary spatial arrangements of cells at a resolution of nm and precise rotation of both single and multiple cells were demonstrated with an optical control system based on a digital micromirror device (dmd). optical trapping of single dna molecules and dna translocations in licl buffers were demonstrated using a solid-state plasmonic nanopore, as shown in fig. . b [ ] . the nanopore is positioned between the gap of a gold bowtie antenna. the enhancement of the rate of dna translocation events was observed and attributed to the plasmon-assisted local heating and the thermophoresis in the thermal gradient. in another example, single bovine serum albumin (bsa) molecules were trapped onto a double-hole structure with a . -mw laser beam focused by a  oil immersion objective [ ] , as shown in fig. . c. the folding and unfolding states of a trapped bsa molecule were revealed by measuring the intensity of the transmitted light. the same research group demonstrated the protein-antibody co-trapping at the double-hole structure and measured the binding kinetics of the protein-ligand interaction, opening up new avenues for studying intermolecular interactions at the single-molecule level [ , ] . transportation of objects "ideal" tweezers for biological samples in fluidic environments should be able to rapidly deliver target objects and to achieve high-resolution d trapping at any desired locations. to realize d low-power trapping and transportation of sub- nm objects over a long range, quidant's group implemented the siba-based tweezers at the tip extremity of a metal-coated optical fiber, which can be raster-scanned in all three spatial directions for dynamic manipulations and simultaneously used to collect the reflected signal and identify the different trapping regimes [ ] , as shown in fig. . a-b. the optical-fiber-based plasmonic tweezers are attractive for in vivo applications, such as trapping proteins or viruses of interest in cells. to solve the long-standing challenge of rapid and on-demand loading of objects at the plasmonic trapping sites, boltasseva's group has combined the plasmonic heating and ac electrical fields for fast delivery and trapping of nanoobjects within a few seconds [ ] . the coupling of the plasmon-enhanced temperature gradient and an applied ac electrical field induces an electrothermoplasmonic (etp) flow, which leads to fluidic motion two orders of magnitude faster than the thermoplasmonic convection and greatly increases the particle capture efficiency, as shown in fig. . c-d. with their capability of rapid manipulation and concentration of dna and protein samples, the hybrid plasmofluidic tweezers can be applied for nanoscale biosensors to improve the sensing throughput and efficiency. high-performance sensing, analysis, and diagnostics in plasmofluidic systems benefiting from a synergy between plasmonic nanotechnology and micro-/nanofluidics, biosensors based on plasmofluidic platforms are attractive for poc devices [ , , ] . they have been applied for portable sensing, analysis, and diagnostics in biomedicine and healthcare. the analytes include nucleic acids, protein, viruses, bacteria, and drugs. tremendous efforts have been made to improve lod, response speed, and accuracy of multiplexed identification. more innovative approaches are being developed to push proof-of-concept or benchtop prototypes into practical uses in directly analyzing bio-objects in body fluids for disease diagnosis. herein, we focus on plasmofluidic poc sensing for dnas/rnas, proteins, viruses, bacteria, and drugs. detection and analysis of dnas and rnas are important for disease diagnostics as specific bacteria and viruses in complex samples can be distinguished based on their unique dna/rna sequences [ ] . conventional technologies for nucleic acid sensing feature high precision and optimal lod. however, they require sophisticated instrumentation with multiple time-consuming steps, including pathogen isolation, pcr, and target identification. plasmofluidic sensors are extensively explored to achieve precise, multiplexed, and label-free detection [ , [ ] [ ] [ ] [ ] . springer et al. demonstrated a spp sensor with a four-channel flow cell (known as dispersionless microfluidics), as shown in fig. . a-b. the microfluidics suppressed the decrease of analyte concentration at the sensing area when different liquid samples were switched and thus improved the kinetic response and sensitivity [ ] . short sequences of dna ( bases) that are characteristic for e. coli cells were probed in less than min for a concentration down to fm levels, outperforming most spp sensors by one order of magnitude. sers-based plasmofluidic sensors for high-throughput detection of multiple dnas in single assays were also developed. one example is based on selfassembled gold nanoparticles (au nps) on a gold nanowire (au nw) at the presence of target dnas [ ] . raman signal was significantly enhanced by placing the dna molecules in the interstices of the gold-particle-on-wire structure. the structure was constructed by sequential incubation of au nws premodified by thiolated probe dnas in solutions of target dnas and au nps functionalized by reporter dnas with raman dyes (fig. . c) . a high selectivity is assured because only those target dnas with sequences complementary to the probe dnas and the reporter dnas can form the particle-on-wire structure for the enhanced signal of the raman dyes. the sensor was applied for quantitative detection of dna concentrations, multiplexed detection of bacterial dnas, and identification of pathogenic bacteria in real clinical samples. the results on bacteria agreed well with those obtained by conventional culture-based assays, as illustrated in fig. . d. vo-dinh's group devised a label-free dna biosensor based on molecular sentinel (ms) immobilized on a metal film over nanosphere (mfon) [ , [ ] [ ] [ ] . upon dna hybridization, the raman label at the end of the ms is separated from the mfon's surface to exhibit decreased raman signal, which works as the readout and renders high selectivity in multiplexed analysis. with the initial detection of human rsad gene [ ] and ki- dna [ ] and the multiplexed detection of ifi a and ifi l [ ] , the group developed the biosensor into a bioassay-on-chip platform and detected ssdna of dengue virus (lod~ attomoles), which is the culprit of dengue fever that plagues - million people each year. the platform can be combined with microfluidics for on-chip sample preparation and detection, providing a new tool for poc diagnostics and global healthcare. detection of micrornas (mirnas) has been a key topic in cancer research, diagnosis, and prognosis. mirnas can repress gene expression in a sequencedependent manner [ ] and are associated with various human diseases such as diabetes, alzheimer's, and cancers [ ] [ ] [ ] . at an early stage of cancer, extremely low concentrated mirnas circulate in human body fluid, making detection of mirnas with improved lod and sensitivity desirable for early disease diagnosis and implementation of new treatment options [ ] . joshi et al. designed a regenerative lspr-based mirna sensor for early diagnosis of pancreatic ductal adenocarcinoma (pdac), a deadly cancer with an overall -year survival rate of %. the cancer is hard to be detected when the tumor is small and at nonmetastatic stage [ , ] . the sensor is based on gold nanoprisms functionalized with -s-c -ssdnas. direct hybridization between the -s-c -ssdnas and the target mirnas forms dna duplex (fig. . e) , which increases the refractive index in local environment of the nanoprism and redshifts the lspr peak wavelength. the high sensitivity down to À m for specific mirna- b benefits from multiple factors: (a) atomically flat surface of the nanoprisms allowing efficient duplex formation, (b) high charge density of the duplex greatly altering the local refractive index, and (c) strong field enhancement at the tips of nanoprisms. detection of mirna- b at a low concentration was achieved in pancreatic cancer cell lines, derived tissue culture media, human plasma, and plasma exosomes. quantification of mirna- b in highly purified exosomes that were isolated from patients with pdac and chronic pancreatitis (cp) was shown in fig. . f. pdac patients had much higher mirna- b levels than cp patients. integration of the sensor into microfluidics is expected to further enhance the mirna measurements and the disease diagnosis with identifiable mirna signatures. proteins in biofluids such as serum are highly associated with biological functionalities. detection of proteins and analysis of their unique sequences have high clinical relevance [ , ] . for instance, a protein biomarker called svegfr- was increasingly expressed in patients with myelodysplastic syndromes (mds), i.e., a diverse group of clonal disorders of the hematopoietic stem cell. prostate-specific antigen (psa) shows increased levels above the normal limits ( ng ml À ) in serum for possible prostate malignancy, which can be used for diagnosis and prognosis of prostate cancer [ ] . plasmofluidic immunoassays and immunosensors feature parallel, label-free, and real-time detection of proteins with high sensitivity, selectivity, multiplicity, and reproducibility [ , [ ] [ ] [ ] [ ] . they are also applied to investigate protein-protein interaction [ ] , monitor live cell secretory events [ ] , and identify diagnosis-related cells [ ] . spp-based plasmofluidic sensors for label-free detection of proteins have recently been implemented on a smartphone platform [ ] . a plasmonic sensor with dispersionless microfluidics was developed to detect protein biomarker of mds disease (i.e., svegfr- ) via interaction with its high-affinity bio-receptor vegf-a [ ] . a detection limit of ng/ml was achieved in % human blood plasma by using the sequential injection approach. however, the traditional experimental design that relies on a specific bio-receptor (known as "lock-and-key" approach) is limited by interference from molecules that are structurally or chemically alike. moreover, most diseases are associated with multiple biomarkers, which necessitate the detection of a total protein distribution rather than a particular protein. to address these challenges, choi et al. developed a "cross-reactive"-based sensor using a substrate of multiple segments of surfaces that are pre-adsorbed by different types of proteins. a distinctive pattern of spp angle shifts arose from the interaction of the substrate with a sample of variable proteins [ ] . the spp angle pattern was used to clarify the concentration distributions of proteins and a specific biomarker, c-reactive protein (crp), in a cocktail sample. a more powerful way for simultaneous detection of multiple proteins is the use of protein microarrays in conjunction with surface plasmon resonance imaging (spri) [ ] . however, protein microarrays often involve time-consuming fabrication process. dehydrated or denatured proteins suffer a shorter lifetime of normal functionality. to overcome these problems, a multiplexed enzymatic synthesis via surfacecoupled transcription-translation was employed for in vitro fabrication of protein microarrays and for their immediate use in biosensing based on a microfluidic platform, as illustrated in fig. . a-c. the plasmofluidic system includes generator, control, and detector components. multiple rna transcripts were created at the generator through surface reaction of rna polymerase with adsorbed dsdna and translated into proteins by cell-free protein synthesis. the synthesized proteins diffused to the detector element and formed protein microarrays. as a demonstration, binding of anti-gfp and antiluciferase to the protein arrays was characterized with spri and time-resolved absorption kinetic measurement, as shown in fig. . d-g. with multiple surface chemistries, the plasmofluidic system can be further implemented for multiplexed spri biosensing in clinical practices. more challenges arise for spp-based sensing in human serum samples due to (i) high nonspecific interaction between the sensor surface and serum proteins and (ii) matrix effects of serum such as high refractive index and viscosity that mask the binding events between the recognition elements on the sensor surface and the analytes in the serum [ ] . uludag et al. used a matrix elimination buffer to eliminate nonspecific binding of % serum proteins and performed a sandwich assay, in which the target proteins were sandwiched between a bottom layer of capture antibody and a top layer of antibody-modified au nps [ ] . the au nps reduced the refractive-index mismatch between the buffer and the sample, leading to amplified spp signal and enhanced sensitivity. a lod of . ng ml À for tpsa in % human serum was attained, which was comparable to that achieved by a quartz crystal microbalance (qcm) sensor. spp and qcm sensors were further combined to detect sub-attomolar human α-thrombin [ ] . the sandwich immunoassays with spp-based sensors are expected to improve early-stage cancer diagnosis and prognosis. lspr-based plasmofluidic sensors enable high-end miniaturization down to the single-nanoparticle scale [ , [ ] [ ] [ ] [ ] . zijlstra et al. reported real-time, label-free detection of single-protein binding events by monitoring the lspr of a nanorod with an ultrasensitive photothermal assay [ ] . when a single protein binds to the receptors on the surface of the nanorod, a small redshift of the longitudinal lspr wavelength changes the absorption cross section of the nanorod at the wavelength of the heating beam, which can be measured with the transduced temperature change. unlike the resonance rayleigh scattering, the contrast scale of photothermal microscopy permits the measurement of mode volume that is commensurate to the size of a protein. ament et al. utilized single gold nanorods to monitor single-protein conformational dynamics and equilibrium coverage fluctuations [ ] . the latter contain the information on the binding kinetics and nonequilibrium thermodynamics. by using a white light laser, an intensified ccd camera, and an engineered nanoparticle geometry, they achieved considerable improvement of signal-to-noise (snr) ratio and time resolution over previous techniques to identify single-molecule binding events. rather than utilizing passive surface binding to capture the proteins, gordon's group used a double nanohole to actively trap single proteins and to measure the singleprotein binding events [ ] . lspr biosensors based on nanoparticle ensembles and microfluidics are more suitable for clinical applications because the ensembles allow statistically significant (c) schematic illustration of antibody bound onto the synthesized protein array. spri difference images taken before and after the exposure to (d) anti-gfp and (e) antiluciferase solutions. realtime spri adsorption kinetic measurement of (f) anti-gfp-and (g) antiluciferase-specific binding (reproduced with permission [ ] . copyright american chemical society) data analysis, relax constraints by nanoparticle variations, and enable simple instrumentation, fast data acquisition, and improved signal-to-noise ratio [ ] . plasmonic nanotechnologies such as plasmonic arrays [ , , [ ] [ ] [ ] and spri [ , ] and innovative microfluidic techniques such as integrated concentration gradient generator [ ] and multi-well fluidic measurement [ ] have been intensely pursued to detect and quantify cancer biomarkers with enhanced sensitivity, robustness, integrity, high throughput, and multiplexity. quidant's group developed a parallel and high-throughput lspr-based plasmofluidic sensor that can be upgraded to a lab-on-a-chip system and be translated to clinical environments (fig. . a) [ ] . for the sensor, a periodic array of gold nanorods with a bio-recognition layer was integrated with a microfluidic network. the authors applied the sensor to detect afp and psa, which are indicators of prostate cancers, at a lod of as low as ng/ml and a timescale of minutes. chen et al. developed a highly integrated, multiarrayed lspr sensor for massively parallel high-throughput detection of multiple cytokine biomarkers in a low-volume assay, as shown in fig. . b [ ] . consisting of lspr sensing spots with multistep processing for eight different samples, including manual loading, incubation, and washing, the sensor allowed quantitative measurements at concentrations ranging from to , pg/ml in a -μl sample of serum. it also enabled multianalyte detection of ten cycles for each sample in min, which is more than ten times shorter than traditional sandwich immunoassays. the sensor was further applied to measure cytokine levels of il- and il- in two neonates, who received cardiopulmonary bypass surgery for congenital heart disease within h. the measurement was valuable for defining the postsurgery responses and predicting the anticipated assay outcomes. measuring binding affinities between proteins is also of fundamental interest and clinical significance. a "nanospr" method was proposed to simultaneously characterize the binding affinities among many macromolecular partners [ ] . briefly, multiple batches of gold nanorods functionalized with different proteins and one batch without functionalization (as a reference) were driven through the flow cell consecutively to generate a positon-encoded sensor. the encoding of the sensor was carried out by repetitive recording of the position of each randomly deposited gold nanorod, as shown in fig. . c. as a demonstration, the sensor was applied to study the interactions of three bacterial division proteins with a target protein ftsz, which is an essential element of the division machinery in most bacterial systems. the results had an excellent agreement with those by conventional composition gradient static light scattering and fluorescence anisotropy. with the small device size, reduced sample volume, and built-in statistics, the sensor is envisioned to be a powerful tool in drug screening and discovery applications. in light of the important role of cell secretion in a variety of physiological processes, wu et al. developed a label-free, ultrasensitive, plasmofluidic platform based on gold nanoslits with strong fano resonance to monitor dynamic live cell secretory activities [ ] . the thp cells were trapped and cultured in microfluidic channels with the sensing surface of gold nanoslits~ - μm away from the cell membrane. upon stimulation with continuous lipopolysaccharide, the cells were found to secrete mmp- proteins in h. the plasmofluidic sensor needs less than ten cells for the study, while elisa analysis demands at least a few thousand cells. the sensor can be applied for fundamental study of secretory events in different physiological environments and medical diagnosis of various diseases at the single-cell level. sers-based immunoassay is considered another promising approach for protein sensing. one of the most popular strategies uses a sandwich immunocomplex protocol with antibody-conjugated metal particles [ , [ ] [ ] [ ] [ ] . choo's group implemented magnetic tweezers [ ] and optoelectronic tweezers [ ] in a gradient microfluidic channel to form sandwich immunocomplexes for the sensitive and automatic quantitative analysis in less than min. in the immunocomplexes, target antigens were sandwiched between antibody-conjugated microspheres and antibody-conjugated sers-active nanoparticles. the magnetic tweezer-based immunoassay had a lod of - ng/ml for rabbit igg, and the optoelectronic tweezer-based one achieved a lod of pg/ml for alpha-fetoprotein (afp). the same group further integrated sandwich immunoassay [ ] with gradient microfluidics to achieve programmable and automatic analysis of biomarkers with small sample volume, easy sample preparation, and short assay time [ ] . after the afp antigens were captured by the immobilized antibodies on gold microwells, polyclonal anti-afp antibody-conjugated particles were attached to form the sandwich immunocomplexes, as shown in fig. . a. a similar methodology was applied to detect hepatitis b virus antigen in human blood and blood plasma. hepatitis b virus infection is a common cause of chronic liver disease worldwide [ ] . using a sandwich-typed immunoassay, zou et al. detected carcinoembryonic antigen in raw blood samples with a lod of as low as À m. carcinoembryonic antigen is a wide-spectrum biomarker for diagnosis of various cancers [ ] . formation of stable electromagnetic "hot spots" in nanoparticle aggregates is another strategy for sers-based protein analysis with high reproducibility and sensitivity. saha et al. designed a paper-based microfluidic sers system to detect streptavidin and glycoprotein with pico to femtomolar concentration, as shown in fig. . b [ ] . two channels with ag/au nanoparticles and proteins merge into a small round-shaped reaction chamber to generate sers-active nanoparticle aggregates via protein-assisted cross-linking. notably, the porous feature of the microchannels regulates the extent of nanoparticle aggregation and prevents the screening of "hot spots" in large aggregates. [ ] . copyright rsc publishing). (b) a paper-based microfluidic device for sers-based protein detection. it involves the aggregation of ag/au nanoparticles at the presence of proteins (reproduced with permission [ ] . copyright american chemical society). (c) schematic representation of a sbt-based microfluidic system to distinguish cancer cells in a low concentration against background of normal cells. the bar graph compares cancer cell counts by microscopy, pca, and cls. the latter two are deconvolution strategies for analyzing the cancer-normal cell ratios with the composite raman spectrum (reproduced with permission [ ] . copyright american chemical society) sers protein biotags (sbts) have been developed for identification of cells and pathogens in body fluids, which is significant for early disease detection and monitoring of patient response to therapy [ ] . in the microfluidic cell identification system, two spectroscopically distinguishable sbts that target distinct cell epitopes were used to label cells in a single focused line by hydrodynamic flow, as illustrated in fig. . c [ ] . the identification was accomplished by measuring the relative signal from the cancer-specific sbt versus the cell-identifying universal control sbt. the discrimination efficiency can reach cancer cell from a population of normal cells due to spectroscopic richness of the raman bands of the reporter molecules on the two sbts and algorithmic effectiveness of the two deconvolution strategies for the composite spectrum. the sbt approach provides a continuous, low-cost, and nondestructive tumor cell identification and paves the way toward clinical cancer diagnosis at the single-cell level. the detection of infectious agents such as viruses and bacteria is critical for public health, homeland security, and food industry [ , , ] . viruses have been responsible for a number of epidemic outbreaks (e.g., h n flu and sars) in recent years. timely virus detection becomes essential for recognizing and controlling future epidemics. conventional techniques such as cell culture methods, pcr, and elisa can detect and quantify pathogens with high sensitivity and specificity [ , ] . however, they require lab-intensive procedures, expensive equipment, and well-trained operators. alternative biosensing techniques that can achieve reliable, accurate, and sensitive detection and analysis of pathogens under the variable settings, including source-limited settings and primary care settings, are in great need. among the various detection platforms that use different mechanisms such as electrical, mechanical, and optical signal transduction [ , , ] , plasmofluidic biosensors are promising for label-free detection of infectious agents. wang et al. achieved label-free imaging, detection, and mass/size measurement of single viral particles with high-resolution surface plasmon resonance spectroscopy [ ] . the viral particles were imaged as diffraction patterns from their scattering of spp. the particle size and mass were determined from the image intensities. two viruses, i.e., h n influenza a/pr/ / and hcmv, were studied with a mass detection limit of ag, which is four orders of magnitude lower than that by conventional surface plasmon resonance method. to meet the needs for quickly recognizing and controlling epidemics, a plasmofluidic sensor was developed to detect viruses from biologically relevant media, as illustrated in fig. . a [ ] . the sensor exploits plasmon-enhanced extraordinary light transmission through metal nanohole arrays and is applicable to a broad range of pathogens. it was applied to detect pt-ebola virus in pbs buffer solution based on the consistent redshift (> nm) of the plasmon resonance wavelength. a -nm shift in resonance wavelength was observed for virus detection in biological media consisted of cell growth medium and % fetal calf serum. the detectable virus concentration ranges from to pfu/ml, which is relevant to both clinical testing and drug screening. inci et al. developed a plasmofluidic platform for label-free, high-sensitive, highspecific, and reproducible hiv viral load quantification from unprocessed whole blood sample [ ] . surface chemistry and highly specific antibody immobilization were applied to capture the viruses at the electromagnetic "hot spots" and to minimize background signal from nonspecific binding of blood cells. briefly, poly-l-lysine-modified polystyrene with terminal amine group was used as substrates to capture gold nanoparticles. the gold nanoparticles were treated with different chemicals consecutively, including -mercaptoundecanoic acid, n-ethyl-n-( -dimethylaminopropyl)carbodiimide hydrochloride, n-hydroxysulfosuccinimide, neutravidin, and biotinylated anti-gp polyclonal antibody. the antibody was positioned in a favorable orientation to have a high capture efficiency for the viruses. a spp platform based on dna-rna hybridization was also developed to detect viruses plaguing plant food and feed crop plants [ ] . rapid detection of bacteria makes a difference in monitoring and preventing outbreaks of infections [ , ] . for this purpose, walter et al. developed microfluidic sers to discriminate bacteria based on strain levels in a fast and reliable fashion [ ] . in their plasmofluidic system, aqueous analytes and colloid solution form droplets in mineral oil, creating a segmented flow for stable optical measurements and reproducible bacterial identification, as shown in fig. . b. besides colloidal nanoparticles, solid-state sers substrate functionalized with recognition molecules was also employed to capture and analyze e. coli cells with high specificity and sensitivity [ ] . demirci's group developed a portable microfluidic spp platform that can rapidly detect and quantify e. coli and s. aureus [ ] . all the elements, including light source, optical components, cmos sensor, circuitry, and microfluidic chip, were packaged in a portable box with dimensions of . cm  cm  . cm, as shown in fig. . c. effective capture and detection of e. coli at concentrations from~ to .  cfus/ml in pbs and peritoneal dialysis fluid were demonstrated. multiplicity and specificity were tested with s. aureus in pbs solution, implying the potential of the platform for pathogen diagnostics at both poc and primary care settings. quick and cost-effective pharmaceutical analysis can benefit healthcare, biomedicine, and food safety [ ] . for example, drug screening is a critical step in new drug discovery. detection of drug residues in food and human liquids is frequently needed for food safety and healthcare. the capability of monitoring antibody-polypeptide reaction is desirable for multiplexed drug discovery. chen et al. proposed a spribased visualization method for monitoring antibody-polypeptide binding in a labelfree and high-throughput format [ ] . the plasmonic platform can support the dynamic imaging of polypeptide microarray and the time tracing of d histograms for monitoring antibody-polypeptide reaction on the surface. chiral compounds have significant impacts on pharmacological and biological processes. effective strategies for chiral discrimination and enantioseparation are highly needed to meet the increasing need for enantiomerically pure compounds. for a pair of enantiomers, it is possible that one configuration is active drug, while the other configuration remains inactive, contributes to side effects, displays toxicity, or works as an antagonist [ , ] . as an example, guo et al. demonstrated α-thrombin-functionalized lspr sensor integrated with a microfluidic chip for enantioselective analysis of melagatran. recently, we have achieved high-sensitive label-free chiral sensing for drug molecules based on moiré chiral metamaterials [ ] . plasmofluidic sensors were also developed for label-free and rapid detection of drug residues in food and clinical samples [ ] . fernandez et al. used a portable six-channel spp sensor to analyze antibiotic residues from different families, i.e., fluoroquinolones, sulfonamides, and phenicols, in whole milk samples [ ] . the sensor showed good regenerative ability and repeatability and accomplished lods far below the maximum residue levels established by the european union for the antibiotics. zhao et al. used a multichannel spp sensor to analyze the concentrations of methotrexate, an anticancer drug, in the serum of a patient undergoing chemotherapy treatments. the results agreed with those by fluorescence polarization immunoassay [ ] . andreou et al. developed a microfluidic sers to identify drugs at clinical levels without specialized chemicals and bio-reagents [ ] . as shown in fig. . d, a laminar flow consisted of a central stream of analytes (i.e., methamphetamine) and two sheath streams of silver nanoparticle and salt solutions was created. at the interrogation region, sers-active nanoparticle dimers and small-order aggregates with methamphetamine predominantly formed. due to the low affinity of methamphetamine to the silver nanoparticles, the salt was added to induce the aggregation. trace concentrations of methamphetamine in saliva were detected within minutes. the microfluidic sers will find applications in detecting many other health-related molecules such as toxins and pollutants. conclusions and future perspectives tremendous progress has been made in plasmofluidics for biological analysis and medical diagnostics. in particular, plasmonic tweezers achieved noninvasive trapping of biological cells, dnas, and proteins at the single-entity level. d manipulations were demonstrated with plasmonic tweezers based on an optical fiber tip with high-resolution mechanical motion control. long-range and rapid delivery of nanoparticles was achieved with electrothermoplasmonics. most of the current tweezers used microfluidic channels as passive containers for sample solutions. it is anticipated that future plasmonic tweezers will fully reap the benefits of micro-/ nanofluidics to further enhance their functionalities for biosensing and diagnostic applications. in particular, the light-fluid interactions at the nanoscale can be explored to achieve versatile multifunctional plasmofluidic tweezers [ ] . our preliminary study indicated that the plasmon-enhanced photothermal effects such as thermophoresis are one of the promising strategies for optical manipulations in variable fluidic environments. a variety of plasmofluidic sensors has been developed to detect and analyze nucleic acids, proteins, pathogens, and drugs in life sciences, disease diagnostics, and drug discovery. innovative microfluidic design, plasmonic engineering, and surface functionalization have led to sensitive, robust, and label-free poc biosensors. the next-generation plasmofluidic sensors are expected to target at translational and clinical applications [ ] . for the purpose, the sensors need to be a highly integrated, robust, and user-friendly system that can handle a variety of clinical samples such as urine, blood, and saliva and enables poc diagnostics at field settings. in summary, the highly interdisciplinary field of plasmofluidics presents exciting opportunities for new discoveries and poc devices. with the stronger collaborations among researchers from physics, chemistry, biology, pharmacology, and engineering, as well as clinicians and entrepreneurs, plasmofluidics for biosensing and disease diagnostics will continue to grow and contribute to global healthcare. advances in plasmonic technologies for point of care applications portable point-of-care diagnostic devices cancer statistics advances and challenges in biosensor-based diagnosis of infectious diseases point-of-care testing for infectious diseases: diversity, complexity, and barriers in low-and middle-income countries microfluidic chips for immunoassays plasmofluidics: merging light and fluids at the micro-/nanoscale surface plasmon subwavelength optics molecular plasmonics for biology and nanomedicine plasmon nano-optical tweezers microfluidic surface plasmon resonance sensors: from principles to point-of-care applications localized surface plasmon resonance sensors spr biosensors: historical perspectives and current challenges origin and future of plasmonic optical tweezers systematic investigation of localized surface plasmon resonance of long-range ordered au nanodisk arrays extended-area optically induced organization of microparticles on a surface scannable plasmonic trapping using a gold stripe theoretical and experimental study of surface plasmon radiation force on micrometer-sized spheres parallel and selective trapping in a patterned plasmonic landscape nanometric optical tweezers based on nanostructured substrates low-power nano-optical vortex trapping via plasmonic diabolo nanoantennas application of plasmonic bowtie nanoantenna arrays for optical trapping, stacking, and sorting nano-optical conveyor belt, part ii: demonstration of handoff between near-field optical traps bubble-pen lithography self-induced back-action optical trapping of dielectric nanoparticles nanoscale control of optical heating in complex plasmonic systems photoinduced heating of nanoparticle arrays plasmon-assisted optofluidics spr and spr imaging: recent trends in developing nanodevices for detection and real-time monitoring of biomolecular events on-chip synthesis of protein microarrays from dna microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications surface plasmon resonance sensor with dispersionless microfluidics for direct detection of nucleic acids at the low femtomole level tunable directive radiation of surface-plasmon diffraction gratings biosensing using straight long-range surface plasmon waveguides sensing using localised surface plasmon resonance sensors localized surface plasmon resonance biosensing: current challenges and approaches flow-through vs flow-over: analysis of transport and binding in nanohole array plasmonic biosensors optofluidic concentration: plasmonic nanostructure as concentrator and sensor high-fidelity optofluidic on-chip sensors using well-defined gold nanowell crystals plasmon line shaping using nanocrosses for high sensitivity localized surface plasmon resonance sensing substrate-induced fano resonances of a plasmonic nanocube: a route to increased-sensitivity localized surface plasmon resonance sensors revealed tuning the plasmon resonance of a nano-mouth array plasmonic gold mushroom arrays with refractive index sensing figures of merit approaching the theoretical limit engineering of parallel plasmonic-photonic interactions for on-chip refractive index sensors optimizing plasmonic nanoantennas via coordinated multiple coupling localized surface plasmon resonance: nanostructures, bioassays and biosensing-a review moiré nanosphere lithography tunable multiband metasurfaces by moire nanosphere lithography on-chip nanohole array based sensing: a review label-free detection and molecular profiling of exosomes with a nanoplasmonic sensor localized surface plasmon resonance spectroscopy and sensing surface plasmon resonance imaging sensors: a review optofluidic sers: synergizing photonics and microfluidics for chemical and biological analysis surface-enhanced raman spectroscopy (sers): progress and trends surface-enhanced raman spectroscopy: concepts and chemical applications sers-enabled lab-on-a-chip systems convenient formation of nanoparticle aggregates on microfluidic chips for highly sensitive sers detection of biomolecules light-directed reversible assembly of plasmonic nanoparticles using plasmon-enhanced thermophoresis nano-optical trapping of rayleigh particles and escherichia coli bacteria with resonant optical antennas integration of plasmonic trapping in a microfluidic environment permanent fixing or reversible trapping and release of dna micropatterns on a gold nanostructure using continuous-wave or femtosecond-pulsed near-infrared laser light dna translocations through solid-state plasmonic nanopores optical trapping of a single protein double nanohole optical trapping: dynamics and proteinantibody co-trapping and r. gordon a label-free untethered approach to single-molecule protein binding kinetics long-range and rapid transport of individual nano-objects by a hybrid electrothermoplasmonic nanotweezer three-dimensional manipulation with scanning near-field optical nanotweezers plasmonics-turning loss into gain thermophoretic tweezers for low-power and versatile manipulation of biological cells patterned multiplex pathogen dna detection by au particle-on-wire sers sensor gold nanoring as a sensitive plasmonic biosensor for on-chip dna detection label-free dna biosensor based on sers molecular sentinel on nanowave chip plasmonics-based sers nanobiosensor for homogeneous nucleic acid detection surface plasmon resonance sensors for detection of chemical and biological species molecular sentinel-on-chip for sers-based biosensing multiplex detection of disease biomarkers using sers molecular sentinel-on-chip dna bioassay-on-chip using sers detection for dengue diagnosis micrornas can generate thresholds in target gene expression microrna expression profiles classify human cancers a microrna expression signature of human solid tumors defines cancer gene targets cancer biomarker profiling with micrornas rapid sub-attomole microrna detection on a portable microfluidic chip highly specific plasmonic biosensors for ultrasensitive microrna detection in plasma from pancreatic cancer patients label-free nanoplasmonic-based short noncoding rna sensing at attomolar concentrations allows for quantitative and highly specific assay of microrna- b in biological fluids and circulating exosomes monitoring protein distributions based on patterns generated by protein adsorption behavior in a microfluidic channel plasmonic sensors for analysis of proteins and an oncologic drug in human serum cancer biomarker detection in serum samples using surface plasmon resonance and quartz crystal microbalance sensors with nanoparticle signal amplification lspr chip for parallel, rapid, and sensitive detection of cancer markers in serum multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays sers-based immunoassay using a gold array-embedded gradient microfluidic chip patterned plasmonic nanoparticle arrays for microfluidic and multiplexed biological assays plasmonic nanosensors for simultaneous quantification of multiple protein-protein binding affinities optofluidic platform for real-time monitoring of live cell secretory activities using fano resonance in gold nanoslits rapid identification by surface-enhanced raman spectroscopy of cancer cells at low concentrations flowing in a microfluidic channel surface plasmon resonance biosensor based on smart phone platforms surface plasmon resonance biosensor for the detection of vegfr- -a protein marker of myelodysplastic syndromes ultrasensitive detection of thrombin using surface plasmon resonance and quartz crystal microbalance sensors by aptamer-based rolling circle amplification and nanoparticle signal enhancement rational aspect ratio and suitable antibody coverage of gold nanorod for ultra-sensitive detection of a cancer biomarker optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod single unlabeled protein detection on individual plasmonic nanoparticles observing single protein binding by optical transmission through a double nanohole aperture in a metal film nanohole-based surface plasmon resonance instruments with improved spectral resolution quantify a broad range of antibody-ligand binding kinetics handheld high-throughput plasmonic biosensor using computational on-chip imaging label-free nanoplasmonic sensing of tumor-associate autoantibodies for early diagnosis of colorectal cancer quantification of ovarian cancer markers with integrated microfluidic concentration gradient and imaging nanohole surface plasmon resonance a localized surface plasmon resonance imaging instrument for multiplexed biosensing -well plasmonic sensing with nanohole arrays paper-based microfluidic approach for surface-enhanced raman spectroscopy and highly reproducible detection of proteins beyond picomolar concentration on-chip immunoassay using surface-enhanced raman scattering of hollow gold nanospheres optoelectrofluidic sandwich immunoassays for detection of human tumor marker using surface-enhanced raman scattering highly reproducible immunoassay of cancer markers on a gold-patterned microarray chip using surface-enhanced raman scattering imaging picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface-enhanced raman spectroscopy detection of hepatitis b virus antigen from human blood: sers immunoassay in a microfluidic system an optofluidic nanoplasmonic biosensor for direct detection of live viruses from biological media towards a fast, high specific and reliable discrimination of bacteria on strain level by means of sers in a microfluidic device portable microfluidic integrated plasmonic platform for pathogen detection rapid detection of drugs of abuse in saliva using surface enhanced raman spectroscopy and microfluidics nanoplasmonic quantitative detection of intact viruses from unprocessed whole blood portable microfluidic chip for detection of escherichia coli in produce and blood fructose enhanced reduction of bacterial growth on nanorough surfaces manipulating biological agents and cells in micro-scale volumes for applications in medicine label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance phytochip': on-chip detection of phytopathogenic rna viruses by a new surface plasmon resonance platform highly sensitive and specific detection of e. coli by a sers nanobiosensor chip utilizing metallic nanosculptured thin films surface plasmon resonance (spr) biosensors in pharmaceutical analysis visualization of high-throughput and label-free antibody-polypeptide binding for drug screening based on microarrays and surface plasmon resonance imaging chiral discrimination and enantioselective analysis of drugs: an overview enantioselective analysis of melagatran via an lspr biosensor integrated with a microfluidic chip optical biosensors for food quality and safety assurance -a review a label-free and portable multichannel surface plasmon resonance immunosensor for on site analysis of antibiotics in milk samples miniature multi-channel spr instrument for methotrexate monitoring in clinical samples the authors acknowledge the financial support of the beckman young investigator program and the office of naval research young investigator program. key: cord- - fxjti authors: banoub, joseph h.; jahouh, farid title: fundamental principles for luminescence sensing measuring devices used for the detection of biological warfare agents date: - - journal: molecular technologies for detection of chemical and biological agents doi: . / - - - - _ sha: doc_id: cord_uid: fxjti this chapter surveys the current detection technologies used in commercially available luminescence biosensor detection equipments currently employed for identifying warfare biological agents (bas). brief technical descriptions of these technologies are presented with emphasis placed on the principles of detection. much of the content presented was obtained from the open-source literature and is an introduction to biosensor fundamentals biological agents (bas) are widely found in the natural environment (work place, hospitals, hvac, etc.) and as a result of voluntary release of biological warfare or terrorism agents [ , ] . biological agents include series of different virulent bacteria, viruses, fungi (yeasts and moulds) and parasites. all of these agents possess irreversible threat to potentially cause ill health and death to soldiers/humans. the incidents of anthrax-laced letters, the emergence of severe acute respiratory syndrome (sars), and repeated occurrences of illnesses caused by food-borne pathogens highlight the need for rapid and sensitive identification of the responsible biological agents [ , ] . biological agents are usually invisible and possess the ability to infect in very small doses [ , ] . they also have the cunning ability to replicate rapidly and require minimal resources to survive. for these reasons, it is impossible to initially feel their presence or to predict the risks they present. recently, it has been demonstrated that bioterrorism raises the specter of exposure to toxins by devising new deliveries by which bas can be weaponized [ ] [ ] [ ] . it has become apparent that bas weapons pose a real and potentially immediate threat as they are relatively cheap to manufacture and employ, and they have tremendous potential impact as terror weapons [ ] . these features make biological weapons attractive to rogue states and terrorist organizations. in this article we briefly describe the threat of biological weapons [ ] [ ] [ ] . biological agents are categorized according to the code of practice to the safety, health and welfare at work (biological agents) regulations, [ ] . this classification system is based on whether: the biological agent is pathogenic to humans, represents a hazard to soldiers, it is transmissible to nearby community and if there is a possible effective treatment available [ ] [ ] [ ] [ ] . consequently, bas are classified into four risk groups (rg), namely, rg , rg , rg and rg , as follows: • rg that includes bas not linked with diseases in healthy adult humans. examples of rg agents include bacillus subtilis or bacillus licheniformis, escherichia coli-k , and adeno-associated virus (aav) types through [ , ] . • rg that includes bas connected with human diseases which is rarely serious and for which preventive or therapeutic interventions are often available. examples of rg agents include salmonella sp., chlamydia psittaci, measles virus, and hepatitis a, b, c, d, and e viruses [ , ] . • rg that contains bas associated with serious or lethal human diseases for which preventive or therapeutic interventions may be available (high individual risk but low community risk). examples of rg agents include brucella, mycobacterium tuberculosis, coccidioides immitis, yellow fever virus and human immunodeficiency virus (hiv) types and [ , ] . • rg includes bas that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk). rg agents only include viruses. examples of rg agents consist of crimean-congo hemorrhagic fever virus, ebola virus and herpesvirus simiae (b-virus). under the classification system, these risk group agents are the least hazardous whilst group are the most hazardous [ , ] . field bas and urban public health surveillance systems usually provide rapid determination of the presence of bas in the atmosphere and may in time provide an indication of when and where the biological agent was released [ ] [ ] [ ] [ ] [ ] . once, the point source is revealed and the ba identified, rapid clean-up effort could be initiated, after the release of the compound. moreover, it is essential to monitor the presence of bas in the environment, to provide people at risk with the means of rapidly identifying contaminated air, water, food and equipment [ ] [ ] [ ] [ ] [ ] . it is well known that antibodies specifically targeting proteins or pathogens, can be generated to a wide variety of target cell bacterial analytes and are the most popular choices for the recognition element in many biosensors. typical immunoassay formats include competitive, displacement, sandwich, and enzyme-linked immunosorbent assays (elisa) [ ] . elisa, for example, uses colorimetric or chemiluminescent enzyme substrates for signal transduction and is more suited to automated instruments because of the multiple incubations and washes required. the classical approach to detect the bacteria species type or microbe involves the use of differential metabolic assays, monitored colorimetrically and on immunochemical routine tests. in addition, the use of cell culture and electron microscopy are vital for the diagnosis of viruses, bacteria and intracellular parasites. consequently, all samples retrieved from the affected environment must ne cultured in order to obtain sufficient numbers of various cell types for reliable identification. unfortunately, one of the major drawbacks is that the time required for the microbial outgrowth is long and some bacteria obtained are not culturable, as a result of genetic mutation. this chapter has been drafted as a "comptes rendus" and general modi operandi of novel biodetection approaches using luminescence biosensing approaches, published recently, for the detection of warfare potential ba weapons; it will exclude all the conventional approaches. the readers are encouraged to peruse the references included within the text, to search for the applied commercial used biosensors, that for simplification reasons, we did not discuss herewith. according to a recently proposed iupac definition a biosensor is "a self-contained integrated device which is capable of providing specific quantitative or semiquantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transducer element [ ] . a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. the term "biosensor" is short for "biological sensor." the device is made up of a transducer and a biological element that can be an enzyme, an antibody or a nucleic acid. the bioelement interacts with the analyte being tested and the biological response is converted into an electrical signal by the transducer (fig. . ) [ , ] sensing can be explained as the use of recognition elements (biological in origin) for binding to the biothreat molecule of interest. the binding event must be transduced in a manner that signals the presence of the targeted analyte. biosensor probes are becoming increasingly sophisticated, mainly owing to combination of advances in two technological fields: microelectronics and biotechnology. biosensors are highly valuables devices in measuring a wide spectrum of ba analytes [ , ] . ideally each sensing detection technology should contain the following characteristics: • specific and able to discriminate between closely related pathogenic and nonpathogenic organisms or toxins. • sensitive and able to detect small amounts of target within a high background matrix. • possess high affinity and being able to maintain binding even through repeated washing steps. • stable enough to allow long-term use. it should be understood that luminescence biosensing technology is totally distinct from other physiochemical methods, such as mass spectrometry (ms) or fourier transform infrared spectroscopy (ftir) and raman based analysis. these methods of course, have their merits and are very sensitive and specific. therefore, in the following sections, we will provide an overview of the state-of-the-art prime molecular sensing technologies for the detection of bas. it should be noticed that the current methods for detecting pathogenic bacteria, which include elisa and pcr [ , ] are assays that exploit antibodies as molecular recognition elements due to their highly specific targeting of antigenic sites. nonetheless, these antibodies lack the stability needed to detect pathogenic species under harsh environments, and requires a one-to-one pairing of antibody-based sensors for each target to be detected. whereas, nucleic acid probe-based techniques such as pcr can reach single-cell detection limits, they still require the extraction of nucleic acids and are limited in portability [ , ] . mcalpine and coworkers developed a robust and portable biosensor for the detection of pathogenic bacteria that could impact water quality monitoring for bacterial contamination [ ] [ ] [ ] . the particular interest of the developed biosensor was that it combined the natural specificity of biological recognition to label-free sensors providing sensitive electronic readout. thus, mcalpine et al. reported the selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays [ ] . the semi-selective antimicrobial peptide magainin i, which occurs naturally on the skin of african clawed frogs, was immobilized on gold microelectrodes via a c-terminal cysteine residue. significantly, exposing the sensor to various concentrations of pathogenic escherichia coli revealed detection limits of approximately bacterium∕μl, a clinically useful detection range. the peptide-microcapacitive hybrid device was further able to demonstrate both gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of e. coli and salmonella [ ] . it is well known that the synthesis of antimicrobial peptides (amps) and their resulting intrinsic stabilities render them particularly interesting candidates for the use as molecular recognition elements in electronic biosensing platforms [ ] [ ] [ ] . amps do exist in nature and are located either in the skin of higher organisms and/ or in the extracellular milieu of bacteria [ ] . the replacements of current antibodybased affinity probes, with more stable and durable amps in biological sensors, have a major advantage as recognition elements. this advantage stems from the amps semi-selective binding nature to target cells of a variety of pathogens. the bioactivity of amps toward microbial cells can be classified into groups according to their secondary structures [ , ] . many amps adopt amphipathic conformations that spatially shield the hydrophobic group of the cationic amino acids, thereby targeting the negatively charged head groups of lipids in the bacterial membrane. in contrast, the membranes of plants and animals separate negative charges to the inner leaflet and contain cholesterols that reduce amp activity [ ] . the amps, linear cationic peptides such as magainins, are particularly attractive for microbial sensing applications because of their small molecular size and intrinsic stability [ , ] . in particular, the positively charged amp magainin i (gigkflhsagkfgkafvgeimks) binds most selectively to the bacterial cell e. coli o ∶h as a precursor to bactericidal activity [ ] [ ] [ ] magainin i displays a broad-spectrum activity toward other gram-negative bacteria, which comprise the majority of pathogenic infection in humans [ ] [ ] [ ] [ ] . the first step toward the development of an amp-based, label-free electronic biosensor consisted of targeting the microbial cells by magainin i using impedance spectroscopy. it should be repeated that electrical impedance measures the total opposition to a circuit or a part of the circuit presented to an electric current. usually the impedance is the results for both resistance and reactance. it is important to remember that the resistance component arises from collisions of the current-carrying charged particles with the internal structure of the conductor. furthermore, the reactance component is an additional opposition to the movement of the electric charge that result from the changing magnetic and electric fields in circuits carrying alternating current. figure . outlines the sensing platform. first, the amps are immobilized on the micro-fabricated interdigitated gold electrodes ( fig. . a) . it should be specified that magainin i contains a cysteine residue on the c terminus ( fig. . b) , which allows the facile site-specific covalent attachment to the gold electrodes. next, the heat-killed bacterial cells are injected and incubated on the amp-modified electrodes [ ] . when, the bacteria are recognized by the amps, binding will ensue ( fig. . c), causing the dielectric property to changes that can be monitored by a spectrum analyzer. usually, the impedance is measured over a frequency range of hz to khz. figure . d shows an optical micrograph of the device, which is made using standard microfabrication techniques. the results of measurements performed after incubation of the immobilized amps with pathogenic e. coli o ∶h cells concentrations ranging from to cfu∕ml are shown in fig. . . when, a blank device with no immobilized amps was also tested for comparison, it was found that there was no change in the impedance of the blank device without immobilized amps, upon exposure to various bacterial concentrations. figure . a shows that at low frequencies, the different concentrations of bacterial cells have the effect of increasing the impedance in proportion to the number of cells present in the sample. as the frequency increases, the contribution to the impedance from the bacterial cells decreases, leaving only the dielectric relaxation of small dipoles including water molecules in the buffer solution to affect the measured impedance. figure . b depicts the impedance change at a fixed frequency of hz. the variation in the impedance is directly proportional to the number of bacterial cells bound to the immobilized amps and manifested in a logarithmic increase with respect to serially diluted bacterial concentrations. significantly, the detection limit of response of the hybrid amp-microelectrode device to e. coli was found to be cfu∕ml ( bacterium∕μl). this lowest limit of detection appears to be limited by the presence of impedance due to the electrical double layer resulting from the electrode polarization effect at low frequencies. notably, these sensitivity limits is clinically relevant [ ] and compares favorably to amp-based fluorescent assays [ ] , antibody-based impedance sensors [ ] , and to the lal test [ ] . to simulate the use of the amp microelectrodes in everyday applications, such as direct water sampling, the biosensor response was investigated in real time, as shown in fig. . . first, a microfluidic cell was bonded to the interdigitated biosensor chip ( fig. . a), such that the electrodes were perpendicular to the direction of the sample flow ( fig. . b) [ ] . next, the fluid was injected using a syringe pump connected to the inlet port and allowed to flow through to the outlet port at a flow rate of μl∕ min. the flow cell was first flushed with buffer to establish a baseline. this was followed by injection of various dilutions ( - cfu∕ml) of the pathogenic e. coli cells in pbs to the channel at a reduced flow rate of μl∕ min for min. for example, fig. . c shows the microelectrode array after exposure to cfu∕ml bacterial cells. simultaneously, the impedance response was continuously monitored during the sample flow-through process ( fig. . d) . all samples produced a measurable response relative to the control sample within min, with the highest concentration sample yielding a response within s; the responses saturated after after min. these results augured well for the implementation of this sensor in continuous monitoring of flowing water supplies. mcalpine also examined the selectivity o the amp-functionalized biosensors toward the following various bacterial species (i) gram-negative pathogenic e. coli o ∶h , (ii) the nonpathogenic e. coli strain american type cell culture (atcc) , , (iii) gram-negative pathogenic salmonella typhimurium, and (iv) the gram positive listeria monocytogenes. the selectivity was first investigated using fluorescent microscopy methods, by staining bacterial cells and optically mapping their binding density to gold films hybridized with amps. the discriminative binding patterns of immobilized magainin i to and the surface density of the various bacterial cells (all cfu/ml) stained with propidium iodide (pi) nucleic acid stain are showed in fig. . . in summary, coupling of amps with microcapacitive biosensors has resulted in the implementation of a portable, label-free sensing platform for the detection of infectious agents. the achievable sensitivity approached bacterium∕μl-a clinically relevant limit-and the amps allowed for sufficient selectivity to distinguish pathogenic and gram-negative bacteria, while retaining broadband detection capabilities [ ] [ ] [ ] [ ] . in addition, the simulated water sampling chip which consisted of a microfluidic flow cell integrated onto the hybrid sensor, demonstrated the potential of real-time on-chip monitoring of the interaction of e. coli cells with the antimicrobial peptides [ ] . this following part discusses the progress made with the nrl array biosensor, which is a portable instrument for rapid and simultaneous detection of multiple targets which was developed, automated and miniaturized for operation at the pointof-use by the us navy research laboratories. this array biosensor has been used for the quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and for its usefulness as semi-selective molecules which can be used as alternative recognition moieties [ ] [ ] [ ] . in this sensor, the antibodies or other capture molecules are immobilized in a two-dimensional array on an optical waveguide (as either stripes or spots) and standard fluoroimmunoassays are performed within the channels of a multi-channel flow cell, which is placed on the waveguide surface ( fig. . , left). the spots are interrogated using evanescent wave technology: light from a nm diode laser is focused into the edge of the patterned slide/waveguide and after propagation and mixing within the waveguide; the confined beam produces an evanescent field within the sensing portion of the waveguide [ ] . in the nrl array biosensor the spots are interrogated using evanescent wave technology that is specifically: a light issued from a nm diode laser that is focused into the edge of the patterned slide/waveguide and after propagation and mixing within the waveguide, the confined beam produces an evanescent field within the sensing portion of the waveguide [ , ] . the definition of an evanescent field or wave is: an oscillating electric and/or magnetic field, which does not propagate as an electromagnetic wave but whose energy is spatially concentrated in the vicinity of the source (oscillating charges and currents) [ ] . the surface bound molecules labeled with fluorophore are excited by this evanescent field, producing a fluorescence signal; this fluorescence is then detected using a ccd camera fitted with appropriate bandpass and longpass filters ( fig. . , right). since the penetration depth of the evanescent field is limited, only surfacebound fluorophores are excited, enabling analysis of non-homogeneous or turbid samples. the locations and intensities of the fluorescent spots indicate the identity and concentration of the target sample in each lane [ , ] . the nrl array biosensor was used successfully for the detection of toxins and for multiple toxins simultaneously in multiple samples, it could also detect toxin levels as low as pg/ml and quantify the toxin concentration, and finally it could perform toxin assays in clinical, food, and environmental samples [ ] . furthermore, both sandwich immunoassays for protein toxins (e.g., staphylococcal enterotoxin b [seb] and ricin) and competitive immunoassays for low molecular weight toxins (e.g., trinitrotoluene and fumonisin b ) were reported ( determination of bacteria and large toxins in foods and air by the nra array biosensor assays normally employ a sandwich immunoassay format. however, mycotoxins are smaller in size and are therefore better assayed using an indirect competitive immunoassay [ ] [ ] [ ] [ ] . the validity of the nrl array biosensor was demonstrated for the detection of mycotoxins (ochratoxin a, deoxynivalenol and aflatoxin b ) in various food matrices and in air [ ] [ ] [ ] [ ] . this competitive assay protocol involved attaching the biotinylated mycotoxin derivatives onto the waveguide, this was followed by incubating the test sample with cyanine (cy )-labeled anti-toxin antibodies and then passing the pre-incubated mix over the immobilized mycotoxin derivatives (fig. . ) . since, the immobilized mycotoxin derivatives competed with the toxin in the test sample for binding to the fluorescent antibodies; it was found that the resulting fluorescent signal of the immunocomplex on the waveguide surface was inversely proportional to the concentration of toxin in the sample (decrease in signal with increasing concentration [ ] [ ] [ ] [ ] . this type of array biosensor has been automated and miniaturized for operation as point-of-use quantitative immunoassay arrays. this methodology was utilized to measure an expanded number of toxins and toxin indicators in food and clinical fluids. in addition, semi-selective recognition molecules were also used to expand the repertoire of toxins that can be detected on a single array. in the automated system, up to samples can be analyzed simultaneously while the non-automated system can test up to samples [ b]. taitt and coworkers investigated the possibility to use antimicrobial peptides (amps) for the detection of inactivated botulinum toxins a, b, and e as well as other toxins in assays analogous to the amp-based bacterial assays [ ] . they observed clear differences in the patterns of binding between botulinum neurotoxoids a, b, and e. it was found that the detection limits were improved when immobilized amps were used for target capture [ ] . the love wave (lw) physical effect was originally discovered by the mathematician augustus edward hough love [ , ] . typically, lw sensors consist of a transducing area and a sensing area. the transducing area consists of the interdigital transducers (idts), which are metal electrodes, sandwiched between the piezoelectric substrate and the guiding layer [ ] [ ] [ ] . the input idt is excited electrically (applying an rf signal) and launches a mechanical acoustic wave into the piezoelectric material which is guided through the guiding layer up to the output idt, where it gets transformed back to a measurable electrical signal ( fig. . ) . it should be understood that the sensing area, is the area of the sensor surface, located between the input and output idt, which is exposed to the analyte. consequently, to permits the use of a saw device as a biosensor, the device has to be coated with a biospecific layer corresponding to the analyte. the immobilization chemistry strongly depends upon the underlying saw substrate with or without a guiding layer and hence on the chemical environment available. gold surfaces, for example, allow the use of functionalized thiols, whereas quartz or sio surfaces enable the use of various silanes [ ] . analyte-specific molecules (e.g., antibodies) are immobilized on the saw device to catch analyte molecules (e.g., antigens) from the sample stream. analytes binding to the immobilized capture molecules will influence the velocity of the saw and hence the output signal generated by the driving electronics [ ] . saw detectors have the ability to identify and measure many bas simultaneously and are relatively inexpensive, making them a popular choice amongst civilian response units [ ] [ ] [ ] [ ] . saw detect changes in the properties of acoustic waves as they travel at ultrasonic frequencies in piezoelectric materials. the basic transduction mechanism involves interaction of these waves with surface-attached matter. multiple sensor arrays with multiple coatings and pattern recognition algorithms provide the means to identify agent classes and reject interferant responses that could cause false alarms [ ] [ ] [ ] [ ] [ ] . recently, saw immunosensors were successfully applied to detect e. coli, legionella, the anthracis simulant b bacillus thuringiensis (b ) and m bacteriophage (m ) acting as model analyte for bacteria or viruses. tamarin et al. used love wave sensors based on quartz substrate with a sio wave-guiding layer. antibodies against m were immobilized on the sensor surface to detect the bacteriophage directly [ ] . stubbs et al. developed a saw immunoassay for the detection of analytes in the gas phase, e.g., cocaine plumes [ ] . for this purpose, saw devices based on quartz were used. antibodies were coupled to the saw device via adsorbed protein a and coated with a hydrogel layer to overcome the problem of hydration of the biomolecule [ ] . benzoylecgonine, the major metabolite of cocaine, could be detected in vapor [ ] . in general, it can be specified that saw based biosensors offer the possibility of observing real-time binding events of proteins at relevant sensitivity levels [ , ] . recently, luminescent semiconductor nanocrystals or quantum dots (qds) have become a successful novel nanomaterial possessing unique photophysical fluorescent properties, which have helped create a new generation of robust fluorescent biosensors [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it should be stated that the fluorescent properties of qds have overcome most of the liabilities of conventional organic and protein-based fluorophores. the biosensing qd properties of interest include high quantum yields, broad absorption spectra coupled to narrow size tunable photo-luminescent emissions and exceptional resistance to both photo-bleaching and chemical degradation. in this section, we will examine the progress in adapting qds for several predominantly in vitro biosensing applications including use in immunoassays, as generalized probes [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the unique advantages of using qds are owed to their inherent photostability, their improved sensitivity, and size tunable photoluminescence coupled to their broad absorption spectra. these unique advantages have allowed them to serve as multicolor or multiplexed immunoassays. it should be mentioned that in terms of coupling qds to antibodies, the most common method reported in the literature utilizes biotinavidin interactions [ ] . the avidin/streptavidin coated qds are commercially available, while biotin-labeling of antibodies are usually prepared in-house. qds presenting available carboxylic acids from their capping agents may also be covalently attached to the epsilon amine of an antibody's lysine residues by using edc/nhs coupling chemistry [ ] [ ] [ ] [ ] [ ] [ ] [ ] . alternatively, simple electrostatic interactions can be used depending on the overall protein charge at the ph of conjugation [ ] . goldman et al. used sandwich immunoassays for the simultaneous detection of four toxins: cholera toxin, ricin, shiga-like toxin and staphylococcal enterotoxin b (seb), in a single microtiter well, see fig. . [ , , ] . in this assay capture antibodies immobilized in a microtiter well plate were first exposed to the mixed toxin sample. antibodies specific for each of the toxins coupled to a different color qd were then added to the microtiter well plate. the resulting signal from the mixed toxin samples was then deconvoluted using a simple algorithm. similarly, qd-antibody bioconjugates were used to identify and differentiate between diphtheria toxin and tetanus toxin proteins which were nonspecifically immobilized onto poly-l-lysine coated cover slips and for the simultaneous detection of escherichia coli o :h and salmonella typhimurium bacteria using different colored qds as immunoassay labels [ ] . liang et al. applied qds to mirna microarray assays by using streptavidin qds probes to label biotinylated mirna targets derived from rice, see fig. . [ ] . initially, mirnas were oxidized with sodium periodate to oxidize the ribose ′-and ′-hydroxyl groups into aldehydes. the resulting dialdehyde was then reacted with biotin-x-hydrazide resulting in biotinylated mirna. this was followed by immobilizing the ′ amine-modified oligonucleotide probes antisense to mirnas on the amine-reactive glass slides. the biotinylated mirnas were captured on the microarray by oligonucleotide probes in hybridization. quantum dots were labeled on the captured mirnas through the strong specific interaction of streptavidin and biotin. as qds have a high extinction coefficient and a high quantum yield, so trace amounts of mirnas are easily detected with a laser confocal scanner. in addition as alternative, the colorimetric gold-silver detection method was used in which captured mirnas were labeled with streptavidin-conjugated gold followed by silver enhancement. during silver enhancement, the gold nanoparticles bound to mirnas catalyzed the reduction of silver ions to metallic silver, which further autocatalyzed the reduction of silver ions to form metallic silver precipitation on gold, resulting in a signal enhancement [ ] . this process allowed straightforward detection of the microarray with an ordinary charge-coupled device (ccd) camera mounted on a microscope. they found that qd probes provided good sensitivity down to sub-femtomolar concentrations and dynamic range over several orders of magnitude. this was far better than other dye-based methods and further obviated the use of amplification while allowing a semi-quantitative comparison of the amount of mirna in different samples [ ] . figure . displays a set of images for various concentrations of mirna ( nt sirna) detected by qd [ ] . it should be noted that the signals become gradually weaker with the decrease in mirna concentration (fig. . a) . when the mirna concentration was as low as pm, the fluorescence signal could be detected, indi- cating that the lower detection limit of mirna microarrays is at least . fmol. as shown in fig. . b, the fluorescence intensity of the spots is linear to the model mirna in a logarithmic fashion from to , pm, and the dynamic range is about orders of magnitude. this implies that this method can be used to quantify mirnas with broad concentration range [ ] . fluorescence resonance energy transfer (fret) is a physical radiationless transmission of energy phenomenon which relies on the distance-dependent transfer of energy from a donor molecule to an acceptor molecule. fret has been extensively used in biophysical and biochemical studies to probe ligand-receptor binding and molecular structural changes [ ] [ ] [ ] . in the following example, the authors incubated the dye-labeled dna targets with biotinylated capture dna probes, which were allowed to be conjugated to streptavidin qds, only when the two dna sequences hybridize. the resulting hybridization was then detected via fret between the qd and the dye acceptor (fig. . ) [ ] . it should be noted that the additional background caused by the acceptor direct excitation is virtually eliminated through the choice of an appropriate excitation wavelength; this led to a fold improvement in sensitivity compared to single organic dye molecular beacon-based detection. this type of sensing schemes can also be amenable to use in a multiplex format. the narrow and symmetric qd emissions allow easy spectral petrovick et al. have developed a novel inexpensive genetically engineered whiteblood cells biosensor for the rapid identification of warfare ba pathogens and toxins [ ] . this new sensor was named and abridged as canary for "cellular analysis and notification of antigen risks and yields". canary sensors are capable to detect soluble protein toxins, which are an important class of potential bioweapon, and can also be used for sequencing dna and rna [ ] . the canary technology is based on genetically engineered white blood b cells, which has the ability to bind to and to recognize pathogens quickly and assists other parts of the immune system to fight the infection. it is well known that b cells are the fastest known pathogen identifiers (intrinsic response less than sec). the b-lymphocytes recombinantly express cytosolic aequorin, a ca-sensitive bioluminescent protein from the jellyfish aequoria victoria, which emits light in response to elevations of intracellular ca, along when membrane-bound by antibodies. binding of the pathogen to the cell surface antibodies causes an increase in intracellular ca levels, resulting in the emission of light from the cytosolic aequorin [ ] [ ] [ ] [ ] . two routine genetic modifications enable engineered b-cell lines to express cytosolic aequorin, a calcium sensitive bioluminescent protein, as well as membrane bound antibodies specific for pathogens of interest [ , ] . this was achieved by crosslinking the membrane-bound antibodies to a polyvalent antigen that induces a signal-transduction cascade. this latter, sequentially involves tyrosine kinases, phospholipase c and inositol triphosphate (ip ). the ip activates calcium channels, thereby increasing cytosolic calcium from both internal stores and the extracellular medium, which activates the aequorin, causing it to emit light (fig. . ) [ , [ ] [ ] [ ] . the canary sensor can detect less than colony-forming units (cfu) of pathogen in less than min, which include the time required to concentrate the sample [ ] . it should be mentioned that state-of-the-art immunoassays take at least min, whereas polymerase chain reaction (pcr) takes longer than min. the novel genetic-engineering system developed by petrovick et al. have consisted of the efficient production of b-cell lines that can react specifically and rapidly to a variety of pathogens [ ] . the antibody genes were cloned from hybridomas and inserted into expression vectors. these were transfected into a parental b cell line that expresses active aequorin, and the cells are screened for their response to patho-gen. the genetically engineered canary cells can be used separately in a single identification assay, or as many as three can be combined to achieve a multiplexed assay. alternatively, several antibodies can be expressed in a single cell line to provide a classification assay [ ] . it is also feasible to create b cells that emit at different wavelengths of light, enabling multiplexed assays that simultaneously distinguish among several targets [ ] . a novel effective method described by petrovick et al. for the immobilization of a toxin was to capture it on beads coated with antibodies against that specific toxin [ ] . the antibody-coated beads were then incubated in a solution containing the suspected toxin, and washed to remove the contaminating proteins and other materials. the toxin-obtained decorated beads are excellent candidates for the purification and immobilization of toxin analytes by the canary cells, as illustrated in fig. . [ ] . it should be mentioned, that the canary cells should express an antibody that binds to the toxin at a different site from that of the capture antibody. because the toxin is immobilized on the bead, the antibodies on the canary cell that bind to the toxin are also immobilized, and therefore light emission is stimulated [ ] . this approach has been used to develop a very effective canary assay for botulinum neurotoxin type a (bont/a) which is the most poisonous toxin known to man, with the detection of soluble macromolecules has a second interesting application: identification of dna and rna sequences. the ability to identify nucleic acid (na: dna or rna) sequences is central in the deduction of the first hard na sequence information, concerning a new or genetically modified pathogen [ ] . it is important to develop assays that have the flexibility to respond quickly to new threats. for this reason, developing na probes allow for the rapid and quick examination of the actual genetics of the target organism [ ] . once the na sequence of a pathogen is determined, multiple short probes are synthesized that bind adjacent to each other along a specific sequence on the target na [ ] . consequently, petrovick et al. developed a novel assay that uses a single canary cell line that expresses an antibody against digoxigenin. each of these probes is labeled with a single digoxigenin molecule. if these probes are added to solution containing the target na sequence, the binding of multiple digoxigenin-containing probes produces a tight cluster of immobilized digoxigenin molecules, which will stimulate light production from the canary cell (see fig. . ) [ ] . in the absence of target na, each digoxigenin-labeled probe remains monomeric, and therefore cannot crosslink antibodies on the surface of canary cells. there are advantages to detecting rna as compared to dna. primarily, while there is only one copy of genomic dna per bacterium, there can be thousands of copies of a single rna; so the number of target molecules per bacterium is much higher. furthermore, since probe binding requires that the target na be single stranded, a denaturing step must be performed to separate the two constituent strands of dna. rna, however, is normally single stranded [ ] . the canary approach takes advantage of a receptor that binds to the constant region of antibodies, leaving the antigen-binding region of the antibody free. after binding bacteria with the captured antibodies, the receptor initiates a signal cascade, similar to the one induced by the crosslinking of membrane-bound antibodies on b cells, which activates aequorin [ ] . the excellent combination of speed and sensitivity of the canary system was demonstrated with cell lines expressing an antibody specific for the f antigen of yersinia pestis (yp), shown in fig. . [ ] . when concentrated in the centrifuge luminometer, as little as cfu of formalin-inactivated yp are detected. however, there was no response to relatively large numbers of f. tularensis [ ] . for biological defense applications, the canary technology was incorporated into a flexible biological-aerosol sensor platform called panther that can form the core of a family of mission-specific bio-aerosol identification sensors useful as standalone sensors for site/building protection, emergency response, rapid screening, and environmental monitoring [ ] . to summarize, the canary's capabilities open possible applications in pathogen genotyping, virulence testing, antibiotic resistance screening, and viability assessment. moreover, using these cells demonstrate the best known combination of speed and sensitivity. other applications of canary technology include biological aerosol sampling, point-of-care diagnostics, pre-symptomatic diagnosis in the aftermath of a biowarfare attack, detection of agricultural pathogens at ports of entry, or screening of perishable food supplies medium [ ] , which activates the aequorin, causing it to emit light [ , ] . it is well known that an optical fiber or optical fibre is a flexible, transparent fiber made by drawing glass (silica) or plastic to a diameter slightly thicker than that of a human hair [ , ] . optical fibers consist of an inner core which is surrounded by a clad material of lower refractive index. because of the differences in refractive index light is totally reflected. a fiber optic bundle consists of thousands of individual fibers fused together such that each fiber retains its ability to transmit light independently of its neighbors (fig. . ) [ ] . pantano and walt showed that by selectively etching the fiber core, an array of microwells can be formed [ ] . these microwells can be filled with oligonucleotidefunctionalized microspheres. the array dimensions can be tailored to suit any size of oligonucleotide-functionalized microsphere. the well diameters are equal to those of the fiber cores, and the depths are dependent on the etchant concentration, the exposure time, and the fiber composition. because each microsphere is optically wired to a fiber, the specific interactions on each microsphere surface can be independently monitored. walt and coworkers developed a high-density fiber optic dna microarray consisting of oligonucleotide-functionalized, . -μm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle [ ] . usually these fiber bundles are composed of around / fused optical fibers, in which each fiber -contains an etched well [ ] . the desired oligonucleotide sequences are attached to individual microspheres, than added to each etched wells on the fiber optic bundle face. the produced microwell arrays are capable of casing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. walt and coworkers showed that the array fabrication process resulted in random microsphere placement. it should be understood that the determination of the position of microspheres in the random array, essentially required an optical encoding scheme. the detection schemes, which are combined the intrinsic recognition abilities of nucleic acids, are usually measured with fluorescence-based detection methods. this "optical bar code" obtained is simply a combination of fluorescent dyes with different excitation and emission wave lengths and intensities that allows each bead to be independently identified [ ] . nonetheless, additional degrees of freedom are available in the fiber-optic format, that allows additional number of excitation and emission wavelengths that can be used. it should be mentioned that the optically bar-coded arrays, can be decoded in a matter of seconds. this occurs byh conventional image processing software that collect series of fluorescence images at different excitation and emission wavelengths and then analyze the relative intensities of each bead. alternatively, preformed oligonucleotides may be added directly to surface-activated microspheres [ ] . needless to say those fluorescence-based assays are more desirable than traditional radiolabeled methods due to their increased safety and experimental versatil-ity. fluorescence can be incorporated into microarray assays by fluorescent intercalating dyes, fluorescently labeled targets, or label-less methods employing fluorescence resonance energy transfer (fret). fluorescence-based assays enable the measurement of multiple wavelengths independently and simultaneously. the use of multiple fluorophores enables parallel interrogation schemes [ ] [ ] [ ] [ ] . a general array protocol entails immobilizing a probe sequence (primer) that can hybridize to its fluorescently-labeled complementary target. the fluorescent tag is commonly incorporated into the target molecules, via polymerase chain reaction (pcr) [ ] . this primer labeling method is convenient when amplification is needed for detection, or when it is used to construct a cdna library from a genomic rna pool via reverse transcription. in addition, the derivatized fiber core experiments are able to detect unlabeled (non-fluorescent) target solutions ]. this is achieved by competitive hybridization with fluorescent target samples. in this method, the fluorescent synthetic target complements are synthesized and initially hybridized to the array to saturate the array probe elements [ ] . the unlabeled target solution is then hybridized to the same array, competing with the prehybridized synthetic targets. the presence of unlabeled target is determined by a fluorescence decrease caused by displacement of the fluorescent synthetic target by the unlabeled species. this procedure eliminates the need to incorporate fluorescence into the target and allows quantitative measurements to be performed. these fiber optic platforms are the basis for microsphere array designs that improved array fabrication and allowed extremely high-density sensor placement [ ] . the imaging system consists of a light source, an inverted microscope, and a modified olympus epifluorescence microscope/charge-coupled device camera (photometrics pxl). a fiber chuck held the imaging fiber in a fixed position while electronically controlled filter wheels switched between the analytical wavelength and the encoding wavelengths, enabling complete analysis and identification of the microspheres within minutes. excitation light is sent into the proximal tip of the imaging fiber, and emission from the fluorescing molecules captured and directed onto the ccd camera detector (fig. . ) [ ] . the multiplex analysis images were acquired for and . s at wavelengths specific to each encoding dye. a -nm excitation filter and a -nm long-pass emission filter were used for the eu-dye. a -nm excitation filter and a -nm emission filter were used for the cy dye. a -nm excitation filter and a -nm emission filter were used for tamra. that is detection of amplified dna fragments which were incubated with a fluorescein-labeled sequencing primer in the presence of the two allelic rox-ddntp or tamra-ddntp terminators. all targets were labeled with fluorescein. it was shown that the fluorescence intensity was proportional to the extent of hybridization at each probe position [ a]. in addition, the camera was equipped with an internal chip that provides megapixel resolution ( / ). this megapixel chip was able to resolve the arrays miniatured feature sizes ( mm) and provided multiple pixels for each optical channel in the fiber bundle (fig. . ) . finally, it can be surmised that the described fiber optic microsphere-based biosensor is a versatile platform that possesses micro-scale features and an overall array size that enables rapid analysis and extremely low detection limits. redundant detection elements in the array increase the signal-to-noise ratio and avoid the potential for false positive and false negative results. microsphere-based arrays are reusable, and are easy to fabricate. the fiber optic platform has also been applied to other applications including artificial olfaction and cell-based array sensing [ , ] . recently, numerous strategies for protein labeling were developed and they allowed the characterization of proteins regarding their structure, folding, or interaction with other proteins [ ] . surface plasmon resonance (spr) is a label-free detection method which is a suitable and reliable platform in clinical analysis for biomolecular interactions. undeniably, spr has been proven to be one of the most powerful technologies to determine the specificity, affinity and kinetic parameters displayed during the binding of macromolecules. these binding of macromolecules include, but are not limited to, protein-protein, protein-dna, enzyme-substrate or inhibitor, receptor-drug, lipid membrane-protein, protein-polysaccharide and cell or virusprotein [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . spr is an optical technique which measures the refractive index changes in the vicinity of thin metal layers (i.e., gold, silver, or aluminum films) in response to biomolecular interactions. when the photon of an incident light strikes a metal surface, typically a gold surface, surface plasmon resonance occurs. when a portion of the light energy excites the electrons of the metal surface layer, at a certain angle of incidence, it creates electron movements which propagate parallel to the metal surface. this electric oscillation is termed as a "plasmon". this oscillation, in turns, generates an electric field whose range is around nm from the boundary between the metal surface and sample solution [ , ] . in a commercial spr biosensor configuration, the incident light employed is generated by a high-reflective index glass prism of the kretschmann geometry of the attenuated total reflection (atr) method (fig. . ) [ ] . the defined spr angle, at which resonance occurs, relies on the refractive index of the material coating the metal surface, and the constant light source wavelength. when there is no change in the reflective index of the sensing medium, the plasmon oscillation cannot be formed (fig. . a) . in addition, it should be noted that when the surface of the sensing material has been coated through biomolecule attachment only, there will be perhaps an unnoticed small change in the reflective index of the sensing medium, and as a result, the plasmon oscillation cannot be formed ( fig. . c, left side) [ ] . however, when the metal surface has been coated with an analyte-biorecognition couple of biomolecules, detection is accomplished by measuring the changes in the reflected light obtained on a detector (fig. . c, right side). in addition, the amount of surface concentration can be quantified by monitoring the reflected light intensity or tracking the resonance angle shifts. typically, an spr biosensor has a detection limit of pg/ml [ ] [ ] [ ] [ ] . in all commercial spr biosensors, probe molecules are firstly immobilized on to the sensor surface. when the solution of target molecules is flown into contact with the surface, a probe-target binding via affinity interaction occurs, which consequently induces an increase in the refractive index at the spr sensor surface (fig. . d ) [ ] . in spr experiments, resonance or response units (ru) are used to describe the signal change, where ru is equivalent to a critical angle shift of − degree [ ] [ ] [ ] [ ] [ ] . at the start of the experiment whereas probe target interactions have not occurred, the initial ru value corresponds to the starting critical angle. the change in refractive index Δnd arisen within a layer of thickness h can be calculated as: where (dn/dc) vol is the increase of refractive index n with the volume concentration of analyte c, and ΔΓ is the concentration of the bound target on the surface [ ] . there are several different formats of spr biosensors. these comprise the array format, multi-channel unit format, and spr imaging format, which allow simultaneous and continuous detection to analyze the performance of hundreds to thousands of affinity binding events on a chip surface [ ] [ ] [ ] . in spr imaging, the incidence angle remains fixed, and the binding of biomolecules on a gold surface is measured as the change in reflectivity (or reflectance) in relation to the incident ray intensity, unlike spr sensors that depend on the measurement of the absorption dip in the spr angle or spr wavelength [ ] [ ] [ ] [ ] [ ] . recently, it was shown that spr imaging technology using a multi-analyte biosensor allows the measurement of a high-throughput approach, and it achieves a similar degree of sensitivity of conventional spr biosensors ( fig. consequently, spr imaging systems without any labeling requirements can be used for high-throughput screening (hts), particularly in drug discovery, than any other optics-based detection techniques [ , ] . dhaked and coworkers developed a label free real time method for the detection and quantification of botulinum neurotoxin a (bont/a) using surface plasmon resonance (spr) [ ] . the authors used antibody against rbont/a-hcc fragment and synaptic vesicles (sv), which were immobilized on a carboxymethyldextran modified gold chip. the immobilization of bont/a antibody and interaction of bont/a with immobilized antibody were characterized in-situ by spr and electrochemical impedance spectroscopy. a sample solution containing bont/a antigen in concentrations ranging from . to . fm and . to . fm was interacted with the immobilized antibody and immobilized sv, respectively [ ] . dhaked and coworkers described the stepwise immobilization of bont/a antibody and sv protein on a cm chip, respectively in nine steps. these steps involved the stabilization of the baseline, the activation of carboxyl groups on the cm chip, and converting them into activated carboxymethylated groups on the sensor chip for future bounding to the free amino groups of bont/a antibody. this was followed by washing with pbs and measuring the spr angle that has shifted nearly to the baseline [ ] . when the bont/a antibodies are injected on the cm chip an increase in spr angle was observed. finally, washing with mm ethanolamine was performed to prevent non-specific binding and to block the unreacted nhsester groups on cm chip. most of the sensing measuring devices used for the detection of biological warfare agents are based on luminescence immunoassay signal transduction mechanisms which are optical. in this chapter, we have discussed the following subjects: impedance spectroscopy, evanescent wave technology and internal reflection fluorescence (tirf) excitation, surface acoustic wave sensors, fluorescent biosensors, fluorescence resonance energy transfer (fret), light emission (canary), microsphere-based fiber optic biosensors, and surface plasmon resonance. it should be noted that although fluorescence detection is the most popular, surface plasmon resonance (spr)-based immunoassay formats with surface imaging capabilities are growing in popularity [ [ ] [ ] [ ] [ ] [ ] [ ] [ ] . multiplexing, or simultaneous detection of multiple analytes, is one of the most important prerequisites for biothreat agent detection. as mentioned in our introduction, the presented work is more a "comptes rendus" and an introduction to this series of sensing measuring devices used for the detection of biological warfare agents that were presented to the participants of this nato-asi meeting. medical aspects of chemical and biological warfare biological warfare and bioterrorism: a historical review another bloody century: future warfare strengthening global preparedness for defense against infectious disease threats. committee on foreign relations, united states senate. hearing on the threat of bioterrorism and the spread of infectious diseases lessons learned from a full-scale bioterrorism exercise new world coming: american security in the st century, supporting research and analysis history of chemical and biological warfare agents biological and chemical terrorism: strategic plan for preparedness and response ( ) recommendatons of the cdc strategic planning workgroup bioweapons and bioterrorism: a review of history and biological agents assessment of biological agent detection equipment for emergency responders chemical and biological terrorism: research and development to improve ivilian medical response to chemical and biological terrorism incidents, national academy of sciences final report on the assessment of biological agent detection equipment for emergency responders state of the art and recent advances in immunoanalytical systems sensors for detecting biological agents electrochemical biosensors: recommended definitions and classification electrochemical biosensors: recommended definitions and classification electrochemical biosensors: recommended definitions and classification pcr-elisa detection of escherichia coli in milk determination of the sensitivity of a rapid escherichia coli o :h assay for testing -gram composite samples peptides as weapons against microorganisms in the chemical defense system of vertebrates antimicrobial peptides of multicellular organisms antimicrobial activity and protease stability of peptides containing fluorinated amino acids electrical detection of pathogenic bacteria via immobilized antimicrobial peptides antimicrobial activity of synthetic magainin peptides and several analogues magainins, a class of antimicrobial peptides from xenopus skin: isolation, characterization of two active forms, and partial cdna sequence of a precursor interactions of an antimicrobial peptide, magainin , with outer and inner membranes of gram-negative bacteria array biosensor for toxin detection: continued advances array biosensor for detection of toxins reflectively coated optical waveguide and fluidics cell integration a portable automated multianalyte biosensor detection of deoxynivalenol in foods and indoor air using an array biosensor shriver lake lc ( ) indirect competitive immunoassay for detection of aflatoxin b- in corn and nut products using the array biosensor an array biosensor for detection of bacterial and toxic contaminants of foods blind laboratory trials for multiple pathogens in spiked food matrices augustus edward hough love. - . obituary notices of fellows of the augustus edward hough love surface acoustic wave biosensors: a review acoustic wave sensors fluorescence resonance energy transfer concepts, applications and advances luminescent quantum dots in immunoassays avidin: a natural bridge for quantum dot-antibody conjugates antigen/antibody immunocomplex from cdte nanoparticle bioconjugates microminiaturized immunoassays using quantum dots as fluorescent label by laser confocal scanning fluorescence detection simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy quantum dots as a novel immunofluorescent detection system for cryptosporidium parvum and giardia lamblia an oligonucleotide microarray for microrna expression analysis based on labeling rna with quantum dot and nanogold probe biosensing with luminescent semiconductor quantum dots quantum dot-labeled trichosanthin fret imaging fluorescence resonance energy transfer concepts, applications and advances selective, reversible, reagentless maltose biosensing with core-shell semiconducting nanoparticles rapid sensors for biological-agent identification the enzymology and molecular biology of the ca +−activated photoprotein, aequorin, photochem semi-synthetic aequorins with improved sensitivity to ca + ions cross-linkage of b lymphocyte surface immunoglobulin by anti-ig or antigen induces prolonged oscillation of intracellular ionized calcium site-specific mutagenesis of the calcium-binding photoprotein aequorin peroxidized coelenterazine, the active group in the photoprotein aequorin nargi fe a b cell-based sensor for rapid identification of pathogens optical fiber communications: principles and practice high-density, microsphere-based fiber optic dna microarrays ordered nanowell arrays light-directed, spatially addressable parallel chemical synthesis quantitative monitoring of gene expression patterns with a complementary dna microarray high-density fiber-optic genosensor microsphere array capable of zeptomole detection limits high density fiber optic dna random microsphere array image processing for cameras with fiber bundle image relay polymerase chain reaction (nobel prize) surface plasmon resonance: a versatile technique for biosensor applications choosing an effective protein bioconjugation strategy isotope labeling strategies for the study of highmolecular-weight proteins by solution nmr spectroscopy generation of bioreagents for protein chips surface plasmon resonance sensing of nucleic acids: a review ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the air-water interface surface plasmon resonance imaging as a tool to monitor biomolecular interactions in an array based forma surface plasmon resonance imaging surface plasmon resonance imaging-based protein arrays for high-throughput screening of protein-protein interaction inhibitors surface plasmon resonance biosensor chips surface plasmon resonance sensing of biological warfare agent botulinum neurotoxin a spr-based immunosensor for determining staphylococcal enterotoxin a quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins surface plasmon resonance sensing of biological warfare agents key: cord- -mmmm pmv authors: mujahid, adnan; dickert, franz l. title: surface nano-patterning of polymers for mass-sensitive biodetection date: - - journal: nano-bio-sensing doi: . / - - - - _ sha: doc_id: cord_uid: mmmm pmv the crafting of sensor material of desired features has always remained a challenging task in the field of material designing and predominantly becomes more interesting when analyte belongs to biospecies. label-free detection of different bioanalytes such as enzymes, viruses, microorganisms, and blood groups through mass-sensitive transducers has gained considerable importance in the development of modern biosensors. analyte molecules interact with the surface of sensitive layer coated on these devices and as a result of this interaction, the frequency change is determined, which provides quantitative information about the mass of analyte. one of the most vital elements of these detection systems is to design selective sensor coatings through control surface structuring at nanoscale. molecular imprinting has proven to be a highly suitable technique to generate selective surfaces that are capable of detecting different analytes, quantitatively and qualitatively as well. the tailor-made synthetic antibody cavities are rigid and stable, which are not immediately collapsed upon analyte interaction; moreover, the different bioanalytes do not undergo any phase change and maintain their original identity during analysis. this chapter will discuss the contribution of imprinting methods to design optimized surfaces for mass-sensitive detection of diverse biological species. detection of biological species such as enzymes, microorganisms, proteins, dna, etc., has gained substantial importance in various fields, which include health care, industrial and environmental analysis, and biotechnology and process control. currently, most of the detection systems used to monitor binding of biological molecules on sensor surface need some fluorescent or enzymatic labeling. this labeling step charges additional cost and time to the bioanalysis. the other concerned problem regarding this type of analysis is that, in some cases, the labeling reagent itself interferes with the analyte molecule, which then leads to false measurements. this makes the analysis more complex and less reliable. considering the cost, time consumption, and reliability of the measuring system, this strategy has some disadvantages in monitoring the binding of biological species at the surfaces. there is a growing interest in designing modern bio-recognition systems because of the rapid developments in this field. to obtain the desired information about the analyte molecule with certain degree of confidence, we have to develop suitable sensitive, selective, fast, inexpensive, and label-free detection systems. this problem needs to be addressed mainly in two parts: the first part is associated with the design of selective antibody surfaces that are capable of interacting only specifically with analyte molecules in complex matrices; and the second part is related with the design of transducer that can generate signals more sensitively for very low concentrations of analytes. mergence of these two strategies can produce innovative detection systems, which can perform monitoring of different bioanalytes with appropriate selectivity and sensitivity. molecular imprinted polymers (mips) along with acoustic or mass-sensitive devices provide a highly favorable route to carry out various types of bioanalysis. although there are some other surface structuring techniques that have some applications in mass-sensitive biodetections, molecular imprinting has high repute in this regard. the imprinted polymers are of rigid and robustness nature, which is not damaged by exposing them into extreme conditions, and, in contrast, acoustic devices give a direct change in frequency corresponding to the bounded mass on their surface. such detection systems have gained substantial importance during the last decade because of their widespread applications in different fields such as degradation analysis, compost monitoring, enantio-selective sensors, and environmental monitoring. to understand how to design highly selective surfaces for bioanalytes and for their detection by mass-sensitive devices, one should follow the concept of molecular imprinting along with other surface crafting schemes and fundamental principles of acoustic devices. in this chapter, coming sections will focus on these issues and discuss their potential applications in bioanalytics. crafting of innovative materials for selective detection of different species is clearly a key assignment in bioanalytics, and especially their surface structuring is even more a challenging task at micro and nanoscale. the issue can be resolved by considering natural antibodies [ ] as a direct tool for monitoring immunochemical reactions for detection purposes. one of the major advantages of this scheme is that natural antibodies provide sufficient degree of specificity for desired target molecule in complex mixtures. these materials can be immobilized on an appropriate transducer and be used to design biosensors [ , ] . a very recent example of this strategy has been explained in a review article in which a thin layer of enzymes is immobilized on an electrochemical probe. this sort of detection systems has shown good results regarding sensitivity and selectivity. the major drawback of this technique is the lack of ruggedness and long-term stability of the designed surface, which limits their applications while crafting materials that can face severe conditions and retain their properties for longer period of time. the other concerned problem regarding these materials is that the interaction between antibody and antigen is usually very strong, which significantly reduces the reversibility and thus restricts their reuse. one of the modern trends in material designing to synthesize very thin films of layer heights ranging from one micrometer to several nanometers is set by the layer-by-layer (lbl) or electrostatic self assembly (esa) methods. it was first introduced by decher et al. [ ] in during the synthesis of controlled thin films [ ] . the synthetic procedure for developing lbl assembly is relatively easy and convenient. a schematic diagram for the preparation of lbl thin films is shown in fig. this is a four-step procedure as shown in the figure, which is continuously repeated until the required numbers of layers are generated. this technique is quite new but still promising to craft the different materials to get the desired properties such as chemical, mechanical, thermal, and biocompatibility having sufficient degree of roughness. the synthesized lbl assemblies are of robust nature, which can effectively perform in severing conditions. the stimulating feature about these materials is that the layer-to-layer attractions are not restricted to electrostatic forces but assemblies can also be formed by hydrogen bonding [ ] , covalent bonding [ , ] , and some hydrophobic interactions [ , ] as well. lbl assembly methods are very advantageous to organize the surface structures at nanometer scale and to incorporate the desired biomolecules. the versatility of this strategy enables us to launch any kind of charge species such as proteins [ ] [ ] [ ] [ ] , polypeptides [ ] , dna [ ] , viruses [ ] , antibodies [ ] , and various other biological compounds in thin films preparation. the introduction of different biological species in lbl assembly generates very sensitive biological receptors having tunable selectivity. the induced functionalities in thin films truly favor the production of biorecognition materials. these materials can also be patterned by fabricating different components such as nanoparticles [ ] , nanotubes [ ] , and some other nanoplates [ ] [ ] . this is of course of great interest in designing the biorecognition [ ] of thin films at nanoscale [ ] , which can be deposited on a suitable transducer to conduct various bioanalyses. besides many significant advantages of lbl technique, there are certain limitations. the major drawback of this method is that it requires quite a long deposition time of layer formation, which makes synthetic procedure too lengthy. moreover, very high concentrations of deposition reactants are needed in order to coat appropriate layer material. certain lbl assembly formation steps are very complex, which needs special masking and is time consuming. although the binding of lbl receptors are of selective nature, sometime it is nonspecific due to exposition of the whole substrate surface to reactant solutions. the practical examples of lbl assemblies in mass-sensitive biodetection applications are still infancy but, nevertheless, talented enough to meet the challenges of modern bioanalytics. supramolecular chemistry provides an alternative synthetic route to design these materials, according to the technical needs. in these artificial materials, intramolecular and intermolecular noncovalent forces operate between the analyte and the host molecule. this strategy has been proven to be very successful in generating selective recognition systems for biomolecules [ ] . it has solved the problem of reversibility and reusability when compared with the natural antibodies. the major drawback of this scheme is that the synthetic procedure is complicated and relatively time consuming. it is also a tedious task to find out the optimal interaction conditions between host and guest molecule. therefore, to design an optimal recognitions system with minimum effort and technical expertise, we have to consider other strategies. the problem can be solved by introducing artificial recognition layers modified according to the shape and size of bioanalyte. these artificial recognition materials offer considerable flexibility in terms of tailoring the interaction sites in polymer surfaces. mips [ ] promise to solve all the concerned problems and meet the desired needs in designing of novel materials. the most exciting aspect of molecular imprinting is that it provides molecular recognition to polymer matrix, which generates selectivity for very similar class of biomolecules. this technique was first discovered by the groups of kiefer [ ] and wulff [ ] independently in , during the synthesis of organic polymers. initial applications of molecular imprinting were found in separation processes and since after that the scope of this technique was extended to other fields such as chemical and biosensors [ ] , artificial biorecognition surfaces, and artificial synthetic antibodies. the principle of molecular imprinting can be better understood from the following example in fig. . where the model compound as template (i.e., usually analyte molecule) is introduced in polymer mixture along with monomers and cross linker. the required degree of polymerization is achieved by carefully controlling reaction conditions. during polymerization reaction, the polymer chains are self organized around the template compound, which are fixed at the end of reaction. the template or analyte molecules are finally removed from polymer matrix by heating or washing methods, which leaves behind geometrically adapted cavities for analyte reinclusion. generally imprinting procedure can be accomplished as a three-step process: the first step is the prearrangement of template, monomer, and cross linker; second is the engulfing of polymer chains around template; and the third is the extraction of the template molecules. in fact imprinted polymer possesses the memory of removed analyte molecule and can recognize and extract it selectively from a complex mixture of closely related compounds. the geometrical shape, size, and configuration of cavities can be improved for optimal template interaction by modifying the reaction conditions [ , ] such as pressure, temperature, amount of cross linker and monomer to template proportion [ ] . the amount of cross linker is very important as the higher degree of cross linking ensures the generation of cavities of very fine shape for optimal selectivity. some post-imprinting modifications can also be employed to further improve the cavities shapes for reinclusion. the basic requirements to conduct imprinting process are the high cross linking of polymer and the noninterfering nature of template with polymer matrix. molecular imprinting can be classified into two types, according to the interaction of monomers with template molecules (i.e., covalent and noncovalent imprinting). noncovalent imprinting was first introduced by mosbach [ ] , where the template molecules develop affinity with function groups of monomer through noncovalent associations such as hydrogen boding, dipole-dipole interactions, van der waals forces, etc. prior to polymerization. the removal of template molecules takes place very fast as it does not require any bond rupture. this is a fast and straightforward way to design recognition materials for different detection purposes or developing chemical sensors. the only requirement for this process as earlier mentioned is that the template should not interfere with polymer, so there are no more restrictions for analyte size and shape. the only drawback of this technique is that it cannot be applied to those template molecules that do not have any functional group to interact with polymer system, which limits the construction of binding sites for analyte interactions. this problem is solved by later technique in which the model compound is covalently linked with polymer chain. after polymerization, the template molecules are removed by bond rupturing between template and polymer. the imprinted polymer can rebind the analyte molecules reversibly, and the selectivity is achieved by reactive groups in polymer matrix. covalent imprinting is no doubt a practical method to various templates not having functional group but is limited due to the small number of useful applications of reversible covalent bonding. a hybrid imprinting technique was developed by whitecombe [ ] in in which template molecule is covalently bonded to polymer matrix, whereas after its removal reversible and noncovalent interactions takes place. tepper [ ] has adopted a different imprinting approach in which the polymerization was carried out on transducer surface in solid phase rather than in solution form in the presence of alkaline vapors that contribute to generate imprinted sites for analyte reinclusion. the sensitivity achieved by mips is accessed by number of imprinting sites available for analyte interactions (i.e., more the imprinted sites available higher will be the sensitivity). nanoporous polymers possess more imprinted sites when compared with microporous polymers. the other important aspect regarding the sensitivity is the distribution of imprinted sites in polymer layer prior to use for detection purposes. the size of analyte molecules governs the imprinting strategy whether bulk imprinting [ ] or surface imprinting is suitable [ ] . in bulk imprinting (as shown in fig. . ), the template molecule is added along with monomer and cross linker at the start of reaction and after polymerization is removed. this strategy is useful for relatively smaller analyte molecules having molecular mass < g mol - but while considering the larger analytes such as biomolecules, the bulk imprinting is not always complimentary. although number of interaction sites in bulk imprinting are high in comparison with surface imprinting, keeping in mind the larger size of biomolecules, incomplete reversibility, relatively longer diffusion pathways, and longer time for measurement make their detection highly unfavorable. therefore, for larger biomolecules, surface imprinting, as shown in fig. . , is proposed where template molecules are directly imprinted on the prepolymerized surface by stamping method [ ] with a little force. thus, the patterned polymer surface possesses the dimensions from one to several hundred nanometers that can exclusively extract target molecule from the complex mixture. molecular imprinting provides a straightforward, versatile, and unproblematic way to synthesize selective coating materials for the detection of various biospecies. the most beneficial aspect of this scheme is that it is not limited to a certain class of compounds unlike the natural antibodies detection systems. the other significant feature of these materials is that they exhibit long-term stability and do not undergo degradation over the course of time, which makes use of these materials for extended period of time. reversibility of surface imprinted materials makes reusable these materials for several analyses, which reduces the cost. the synthetic route of imprinting procedure is relatively easy as compared to the scheme followed for host-guest interactions such as in the case of cyclodextrines, paracyclophanes, and calixarenes. considering the versatility in synthetic approach, ruggedness of designed imprinted material to severe conditions, flexibility regarding choice of analyte, and long-term stability make these materials superior for rapid, inexpensive, and selective detections of various bioanalytes over other strategies. in order to make use of surface strategies for the detection of biospecies, first we have to understand the fundamental principles involved in transducers (i.e., acoustic or mass-sensitive devices). there are different types of these devices, which are employed in different working environments, according to their specific job to get desired information with minimum error. thus, it is very important to select a right device for the dedicated task. in the coming section, we will focus on the basic principles of these devices and their operating modes in diverse mediums to get an optimized detection signal of different analytes. the fundamental principle involved in acoustic or mass-sensitive devices can be explained by piezoelectric effect, which was first discovered in [ ] by two brothers pierre curie and jacques curie, in some crystalline materials such as quartz, rockchille salt, and ceramic. they had observed that if stress is applied on such crystalline materials in certain dimensions, it separates the negative and positive charges from their center creating a dipole, which leads to the generation of electrical voltage. the same is true if we apply an electrical voltage to such materials, it causes mechanical deformation in their shape. former phenomenon is called piezoelectric effect, while the later one is known as inverse piezoelectric effect. the common examples of such materials that possess piezoelectric character are cane sugar, rochelle salt, berlinite (alpo ), and quartz, which are natural materials, while synthetic examples are gallium orthophosphate (gapo ), langasite (la ga sio ), and lithium tantalate (litao ). the selection of these materials depends upon their application in a typical medium and working environment: for example, gallium orthophosphate has high temperature coefficient than quartz and can be employed in working environment having temperature up to c. quartz crystals are well-known acoustic device, which are widely used as masssensitive transducers for different sensing purposes. they are capable of measuring the mass changes in nanogram range. to have the desired properties of piezoelectric material, the crystal plate is cut at a specific angle, which is at-cut ( ) and btcut ( ) . the at-cut quartz is very famous and most frequently used as masssensitive transducer, as it has remarkable temperature and frequency properties. acoustic devices are considered as mass-sensitive devices because when a certain mass is loaded on an oscillating device, there is a corresponding change in the frequency of these devices. this phenomenon was first reported by sauerbrey in [ ] in his famous article. sauerbrey has developed a relationship of frequency change in acoustic resonators upon mass deposition considering all the physical parameters of crystal material. the relationship is best described by the following equation. in this relationship, the symbols represent the following: df is the frequency change, f o is the resonant frequency, dm is the change in mass, a cr is the active piezoelectric area of quartz (usually electrode area), c q is the shear modulus of quartz crystal, and r m is the density of quartz. this equation clearly indicates that the frequency change is directly related to the mass load on the surface of substrate. this equation is designed for typical gas phase environment. if we shift from gaseous phase to liquid phase, the equation is modified as described, considering the certain physical parameters associated with liquids such as viscosity and density. the other important information revealed from sauerbrey equation is that the frequency change also depends upon the square of fundamental resonance frequency of device. it means by doubling the fundamental frequency of the device, the frequency change would be four times. this is very important in the design of device because extremely low concentrations of analyte can be determined by simply selecting a device of highest available frequency. the frequency of a piezoelectric material is mainly determined by its thickness. the characteristic frequency of these materials is given by the following relationship. in this relation, c is the velocity of sound in the material and d is the thickness of the sheet. the quartz sheet undergoes resonance oscillations when connected with circuit oscillator, where an electrical and mechanical oscillation comes close to the fundamental frequency of quartz. an equivalence circuit for quartz resonator is designed to understand the oscillation phenomena and to calculate the quality factor (q) of oscillations or dissipation (d) (i.e., mass accumulation on quartz surface and visco elastic behavior of deposited polymer). this equivalence circuit is shown in fig. the oscillations of resonating quartz having two electrodes can be compared with this circuit in such a way that l stands for deposited mass, c shows elastic behavior, r represents the damping loss, and c o shows the capacity between two electrodes. the impedance is calculated by taking the ratio of applied electrical voltage and the current flowing through it. this value of impedance gives information about the quality of oscillations and explain about visco elastic nature of deposited layer on quartz surface. the different resonating modes of a mhz quartz wafer (i.e., serial resonance and parallel resonance) can be compared with this circuit along with the phase shift. generally, acoustic devices can be classified in two main types depending upon the nature of wave propagation mode (i.e., bulk acoustic wave (baw) devices as discussed earlier and surface acoustic wave (saw) devices, which is explained later). as from the name, it is obvious these are the devices in which oscillation phenomena is related to the surface of the material. rayleigh [ ] had discovered the phenomena of surface waves and explained their propagation mode in , since then they are known rayleigh waves. saws have both components shear vertical and shear horizontal, which can couple with the surface of substrate and propagate in a nondispersive manner. the coupling of these components determines the velocity and amplitude of the waves that travel along the surface, and hence, make these devices a useful tool for mass sensing purposes. a typical setup for saw resonator has been shown in fig. . . saw devices are typically made of st-cut quartz crystal on which comb-shaped electrodes are generated by lift off process. the distance between these comb-shaped electrodes determines the wavelength, which leads to a distinct resonance frequency of saw devices. although the maximum frequency achieved for saw devices is . ghz, the analytically reported frequency for chemical sensors is about ghz [ ] . such a high resonating frequency of these devices make themselves exceptionally valuable for detecting very low mass of analytes (e.g., for mhz device mass changes up to pg can be detected). apart from their successful sensor applications at higher frequencies, there are some limitations about their use in liquid phase. saw resonators are influenced by the viscosity of surrounding liquid, which increases the damping of device significantly such that it becomes almost impossible to conduct measurements in liquid phase. this problem can be solved by introducing shear transverse wave (stw) resonators [ , ] . stw resonators are made of lithium tantalate (litao ) in contrast to saw devices, which are made of quartz substrate. the high dielectric )) is somehow similar to that of water, which is ( ) . this makes the stw devices operational in highly polar mediums avoiding any short circuiting. the other modification made in these devices is that the st-cutting angle of crystal material is rotated in such a way that it demonstrates a complete shear horizontal wave that dissipates a very small amount of energy when subjected to liquid phase analysis. stw devices have established themselves a highly useful tool for various analysis particularly where the analyte concentration is too low to generate a reasonable signal. different surface modifications techniques for the sensitive and selective detection of various biospecies have already been discussed including natural antibodies, host-guest biointeractions, lbl strategy, and surface imprinting. among them, surface imprinting seems to be very attractive to get the desired specificity especially for bioanalytes. on the other hand, the basic principle and theory of mass-sensitive transducers have also been explained in previous section. the next headings will focus on the combination of surface modification techniques along with mass-sensitive transducers to design suitable biodetection systems. microorganisms have key role in almost every field of human life and particularly toward the environment. the microbial activities have significant applications in different areas such as genetic engineering, food processing, photo synthesis, biotechnology, bioremediation, and many more. besides the numerous useful applications of microorganisms, there are some serious threats to health as some of them are source of infectious diseases. classical methods for the analysis of dangerous microorganisms require very long time, which restricts the rapid determination. it is the beauty of molecular imprinting that design sophisticated sensitive surfaces for the quick detection of different microorganisms such as yeast, bacteria, different blood groups, viruses, and others. some examples of mass-sensitive detection of unicellular and multicellular species have been discussed further. yeast [ ] are very important microorganisms that have several applications in biotechnology, among them fermentation of sugar is the most widely known. basically, these are unicellular eukaryotic microbes having the length from to mm and diameter of - mm. production of different wines through fermentation process, bread baking, and synthesis of ethanol at industrial scale are the well-established applications of yeasts. saccharomyces cerevisiae [ ] are the most widely used yeast species that have been extensively examined in microbiology laboratories because they are considered as the model microorganism [ ] in the development of useful biosensors and to study certain biological phenomena. in different microbiology laboratories and other production plants, their growth in cultured medium is controlled at defined conditions. the growth and reproduction of yeast cells largely depends upon the composition of prepared medium, humidity, and temperature that demands constant surveillance. it has been noted that during the phase of production, concentrations of different reactants are changing with the passage of time that makes the mixture more complex. already established methods for the monitoring of yeast cells in different production processes are based on indirect measurements or some labeling techniques. the online monitoring in fermentation plants at larger scale has always been of fundamental importance for process control. this can be achieved by employing mass-sensitive transducers such as quartz crystal microbalances (qcm) having patterned coatings on them for selective detection of yeast cells. highly sensitive and selective coatings can be prepared by surface imprinting strategy, which has already been explained. the selection of polymer for surface imprinting is also very important as the cavities generated by yeast imprints should retain the structural characteristics in terms of size and dimension after the interaction of analytes with surface. polyurethane is a robust polymer that has been used widely for imprinting purposes. this polymer is synthesized using higher ratios of cross linker at defined reaction conditions. the image of s. cerevisiae is casted on polymer layer by pressing glass stamp to make highly packed and flat imprinted surface. the qcm measurement performed at suitable conditions on such surface shows remarkable sensor effect. while considering cross sensitive, it has been noticed that designed imprinted polymer surface shows highest frequency shift for the target molecule when compared with other similar yeast strands [ ] as shown in fig. . . although some of them have a size smaller than the cavity, they still do not have much impression on the same surface, which indicates the selective nature of the imprinted polymer. this reveals that the surface structure is patterned down to molecular/nanolevel, whereas the cavity shape and whole dimensions are optimized to the target analyte. an afm image of imprinted surfaces has been shown in fig. . . this measurement scheme does not stop here but it further investigates to monitor the growth stages [ ] of yeast cells in order to study the cell behavior, which is of great interest in different pharmaceutical industrial applications, drug release, and many other biological processes. especially in food processing industries, molecularly imprinted polyurethane surfaces of suitable layer heights can be used to inspect the changes taking place in surroundings such as concentration of nutrients, humidity effect, and variable ph. this is indeed a significant achievement by biomimetic surfaces when realizing the concept of process control [ ] for online monitoring purposes. recent developments shows that individual growth stages of yeast cells can also be monitored simultaneously by employing a multichannel qcm [ ] following the similar synthetic strategy. in comparison with classical methods for yeast detections, surface imprinted polymers along with mass-sensitive transducers are highly favorable in many regards. as already stated that classical methods are focused on indirect measuring techniques that are not much reliable in complex matrix where different chemical and physical parameters are varying continuously. the side-by-side selective detection of yeast cells at different growth stages is made possible by surface modified coatings on qcms, but other methods lack this advance feature, and thus cannot be applied for online monitoring. mass-sensitive devices having tailor-made surfaces have established themselves as promising tool to study cell properties and behavior in various biotechnology applications. the detection of different pathogenic bacteria is of great concern in microbiology and modern clinical analysis owing to the public health and safety. the contamination of harmful microbes in drinking water and food can lead to severe diseases. for example, escherichia coli (e. coli) are gram-negative pathogenic [ ] bacteria, which causes contamination in food and beverages. although not all of e. coli are toxic for human beings, still some of them are a potential hazard to public health. among the dangerous species, e. coli o :h is the most fatal one, which can cause severe diseases such as diarrhea, kidney failure, and even death in certain cases. in the united states, there are about , cases [ ] related to different diseases spread by e. coli annually. therefore, accurate, quick, cost-effective, and direct determination methods are needed in order to meet the requirements of environmental monitoring and other diagnostic purposes. the development of such detection systems are highly desirable in various industrial applications particularly related to food stuff [ ] . already established methods for bacterial detection are related to culturing, microscopy, and some other biochemical test. these methods are unfit for rapid analysis and particularly inappropriate for screening purposes, where prompt decision has to be made. the latest technology such as dna chips has solved this problem to make faster analysis and reach toward conclusion. for a very sensitive and sophisticated detection of e. coli, polymerized chain reaction (pcr) [ , ] technique is also very useful. masssensitive devices such as qcm [ , ] and surface plasmon resonance (spr) [ ] are also used for the characterization of pcr products. these detection methods are time consuming and not widely adoptable because of the high expertise required for handling. some other sensing schemes based on electrochemical impedance [ , ] , chemiluminescence [ ] , spr [ ] [ ] [ ] , and optical absorbance spectroscopy [ ] are also reported in literature for e. coli o :h detection. the direct use of mass-sensitive devices as transducer for e. coli detection [ , ] is not very general but still good enough to give a direct change in the frequency corresponding to the binding mass of analyte. the idea of using functionalized surfaces with acoustic or mass-sensitive devices seems to be promising for developing a sensitive, selective, rapid, and relatively inexpensive detection system. in literature, there are different surface techniques that have been used with mass-sensitive transducers for detection of different types of bacteria. during the last decade, different groups tried to develop various biosensors based on natural antibodies for the detection of e. coli using different transducers. most of them had applied specific antibodies immobilized on gold electrode of piezoelectric device such as quartz crystal microbalance. the strategy is very attractive in terms of getting selectivity as natural antibodies specifically bound to particular bacteria with sufficient sensitivity. zhihong [ ] has developed interestingly an unusual strategy for mass-sensitive bacterial detection. he has exploited the carbohydrates and protein recognition interactions for a very sensitive and specific e. coli detection. this method is not purely synthetic but in fact a hybrid approach by designing suitable mannose surfaces for a rigid and strong binding of e. coli. it has been noticed that the detection of e. coli on lectin modified mannose surface has much higher sensitivity about times better than mannose alone detection. this in terms of sensitivity is quite good as it improves the detection limit. as the binding of bacteria is strong that does not increase damping drastically, which is good for smooth qcm measurements without too much loss. the sensor specificity was also characterized by exposing different electrodes coated with different chemical layers to known concentration (i.e., .  cells/ml of e. coli w ). the frequency shift for different layers, that is, lectin-modified mannose layer (a) and recombinant antibody ( e scfv-cys-) treated electrode qcm surface (b) was determined. the control antigen test was also conducted by adding staphylococcus aureus (c), and the results have been shown in fig. . . the lectin-modified sensor layer exhibits highest sensor response for e. coli w in comparison with other layer and control antigen, which indicates the enhanced sensor specificity. recently, a new strategy has been introduced where gold nanoparticles [ ] are immobilized on thiol modified qcm electrode to develop piezoelectric sensor for real time detection of e. coli o :h detection. the main problem is related to the low stability of antibodies deposited on the transducer surface. these antibodies cannot perform for a longer period of time as they tend to deteriorate with the passage of time and so cannot be stored for longterm use. some antibody coatings are good for sensing purpose but most of them are of a flexible nature that can cause severe damping during mass-sensitive measurements, which leads to an unreliable data both in terms of qualitatively and quantitatively. moreover, some natural antibodies once bound to microorganisms then afterward they are reluctant to release them, which reduce the reversibility of mass-sensitive measurements. the solution of these problems can be made by following a different approach in which artificial recognition materials are used. surface imprinted layers can be used as artificial antibodies [ ] for bacterial detection more effectively when compared with natural antibodies. polyurethane layers that are robust and rigid in nature have been utilized for surface imprinting of e. coli. the imprinting is done on prepolymerized surface of polyurethane with a suitable stamp made of densely packed cells. the afm image of e. coli imprinted polyurethane surface has been shown in fig. . . these layers coated on qcm exhibit significant sensor response to e. coli. although there was anti-sauerbrey effect, the reported sensitivity and the reversibility of the measurement is quite good for e. coli detection. surface studies of imprinted polyurethane layers reveal that excess of phenolic groups hindered the covalent linkage with polymer surface. this indeed is very useful to improve the imprinting effects for bacteria sensing and to develop a completely reversible detection system. molecular imprinting has introduced a new aspect in material science for designing sensor surfaces for exceptionally small entities. this becomes highly useful for the recognition of viruses, which have size in the range of some nm. their extremely small size makes their detection virtually impossible on normal optical devices. viruses have serious health effects on human life as they can cause various diseases such as common cold, influenza, chicken pox, and some severe ones are acquired immune deficiency syndrome (aids), severe acute respiratory syndrome (sars), hepatitis, etc. enzyme-linked immuno sorbent assay (elisa) [ ] and pcr [ ] are highly adopted methods for determining the exact stage of viral disease but their cost is too high and they need highly trained personnel to operate. some inexpensive strips are available in the market for screening purposes for hepatitis c virus and hiv detection [ ] in blood. this screening technology is of low cost and gives quick results, which are very suitable for rapid detection but these strips are limited to certain viruses and not generally available for a broad variety. at the moment, there are not much rapid and online analysis methods available for the viral detection, which have low cost, easily operable, and can be applied to a wider range of these species. this is a highly demanding task in which one has to make quick decisions about fatal viral diseases and reach a conclusion as some viral diseases are incurable and require immediate diagnosis. so, express detection systems need to be developed for fast and accurate analysis of viruses that have relatively low cost. tobacco mosaic virus (tmv) is the first discovered species of this class, which has the potential to damage the leaves of different plants particularly of tobacco. the structure of tmv [ ] is surprisingly rigid and it can survive in a very broad range of ph . - . and can tolerate the temperature up to c. afm picture shows that tmv rods arrange themselves linearly over designed surface. it has been reported that the immobilization of tmv should be done on nonpolar surfaces, which prevent any structural distortion due to virus surface interaction [ ] (fig. . ) . surface imprinted polyacrylic [ ] acid has been applied successfully for casting tmv image. mass-sensitive measurement performed on mhz qcm shows excellent response for imprinted channel, whereas nonimprinted exhibits very small effect, which is due to unspecific binding. the frequency shift for mip and nip has been demonstrated in fig. . . the surface modified polyurethane layers [ ] offer better sensitivity for tmv than polyacrylic acid, which is very valuable to detect lower concentrations and ultimately avoid production losses. although tmv have not been considered widely as a model for designing virus sensitive layers, still it has found some important functions in other areas. an exciting application of tmv imprints has been reported by kenz et al. [ ] in which they have used this virus as template to generate highly selective nanostructured inorganic matrices for binding metal ions. this not only offers a true nano pattering of surfaces but also induces recognition properties to design functionalized materials. similarly, another interesting example of tmv imprints has been reported by bolisay et al. [ ] in which they have used this virus to improve the binding affinities for itself against the other non-target virus. although the detection was not made on acoustic devices, still this synthetic approach is useful to avoid nonspecific binding and is a step toward improving imprinting factor for tmv. surface pattering can also be used to differentiate the different viruses of same group (e.g., human rhinovirus, hrv), which is an infectious virus that causes common cold. this class of virus has a very small diameter of about nm but has a length of nm for surface patterning of polymers. the study [ ] has been conducted on different types of rhinoviruses (i.e., hrv a, hrv , and hrv ) to examine cross sensitivity. the reported results in table . show that polymer surface imprinted with one specie prefer to bind selectively the very same virus over the others. in other words, hrv a imprinted polymer surface exhibit comparatively higher frequency shift for hrv a than the others. this demonstrates that imprinted structure of polymer surface is competent enough to recognize the target virus among similar species at nanometer scale. the imprinting of hrv along with other viruses of same class and their selective detection on qcm is a model example of intragroup selectivity. the study can also be extended to attain intergroup selectivity pattern among different classes of viruses. suitable imprinting procedures can also be employed to distinguish between two different groups of viruses, using acoustic devices. the detection system [ ] has been designed for selectively determining tmv and hrv in controlled conditions. these viruses have entirely different structural dimensions, for example, tmv has cylindrical shape whereas hrv is found mostly in icosahedral form. mass-sensitive measurements performed on premeditated polymer surfaces show excellent selectivity pattern as shown in fig. . for the respective imprinted virus. this detection scheme is very useful as it gives rapid results about different virus species including cross sensitivity studies in comparison with other modern techniques such as pcr. recently, an outstanding advancement has been made in this regard where a suitable mip chip [ ] is designed for continuous monitoring of viral contamination. this development has introduced an innovative sensor platform for screening diverse biological complex mixtures and for micro total analysis system (mtas). these artificial receptors [ ] are used to design more selective matrices for different pico viruses (i.e., hrv, foot and mouth disease viruses, fmdv). although fmdv rarely infects humans however, it has strong impact on animals which is dangerous for agriculture industry and livestock. molecular imprinting promise to achieve desired intergroup selectivity between hrv and fmdv. masssensitive measurements performed on hrv stamped polyurethane layers demonstrate substantial frequency change for the imprinted picovirus, whereas the same surface shows very little effect for fmdv. one interesting strategy has been developed in which the whole analyte is not used as a template for the detection of biological analytes. this strategy is named as epitope approach [ ] in which a certain protein sequence is used as template that acts as antigen for the detection of target microorganism. the method is somewhat inspired from natural antibodies where only a small part of antigen interacts (e.g., instead of whole protein molecule only amino acid sequence is detected). although there are not too many applications of this approach in literature for biological detection, still this scheme can be introduced where practically whole structure cannot be used as a template. one successful example of this strategy is reported in dengue virus [ ] detection, which is an infectious pathogen virus that has been a serious threat to human life. a similar approach as epitope has been used to design biological sensor for bovine leukemia virus [ ] (blv). the overall studies conducted on surface imprinted polymers along with masssensitive devices for viral detection open a new dimension for rapid, inexpensive, selective, and precise analysis methods, which is not provided by other analytical tools. proteins are highly significant macromolecules that possess very complex structure, and their selective recognition is also very important in biochemistry. molecular imprinting has widely adopted technique to generate suitable polymer systems for protein sensing. in recent years, a number of review articles are presented for the imprinting of proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] in which different strategies such as bulk imprinting, surface imprinting, epitope approach, and hydro gels are used. the protein imprinted surfaces are characterized by different techniques such as afm and scanning electron microscopy (sem). the images obtained from these methods reveal that the artificially imprinted protein materials have nano-patterned structures that possess specificity for template protein, which can be confirmed by other methods. it has been noted that the imprinted surface retains the utmost amount of that protein molecules, which were being used as model template while the nonspecific binding were very less when exposed to other protein solutions. despite the numerous applications of molecular imprinting for proteins and other macromolecules, their detection by mass-sensitive devices is not fully explored. zahang et al. [ ] has reported the piezoelectric detection of proteins using molecularly imprinted self-assembled thin films. they have selected human serum albumin (hsa) (i.e., a major plasma protein taken from urine) as a template and used silane solgel layers for imprinting. the inherent features of sol-gel technology provide a better platform for successful imprinting of biomolecules without modifying their original functionality. the imprinted sol-gel films were examined by sem to observe the conductivity difference between coated and uncoated quartz crystal gold electrode. they have found that the impedance of coated electrode increases with increasing layer height. authors have also studied the other parameters such as temperature, effect of different salts and solvents on binding capacity, and concentration effect to develop an optimize detection system for has. the thin sol-gel films have shown suitable selectivity pattern in the presence of bovine serum albumin (bsa), horseradish peroxidase (hrp), and trypsin. the detection procedure of has was not fully directly based on mass change by quartz crystal, but rather based on electrochemical impedance, which is good enough for selective protein detection. surface imprinted polymers [ ] are very effective for selective recognition of different proteins induction for crystallization. an example of such approach has been reported where methacrylic acid along with divinyl benzene (dvb) was polymerized at suitable conditions, and crystallized lysozyme was stamped for surface imprinting. mass-sensitive measurements conducted on dual electrode qcm show that the imprinted channel shows significant frequency shift, while the nonimprinted channel remain unaffected. the growing of lysozyme crystals on imprinted polymer surface have been demonstrated by afm image shown in fig. . . another very interesting example of trypsin imprinting [ ] and its detection on qcm has been reported where template has been used in three different forms: amorphous, crystalline, and solubilized. although the surface imprinting is possible with all three template models, experimental results suggest that the solubilized form of trypsin is highly appreciable concerning sensitivity point of view. these data obtained by exposing a set of different concentrations to three differently imprinted forms of trypsin have been presented in table . . trypsin imprinted surface was exposed to different other enzymes such as lysozyme and pepsin, and their relative mass change is noted. an excellent selectivity pattern is observed where the mass change is higher for trypsin than that for lysozyme and pepsin. this scheme was extended for lysozyme imprinting, and then a differential measurement was carried out for trypsin and lysozyme. it was evident from qcm results that surfaces imprinted with lysozyme and trypsin exhibit higher frequency response toward their original imprints in the presence of other (fig. . ) . in all the mass-sensitive measurements, the response of nonimprinted channel was almost negligible to different enzymes. the effect of ph on trypsin detection was also monitored by performing the measurement in different conditions. the frequency shift for trypsin imprinted surface was calculated for three different ph values, , , and . and found to be highest at a ph value . this could be explained in a way that the varying conformations of enzymes and the columbic interactions between surface and analyte seriously distort the imprinted surface structure. the most remarkable feature of this sensing technique is that the detection limit of ng/ml can be achieved. nicholas et al. [ ] has also reported protein imprinting and their very sensitive and specific detection by qcm. a very interesting approach about protein imprinting has been presented by john rick and tse-chuan chou in which they have used single and double protein molecules and observed protein-protein interactions [ ] by masssensitive devices. they have used lysozyme and cytochrome c as individual templates . cross sensitivity comparison by differential mass-sensitive measurements on trypsin and lysozyme imprinted surfaces (adapted from [ ] ) and together in different molar ratios for imprinting using poly-aminophenylboronic acid as a monomer. experimental results have revealed that the lysozyme imprinted layer shows fairly selective binding of the own template, while cytochrome c imprinted layer does not exhibit recognition properties. regarding the double imprinted protein structure, both species were used as templates in different molar ratios and their response toward different proteins was monitored. it has been found that when lysozyme and cytochrome c were used in equal molar ratios for imprinting, the layer has shown maximum frequency change for the imprinted proteins. these observations provide a new route for multiple imprinting procedures for studying the interactions between same biomolecules on quartz surface. erythrocytes are basically red blood cells (rbc) that have hemoglobin and act as transporter for oxygen in blood. erythrocytes are very flexible cells shaped like disks, having a diameter of approximately mm, and a thickness of mm. in previous examples, we already have knowledge of molecular imprinting for generating sensitive and selective materials for recognizing various microorganisms including bacteria and viruses. as erythrocytes have four different blood groups,a, b, o, and ab, for which molecular imprinting [ ] can be handy to generate selective polymer layers capable of recognizing the respective blood group. the three blood groups, a, b, and ab, are recognized by the antigen on their surfaces and composed of oligosaccharides having six sugar molecules excluding blood group o, which has one less. the difference between blood group a and b is by only sixth sugar molecule at terminal position, whereas in the case of a, it is n-acteyl-d-galactosamine, and dgalactoside for blood group b. molecular imprinting provides a straightforward approach to design suitable recognition materials for the detection of different blood groups on acoustic resonators. the blood groups a, b, ab, and o can be used as template model for imprinting on highly cross linked polyurethane layer. typical polyurethane is composed of diphenyl diisocyanate as monomer, whereas bis phenol a and phloroglucinol are used as cross linkers. these cross linkers have excess of hydroxyl groups that play a very important role in imprinting as they offer suitable hydrogen bonding to the sugars present on blood group surface. although the geometrical shapes of all these blood groups are almost identical, the predefined interaction of cells through hydrogen bonds with polyurethane surface offers suitable recognition. afm image of imprinted erythrocytes on polymer surface have been shown in fig. . . different blood groups a, b, ab, and o have been used as templates [ ] for surface imprinting by stamping method. the modified polyurethane surface was exposed to equal concentrations of different blood group samples. mass-sensitive measurements reveal that the polyurethane surface imprinted with certain blood group (bg) shows utmost mass loads, particularly for bg that was used as template in surface imprinting procedures. generally, in each case, the relative sensor response of a polyurethane layer is highest for its own template. these data obtained from mass-sensitive measurements have been presented in table . . these findings show that these studies can be extended to real-life samples. despite the fact that the structural dimensions of erythrocytes are in micrometer range, the recognition of antigens present on their interface ensures the nano structuring pattern on polymer surface. the conventional methods for blood group detection are based on chemical reactions with specific antigens that form precipitates, whereas the latest methods give a change in the reflectance of laser beam when the surface is exposed to the samples. on the other hand, molecular imprinting in combination with masssensitive devices offers much cheaper and rapid detection scheme for different blood group samples. mips can be designed in a more sophisticated way to differentiate the subblood groups (i.e., a and a ) by mass-sensitive transducers. recently [ ] , polyvinyl pyrrolidone has successfully been used for surface imprinting of a and a and were put under mass-sensitive measurements. experimental results showed that the mip layer gives larger signal for that subgroup, which was used for imprinting. for example, the mass effect for a imprinted layer is higher by a factor of approximately three when compared with a (fig. . ) . the results obtained from mass-sensitive measurements show remarkable selectivity as they have similar geometrical dimensions and belong to the same abo system. they differ from each other by the amount of antigen density on their surface as in the case of a it is . - .  a-antigens and for a it is . - .  . it is the beauty of molecular imprinting that provides patterned surfaces for selective incorporation of antigens. this study also reveals that mip surfaces does not interact with analyte partially but shows affiliation with the whole cell to get complete information about surface density. these results are very appreciable and provide a complete understanding about the interactions of whole cell with modified polyvinyl pyrrolidone surfaces. a slightly different synthetic strategy has been proposed for monitoring [ ] of different blood groups and immunohematological reactions, using mass-sensitive devices (i.e., qcm) as transducers. hormones are basically chemical envoys in the body that carry the commands from one part to the other and are released by body cells in response to certain metabolic reactions. the excess or deficiency of any hormone in the body is called hormone disorder, which can lead to severe problems in functioning of cells. different hormones in human body can be detected by examining blood, urine, and saliva samples, according to the desired hormone test. among different hormones, insulin is very important, which takes the glucose from blood and stocks it up as glycogen in the liver and other cells and does not allow the use of body fat as energy resource. the disorder of insulin can cause serious diseases to the body such as diabetes, insulinoma, and others. in market, synthetic insulin can be produced for diabetic patients by fermentation process governed by yeast and bacteria. so, the detection of insulin is very important not only in human body to control metabolic reactions but also highly desirable in commercial point of view. the application of artificial materials for masssensitive detection of insulin and other hormones is very rare. the versatility of surface imprinting makes promise to design suitable sensor materials for insulin detection [ ] with a broad concentration range. highly cross-linked polyurethane was prepared and coated on suitable mass-sensitive transducer (i.e., qcm) having mhz fundamental resonance frequency. as mentioned already, solution phase stamping procedure was followed for imprinting of insulin on polyurethane surface. a series of different concentration solutions of insulin ranging from mg/ml to mg/ml has been exposed to imprinted channel and corresponding frequency change was monitored. it has been observed that the sensor response for different insulin solutions was linear for such a broad range, which suggests that this system can be used for quantitative determination of insulin in pharmaceutical formulations (i.e., iu or iu). iu stands for international unit for pharmacology activity that is mg/ml of insulin. interestingly, different experiments have shown that the sensor response for insulin does not depend upon the layer height of the polymer on quartz, which is very surprising while considering surface imprinted layers. the sensor signal was reversible and reproducible for several measurements performed on different layer heights of polyurethane. the temperature effect on insulin has also been monitored to examine its behavior by performing mass-sensitive measurements at different temperatures (i.e., , , , and c) . at all these temperatures, same concentration of insulin (i.e., mg/ml) was used for analysis. it has been observed that the frequency shift is highest for that measurement, which was conducted at c and then gradually decreases until at c where it is lowest. although the concentrations are same, the effect is different at different temperatures. this is probably due to the denaturing of insulin in which the structural features and functionality is lost, which ultimately cuts down the surface interactions between the analyte and the polymer layer. it signifies that by selecting proper storage temperature, the shelf life of insulin drugs can be enhanced. these findings are very significant for monitoring the quality of insulin products in pharmaceutical industry (fig. . ) . another strategy has also been proposed by satish et al. [ ] , where a sandwich layer material has been crafted and coated on qcm for insulin detection. they have sandwiched an antigen between two specific antibodies and used it as sensing element. the lower antibody layer is deposited on quartz surface, while the upper antibody layer is used for insulin recognition and in between these layers an antigen is pressed. this layer material is exposed to different insulin concentrations and corresponding frequency shifts were noted. although the lowest detection limit was achieved (i.e., ng/ml), the sensor signal does not remain linear after mg/ml of insulin, which is not favorable for commercial use. the effect of temperature on mass-sensitive measurement has not been discussed. the synthetic scheme is also very complex for layer structuring that requires relatively more care, and more importantly low stability of natural antibodies restrict their use for long-term analysis. catecholamine is a very important class of hormones, which acts as neurotransmitters in the body and is released in response to certain psychological reactions. major catecholamines are dopamine, norepinephrine, and epinephrine, which are excreted in urine. disorder in this hormone can lead to serious complications in the central nervous system. the detection of these hormones can be made by fluorescent technique and other methods such as ion exchange chromatography and high performance liquid chromatorgraphy (hplc), where different catecholamines are isolated and characterized from urine samples. suitable sensor materials can be generated through molecular imprinting for selective detection of catecholamines in a complex urine solution. tzong et al. [ ] designed such materials by imprinting dopamine and developed a suitable sensor system using qcm as transducer. authors have synthesized dopamine mip powder and examined their binding properties at different ph values by hplc studies. dopamine mip films were also generated by following a different reaction route and the effect of ph was also monitored on rebinding capacity of these films. these studies reveal that dopamine imprinted film preferably binds dopamine when compared with norepinephrine and epinephrine. similarly other catecholamine imprinted films (i.e., norepinephrine and epinephrine) specifically bind the original imprint molecule as shown in table . . dopamine mip layer coated on mhz quartz exhibits maximum frequency shift for dopamine itself, while for norepinephrine and epinephrine it is negligible. the results are summarized in table . . these studies are valuable and encouraging in designing a suitable detection model for catecholamines. mips with acoustic devices have also been employed for detection of plant hormones, for example, indole- -acetic acid (iaa) plays a vital role in plant development. akimitsu and toshifumi [ ] had designed molecularly imprinted meth acrylic acid (maa) polymer for selective recognition of iaa, using mhz qcm to perform mass-sensitive measurement. a series of different concentrations of iaa ranging from to nm has been used on imprinted and the nonimprinted qcm. the change in the frequency is linear in the defined range upon mass loading of iaa. this experiment was performed on three different qcms to observe the relative variation in the frequency shifts on same concentrations of iaa. the reported results show a coefficient of variation for three different sensors . %, . %, and . %, respectively, which means that there is no significant difference in their performance. this is an evidence for a sensor system to generate reproducible results. the selectivity of this sensor has also been monitored by performing measurements with indole- -butyric acid (iba), indole, and indole- -ethanol (iet) of same concentrations. sensor results clearly demonstrate that the frequency change for iaa is maximum, while for iba, indole, and iet, the response is quite low, which shows the selective nature of designed sensor material. the reason of highly selective nature of imprinted maa polymer is could be due to the suitable electrostatic interactions between n,n-dimethylamino group from monomer to carboxylic group of iaa ( fig. . ). synthetic receptors derived from molecular imprinting possess desired degree of selectivity in comparison with other schemes. in combination with mass-sensitive devices, molecular imprinting is very advantageous for developing suitable sensor systems for hormones detection. despite of the fact that mip applications for hormones detection are very few but still promising to replace conventional methods for their detection. molecularly imprinted polymers have been proved very effective for bilirubin detection via mass-sensitive devices. bilirubin is a yellow color waste material that is produced from the breakdown of hemoglobin molecules of rbc. bilirubin tests are often conducted to examine its production and excretion from body, which ultimately refers to the working of the liver cells. elevated amounts of bilirubin in the body indicate the improper functioning of liver, which causes hepatic diseases and in severe cases permanent damage to the brain or even results in death [ ] . various voltammetric [ ] and flourometric [ ] methods have been reported for bilirubin analysis with substantial sensitivity. conventional method for bilirubin detection is based on dizao reaction, where azobilirubin is produced by the condensation reaction of diazotized sulfanilic with bilirubin. authors [ ] have successfully prepared bilirubin imprinted polymer, using -vinyl pyrrolidone as functional monomer along with dvb as cross linker. the polymerization reaction was initiated by benzophenone and carried out under uv light. the surface of gold electrodes on qcm is properly cleaned and is modified by treating it with thiols before coating mip layer. different topographical images of the coated film had been recorded by sem that exhibit a uniform surface structure of bilirubin mip. mass-sensitive measurements performed on mhz qcm exhibit suitable sensor response for defined amounts of bilirubin samples. the effect of ph on bilirubin detection was also examined by performing mass-sensitive measurements at different ph values ranging from . to . . it has been found that at higher ph value (i.e., . ), the frequency shift is also higher. the bilirubin mip surface was exposed to a set of different concentrations (i.e.) from . to . mg/dl of bilirubin and found that the sensor response is linear in this range. biliverdin, which is a similar compound to bilirubin, was selected as a model to examine the selectivity of bilirubin mip. the sensor signal for bilirubin is much higher when compared with biliverdin, which shows that bilirubin imprinted surface preferably binds the own template compared with other analogous species. mip coated on qcm has proven itself as an effective tool for clinical analysis of bilirubin, which offers a reliable, simple, and fairly cheap detection system in comparison with other methods. molecular imprinting approach can also be extended for another very important class of materials (i.e., nanoparticles) to generate more sophisticated surfaces for sensing principles. kristina et al. [ ] prepared imprinted nanoparticles and introduced them in to a thin poly ethylene terephthalate (pet) layer coated on quartz wafer. the afm study of imprinted nanoparticles shows a fine and uniform distribution that has potential to recognize pair (r)-and (s)-propranolol in aqueous buffer. propranolol is a nonselective beta blocker drug that is very useful for hypertension treatment. the imprinted nanoparticles show a definite degree of chiral selectivity for enantiomeric pair of (r)-and (s)-propranolol. this indeed is a positive step toward designing enantiomeric biosensor materials and can only be achieved by careful controlling of the morphology of nanoparticles. the applications of molecular imprinting with mass-sensitive devices such as qcm are very limited for dna identification. recently, a new biosensor has been designed for dna recognition [ ] , based on molecularly imprinted polymers coated on qcm. authors had synthesized thymine-mip by using methacryloylamido, adenine, thymine (ma-ade-thymine) as functional monomer along with edma as cross linker with aibn initiator. the nonimprinted polymer was prepared in a similar fashion excluding thymine. they had modified the gold electrode surface of qcm before coating thymine-mip layer. the surface of bare electrode, thiol-treated gold surface, and thymine layer was characterized by afm on wave mode. mip generates very suitable tailor-made cavities for thymine interactions through hydrogen bonding with functional groups of polymer. mass-sensitive measurements performed on imprinted and nonimprinted channels exhibit a substantial difference in frequency shifts for a defined concentration range. the selectivity of mip layer was also examined by calculating the sensor response for same concentrations of thymine, uracil, poly(dt) (ssdna), and poly(u) (ssrna). the sensor signal for thymine at all concentration points was higher in comparison with others. some other strategies are also reported where instead of a synthetic polymer, gold nanoparticles act as sensing material [ ] for mass-sensitive detection of dna molecules. although these materials are very rarely used for biorecognition, they have very good sensitivity (e.g., À m and À mol/l). another more advanced design has been reported [ ] where  qcm matrix is assembled on which specific antibody is coated for dna sensing. this detection mechanism is very attractive as it provides a suitable platform for online monitoring of various biological analytes. colloidal gold nanoparticles have also been reported [ ] to improve the gold-coated qcm surface. these gold particles possess high surface area and enhanced interaction sites that exhibit appreciable sensor signal for target dna strands. despite the fact that these schemes do not hold any modified polymer interface on quartz surface, they are good enough for designing dna sensing systems. the lbl technique also contributes to the detection of various bioanalytes but along with mass-sensitive devices, their applications are relatively limited. a lbl assembling of liposomes and protein membrane [ ] was made on qcm surface for biological recognition. the sensitive layer was characterized by afm and put under detection of nonylphenols where it had showed satisfactory results in low ppm concentration range. some other examples are also reported where lbl films with qcm have proven very handy for sensitive detection of dna strains. a very exciting example of dengue virus detection has been reported using lbl hybridized gold nanoparticles [ ] as sensing element. these modified nanoparticles provide high sensitivity through qcm for the detection of dengue virus. in comparison with other polymer surfaces, nanoparticles provide more binding sites that has affinity for target analyte. sem images give a clear picture of lbl assembling of gold nano particles on quartz surface. the modified oligonucleotide gold particles are not only sensitive for viral detection but also very specific for target specie. along with mass-sensitive devices, they have proven themselves to be very successful for diagnostic purposes in real blood samples of dengue viral infections. almost more than hundred countries had suffered from this virus and very high rate of causalities have been reported in different regions. such detection methods are very encouraging in developing more effective systems that are low cost and suitable for online surveillance. multi-sensor strategy is very exciting and innovative regarding the detection of diverse bioanalytes on a single platform. in this way, we would be able to immobilize different mip on a single qcm that would be competent to bind specifically target analytes. one such approach has already been mentioned. considering the material designing for biosensors, molecular imprinting has shown potential for recognition of various bioanalytes including microorganisms such as yeast, bacteria, viruses, and others such as proteins, enzymes, erythrocytes, etc. the tailor-made structures of imprinted polymers provide enhanced selectivity for target analyte molecules at nanometer scale where the size, shape, and dimensions of imprinted cavities are optimized for a desired biomolecule. the most significant aspect is that molecular imprinting is virtually applicable to almost all biological species unless template does not chemically react with polymer matrix. the synthetic procedure of imprinting is much more convenient when compare with other methods that are more complex, time consuming, and relatively need expansive chemical reagents. as we have learnt from the previous study that surface imprinted polymers have very broad spectrum for biosensors when compared with other material crafting technique. unlike natural antibodies, mips bind analyte molecules reversibly through weak attraction forces and thus makes reusability of these materials that can be used for several analysis. the other material designing strategies such as natural antibodies, lbl, and host-guest interactions have some contributions in developing biorecognition materials but not exploited for commercial use of biosensors. the other part of biosensors concerning transducers is accomplished by acoustic or mass-sensitive devices such as qcm, which furnishes high sensitivity to designed biosensors. these devices are capable of sensing the mass of analyte in nanogram or even in picogram range (i.e., saw devices, which are very valuable while dealing with extremely low concentrations). the sensitive and precise detection of different bioanalytes can be enhanced in two ways: first, optimizing the synthetic procedures for imprinting, and second by tuning mass-sensitive devices for achieving best possible sensitivity. the first part of sensor design deals with imprinting technique where the analyte interactions with mip surface need to be made better adjusting monomer cross linker ratio, establishing proper reaction conditions such as favorable solvent, temperature, and heating time. the careful monitoring of different reaction parameters not only provides maximum binding sites but also constructs template-oriented cavities for selective detection. another very important aspect (i.e., response time) can be improved by controlling the surface structures of mips so that the analyte molecule readily be attached or detached without consuming much time. these modifications in synthetic receptors enhance the sensitivity and permit only target analyte to be bounded specifically with mip surface. concerning the reusability of the sensor materials, the template mip interaction should be entirely reversible that can only be achieved by developing noncovalent interface. the other part is concerned with the design of transducers. at the moment, qcms having frequency - mhz are used widely as mass-sensitive transducers. the fundamental resonance frequency depends on thickness of quartz sheet, which is a key parameter to amplify the mass sensing capabilities of acoustic devices. the sensitivity can be made better by producing more thin qcms, which is not feasible from a mechanical point of view as these devices would turn out to be very fragile. this restricts the use of acoustic devices to extend the detection limits of acoustic biosensors. an alternative can be used to overcome this deficiency of acoustic resonators by introducing film bulk acoustic resonators (fbar). these devices operate in the ghz range, which is higher by a factor of than typical qcm resonance frequency. they not only offer enhanced sensitivity but also exhibit fine linearity, small and compact size, and less damping loss in viscoelastic medium. the overall objective of this study is to demonstrate patterned polymer surfaces for selective recognition of different bioanalytes through acoustic resonators, thus providing a suitable, economic detection system to improve public health and ensure a safe environment. orientational aspects of antibody immobilization and immunological activity on quartz crystal microbalance electrodes piezoelectric sensors rapid detection of bacterial spores using a quartz crystal microbalance (qcm) immunoassay buildup of ultrathin multilayer films by a selfassembly process: iii. consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces fuzzy nanoassemblies: toward layered polymeric multi composites molecular layer processing of polyaniline via the use of hydrogen bonding interactions multiple alternating molecular layers of albumin and heparin on solid surfaces of medical devices fabrication of a covalently attached multilayer via photolysis of layer-by-layer self-assembled films containing diazo-resins layer-by-layer self-assembly: the contribution of hydrophobic interactions buildup of polyelectrolyte-protein multilayer assemblies on gold electrodes. role of the hydrophobic effect layer-by-layer deposited multilayer assemblies of polyelectrolytes and proteins: from ultrathin films to protein arrays layer-by-layer assembly of alternate protein/polyion ultrathin films a new kind of immobilized enzyme multilayer based on cationic and anionic interaction assembly of multicomponent protein films by means of electrostatic layer-by-layer adsorption buildup of exponentially growing multilayer polypeptide films with internal secondary structure assembly of thin films by means of successive deposition of alternate layers of dna and poly(allylamine) spontaneous assembly of viruses on multilayered polymer surfaces assembly of alternating polyelectrolyte and protein multilayer films for immunosensing layer-by-layer self-assembly of polyelectrolytesemiconductor nanoparticle composite films strain-sensitive raman modes of carbon nanotubes in deflecting freely suspended nanomembranes layer-by-layer assembly of intercalation compounds and heterostructures on surfaces: toward molecular "beaker" epitaxy stepwise formation of multilayered nanostructural films from macromolecular precursors layer-by-layer construction of multilayer thin films composed of avidin and biotin-labeled poly(amine)s biomaterials by design: layer-by-layer assembled ion-selective and biocompatible films of tio nanoshells for neurochemical monitoring synthetic self-assembled models with biomimetic functions molecularly imprinted polymers -techniques and instrumentation in analytical chemistry catalytic accelerations of -fold by an enzyme-like synthetic polymer the use of polymers with enzyme-analogous structures for the resolution of racemates molecularly imprinted polymers and their use in biomimetic sensors polymer cookery: influence of polymerization conditions on the performance of molecularly imprinted polymers polymer cookery. . influence of polymerization pressure and polymer swelling on the performance of molecularly imprinted polymers study of the nature of recognition in molecularly imprinted polymers ii. influence of monomer-template ratio and sample load on retention and selectivity molecular imprinting a new method for the introduction of recognition site functionality into polymers prepared by molecular imprinting: synthesis and characterization of polymeric receptors for cholesterol molecular imprinting using monomers with solid-state polymerization synthesis of a confined class of chiral organic catalysts via bulk imprinting of silica surface imprinting strategies for the detection of trypsin combining atomic force microscope and quartz crystal microbalance studies for cell detection développement, par pression, de l'électricité polaire dans les cristaux hémièdres à faces inclinées verwendung von schwingquarzen zur wangung dunner schichten und zur mikrowagung on waves propagated along the plane surface of an elastic solid saw devices -sensitivity enhancement in going from mhz to ghz atrazine measurements using surface transverse wave devices gravimetric sensitivity of transverse waves trapped by metal gratings on thin quartz plates beginnings of microbiology and biochemistry: the contribution of yeast research metabolic engineering of saccharomyces cerevisiae the use of yeast and moulds as sensing elements in biosensors selective microorganism detection with cell surface imprinted polymers application of yeast imprinting in biotechnology and process control biomimetic yeast cell typingapplication of qcms h outbreaks coliforms and e. coli: problem or solution? lessons from a large outbreak of escherichia coli o :h infections: insights into the infectious dose and method of widespread contamination of hamburger patties rapid sample preparation method for pcrbased detection of escherichia coli o :h in ground beef direct pcr detection of escherichia coli o :h polymers for use on bulk acoustic wave dna hybridization biosensors sequences of e. coli o :h detected by a pcracoustic wave sensor combination detection of pcr products of e. coli o :h in human stool samples using surface plasmon resonance immunobiosensor chips for detection of e. coli o :h using electrochemical impedance spectroscopy the novel immunobiosensors for detection of escherichia coli o :h using electrochemical impedance spectroscopy rapid detection of e. coli o :h in ground beef, chicken carcass, and lettuce samples using an immunomagnetic chemiluminescence fiber optic biosensor surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel spr sensor a mixed self-assembled monolayer-based surface plasmon immunosensor for detection of e. coli o :h an antibody-immobilized capillary column as a bioseparator/ bioreactor for detection of e. coli o :h with absorbance measurement a self-assembled monolayer-based piezoelectric immunosensorfor rapid detection of escherichia coli o :h a comparison of protocols for the optimisation of detection of bacteria using a surface acoustic wave (saw) biosensor nonlabeled quartz crystal microbalance biosensor for bacterial detection using carbohydrate and lectin recognitions the escherichia coli o :h dna detection on a gold nanoparticle-enhanced piezoelectric biosensor borderline applications of qcm-devices: synthetic antibodies for analytes in both nm-and mm-dimensions an antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein ns in the sera of infected patients real time quantitative pcr as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification rapid tests for hiv antibody artificial antibodies for bioanalyte detectionsensing viruses and proteins binding the tobacco mosaic virus to inorganic surfaces nano-and micro-structuring of sensor materials -from molecule to cell detection spatially selective nucleation of metal clusters on the tobacco mosaic virus optimization of virus imprinting methods to improve selectivity and reduce nonspecific binding sensing picornaviruses using molecular imprinting techniques on a quartz crystal microbalance detection of viruses with molecularly imprinted polymers integrated on a microfluidic biochip using contact-less dielectric microsensors towards molecularly imprinted polymers selective to peptides and proteins. the epitope approach recognition of dengue virus protein using epitopemediated molecularly imprinted film molecularly imprinted polypyrrole-based synthetic receptor for direct detection of bovine leukemia virus glycoproteins too large to fit? recent developments in macromolecular imprinting from d to d: a review of the molecular imprinting of proteins molecularly imprinted polymers for the recognition of proteins: the state of the art molecular imprinting of peptides and proteins in aqueous media recent developments in the molecular imprinting of proteins molecular imprinting within hydrogels biomolecule imprinting: developments in mimicking dynamic natural recognition systems molecularly imprinted thin film self-assembled on piezoelectric quartz crystal surface by the sol-gel process for protein recognition mass-sensitive detection of cells, viruses and enzymes with artificial receptors surface imprinting strategies for the detection of trypsin formation of protein molecular imprints within langmuir monolayers: a quartz crystal microbalance study imprinting unique motifs formed from protein-protein associations synthetic receptors for chemical sensors -subnano-and micrometer patterning by imprinting techniques biomimetic abo blood-group typing synthetic receptors for selectively detecting erythrocyte abo subgroups rapid monitoring system for blood groups and immunohematological reaction detection printing materials in micro-and nano-scale: systems for process control sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal size-selective recognition of catecholamines by molecular imprinting on silica-alumina gel molecularly imprinted polymer-coated quartz crystal microbalance for detection of biological hormone hyperbilirubinemia and cholestatic liver injury in hepatitis c-infected liver transplant recipients measurement of direct bilirubin by use of bilirubin oxidase light emission from bilirubin using the peroxyoxalate chemiluminescence reaction synthesis of bilirubin imprinted polymer thin film for the continuous detection of bilirubin in an mip/qcm/fia system characterization of qcm sensor surfaces coated with molecularly imprinted nanoparticles designing of mip based qcm sensor having thymine recognition sites based on biomimicking dna approach dna biosensor with high sensitivity amplified by gold nanoparticles a versatile qcm matrix system for online and highthroughput bio-sensing the enhancement effect of gold nanoparticles as a surface modifier on dna sensor sensitivity layer-by-layer of liposomes and membrane protein as a recognition element of biosensor a method of layer-by-layer gold nanoparticle hybridization in a quartz crystal microbalance dna sensing system used to detect dengue virus acknowledgments i thank higher education commission (hec) of pakistan for providing funding for my phd studies. key: cord- - n s bl authors: colin, marius; klingelschmitt, flora; charpentier, emilie; josse, jérôme; kanagaratnam, lukshe; de champs, christophe; gangloff, sophie c. title: copper alloy touch surfaces in healthcare facilities: an effective solution to prevent bacterial spreading date: - - journal: materials (basel) doi: . /ma sha: doc_id: cord_uid: n s bl in the healthcare environment, microorganisms’ cross-transmission between inanimate surfaces and patients or healthcare workers can lead to healthcare-associated infections. a recent interest has grown to create antimicrobial copper touch surfaces, in order to counteract microbial spread in the healthcare environment. for the first time, five french long-term care facilities were at % fitted with copper alloys door handles and handrails. related to the environmental bacterial contamination, samples were carried out on copper and control surfaces over three years after copper installation. in addition, some copper door handles were taken from the different facilities, and their specific activity against methicillin-resistant s. aureus (mrsa) was tested in vitro. in comparison to control surfaces, copper door handles and handrails revealed significantly lower contamination levels. this difference was observed in the five long-term care facilities and it persists through the three years of the study. high and extreme levels of bacterial contamination were less frequent on copper surfaces. although, the antibacterial activity of copper surfaces against mrsa was lowered after three years of regular use, it was still significant as compared to inert control surfaces. therefore, copper containing surfaces are promising actors in the non-spreading of environmental bacterial contamination in healthcare facilities. among the various problems to deal with in medicalized environment, healthcare-associated infections (hai) currently represent one of the biggest threats for resident and hospitalized persons all around the world. a prevalence survey estimated the number of hai in the united states as around , cases during the year , corresponding to a prevalence of % [ ] . in , in europe, the hai prevalence was around %, ranging from % to % for overall european countries [ ] . in the healthcare environment, microorganisms can easily spread from resident to resident [ ] and from resident to staff through different pathways. direct contact between people is the most obvious factor of microbial cross-contamination. the hands of a healthcare worker can have a high risk of being contaminated following a direct skin contact with infected patient [ ] [ ] [ ] , and hand hygiene is the first strategy for preventing healthcare-associated infections [ ] . however, pathogens can also the study was conducted in five long-term care facilities located in the marne territory, france, named a-e afterwards in the text: . each facility employs its own staff and uses its own protocol for disinfection and cleaning. those protocols have not been changed throughout the study. in each facility, half of the resident's room and corridors were fitted out with steriall ® copper alloys (lebronze alloys, suippes, france). briefly, half of the original door handles of residents' room and original corridors' handrails were replaced by copper ones, the other half remained as before and was used as control surfaces. two different copper alloys were used, one for the rooms' door handles (around % of copper) and one for the handrails (around % of copper) in every corridor adjoining copper fitted rooms. the sampling series were conducted over an months period and executed with at least one week of delay between them. the number of rooms involved, surface cleaning protocol and frequency, date of the copper alloys set-up, and sequences of sampling are summarized in table . for each series, ten copper as well as ten non-copper fitted patient rooms were randomly selected and excluded from the next series of the same sequence (except in facility c were the total number of rooms was below ). for each selected room, the samplings were performed on the external door handle (corridor side) and on the closest handrail in the corridor, at a distance of centimeters from the edge of the handrail. samplings were performed on monday or tuesday between : a.m. and a.m., during staff operating time and before surfaces daily cleaning. each door handle or handrail was sampled using a cm ( cm × cm) sterile silicon template and a sterile swab (copan, murrieta, ca, usa). the swab was moistened in sterile peptone water (sigma-aldrich, dutscher, brumath, france) and was applied five times longitudinally and times transversally. then after, each swab was immediately transferred in a ml tube (falcon, dutscher, brumath, france) containing ml of sterile peptone water. samples were stored at • c during transport to the laboratory and were then inoculated on agar plates' surface within the h following sampling. to release the collected bacteria from the swab to the resuspension medium, samples were placed in ultrasonic bath at khz for two minutes, and then vortexed for s. hundred µl of each sample were then inoculated onto tryptic soy agar plates (tsa, biokar diagnostics, allonne, france). plates were incubated aerobically at • c. the number of bacterial colony-forming units (cfu) on each plate was determined after h and confirmed after h and h. the lower detection limit was one cfu per cm . the antimicrobial activity of the door handles on mrsa was studied in vitro on different sets of door handles (lebronze alloys, suippes, france). unused door handles came directly from the factory. used door handles were collected from the five facilities approximately one year (beginning of the in situ study) or three years (end of the in situ study) after their set-up in the long-term care facilities. in order to preserve the characteristics of the in-use copper door handles, they were carefully removed from the doors of the residents' room. to avoid any changes in the characteristics of the handles, no drastic physical or chemical treatments were performed on their surface before the in vitro tests. three hours before the antibacterial activity assay, a sterile swab was moistened with µl of peptone water and was applied gently on all the surface of the handle to remove non-adherent particles if any. glass slides (ghäasel, dutscher, brumath, france) and stainless steel door handles (trenois decamps, wasquehal, france) were used as control surfaces. the mrsa strain cip was maintained for long-term conservation at − • c in glycerol in cryotubes and thawed just before use. two hundred µl were resuspended in ml of tryptic soy broth (tsb, sigma-aldrich, dutscher, brumath, france) and incubated at • c over night. ten µl of the culture were then stripped on tsa to create a reference petri dish. the strain purity was checked by gram staining on colonies, and the resistance to methicillin was confirmed by the disc diffusion method according to the european committee on antimicrobial susceptibility testing guidelines [ ] . for each assay, a single colony was resuspended in ml of tsb and incubated at • c over night. subsequently, ml of the culture were added to ml of tsb, and incubated at • c, stirring at rpm, during four hours. after three washes with ml peptone water and centrifugation ( g, min), the bacterial pellet was resuspended in peptone water to reach a concentration of cfu/ml. under sterile conditions, three independent droplets of µl (around cfu) were inoculated on the tested surface and then incubated for two hours at room temperature. bacteria of each inoculum were then carefully harvested using a sterile swab moistened with µl of peptone water. the swab was firmly applied on area of the inoculum and rotated. the swab was then placed in a ml sterile tube containing . ml of peptone water. tubes were placed in ultrasonic bath ( khz) during min and then briefly vortexed for s. serial dilutions of each tube suspension were performed in peptone water and µl of each dilution was exponentially seeded (easyspiral pro, interscience, saint-nom-la-bretèche, france) on tsa. plates were incubated at • c for h. colonies were counted with the interscience scan to determine the number of viable mrsa remaining in each inoculum. for each test, these numbers were streamlined to the mean number of viable mrsa remaining on the glass slide. the non-parametric mann-whitney test was used to compare the copper and control surfaces, in both in vitro and in situ analysis, using the statxact software (version . . , cytel studio, cambridge, ma, usa). kruskal-wallis was used to compare contamination between the five long-term care facilities, also using the statxact software. for every test, a p-value < . was considered as statistically significant (two-sided). during the sampling sequences in the five long-term care facilities, a total of copper surfaces and control surfaces were sampled, with samples being obtained from door handles of resident's room and obtained from handrails in corridors. as seen in figure a , the profiles obtained for the door handles and the handrails are similar, the bacterial burden being significantly lower on copper surfaces than on control surfaces. copper door handle showed an average of % reduction (median difference, − . cfu/cm ), while copper handrails showed an average of % reduction (median difference, − . cfu/cm ). looking more specifically at the frequencies of low and high levels of contaminations (figure b) , we can see that the copper door handles and handrails are much less frequently contaminated than the controls. looking at very low contaminations, % of the copper door handles showed less than cfu/cm on their surface, as compared to only % for the control door handles. looking at the high contaminations, superior to cfu/cm , only % for the copper alloys handles, while more than % of the control door handles were concerned. furthermore, extreme contaminations, defined as sample presenting ≥ cfu/cm , have been observed but occurred significantly less frequently on copper surfaces [three copper door handles ( %) vs. control door handles ( %) and three copper handrails ( %) vs. six control handrails ( %)]. to get a more precise view, the contamination distribution in each long-term care facilities was analyzed independently. as seen in figure , the surface contamination was different from one establishment to another, with significant differences between the five long-term care facilities (p < . ). nevertheless, in each facility and for both door handle and handrail, the median bacterial burden was lower on copper surface than on the control. concerning door handles (figure a) , the differences between copper and control median burdens were significant in every establishment, and ranged from . cfu/cm (a) to . cfu/cm (e). for handrails, the median differences ranged from . cfu/cm (c and d) to . cfu/cm (b and e) and they were statistically significant only for b and e. as different series of sampling have been realised over the three years in each long-term care facilities, the persistence over time of the mean antibacterial activity was analysed (figure ). looking at the door handle (figure a) , a stronger disparity could be observed in contaminations sampled from control elements than from copper elements. comparison between copper and control surfaces revealed that, in each long-term care facilities and for both sequences, the median bacterial burden was lower on copper door handles. looking at the median bacterial burden on handrail (figure b) , it was once again systematically lower on copper, except for one series for c and d, where the median on copper and control surfaces were equal. one and three years after copper alloys set-up, several copper door handles, randomly selected, were removed from the five long-term care facilities and transferred to the laboratory to evaluate their efficiency against a mrsa strain. in vitro tests were also performed on unused copper door handles, stainless steel door handles, and glass slides, to compare the residual bacterial burden after two hours of contact (figure ). no significant difference was observed in the distribution of the residual burden that was recovered from glass slides or stainless steel door handles. even so, the mrsa burden was significantly reduced as compared to control; there was more heterogeneity in the residual burden distribution on the copper handles. unused copper door handles showed an average . (± . ) log reduction compared to glass, while one year used copper handles showed a . (± . ) log reduction average, and the three years used copper handles a . (± . ) log reduction average. the door handles that were used for three years presented the higher heterogeneity with efficiency ranging from . log reduction to . log reduction. the spread of infections in healthcare facilities is an accurate problem, notably through inanimate surfaces contamination [ , ] . a simple gesture such as "holding a door handle" can participate to a wide and fast dispersion of pathogens and favor the cross-contamination between inanimate surfaces and patients. indeed, inanimate surfaces play an important role as a microbial beholder, despite hand-washing, which currently remains the primordial step against pathogens dissemination. to fight against microbial dissemination, new disinfection methods are being developed. nevertheless, bacteria can still rapidly recolonize touch surfaces, especially multidrug resistant bacteria that are frequently found in healthcare facilities [ ] . given this context, the use of copper surfaces brings a new perspective for constant and inherent disinfection. in this study, we investigated the antimicrobial properties of door handles and handrails containing copper that have been used on a daily base in five long-term care facilities. independently of the type, size, or localization of the establishments (city, countryside), the median contaminations levels observed were always lower on copper surfaces than on the control surfaces ( figure ). the estimate average bacterial burden reduction on copper was % for door handles and % for handrails. when compared to similar in situ investigations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , these reduction levels may seem lower than those that were observed in several other studies [ ] [ ] [ ] [ ] , ] reaching, for some types of furniture, % of reduction [ ] , but obviously, several different experimental points between these studies and ours can potentially explain these differences. first, those studies mainly focused on hospital wards, while our study focused on five long-term care facilities, where different types of peoples are moving and meeting (patients-residents/staff/visitors). different human populations can convey different microbiomes and bacterial burdens, directly impacting bacterial contamination on touch surfaces. second, the sampling protocols are quite different. the difference in sampling protocols may induce performance variations in the bacterial recovery. to collect bacteria from touch surfaces, we used a moistened cotton swab. among others techniques, such as contact agar plates or wipes [ , , [ ] [ ] [ ] , swabbing is preferred to perform samplings on small area and non-flat surfaces [ , , , ] , like the door handles and handrails of this study. due to the size and form of the copper door handles, the area of cm was used here as the standard area to sample, which is a smaller surface than in several other studies where sampled areas were frequently up to cm or more [ ] [ ] [ ] [ ] [ ] [ ] . also, peptone water was used as resuspension medium for bacteria, while other studies used media like saline water ( . %) [ ] [ ] [ ] [ ] [ ] or neutralizing buffers [ ] [ ] [ ] , ] . all of these experimental factors combined can lead to differences in the results. third and maybe the most important, copper alloys compositions are probably very different from one study to another. even if the percentage of copper is known, minor elements can have a huge importance in the antimicrobial efficiency of the alloy [ , ] . in our study, the cleaning protocols remained unchanged after the copper surfaces set-up in four of the five long-term care facilities and are different when compared to those that were used in other studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . indeed, the regular use of classic disinfectants or cleaning solutions can somehow modify the copper surface and lead to a loss of the surface antibacterial activity. such antagonist effects between cleaning solutions and copper surfaces have already been pointed out [ , ] . mikolay et al. [ ] observed low bacterial burden reductions by copper surface, ranging from % to %. they suggested that these values were due to the daily cleaning solution used in the hospital, proposing that the glucoprotamin, a bactericidal compound that is present in the solution, may form a thin layer on copper surface, thus protecting environmental bacteria from direct contact with copper. these results indicate that the use of an appropriate disinfectant or cleaning solution for copper surfaces and the application of a specific upkeep protocol could lead to an even better and stable copper antimicrobial activity. looking individually at the five establishments, singularities in the bacterial burden levels have been observed. highest global contaminations were found in long-term care facilities e, on both control (median of door handles, cfu/cm ; median of handrails, cfu/cm ) and copper (median on door handles, cfu/cm ; median on handrails, cfu/cm ). interestingly, it is, by far, the largest long-term care facility of the study, with a capacity of residents and a number of employees. potentially, a higher number of people in an establishment may induce a more intensive use of surfaces, and then results in higher levels of surface contaminations, but also on a faster lowering of the antibacterial activity of copper by oxidation or dirt accumulation. the values of bacterial burden were very variable between the establishments and inside each establishment, ranging from < cfu/cm to > cfu/cm . high-level contaminations, depending on numerous parameters (humidity, temperature, time delay between contamination, and sampling) stochastically occurred on touch surfaces in long-term care facilities during the study. our results showed that copper surfaces are less subject to these extreme contaminations, this frequency being reduced by % on copper handrail and by % on door handles. the average reductions of bacterial burden, as well as the , times decrease of high contaminations (> cfu/cm ) on copper door handles as compared to control handles, (figure ) suggest that door handles display a stronger antibacterial activity than handrails. these differences in activity between the door handles and handrails may depend in many factors, including the cleaning protocols that are different for door handle and handrail, the way the two types of items are used by the persons and most probably the difference in the percentage of copper, door handles having a higher copper percentage ( % vs. %) and being more active against bacteria than handrails. no correlation was highlighted between contaminations levels and external temperature when sampling was carried out (data not shown). nevertheless, it cannot be excluded that the period of the year has a slight effect on the global bacterial populations (temperature, humidity, wintry and summer infections, percentage of sick residents). in addition, statistical correlation indicates that the medians of bacterial burden on copper surfaces correlates with the medians that were observed on control, demonstrating that the global bacterial burden levels on touch surfaces are influenced by forces depending on the facility and the period of time. taking all into account, the overall bacterial burden levels were lower on copper surfaces, and this was observed in the five establishments, one year and three years after copper set-up, for both door handle and handrails ( figure ) . thereby, regardless of the healthcare facilities, copper surfaces maintained their antibacterial activity. to see if this antibacterial activity was still relevant against mrsa, in vitro contaminations were assayed (figure ). mrsa is a pathogen that is frequently involved in healthcare-associated infections [ , ] and is regularly found on healthcare touch surfaces [ ] , where it can potentially persist under environmental conditions for months [ ] . here, we highlighted that, after three years of normal use in healthcare facilities, the copper handles maintain their activity against this pathogen, with a reduction superior to % for most of the tested copper door handles, as compared to the residual burden on glass and stainless steel door handles. the efficiency of the copper surfaces, however, seems to slowly decrease with time. while new copper door handles allowed an average . logs reduction of number of mrsa within two hours, the reduction dropped to . logs after one year and . logs after three years of use. still, copper surfaces remain an interesting long-term solution to fight against touch surface contaminations, especially if installed in the whole facility. also, as mentioned before, the cleaning protocols remained identical than before the set-up of copper surfaces and other studies have already suggested that cleaning solutions may have an antagonist effect on copper antimicrobial activity [ , ] . yet, even if using these inadequate solutions for more than three years, copper surfaces still demonstrate a significant impact against mrsa. this study shows for the first time in long-term care facilities the antibacterial activity of steriall ® copper alloys over more than three year of use, as well as the persistence of their activity against mrsa. although some ameliorations can be proposed in term of cleaning solution or protocols to keep the activity at its highest level, these copper surface already play an effective role in the thwarting of the bacterial spread, even in non-optimal use. working on the aging of the copper surface could bring new insight to further increase the antimicrobial activity of the copper alloys and to develop copper surfaces that completely sustain this activity over years of use. multistate point-prevalence survey of health care-associated infections european centre for disease prevention and control. point prevalence survey of healthcare-associated infections and antimicrobial use in european acute care hospitals patient-to-patient transmission is important in extended-spectrum β-lactamase-producing klebsiella pneumoniae acquisition the role of the surface environment in healthcare-associated infections risk of hand or glove contamination after contact with patients colonized with vancomycin-resistant enterococcus or the colonized patients' environment an investigation of contact transmission of methicillin-resistant staphylococcus aureus everybody hands-on to avoid eskape: effect of sustained hand hygiene compliance on healthcare-associated infections and multidrug resistance in a paediatric hospital evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings environmental reservoirs of methicillin-resistant staphylococcus aureus in isolation rooms: correlation with patient isolates and implications for hospital hygiene risk of organism acquisition from prior room occupants: a systematic review and meta-analysis intrinsic bacterial burden associated with intensive care unit hospital beds: effects of disinfection on population recovery and mitigation of potential infection risk how long do nosocomial pathogens persist on inanimate surfaces? a systematic review rapid recontamination with mrsa of the environment of an intensive care unit after decontamination with hydrogen peroxide vapour linear correlation between inactivation of e. coli and oh radical concentration in tio photocatalytic disinfection different inactivation behaviors of ms- phage and escherichia coli in tio photocatalytic disinfection photocatalytic bactericidal effect of tio thin films: dynamic view of the active oxygen species responsible for the effect influence of surface morphology on the antimicrobial effect of transistion metal oxides in polymer surface highly efficient antiviral and antibacterial activities of solid-state cuprous compounds the antimicrobial activity of copper and copper alloys against nosocomial pathogens and mycobacterium tuberculosis isolated from healthcare facilities in the western cape: an in-vitro study mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces bacterial killing by dry metallic copper surfaces inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity human coronavirus e remains infectious on common touch surface materials inactivation of norovirus on dry copper alloy surfaces potential action of copper surfaces on meticillin-resistant staphylococcus aureus survival of clostridium difficile on copper and steel: futuristic options for hospital hygiene antimicrobial efficacy of copper surfaces against spores and vegetative cells of clostridium difficile: the germination theory metallic copper as an antimicrobial surface small colony variants are more susceptible to copper-mediated contact killing for pseudomonas aeruginosa and staphylococcus aureus contact killing of bacteria on copper is suppressed if bacteria-metal contact is prevented and is induced on iron by copper ions société française de microbiologie. staphylococcus spp.; casfm/eucast a survey of gram-negative bacteria survival on hospital fabrics and plastics role of copper in reducing hospital environment contamination copper alloy surfaces sustain terminal cleaning levels in a rural hospital reduction of bacterial burden by copper alloys on high-touch athletic center surfaces copper as an antibacterial material in different facilities the antimicrobial efficacy of copper alloy furnishing in the clinical environment: a crossover study survival of bacteria on metallic copper surfaces in a hospital trial evaluation of the antimicrobial properties of copper surfaces in an outpatient infectious disease practice copper surfaces are associated with significantly lower concentrations of bacteria on selected surfaces within a pediatric intensive care unit sustained reduction of microbial burden on common hospital surfaces through introduction of copper reduction of environmental contamination with multidrug-resistant bacteria by copper-alloy coating of surfaces in a highly endemic setting potential use of copper as a hygienic surface; problems associated with cumulative soiling and cleaning enquête nationale de prévalence des infections associées aux soins et des traitements antibiotiques en Établissements d'hébergement pour personnes âgées dépendantes (ehpad); résultats nationaux evidence that hospital hygiene is important in the control of methicillin-resistant staphylococcus aureus the authors would like to warmly thank velard f. (ea bios, reims), reffuveille f. key: cord- - md xox authors: lang, hans peter; hegner, martin; gerber, christoph title: nanomechanical cantilever array sensors date: journal: springer handbook of nanotechnology doi: . / - - - - _ sha: doc_id: cord_uid: md xox microfabricated cantilever sensors have attracted much interest in recent years as devices for the fast and reliable detection of small concentrations of molecules in air and solution. in addition to application of such sensors for gas and chemical-vapor sensing, for example as an artificial nose, they have also been employed to measure physical properties of tiny amounts of materials in miniaturized versions of conventional standard techniques such as calorimetry, thermogravimetry, weighing, photothermal spectroscopy, as well as for monitoring chemical reactions such as catalysis on small surfaces. in the past few years, the cantilever-sensor concept has been extended to biochemical applications and as an analytical device for measurements of biomaterials. because of the label-free detection principle of cantilever sensors, their small size and scalability, this kind of device is advantageous for diagnostic applications and disease monitoring, as well as for genomics or proteomics purposes. the use of microcantilever arrays enables detection of several analytes simultaneously and solves the inherent problem of thermal drift often present when using single microcantilever sensors, as some of the cantilevers can be used as sensor cantilevers for detection, and other cantilevers serve as passivated reference cantilevers that do not exhibit affinity to the molecules to be detected. microfabricated cantilever sensors have attracted much interest in recent years as devices for the fast and reliable detection of small concentrations of molecules in air and solution. in addition to application of such sensors for gas and chemicalvapor sensing, for example as an artificial nose, they have also been employed to measure physical properties of tiny amounts of materials in miniaturized versions of conventional standard techniques such as calorimetry, thermogravimetry, weighing, photothermal spectroscopy, as well as for monitoring chemical reactions such as catalysis on small surfaces. in the past few years, the cantilever-sensor concept has been extended to biochemical applications and as an analytical device for measurements of biomaterials. because of the label-free detection principle of cantilever sensors, their small size and scalability, this kind of device is advantageous for diagnostic applications and disease monitoring, as well as for genomics or proteomics purposes. the use of microcantilever arrays enables detection of several analytes simultaneously and solves the inherent problem of thermal drift often present when using single microcantilever sensors, as some of the cantilevers can be used as sensor cantilevers for detection, and other cantilevers serve as passivated reference cantilevers that do not exhibit affinity to the molecules to be detected. pick-up, microphone), photoelectric (photodiode, solar cell), electromagnetic (antenna), magnetic (hall-effect sensor, tape or hard-disk head for storage applications), electrostatic (electrometer), thermoelectric (thermocouple, thermoresistors), and electrical (capacitor, resistor). here we want to concentrate on a further type of sensor not yet mentioned: the mechanical sensor. it responds to changes of an external parameter, such as temperature changes or molecule adsorption, by a mechanical response, e.g. by bending or deflection. mechanical sensors consist of a fixed and a movable part. the movable part can be a thin membrane, a plate or a beam, fixed at one or both ends. the structures described here are called cantilevers. a cantilever is regarded here as a microfabricated rectangular bar-shaped structure that is longer than it is wide and has a thickness that is much smaller than its length or width. it is a horizontal structural element supported only at one end on a chip body; the other end is free (fig. . ). most often it is used as a mechanical probe to image the topography of a sample using a technique called atomic force microscopy (afm) or scanning force microscopy (sfm) [ ( ) rigid chip body, ( ) solid cantilever-support structure, ( ) hinge of cantilever, ( ) upper surface of the cantilever, which is usually functionalized with a sensor layer for detection of molecules, ( ) lower surface of the cantilever, usually passivated in order not to show affinity to the molecules to be detected. the geometrical dimensions, length l, width w and thickness t, are indicated by scanning the tip across a conductive or nonconductive surface using an x-y-z actuator system (e.g. a piezoelectric scanner), an image of the topography is obtained by recording the correction signal that has to be applied to the z-actuation drive to keep the interaction between tip and sample surface constant. sfm methods are nowadays well established in scientific research, education and, to a certain extent, also in industry. beyond imaging of surfaces, cantilevers have been used for many other purposes. however, here we focus on their application as sensor devices. the idea of using beams of silicon as sensors to measure deflections or changes in resonance frequency is actually quite old. first reports go back to , when wilfinger et al. [ . ] investigated silicon cantilever structures of × × mm , i. e. quite large structures, for detecting resonances. on the one hand, they used localized thermal expansion in diffused resistors (piezoresistors) located near the cantilever support to create a temperature gradient for actuating the cantilever at its resonance frequency. on the other hand, the piezoresistors could also be used to sense mechanical deflection of the cantilever. this early report already contains concepts for sensing and actuation of cantilevers. in the following years only a few reports are available on the use of cantilevers as sensors, e.g. heng [ . ] , who fabricated gold cantilevers capacitively coupled to microstrip lines in to mechanically trim high-frequency oscillator circuits. in , petersen [ . ] constructed cantilever-type micromechanical membrane switches in silicon that should have filled the gap between silicon transistors and mechanical electromagnetic relays. kolesar [ . ] suggested the use of cantilever structures as electronic nerve-agent detectors in . only with the availability of microfabricated cantilevers for afm [ ] observed changes in the resonance frequency of microcantilevers due to adsorption of analyte vapor on exposed surfaces. frequency changes have been found to be caused by mass loading or adsorption-induced changes in the cantilever spring constant. by coating cantilever surfaces with hygroscopic materials, such as phosphoric acid or gelatin, the cantilever can sense water vapor with picogram mass resolution. the deflection of individual cantilevers can easily be determined using afm-like optical beam-deflection electronics. however, single cantilever responses can be prone to artifacts such as thermal drift or unspecific adsorption. for this reason the use of passivated reference cantilevers is desirable. the first use of cantilever arrays with sensor and reference cantilevers was reported in [ . ], and represented significant progress for the understanding of true (difference) cantilever responses. for the use of a cantilever as a sensor, neither a sharp tip at the cantilever apex nor a sample surface is required. the cantilever surfaces serve as sensor surfaces and allow the processes taking place on the surface of the beam to be monitored with unprecedented accuracy, in particular the adsorption of molecules. the formation of molecule layers on the cantilever surface will generate surface stress, eventually resulting in a bending of the cantilever, provided the adsorption preferentially occurs on one surface of the cantilever. adsorption is controlled by coating one surface (typically the upper surface) of a cantilever with a thin layer of a material that exhibits affinity to molecules in the environment (sensor surface). this surface of the cantilever is referred to as the functionalized surface. the other surface of the cantilever (typically the lower surface) may be left uncoated or be coated with a passivation layer, i. e. a chemical surface that does not exhibit significant affinity to the molecules in the environment to be detected. to enable functionalized surfaces to be established, often a metal layer is evaporated onto the surface designed as sensor surface. metal surfaces, e.g. gold, may be used to covalently bind a monolayer that represents the chemical surface sensitive to the molecules to be detected from environment. frequently, a monolayer of thiol molecules covalently bound to a gold surface is used. the gold layer is also favorable for use as a reflection layer if the bending of the cantilever is read out via an optical beam-deflection method. given a cantilever coated with gold on its upper surface for adsorption of alkanethiol molecules and left uncoated on its lower surface (consisting of silicon and silicon oxide), the adsorption of thiol molecules will take place on the upper surface of the cantilever, resulting in a downward bending of the cantilever due to the formation of surface stress. we will call this process development of compressive surface stress, because the forming self-assembled monolayer produces a downward bending of the cantilever (away from the gold coating). in the opposite situation, i. e. when the cantilever bends upwards, we would speak of tensile stress. if both the upper and lower surfaces of the cantilevers are involved in the reaction, then the situation will be much more complex, as a predominant compressive stress formation on the lower cantilever surface might appear like tensile stress on the upper surface. for this reason, it is of utmost importance that the lower cantilever surface is passivated in order that ideally no processes take place on the lower surface of the cantilever. single microcantilevers are susceptible to parasitic deflections that may be caused by thermal drift or chemical interaction of a cantilever with its environment, in particular if the cantilever is operated in a liquid. to exclude such influences, simultaneous measurement of reference cantilevers aligned in the same array as the sensing cantilevers is crucial [ . ]. as the difference in signals from the reference and sensor cantilevers shows the net cantilever response, even small sensor responses can be extracted from large cantilever deflections without being dominated by undesired effects. when only single microcantilevers are used, no thermal-drift compensation is possible. to obtain useful data under these circumstances, both microcantilever surfaces have to be chemically well defined. one of the surfaces, typically the lower one, has to be passivated; otherwise the cantilever response will be convoluted with undesired effects originating from uncontrolled reactions taking place on the lower surface ( fig. . a) . with a pair of cantilevers, reliable measurements are obtained. one cantilever is used as the sensor cantilever (typically coated on the upper side with a molecule layer exhibiting affinity to the molecules to be detected), whereas the other cantilever serves as the reference cantilever. it should be coated with a passivation layer on the upper surface so as not to exhibit affinity to the molecules to be detected. thermal drifts are canceled out if difference responses, i. e. difference in deflections of sensor and reference cantilevers, are taken. alternatively, both cantilevers are used as sensor cantilevers (sensor layer on the upper surfaces), and the lower surface has to be passivated ( fig. . b). it is best to use a cantilever array ( fig. . c), in which several cantilevers are used either as sensor or as reference cantilevers so that multiple difference signals can be evaluated simultaneously. thermal drift is canceled out as one surface of all cantilevers, typically the lower one, is left uncoated or coated with the same passivation layer. in analogy to afm, various operating modes for cantilevers are described in the literature. the continuous bending of a cantilever with increasing coverage by molecules is referred to as operation in the static mode ( fig. . a) . adsorption of molecules onto the functional layer produces stress at the interface between the functional layer and the molecular layer forming. because the forces within the functional layer try to keep the distance between molecules constant, the cantilever beam responds by bending because of its extreme flexibility. this property is described by the spring constant k of the cantilever, which for a rectangu- (b) diffusion of molecules into a polymer layer leads to swelling of the polymer and eventually to a bending of the cantilever. (c) highly specific molecular recognition of biomolecules by receptors changes the surface stress on the upper surface of the cantilever and results in bending. (d) oscillation of a cantilever at its resonance frequency (dynamic mode) allows information on mass changes taking place on the cantilever surface to be obtained (application as a microbalance). (e) changing the temperature while a sample is attached to the apex of the cantilever allows information to be gathered on decomposition or oxidation process. (f) dynamic-mode measurements in liquids yield details on mass changes during biochemical processes. (g) in the heat mode, a bimetallic cantilever is employed. here bending is due to the difference in the thermal expansion coefficients of the two materials. (h) a bimetallic cantilever with a catalytically active surface bends due to heat production during a catalytic reaction. (i) a tiny sample attached to the apex of the cantilever is investigated, taking advantage of the bimetallic effect. tracking the deflection as a function of temperature allows the observation of phase transitions in the sample in a calorimeter mode lar microcantilever of length l, thickness t and width w is calculated as where e is the young's modulus [e si = . × n/m for si( )]. as a response to surface stress, e.g. owing to adsorption of a molecular layer, the microcantilever bends, and its shape can be approximated as part of a circle with radius r. this radius of curvature is given by [ where e is young's modulus, t the thickness of the cantilever, ν the poisson's ratio (ν si = . ), and r the bending radius of the cantilever. static-mode operation has been reported in various environments. in its simplest configuration, molecules from the gaseous environment adsorb on the functionalized sensing surface and form a molecular layer ( fig. . a) , provided the molecules exhibit some affinity to the surface. in the case of alkanethiol covalently binding to gold, the affinity is very high, resulting in a fast bending response within minutes [ . ]. polymer sensing layers only exhibit a partial sensitivity, i. e. polymer-coated cantilevers always respond to the presence of volatile molecules, but the magnitude and temporal behavior are specific to the chemistry of the polymer. molecules from the environment diffuse into the polymer layer at different rates, mainly depending on the size and solubility of the molecules in the polymer layer ( fig. . b) . a wide range of hydrophilic/hydrophobic polymers can be selected, dif- fering in their affinity to polar/unpolar molecules. thus, the polymers can be chosen according to what an application requires. static-mode operation in liquids, however, usually requires rather specific sensing layers, based on molecular recognition, such as dna hybridization [ . ] or antigen-antibody recognition (fig. . c) . cantilevers functionalized by coating with biochemical sensing layers respond very specifically using biomolecular key-lock principles of molecular recognition. however, whether molecular recognition will actually lead to a bending of the cantilever depends on the efficiency of transduction, because the surface stress has to be generated very close to the cantilever surface to produce bending. by just scaling down standard gene-chip strategies to cantilever geometry utilizing long spacer molecules so that dna molecules become more accessible for hybridization, the hybridization takes place at a distance of several nanometers from the cantilever surface. in such experiments, no cantilever bending was observed [ . ]. mass changes can be determined accurately by using a cantilever actuated at its eigenfrequency. the eigenfrequency is equal to the resonance frequency of an oscillating cantilever if the elastic properties of the cantilever remain unchanged during the moleculeadsorption process and if damping effects are insignificant. this mode of operation is called the dynamic mode (e.g., the use as a microbalance, fig. . d). owing to mass addition on the cantilever surface, the cantilever's eigenfrequency will shift to a lower value. the frequency change per mass change on a rectangular cantilever is calculated [ . ] according to where ρ = m/(lwt) is the mass density of the microcantilever and the deposited mass, and n l ≈ is a geometrical factor. the mass change is calculated [ . ] from the frequency shift using where f is the eigenfrequency before the mass change occurs, and f the eigenfrequency after the mass change. mass-change determination can be combined with varying environment temperature conditions ( fig. . e) to obtain a method introduced in the literature as micromechanical thermogravimetry [ . ] . a tiny piece of sample to be investigated has to be mounted at the apex of the cantilever. its mass should not exceed several hundred nanograms. adsorption, desorption and decomposition processes, occurring while changing the temperature, produce mass changes in the picogram range that can be observed in real time by tracking the resonance-frequency shift. dynamic-mode operation in a liquid environment is more difficult than in air, because of the large damping of the cantilever oscillation due to the high viscosity of the surrounding media ( fig. . f) . this results in a low quality factor q of the oscillation, and thus the resonance frequency shift is difficult to track with high resolution. the quality factor is defined as whereas in air the resonance frequency can easily be determined with a resolution of below hz, only a frequency resolution of about hz is expected for measurements in a liquid environment. the damping or altered elastic properties of the cantilever during the experiment, e.g. by a stiffening or softening of the spring constant caused by the adsorption of a molecule layer, result in the fact that the measured resonance frequency will not be exactly equal to the eigenfrequency of the cantilever, and therefore the mass derived from the frequency shift will be inaccurate. in a medium, the vibration of a cantilever is described by the model of a driven damped harmonic oscillator where m * = const(m c + m l ) is the effective mass of the cantilever (for a rectangular cantilever the constant is . ). especially in liquids, the mass of the comoved liquid m l adds significantly to the mass of the cantilever m c . the term γ dx dt is the drag force due to damping, f cos( π ft) is the driving force executed by the piezo-oscillator, and k is the spring constant of the cantilever. if no damping is present, the eigenfrequencies of the various oscillation modes of a bar-shaped cantilever are calculated according to where f n are the eigenfrequencies of the n-th mode, α n are constants depending on the mode: α = . , α = . , α n = π(n − . ); k is the spring constant of the cantilever, m c the mass of the cantilever, and m l the mass of the medium surrounding the cantilever, e.g. liquid [ . ]. addition of mass to the cantilever due to adsorption will change the effective mass as follows where Δm is the additional mass adsorbed. typically, the comoved mass of the liquid is much larger than the adsorbed mass. figure . clearly shows that the resonance frequency is only equal to the eigenfrequency if no damping is present. with damping, the frequency at which the peak of the resonance curve occurs is no longer identical to that at which the turning point of the phase curve occurs. for example, resonance curve with damping γ has its maximum amplitude at frequency f . the corresponding phase would be ϕ res (γ ), which is not equal to π/ , as would be expected in the undamped case. if direct resonance-frequency tracking or a phase-locked loop is used to determine the frequency of the oscillating cantilever, then only its resonance frequency is detected, but not its eigenfrequency. remember that the eigenfrequency, and not the resonance frequency, is required to determine mass changes. if a cantilever is coated with metal layers, thermal expansion differences in the cantilever and the coating layer will further influence cantilever bending as a function of temperature. this mode of operation is referred to as the heat mode and causes cantilever bending because of differing thermal expansion coefficients in the sensor layer and cantilever materials [ . ] (fig. . g) here α , α are the thermal expansion coefficients of the cantilever and coating materials, respectively, λ , λ their thermal conductivities, t , t the material thicknesses, p is the total power generated on the cantilever, and κ is a geometry parameter of the cantilever device. heat changes are either caused by external influences (change in temperature, fig. . g), occur directly on the surface by exothermal, e.g. catalytic, resonance curve with no damping ( ), and increasing damping ( )-( ). the undamped curve with resonance frequency f exhibits a very high amplitude, whereas the resonance peak amplitude decreases with damping. this also involves a shift in resonance frequencies from f to f to lower values. (b) corresponding phase curves showing no damping ( ), and increasing damping ( )-( ). the steplike phase jump at resonance of the undamped resonance gradually broadens with increasing damping reactions ( fig. . h ), or are due to material properties of a sample attached to the apex of the cantilever (micromechanical calorimetry, fig. photothermal spectroscopy when a material adsorbs photons, a fraction of the energy is converted into heat. this photothermal heating can be measured as a function of the light wavelength to provide optical absorption data of the material. the interaction of light with a bimetallic microcantilever creates heat on the cantilever surface, resulting in a bending of the cantilever [ . ]. such bimetalliccantilever devices are capable of detecting heat flows due to an optical heating power of pw, which is two orders of magnitude better than in conventional photothermal spectroscopy. a cantilever coated with a metallic layer (measurement electrode) on one side is placed in an electrolytic medium, e.g. a salt solution, together with a metallic reference electrode, usually made of a noble metal. if the voltage between the measurement and the reference electrode is changed, electrochemical processes on the measurement electrode (cantilever) are induced, such as adsorption or desorption of ions from the electrolyte solution onto the measurement electrode. these processes lead to a bending of the cantilever due to changes in surface stress and in the electrostatic forces [ . ]. the detection of electrostatic and magnetic forces is possible if charged or magnetic particles are deposited on the cantilever [ . , ]. if the cantilever is placed in the vicinity of electrostatic charges or magnetic particles, attractive or repulsion forces occur according to the polarity of the charges or magnetic particles present on the cantilever. these forces will result in an upward or a downward bending of the cantilever. the magnitude of the bending depends on the distribution of charged or magnetic particles on both the cantilever and in the surrounding environment according to the laws of electrostatics and magnetism. silicon cantilever sensor arrays have been microfabricated using a dry-etching silicon-on-insulator (soi) fabrication technique developed in the micro-/nanomechanics department at the ibm zurich research laboratory. one chip comprises eight cantilevers, having a length of μm, a width of μm, and a thickness of . μm, and arranged on a pitch of μm. for dynamic-mode operation, the cantilever thickness may be up to μm. the resonance frequencies of the cantilevers vary by . % only, demonstrating the high reproducibility and precision of cantilever fabrication. a scanning electron microscopy image of a cantilever sensor-array chip is shown in fig. a measurement set-up for cantilever arrays consists of four major parts: ( ) the measurement chamber containing the cantilever array, ( ) an optical or electrical system to detect the cantilever deflection [e.g. laser sources, collimation lenses and a position-sensitive detector (psd), or piezoresistors and wheatstone-bridge detection electronics], ( ) electronics to amplify, process and acquire the signals from the detector, and ( ) a gas-or liquid-handling system to inject samples reproducibly into the measurement chamber and purge the chamber. figure . shows the schematic set-up for experiments performed in a gaseous ( fig. . a) and a liquid, biochemical ( fig. . b) environment for the optical beam-deflection embodiment of the measurement setup. the cantilever sensor array is located in an analysis chamber with a volume of - μl, which has inlet and outlet ports for gases or liquids. the cantilever deflection is determined by means of an array of eight vertical-cavity surface-emitting lasers (vcsels) arranged at a linear pitch of μm that emit at a wavelength of nm into a narrow cone of to • . the light of each vcsel is collimated and focused onto the apex of the corresponding cantilever by a pair of achromatic doublet lenses, . mm in diameter. this size has to be selected in such a way that all eight laser beams pass through the lens close to its center to minimize scattering, chromatic and spherical aberration artifacts. the light is then reflected off the gold-coated surface of the cantilever and hits the sur-face of a position-sensing detector (psd). psds are light-sensitive photopotentiometer-like devices that produce photocurrents at two opposing electrodes. the magnitude of the photocurrents depends linearly on the distance of the impinging light spot from the electrodes. thus the position of an incident light beam can easily be determined with micrometer precision. the photocurrents are transformed into voltages and amplified in a preamplifier. as only one psd is used, the eight lasers cannot be switched on simultaneously. therefore, a time-multiplexing procedure is used to switch the lasers on and off sequentially at typical intervals of - ms. the resulting deflection signal is digitized and stored together with time information on a personal computer (pc), which also controls the multiplexing of the vcsels as well as the switching of the valves and mass flow controllers used for setting the composition ratio of the analyte mixture. the measurement setup for liquids (fig. . b) consists of a polyetheretherketone (peek) liquid cell, which contains the cantilever array and is sealed by a viton o-ring and a glass plate. the vcsels and the psd are mounted on a metal frame around the liquid cell. after preprocessing the position of the deflected light beam in a current-to-voltage converter and amplifier stage, the signal is digitized in an analog-todigital converter and stored on a pc. the liquid cell is equipped with inlet and outlet ports for liquids. they are connected via . mm inner-diameter teflon tubing to individual thermally equilibrated glass containers, in which the biochemical liquids are stored. a sixposition valve allows the inlet to the liquid chamber to be connected to each of the liquid-sample containers separately. the liquids are pulled (or pushed) through the liquid chamber by means of a syringe pump connected to the outlet of the chamber. a peltier element is situated very close to the lumen of the chamber to allow temperature regulation within the chamber. the entire experimental setup is housed in a temperaturecontrolled box regulated with an accuracy of . k to the target temperature. this section describes various ways to determine the deflection of cantilever sensors. they differ in sensitivity, effort for alignment and setup, robustness and ease of readout as well as their potential for miniaturization. piezoresistive readout piezoresistive cantilevers [ . , ] are usually ushaped, having diffused piezoresistors in both of the legs close to the hinge (fig. . a) . the resistance in the piezoresistors is measured by a wheatstone-bridge technique employing three reference resistors, one of which is adjustable. the current flowing between the two branches of the wheatstone bridge is initially nulled by changing the resistance of the adjustable resistor. if the cantilever bends, the piezoresistor changes its value and a current will flow between the two branches of the wheatstone bridge. this current is converted via a differential amplifier into a voltage for staticmode measurement. for dynamic-mode measurement, part b . the piezoresistive cantilever is externally actuated via a frequency generator connected to a piezocrystal. the alternating current (ac) actuation voltage is fed as reference voltage into a lock-in amplifier and compared with the response of the wheatstone-bridge circuit. this technique allows one to sweep resonance curves and to determine shifts in resonance frequency. piezoelectric cantilevers [ . ] are actuated by applying an electric ac voltage via the inverse piezoelectric effect (self-excitation) to the piezoelectric material (pzt or zno). sensing of bending is performed by recording the piezoelectric current change due to the fact that the pzt layer may produce a sensitive field response to weak stress through the direct piezoelectric effect. such cantilevers are multilayer structures consisting of an sio cantilever and the pzt piezoelectric layer. two electrode layers, insulated from each other, provide electrical contact. the entire structure is protected using passivation layers (fig. . b ). an identical structure is usually integrated into the rigid chip body to provide a reference for the piezoelectric signals from the cantilever. for capacitive readout (fig. . c) , a rigid beam with an electrode mounted on the solid support and a flexible cantilever with another electrode layer are used [ . , ]. both electrodes are insulated from each other. upon bending of the flexible cantilever the capacitance between the two electrodes changes and allows the deflection of the flexible cantilever to be determined. both static-and dynamic-mode measurements are possible. interferometric methods [ . , ] are most accurate for the determination of small movements. a laser beam passes through a polarizer plate (polarization • ) and is partially transmitted by a nonpolarized beam splitter (fig. . d) . the transmitted beam is divided in a wollaston prism into a reference and an object beam. these mutually orthogonally polarized beams are then focused onto the cantilever. both beams (the reference beam from the hinge region and the object beam from the apex region of the cantilever) are reflected back to the objective lens, pass the wollaston prism, where they are recombined into one beam, which is then reflected into the other arm of the interferometer, where after the λ/ plate a phase shift of a quarter wavelength between object and reference beam is established. another wollaston prism separates the reference and object beams again for analysis with a four-quadrant photodiode. a differential amplifier is used to obtain the cantilever deflection with high accuracy. however, the interferometric setup is quite bulky and difficult to handle. the most frequently used approach to read out cantilever deflections is optical beam deflection [ . ], because it is a comparatively simple method with an excellent lateral resolution. a schematic of this method is shown in fig. . e. the actual cantilever deflection Δx scales with the cantilever dimensions; therefore the surface stress Δσ in n/m is a convenient quantity to measure and compare cantilever responses. it takes into account the cantilever material properties, such as poisson's ratio ν, young's modulus e and the cantilever thickness t. the radius of curvature r of the cantilever is a measure of bending, ( . ). as shown in the drawing in fig. . e, the actual cantilever displacement Δx is transformed into a displacement Δd on the psd. the position of a light spot on a psd is determined by measuring the photocurrents from the two facing electrodes. the movement of the light spot on the linear psd is calculated from the two currents i and i and the size l of the psd by as all angles are very small, it can be assumed that the bending angle of the cantilever is equal to half of the angle θ of the deflected laser beam, i. e. θ/ . therefore, the bending angle of the cantilever can be calculated to be where s is the distance between the psd and the cantilever. the actual cantilever deflection Δx is calculated from the cantilever length l and the bending angle θ/ by Δx = θ/ · l . ( . ) combination of ( . ) and ( . ) relates the actual cantilever deflection Δx to the psd signal Δx = lΔd s . ( . ) the relation between the radius of curvature and the deflection angle is to serve as sensors, cantilevers have to be coated with a sensor layer that is either highly specific, i. e. is able to recognize target molecules in a key-lock process, or partially specific, so that the sensor information from several cantilevers yields a pattern that is characteristic of the target molecules. to provide a platform for specific functionalization, the upper surface of these cantilevers is typically coated with nm of titanium and nm of gold, which yields a reflective surface and an interface for attaching functional groups of probe molecules, e.g. for anchoring molecules with a thiol group to the gold surface of the cantilever. such thin metal layers are believed not to contribute significantly to bimetallic bending, because the temperature is kept constant. there are numerous ways to coat a cantilever with material, both simple and more advanced ones. the method of choice should be fast, reproducible, reliable and allow one or both of the surfaces of a cantilever to be coated separately. obvious methods to coat a cantilever are thermal or electron-beam-assisted evaporation of material, electrospray or other standard deposition methods. the disadvantage of these methods is that they only are suitable for coating large areas, but not individual cantilevers in an array, unless shadow masks are used. such masks need to be accurately aligned to the cantilever structures, which is a time-consuming process. other methods to coat cantilevers use manual placement of particles onto the cantilever [ . , , - ], which requires skillful handling of tiny samples. cantilevers can also be coated by directly pipetting solutions of the probe molecules onto the cantilevers [ . ] or by employing air-brush spraying and shadow masks to coat the cantilevers separately [ . ]. all these methods have only limited reproducibility and are very time-consuming if a larger number of cantilever arrays has to be coated. microfluidic networks (μfn) [ . ] are structures of channels and wells, etched several ten to hundred micrometer deep into silicon wafers. the wells can be filled easily using a laboratory pipette, so that the fluid with the probe molecules for coating the cantilever is guided through the channels towards openings at a pitch matched to the distance between individual cantilevers in the array (fig. . a) . the cantilever array is then introduced into the open channels of the μfn that are filled with a solution of the probe molecules. the incubation of the cantilever array in the channels of the μfn takes from a few seconds (self-assembly of alkanethiol monolayers) to several tens of minutes (coating with protein solutions). to prevent evaporation of the solutions, the channels are covered by a slice of poly(dimethylsiloxane) (pdms). in addition, the microfluidic network may be placed in an environment filled with saturated vapor of the solvent used for the probe molecules. a similar approach is insertion of the cantilever array into an array of dimension-matched disposable glass capillaries. the outer diameter of the glass capillaries is μm so that they can be placed neatly next to each other to accommodate the pitch of the cantilevers in the array ( μm). their inner diameter is μm, providing sufficient room to insert the cantilevers (width: μm) safely (fig. . b) inkjet spotting all of the above techniques require manual alignment of the cantilever array and functionalization tool, and are therefore not ideal for coating a large number of cantilever arrays. the inkjet-spotting technique, however, allows rapid and reliable coating of cantilever arrays [ . , ]. an x-y-z positioning system allows a fine nozzle (capillary diameter: μm) to be positioned with an accuracy of approximately μm over a cantilever. individual droplets (diameter: - μm, volume . - . nl) can be dispensed individually by means of a piezo-driven ejection system in the inkjet nozzle. when the droplets are spotted with a pitch smaller than . mm, they merge and form continuous films. by adjusting the number of droplets deposited on the cantilevers, the resulting film thickness can be controlled precisely. the inkjet-spotting technique allows a cantilever to be coated within seconds and yields very homogeneous, reproducibly deposited layers of well-controlled thickness. successful coating of self-assembled alkanethiol hydrogen early reports on detection of gases such as hydrogen involve nanomechancal detection of catalytic reactions of bimetallic microcantilevers coated with aluminum and a top layer of platinum in thermal mode [ . ]. the catalytic reaction of oxygen present in a reaction chamber with hydrogen being introduced into the chamber produces oscillatory chemical reactions resulting in mechanical oscillations of the cantilever due to heat formation related to catalytic conversion of h and o to form h o. by use of an array of four platinum coated and four uncoated microcantilevers, a change of the deflection signal due to bending of the platinum coated cantilever relative to the uncoated cantilevers is observed upon hydrogen adsorption in the presence of oxygen based on the edta-cd(ii) complex and its binding capability to bovine serum albumine (bsa) and antibody-based cd(ii) sensor using microcantilevers is presented [ . ]. a microcantilever-based alcohol vapor sensor is described in [ . ] using the piezoresistive technique and polymer coating. they also present a simple evaporation model that allows determining the concentration. the detection limit found is ppm for methanol, ethanol and -propanol. in [ . ] an integrated complementary metal oxide semiconductor (cmos) chemical microsensor with piezoresistive detection (wheatstone bridge configuration) using poly(etherurethane) (peut) as the sensor layer is presented. they are able to reversibly detect volatile organic compounds (vocs) such as toluene, n-octane, ethyl acetate and ethanol with a sensitivity level down to ppm. an improved version of that device is described in [ . ] . the sensitivity could be increased to ppm for n-octane. later the technique has been refined by using electromagnetic rather than electrothermal actuation and transistor-based readout reducing power dissipation on the cantilever [ . ]. piezoelectric readout in dynamic mode and electromagnetic actuation of cantilevers spray-coated with peut is reported in [ . ], achieving a sensitivity of ppm for ethanol. in [ . ] a study is published how to prepare polyethylene glycol (peg) coated microcantilever sensors using a microcapillary pipette assisted method. peg coating is suitable for ethanol sensing as ethanol quickly forms hydrogen bonds with the oh groups of the peg. sensor operation is reported to be reversible and reproducible. in [ . ] artificial neural networks are used for analyte species and concentration identification with polymer coated optically read-out microcantilevers. the analytes detected are carbon dioxide, dichloromethane, diisopropylmethylphosphonate (dimp), dioxane, ethanol, water, -propanol, methanol, trichloroethylene and trichloromethylene. in [ . ] the chemical sensing performance of a silicon reconant microcantilever sensor is investigated in dependence on the thickness of the sensitive coating. for a coating thickness of , and μm of peut a limit of detection of ppm was found for ethanol. a new concept of parylene micromembrane array for chemical sensing is presented [ . ] using the capacitive method. the parylene membrane is suspended over a metal pad patterned on the substrate. the pad and part of the membrane that is metal-coated serve as electrodes for capacitive measurement. the top electrode located on the membrane is chemically modified by applying a gold layer and self-assembled thiol monolayers (-cooh, -ch and -oh) for detection of analyte molecules. successful detection of -propanol and toluene is reported. in [ . ] a sensitive selfoscillating cantilever array is described for quantitative and qualitative analysis of organic vapor mixtures. the cantilevers are electromagnetically actuated and the resonance frequency is measured using a frequency counter. sensor response is reproducible and reversible. using peut coating the smallest measured concentration is ppm, but the limit of detection is well below ppm. in [ . ] a combination of gas chromatography with a microcantilever sensor array for enhanced selectivity is reported. test voc mixtures composed of acetone, ethanol and trichloroethylene in pentane, as well as methanol with acetonitrile in pentane were first separated in a gas chromatography column and then detected using micocantilevers coated with responsive phases such as -aminopropyltriethoxy silane, copper phtalocyanine and methyl-β-cyclodextrin. analytes detected include pentane, methanol, acetonitrile, acetone, ethanol and trichloroethylene. in [ . ] results are presented on independent component analysis (ica) of ethanol, propanol and dimp using cantilever coated with molecular recognition phases (mrp), whereby ica has proven its feature extraction ablility for components in mixtures. detection of the organochlorine insecticide compound dichlorodiphenyltrichloroethane (ddt) is reported using a synthetic hapten of the pesticide as recognition site conjugated with bovine serum albumin (bsa) covalently immobilised on the gold-coated side of the cantilever by using thiol self assembled monolayers [ . ]. security measures require inexpensive, highly selective and very sensitive small sensors that can be massproduced and microfabricated. such low cost sensors could be arranged as a sensor grid for large area coverage of sensitive infrastructure, like airports, public buildings, or traffic infrastructure. threats can be of chemical, biological, radioactive or explosive nature. microcantilever sensors are reported to offer very high sensitivities of explosives detection. photomechanical chemical microsensors based on adsorption-induced and photoinduced stress changes due to the presence of diisopropyl methyl phosphonate (dimp), which is a model compound for phosphorous-containing chemical warfare agents, and trinitrotoluene (tnt), an explosive are reported [ . ]. further explosives frequently used include pentaerythritol tetranitrate (petn) and hexahydro- , , -triazine (rdx), often also with plastic fillers [ . ]. these compounds are very stable, if no detonator is present. their explosive power, however, is very large, and moreover, the vapor pressures of petn and rdx are very low, in the range of ppb and ppt. by functionalizing microcantilevers with self-assembled monolayers of -mercaptobeonzoic acid ( -mba) petn was detected at a level of ppt and rdx at a level of ppt [ . ]. tnt was found to readily stick to si surfaces, suggesting the use of microcantilevers for tnt detection, taking advantage of the respective adsorption/desorption kinetics [ . , ]. detection of tnt via deflagration on a microcantilever is described by pinnaduwage et al. [ . ] . they used piezoresistive microcantilevers where the cantilever deflection was measured optically via beam deflection. tnt vapor from a generator placed mm away from the microcantilever was observed to adsorb on its surface resulting in a decrease of resonance frequency. application of an electrical pulse ( v, ms) to the piezoresistive cantilever resulted in deflagration of the tnt vapor and a bump in the cantilever bending signal. this bump was found to be related to the heat produced during deflagration. the amount of heat released is proportional to the area of the bump in the time versus bending signal diagram of the process. the deflagration was found to be complete, as the same resonance frequency as before the experiment was observed. the amount of tnt mass involved was determined as pg. the technique was later extended to the detection of petn and rdx, where much slower reaction kinetics was observed [ . , ] . traces of , -dinitrotoluene (dnt) in tnt can also be used for detection of tnt, because it is the major impurity in production grade tnt. furthermore dnt is a decomposition product of tnt. the saturation concentration of dnt in air at • c is times higher than that of tnt. dnt was reported detectable at the ppt level using polysiloxane polymer layers [ . ]. microfabrication of electrostatically actuated resonant microcantilever beams in cmos technology for detection of the nerve agent stimulant dimethylmethylphosphonate (dmmp) using polycarbosilane-coated beams [ . ] is an important step towards an integrated platform based on silicon microcantilevers, which besides compactness might also include telemetry [ . ] . cu + /l-cysteine bilayercoated microcantilever demonstrated high sensitivity and selectivity toward organo-phosphorus compounds in aqueous solution. the microcantilever was found to undergo bending upon exposure to nerve agent simulant dmmp at concentrations as low as − m due to the complexation of the phosphonyl group and the cu + /l-cysteine bilayer on the microcantilever surface [ . , ]. ph control of ph is often important in biochemical reactions. therefore this section concerns measurement of ph using microcantilevers. the interfacial stress of self-assembled monolayers of mercaptohexadecanoic acid and hexadecanethiol depends on ph values and ionic strength [ . ]. sio and silicon nitride microcantilevers were also found to exhibit a deflection dependence with ph when coated with -aminobutyltriethoxysilane, -mercaptoundecanoic acid and au/al-coated over a ph range - . aminosilane-modified sio /au cantilevers performed robustly over ph range - ( nm deflection/ph unit), while si n /au cantilevers performed well at ph - and - ( micromechanical cantilever arrays have been used for quantitative detection of vital fungal spores of aspergillus niger and saccharomyces cerevisiae. the specific adsorption and growth on concanavalin a, fibronectin or immunoglobulin g cantilever surfaces was investigated. maximum spore immobilization, germination and mycelium growth was observed on the immunoglobulin g functionalized cantilever surfaces, as measured from shifts in resonance frequency within a few hours, being much faster than with standard petri dish cultivation [ besides chemical and biochemical sensing, microcantilevers can also detect changes in physical properties of surrounding media, such as gas or liquid, or of layers deposited on the cantilever itself. density and viscosity a piezoelectric unimorph cantilever as a liquid viscosity-and-density sensor was tested using waterglycerol solutions of different compositions, whereby the resonance frequency decreased while the width of the resonance peak increased with increasing glyc- gas sensing does not only involve chemical detection, but also pressure and flow sensing. brown et al. [ . ] have studied the behavior of magnetically actuated oscillating microcantilevers at large deflections and have found hysteresis behavior at resonance. the amplitude at the actuation frequency changes depending on pressure due to damping. the authors have used cantilever in cantilever (cic) structures, and have observed changes in deflection as gas pressure is varied. at atmospheric pressure, damping is large and the oscillation amplitude is relative small and hysteresis effects are absent. at lower pressure, abrupt changes in the oscillation amplitude occur with changes in the driving frequency. since the change of amplitude and driving frequency, at which they occur are pressure dependent, these quantities can be used for accurate determina- tion of gas pressure, demonstrated in the range between − and torr. brown et al. [ . ] emphasize that microelectromechanical system pressure sensors will have a wide range of applications, especially in the automotive industry. piezoresistive cantilever based deflection measurement has major advantages over diaphragms. the pressure range has been extended to - torr by means of design geometry adaptation. su et al. [ . ] present highly sensitive ultrathin piezoresistive silicon microcantilevers for gas velocity sensing, whereby the deflection increases with airflow distribution in a steel pipe. the detection principle is based on normal pressure drag producing bending of the cantilever. the minimum flow speed measured was . cm/s, which is comparable to classical hot-wire anemometers. mertens et al. [ . ] have investigated the effects of temperature and pressure on microcantilever resonance response in helium and nitrogen. resonance response as a function of pressure showed three different regimes, which correspond to molecular flow, transition regimes and viscous flow, whereby the frequency variation of the cantilever is mainly due to changes in the mean free path of gas molecules. effects observed allow measurement of pressures between . × − and torr. mortet et al. [ . ] present a pressure sensor based on a piezoelectric bimorph microcantilever with a measurement range between and torr. the resonance frequency shift is constant for pressures below torr. for higher pressures the sensitivity is typically a few ppm/mbar, but depends on the mode number. sievilä et al. [ . ] present a cantilever paddle within a frame operating like a moving mirror to detect the displacements in the oscillating cantilever using a he-ne laser in a michelson interferometer configuration, whereby the cantilever acts as moving mirror element in one path of the interferometer. a fixed mirror serves a reference in the other arm of the interferometer. the thermal expansion of tao x n y thin films deposited on a microcantilever was measured to examine the residual stress and the thermal expansion coefficient by observsing the changes in radius of curvature [ . ]. thermal drift issues of resonaniting microcantilevers are discussed in detail in [ . ] . microcantilevers can also be used as uncooled, microcantilever-based infrared (ir) imaging devices by monitoring the bending of the microcantilever as a function of the ir radiation intensity incident on the cantilever surface. the infrared (thermal) image of the source is obtained by rastering a single microfabricated cantilever across the image formed at the focal plane of a concave mirror [ . - ]. the method has later been refined such that photons are detected using the stress caused by photoelectrons emitted from a pt film surface in contact with a semiconductor microstructure, which forms a schottky barrier. the photoinduced bending of the schottky barrier microstructure is due to electronic stress produced by photoelectrons diffusing into the microstructure [ . ]. the performance of ir imaging via microcantilevers has been enhanced by one-fold leg and two-fold legs beam structures with absorber plates [ . - ]. cantilever-sensor array techniques have turned out to be a very powerful and highly sensitive tool to study physisorption and chemisorption processes, as well as to determine material-specific properties such as heat transfer during phase transitions. experiments in liquids have provided new insights into such complex biochemical reactions as the hybridization of dna or molecular recognition in antibody-antigen systems or proteomics. future developments must go towards technological applications, in particular to find new ways to characterize real-world samples such as clinical samples. the development of medical diagnosis tools requires an improvement of the sensitivity of a large number of genetic tests to be performed with small amounts of single donor-blood or body-fluid samples at low cost. from a scientific point of view, the challenge lies in optimizing cantilever sensors to improve their sensitivity to the ultimate limit: the detection of individual molecules. several fundamentally new concepts in microcantilever sensing are available in recent literature, which could help to achieve these goals: the issue of low quality factor of resonating microcantilevers in liquid has been elegantly solved by fabrication of a hollow cantilever that can be filled with biochemical liquids. confining the fluid to the inside of a hollow cantilever also allows direct integration with conventional microfluidic systems, and significantly increases sensitivity by eliminating high damping and viscous atomic force microscope the resonistor, a frequency sensitive device utilizing the mechanical resonance of a silicon substrate trimming of microstrip circuits utilizing microcantilever air gaps micromechanical membrane switches on silicon electronic nerve agent detector suga: force sensing microcantilever using sputtered zinc-oxide thin-film a nondestructive method for determining the spring constant of cantilevers for scanning force microscopy allison: thermal, ambient-induced deflections of scanning force microscope cantilevers schlittler: observation of a chemical reaction using a micromechanical sensor vapor detection using resonating microcantilevers sequential position readout from arrays of micromechanical cantilever sensors adsorbate-induced surface stress surface stress in the self-assembly of alkanethiols on gold photothermal spectroscopy with femtojoule sensitivity based on micromechanics ( ) . f.j. von preissig: applicability of the classical curvature-stress relation for thin films on plate substrates translating biomolecular recognition into nanomechanics nanomechanics of the formation of dna self-assembled monolayers, hybridization on microcantilevers scanning force microscopy with applications to electric, magnetic, and atomic forces micromechanical thermogravimetry frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope condensation of isolated metal clusters studied with a calorimeter measuring the surface stresses in an electrochemically deposited monolayer electrostatic forces and their effects on capacitive mechanical sensors obermaier: cantilever beam accelerometer based on surface micromachining technology development of a piezoelectric self-excitation, self-detection mechanism in pzt microcantilevers for dynamic scanning force microscopy in liquid heiden: force microscope with capacitive displace nanomechanical cantilever array sensors references ment detection micromachined atomic force microprobe with integrated capacitive read-out alvarado: a differential interferometer for force microscopy a differential interferometer for scanning force microscopy amer: novel optical approach to atomic force microscopy thermal analysis using a micromechanical calorimeter combination of single crystal zeolites and microfabrication: two applications towards zeolite nanodevices micromechanics: a toolbox for femtoscale science: towards a laboratory on a tip a chemical sensor based on a micromechanical cantilever array for the identification of gases and vapors a cantilever array based artificial nose delamarche: high-sensitivity miniaturized immunoassays for tumor necrosis factor α using microfluidic systems stress at the solid-liquid interface of self-assembled monolayers on gold investigated with a nanomechanical sensor multiple label-free biodetection and quantitative dna-binding assays on a nanomechanical cantilever array label-free protein assay based on a nanomechanical cantilever array inkjet deposition of alkanethiolate monolayers and dna oligonucleotides on gold: evaluation of spot uniformity by wet etching gerber: rapid functionalization of cantilever array sensors by inkjet printing baltes: complementary metal oxide semiconductor cantilever arrays on a single chip: mass-sensitive detection of volatile organic compounds manalis: microfabricated mechanical biosensor with inherently differential readout hydrogen gas sensing using a pd-coated cantilever monitoring the chemical changes in pd induced by hydrogen absorption using microcantilevers warmack: design and performance of a microcantilever-based hydrogen sensor study on pd functionalization of microcantilever for hydrogen detection promotion combination of single crystal zeolites and microfabrication: two applications towards zeolite nanodevices reed: hydration level monitoring using embedded piezoresistive microcantilever sensors al o -coated microcantilevers for detection of moisture at ppm level zeolite-modified microcantilever gas sensor for indoor air quality control a new cantilever system for gas and liquid sensing surface stress in the self-assembly of alkanethiols on gold probed by a force microscopy technique sauers: detection of -mercaptoethanol using gold-coated micromachined cantilevers low-energy ion-induced tensile stress of selfassembled alkanethiol monolayers surface stress, kinetics, and structure of alkanethiol self-assembled monolayers thundat: effect of nanometer surface morphology on surface stress and adsorption kinetics of alkanethiol self-assembled monolayers nanomechanics of a self-assembled monolayer on microcantilever sensors measured by a multiple-point deflection technique method for measuring the self-assembly of alkanethiols on gold at femtomolar concentrations hope-weeks: monitoring the formation of self-assembled monolayers of alkanedithiols using a micromechanical cantilever sensor effect of chain length on nanomechanics of alkanethiol self-assembly minne: mercury vapor detection with a self-sensing, resonating piezoelectric cantilever haynie: , -hexanedithiol monolayer as a receptor for specific recognition of alkylmercury comparison of microcantilever hg sensing behavior with thermal higher order modes for as-deposited sputtered and thermally evaporated au films a novel self-assembled monolayer (sam) coated microcantilever for low level caesium detection measuring the surface stresses in an electrochemically deposited metal monolayer bourillot: detection of gas trace of hydrofluoric acid using microcantilever thundat: detection of femtomolar concentrations of hf using an sio microcantilever changes in surface stress, morphology and chemical composition of silica and silicon nitride surfaces during the etching by gaseous hf acid delinger: a solid-state sensor platform for the detection of hydrogen cyanide gas ultrasensitive detection of cro − using a microcantilever sensor detection of hg + using microcantilever sensors thundat: investigating the mechanical effects of adsorption of ca + ions on a silicon nitride microcantilever surface thundat: detection of heavy metal ions using proteinfunctionalized microcantilever sensors detection of pb + using a hydrogel swelling microcantilever sensor sepaniak: characterization of ligand-functionalized microcantilevers for metal ion sensing selective detection of cr(vi) using a microcantilever electrode coated with a self-assembled monolayer thundat: photochemical hydrosilylation of -undecenyltriethylammonium bromide with hydrogen-terminated si surfaces for the development of robust microcantilever sensors for cr(vi) electrochemical deposition of sol-gel films for enhanced chromium(vi) determination in aqueous solutions detection of cd(ii) using antibody-modified microcantilever sensors boisen: a microcantilever-based alcohol vapor sensor-application and response model baltes: application-specific sensor systems based on cmos chemical microsensors baltes: complementary metal oxide semiconductor cantilever arrays on a single chip: mass-sensitive detection of volatile organic compounds hierlemann: magnetically actuated complementary metal oxide semiconductor resonant cantilever gas sensor systems français: chemical sensing: millimeter size resonant microcantilever performance study of microcapillary pipetteassisted method to prepare polyethylene glycolcoated microcantilever sensors analyte species and concentration identification using differentially functionalized microcantilever arrays and artificial neural networks silicon made resonant microcantilever: dependence of the chemical sensing performances on the sensitive coating thickness parylene micro membrane capacitive sensor array for chemical and biological sensing a highly sensitive self-oscillating cantilever array for the quantitative and qualitative analysis of organic vapor mixtures facile hyphenation of gas chromatography and a microcantilever array sensor for enhanced selectivity independent component analysis of nanomechanical responses of cantilever arrays development of nanomechanical biosensors for detection of the pesticide ddt thundat: sensitive detection of plastic explosives with self-assembled monolayer-coated microcantilevers adams: a microsensor for trinitrotoluene vapour adsorption of trinitrotoluene on uncoated silicon microcantilever surfaces lareau: detection of trinitrotoluene via deflagration on a microcantilever desorption characteristics, of un-coated silicon microcantilever surfaces for explosive and common nonexplosive vapors electrostatically actuated resonant microcantilever beam in cmos technology for the detection of chemical weapons thundat: moore's law in homeland defense: an integrated sensor platform based on silicon microcantilevers thundat: nerve agents detection using a cu + /l-cysteine bilayer-coated microcantilever thundat: molecular recognition of biowarfare agents using micromechanical sensors detection of ph variation using modified microcantilever sensors brown: a ph sensor based on a microcantilever coated with intelligent hydrogel mckendry: investigating the molecular mechanisms of in-plane mechanochemistry on cantilever arrays glucose biosensing using an enzyme-coated microcantilever lvov: modification of microcantilevers using layer-by-layer nanoassembly film for glucose measurement glucose biosensor based on the microcantilever glucose oxidase multilayer modified microcantilevers for glucose measurement microcantilevers modified by horseradish peroxidase intercalated nano-assembly for hydrogen peroxide detection thundat: cantilever-based optical deflection assay for discrimination of dna singlenucleotide mismatches nanomechanical forces generated by surface grafted dna adsorption kinetics and mechanical properties of thiol-modified dna-oligos on gold investigated by microcantilever sensors thundat: nanomechanical effect of enzymatic manipulation of dna on microcantilever surfaces microcantilever resonance-based dna detection with nanoparticle probes nanomechanics of the formation of dna selfassembled monolayers and hybridization on microcantilevers investigation of dna sensing using piezoresistive microcantilever probes, ieee sensor optical sequential readout of microcantilever arrays for biological detection highly sensitive polymerbased cantilever-sensors for dna detection besenbacher: nanomechanical sensing of dna sequences using piezoresistive cantilevers bernad: a highly sensitive microsystem based on nanomechanical biosensors for genomics applications nanomechanical detection of dna melting on microcantilever surfaces chemomechanics of surface stresses induced by dna hybridization label free analysis of transcription factors using microcantilever arrays using a microcantilever array for detecting phase transitions and stability of dna rapid and label-free nanomechanical detection of biomarker transcripts in human rna microcantilever-based biosensors leech: characterisation of an antibody coated microcantilever as a potential immuno-based biosensor investigation of the antigen antibody reaction between anti-bovine serum albumin (a-bsa) and bovine serum albumin (bsa) using piezoresistive microcantilever based sensors label free novel electrical detection using micromachined pzt monolithic thin film cantilever for the detection of c-reactive protein micromechanical measurement of membrane receptor binding for label-free drug discovery a label-free immunosensor array using single-chain antibody fragments novel electrical detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers besenbacher: cantilever sensor for nanomechanical detection of specific protein conformations surface stress changes induced by the conformational change of proteins development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase seshia: investigation of biotin-streptavidin binding interactions using microcantilever sensors antibody-based protein detection using piezoresistive cantilever arrays label-free protein recognition twodimensional array using nanomechanical sensors hegner: conformational change of bacteriorhodopsin quantitatively monitored by microcantilever sensors detection of antibody peptide interaction using microcantilevers as surface stress sensors sensing lipid bilayer formation and expansion with a microfabricated cantilever array mechanical detection of liposomes using piezoresistive cantilever single cell detection with micromechanical oscillators real-time measurement of the contractile forces of self-organized cardiomyocytes on hybrid biopolymer microcantilevers montelius: phospholipid vesicle adsorption measured in situ with resonating cantilevers in a liquid cell hegner: micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection bioassay of prostatespecific antigen (psa) using microcantilevers in-situ quantitative analysis of a prostate-specific antigen (psa) using a nanomechanical pzt cantilever immunoassay of prostate-specific antigen (psa) using resonant frequency shift of piezoelectric nanomechanical microcantilever detection of feline coronavirus using microcantilever sensors method for quantification of a prostate cancer biomarker in urine without sample preparation aksay: simultaneous liquid viscosity and density determination with piezoelectric unimorph cantilevers evaluation of a vibrating micromachined cantilever sensor for measuring the viscosity of complex organic liquids simultaneous determination of density and viscosity of liquids based on resonance curves of uncalibrated microcantilevers rebière: study of viscoelastic effect on the frequency shift of microcantilever chemical sensors microcantilever mechanics in flowing viscous fluids stark: cantilever micro-rheometer for the characterization of sugar solutions microstructural pressure sensor based on an enhanced resonant mode hysteresis effect simple resonating microstructures for gas pressure measurement ensell: characterization of a highly sensitive ultra-thin piezoresistive silicon cantilever probe and its application in gas flow velocity sensing bourillot: effects of temperature and pressure on microcantilever resonance response wide range pressure sensor based on a piezoelectric bimorph microcantilever tittonen: fabrication and characterization of an ultrasensitive acousto-optical cantilever fang: residual stress and thermal expansion behavior of tao x n y films by the micro-cantilever method thermal effects on coated resonant microcantilevers remote infrared radiation detection using piezoresistive microcantilevers uncooled thermal imaging using a piezoresistive microcantilever sharp: remote optical detection using microcantilevers datskou: detection of infrared photons using the electronic stress in metal-semiconductor cantilever interfaces a novel uncooled substrate-free optical-readable infrared detector: design, fabrication and performance design and fabrication of a high sensitivity focal plane array for uncooled ir imaging an optical readout method based uncooled infrared imaging system manalis: suspended microchannel resonators for biomolecular detection enantioselective sensors based on antibody-mediated nanomechanics thundat: spiral springs and microspiral springs for chemical and biological sensing fabrication and mechanical characterization of a force sensor based on an individual carbon nanotube applications of single-layered graphene sheets as mass sensors and atomistic dust detectors key: cord- -tqqt hj authors: alidjinou, enagnon kazali; sane, famara; firquet, swan; lobert, pierre-emmanuel; hober, didier title: resistance of enteric viruses on fomites date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: tqqt hj human enteric viruses are associated with several clinical features, especially gastroenteritis. large amounts of these viruses can be released in the environment and spread to people. enteric viruses are nonenveloped viruses and have displayed good survival in the environment. they can be significantly resistant in food and water but also on fomites, and this is thought to play a role in transmission, leading to sporadic cases or outbreaks. the survival of enteric viruses on fomites relies on many factors including the virus itself, fomite properties, and extrinsic environmental factors such as temperature or relative humidity. several reports in the literature have found an association with gastroenteritis cases or outbreaks and fomites naturally contaminated by enteric viruses. however, the study of virus survival following natural contamination is challenging, and most published studies are laboratory based, using experimental contamination. in addition, recent and detailed data on the resistance of each of the main enteric viruses on fomites are scarce. many approaches, both physical and chemical, can be used to inactivate enteric viruses, the efficacy of which depends on the virus and the disinfection conditions. taminated hands and surfaces [ ] . various surfaces such as door handles, clothes, telephones, toilet seats, walls, thermometers, gloves, and papers can be contaminated and serve as vehicles for the virus [ ] . most fomite contaminations occur through direct contact or deposition of aerosol particles containing the virus. aerosols can be produced for example by toilet flushing [ ] or via vomiting, coughing, sneezing, or talking [ ] . thereafter, the virus can be transferred easily to others fomites through interaction with individuals [ ] . virus transfer from hands to fomites and vice-versa is thought to play a significant role in the spreading of infection. it has been shown that hands can be easily contaminated with norovirus by transfer from fomites, and then the contaminated hands could cross-contaminate up to other surfaces without any recontamination [ ] . in hospitals and others health care centers, further dissemination has been associated with parents and staff trafficking when hygiene measures such as hand washing before and after visiting a patient are not strictly respected [ ] . the prevention of enteric virus-related nosocomial infections or outbreaks requires a good understanding of virus persistence in the environment and of the role of contaminated surfaces and objects in virus transmission. the survival of main enteric viruses on fomites and its implication for virus transmission will be analyzed, and the major disinfection procedures and their impact will be described. the resistance of viruses in the environment, which determines the risk of transmission, is multifactorial, depending not only on virus characteristics but also on fomite properties and extrinsic environmental factors (fig. ) . the virus type is determinant for survival on surfaces, as well as the initial inoculum. generally enteric viruses are resistant and have been reported to persist on fomites for several weeks to months, as compared for example to respiratory viruses which can only resist for a few hours or days [ ] . the higher the contaminating viral titer is, the longer the persistence will be [ ] . some differences between the groups of viruses involved in gastroenteritis have also been described. hav and rotavirus have been shown to be more resistant than adenovirus and enterovirus [ ] . the nature of fomites could influence the survival of viruses. surfaces are generally classified as porous (e.g., papers and clothes) or nonporous (e.g., stainless steel, plastic, and glass). available data suggest that the majority of viruses persist longer on nonporous surfaces [ ] ; however, results are sometimes conflicting, and the effect of fomite properties might also depend on the viral type. for example, rotavirus seem to survive very well on nonporous surfaces, while results are very variable for porous ones [ ] . poliovirus has been shown to be particularly susceptible to aluminum. by comparing hav and rota- virus, it has been shown that hav is more resistant on nonporous materials, while rotavirus is more stable on porous surfaces such as paper [ ] . in similar conditions, astroviruses are able to persist for up to days on paper, while they survive for a maximum of days on nonporous surfaces [ ] . the environmental factors include mainly air temperature and humidity, but the length of virus viability may also depend on the cleanliness of the surface, which can be determined for example by the presence or not of fecal material or by the number of microbes on the surface. these factors largely affect the resistance to desiccation, which appears to be an important determinant of virus survival on fomites [ ] . for most enteric viruses, low temperatures have been reported to promote a longer persistence [ ] . the effect of humidity is less consistent. it has been reported that decreases in humidity could affect the infectivity of hav, rotavirus, and adenovirus [ ] . another study found that hav was further preserved at a low humidity [ ] . conflicting results have also been described for other enteric viruses [ ] . indeed, it has been suggested that viruses have a better survival at both low and high humidity, while intermediate humidity (e.g., between and %) is deleterious [ ] . overall, the humidity level is thought to play an important role in virus persistence in the environment, and it has been suggested to explain the seasonality of rotavirus infections, for example [ ] . nevertheless, in most studies, relative humidity (temperature dependent) has been reported, while some authors have shown more recently that absolute humidity (temperature independent) rather than relative humidity could explain the seasonality of infections [ ] . absolute humidity shows a high correlation between indoor and outdoor conditions [ ] , and it could be more useful to explain infections occurring in closed environments, such as norovirus outbreaks [ ] . the contamination and the survival of enteric viruses on surfaces and objects can undoubtedly explain a significant proportion of acute viral diarrhea. direct evidence of virus transmission via fomites is difficult to obtain because others routes such as person-to-person transmission are also usually suspected. enteric viruses have been detected frequently from naturally contaminated fomites in hospitals, houses, or others community facilities. the early reported preva-lence of rotaviruses on fomites was very high, especially in pediatric settings, ranging from to % [ ] [ ] [ ] [ ] . in a more recent study, a significant rate ( %) was detected on environmental surfaces in a hospital intensive care unit [ ] . toys were associated with a rotavirus outbreak in a pediatric oncology unit [ ] . beyond detection of viral rna by rt-pcr, infectious particles have been reported in some cases [ ] . noroviruses have been found on surfaces and objects around patients during outbreaks in hospitals, hotels, or cruise ships [ , , ] . the role of contamination of environmental surfaces such as dining room tables or elevator buttons used by staff has been reported in a norovirus outbreak in a long-term care center [ ] . touching a reusable grocery bag was linked to cases in a norovirus outbreak in a soccer team [ ] . a high rate of norovirus contamination ( %) in environmental swabs has been observed during outbreaks [ ] . contamination of fomites by adenoviruses has been described [ , ] , including the presence of infectious particles [ ] . detection of astroviruses on environment surfaces was also reported during a hospital outbreak [ , ] . contamination of drinking glasses with hav by a barman during incubation of the infection resulted in an outbreak of hepatitis a in a public house [ ] . the abundant data available in the literature on virus survival come mainly from laboratory investigations and disinfection intervention studies. all of these studies are generally conducted following similar principles. briefly, a sample of a specific virus suspension with a known infectious titer is applied on the surface under selected conditions and exposition times that are close to the studied natural contamination. then, after recovery, the viral titer is determined and compared to the former one, and statistical methods are used to calculate the decline in infectious particles [ ] . the recovery method, especially the swab material used (cotton, polyester, rayon, macrofoam, or an antistatic wipe), can be critical for the results [ , ] . cell culture methods (plaque assay, % tissue culture infective dose) are required for assessment of the viral titer. indeed, viral nucleic acids detected by molecular methods cannot differentiate between infective and noninfective viruses. this could constitute a limit to virus survival investigation because only viruses able to propagate in cell lines can be studied. recently, some authors sug-gested new pcr-based approaches that could correlate to viral infectivity either by assessing the presence of an intact genome or an amplifiable undamaged genome using direct amplification or by coupling a pre-pcr sample enzymatic treatment in order to assess viral capsid integrity prior to nucleic acid extraction and amplification [ ] . results are usually expressed as inactivation coefficients defined as the ratio between the decline of the viral titer and time. alternative ways of expressing viral resistance include t and t values, which represent the time needed for the initial viral titer to decrease by and %, respectively, and they are determined using a survival curve [ ] . in the following section, the experimental data accumulated in the literature about the survival of the main enteric viruses on fomites are presented. norovirus is a leading cause of sporadic and epidemic cases of acute gastroenteritis across all age groups worldwide [ ] . the virus is highly infectious and - virions are enough to cause gastroenteritis. to date, human norovirus cannot be propagated in mammalian cell lines. therefore, survival and inactivation studies are commonly conducted using cultivable surrogates such as feline calicivirus (fcv) or murine norovirus (mnv) [ ] . doultree et al. [ ] assessed fcv survival on glass and glass coverslips. the virus remained infectious for up to days at ° c and the resistance decreased significantly with higher temperatures. d'souza et al. [ ] also found that fcv survived for more than days at room temperature on stainless steel, ceramic, and formica surfaces. a shorter survival ranging from - h (keyboard keys and brass) to - days (telephone buttons and telephone receivers) was reported on uncommon fomites. unlike norovirus, which is an enteric pathogen, fcv causes respiratory diseases in cats and is not an ideal surrogate. mnv has been reported to be more applicable as a norovirus surrogate because it shows environmental stability and is genetically close to human norovirus [ ] . kim et al. [ ] investigated mnv survival on steel and wood, and a better resistance was shown on wood at lower temperature and higher relative humidity values. in another study conducted at room temperature, the authors demonstrated that mnv could survive for up to days on different surfaces and the rank order of infectivity reduction from highest to lowest was stainless steel, plastic, rubber, glass, ceramic, and wood [ ] . human norovirus, mnv, and fcv were found to remain infective beyond days on stainless steel and plastic both at ° c and at room temperature [ ] . rotavirus is the leading cause of diarrhea-related illness worldwide among children aged < years, accounting for the majority of diarrhea-related deaths, especially in low-income countries [ ] . rotavirus has displayed a good resistance in the environment. when rotavirus suspension in culture medium is applied on aluminum and paper, infectious particles can be detected for more than months between and ° c at a high relative humidity. however, in a % fecal suspension, survival was better at ° c [ ] . the impact of relative humidity is not consistent throughout studies [ , ] . human adenoviruses type / , known as enteric adenoviruses, have been associated with sporadic acute diarrhea as well as outbreaks [ , ] . adenovirus has been shown to resist for more than month between and ° c. survival is longer on paper than on aluminum [ ] . human astroviruses are considered gastrointestinal pathogens that affect mainly children worldwide [ ] . the experimental survival of astroviruses has been poorly investigated. it has been shown that the virus can survive for a long time on both porous and nonporous surfaces. resistance is better at low temperatures and on porous surfaces (up to days at ° c on paper) [ ] . hepatitis a viral hepatitis a is a global health problem that affects hundreds of millions of children and adults, especially in low-income countries where food and water hygiene may be of a low standard [ ] . the virus is primarily a human pathogen transmitted by person-to-person contact or ingestion of contaminated food or water, but contaminated fomites can play a role in transmission. hav can survive for more than months on fomites, and a better resistance has been observed on nonporous surfaces. fecal suspension and a low temperature have been shown to have a positive effect on persistence [ ] , but the impact of the relative humidity level has not been confirmed [ ] . a recent study found in similar conditions a better survival on wood [ ] . the genus enterovirus includes a great variety of human pathogens such as polioviruses, coxsackieviruses a and b, echovirus, and ev . enteroviruses can cause gas- intervirology ; : - doi: . / trointestinal symptoms; however, studies investigating the role of nonpolio enteroviruses in acute diarrhea are rare compared to those on major agents such as rotavirus [ ] . enteroviruses are involved in various and numerous acute diseases, and their role in chronic cardiac diseases and type diabetes is highly suspected [ ] . data on the survival of enteroviruses are scarce, with only few old data focusing on the poliovirus prototype. poliovirus can survive for up to month at ° c. feces increase survival, while a rapid decrease has been observed on aluminum surfaces [ ] . recently our team investigated the survival of coxsackievirus b on an inanimate surface. when the virus was dried on a petri dish lid at ° c, infectious particles could be detected for up to weeks. cvb was especially susceptible to repetitive cycles of drying and resuspension, with a significant decrease in the viral titer at each cycle. the viability of cvb was also highly reduced when the virus was dried in a protein-rich medium [ ] . selected data on the survival of enteric viruses are shown in table [ , , , , ] . several physical inactivation methods have been described for enteric viruses, including heating, high-pressure processing, dehydration, freezing, ultraviolet (uv) inactivation, and heavy metal use. however, some of these approaches cannot be easily applied routinely to surfaces and are more appropriate for water or food. this section only focuses on the methods that can be applied on fomites to reduce virus survival. temperature is one of the major factors determining virus survival outside a cell. its effect on virus inactivation has been evaluated under different conditions, including wet and dry. the common mechanism reported for picornaviruses and caliciviruses is destabilization and disruption of the viral capsid leading to its breakdown and the release of viral rna [ ] , while for rotaviruses heat treatment has been shown to primarily target viral transcription functions [ ] . experiments for evaluation of the long-term persistence of enteric viruses outside a cell have shown that, overall, enteric viruses have a long-term survival in suspension at environmental temperatures (e.g., up to ° c) [ ] [ ] [ ] . thermal inactivation is principally used for enteric viruses in suspension and in food samples and less often for fomites. two temperatures have been commonly applied: ° c, recommended for pasteurization and, ° c, recommended for sterilization, with an exposure time of and min, respectively. under experimental conditions these two temperatures would most likely result in efficient virus inactivation. resistance on warmed surfaces of mnv and cvb contained in droplets has been studied [ ] . cvb was inactivated in s at ° c, while min were necessary to inactivate mnv at ° c. uv irradiations are commonly used for viral inactivation in food, in water, on food preparation surfaces, and in microbiology laboratories [ , ] . uv-c radiation at wavelengths of - nm has been approved for disinfection purposes by the us food and drug administra- tion (fda). its antiviral activity is well known and can be applied to food, water, and surfaces [ , ] . susceptibility may vary between viruses. enteroviruses have been inactivated with commonly recommended uv-c doses, while adenoviruses have been reported to be more resistant [ , ] . park and al. [ ] evaluated the effect of continuous uv-c treatment on stainless steel experimentally contaminated with mnv and hav. their data showed that hav was more resistant to uv-c radiation than to mnv. using various uv-c dosages, they concluded that only mw/cm was needed to reduce mnv infectivity by logs, while this result was achieved beyond mw/cm for hav [ ] . pulsed uv light has been reported to be more effective than conventional (continuous) uv light, inducing a more rapid inactivation of infectious agents. one -s treatment ( pulses) can result in a -log decrease in the viral load for both mnv and hav. effectiveness is reduced in the presence of organic matter (fetal bovine serum) [ ] . the effects of uv inactivation have been observed at the protein and rna levels. it has been shown that uv disrupts the mnv- structure and degrades both viral protein and rna [ ] . heavy metal-impregnated surfaces certain heavy metals have been shown to have intrinsic antimicrobial effects. the most known include copper and gold. the effect of copper and copper alloy surfaces has been extensively investigated [ , ] . studies have shown a rapid inactivation of bacterial, fungal, and viral pathogens on copper and copper alloy surfaces, and these have led to clinical trials and real strategies for reduction of the microbial burden in some health settings [ , ] . reports on enteric viruses have focused on noroviruses. mnv has been rapidly inactivated on copper and copper alloys containing over % of copper in both simulated wet fomite and dry touch contamination, while no reduction of infectivity has been observed on stainless steel. mnv is completely inactivated within min on copper. the highest rate of inactivation occurs upon immediate contact and is proportional to the copper content of alloys. it has also been shown that cu + and cu + ions but not superoxide and hydroxyl radicals are the primary effectors of toxicity, and the targets are both viral capsid and rna [ , ] . similar findings have also been reported for human norovirus [ ] . disinfectants are commonly used for virus inactivation, especially in the health care settings and the food industry, to prevent outbreaks due to enteric viruses. the most popular disinfectants are ethanol, peroxyacids, chlorine, and quaternary ammonium. laboratory or field investigations have evaluated the efficacy of these disinfectants for the inactivation of enteric viruses [ , ] . however, most of these studies have focused on human norovirus and cultivable surrogates as enteric virus models because human norovirus combines many of the epidemiological characteristics of enteric viruses [ ] . mnv and fcv are to date the most common surrogates and the latter is still considered the gold standard by the us environmental protection agency for evaluating the antinoroviral activity of new products. however, the use of multiple surrogate viruses rather than one is more judicious and recommended to obtain more reliable information on the effectiveness of disinfectants and this has been well documented in some studies [ , ] . indeed, differences in the sensitivity of the surrogates to experimental conditions and inactivation treatments have been reported. some studies have shown that mnv is more susceptible to alcohols than fcv, whereas fcv is more susceptible to chlorine [ , ] . in a systematic meta-analysis, hoelzer et al. [ ] showed that hav and mnv were significantly more resistant to disinfection than fcv, even if the differences in viral titer reduction appeared to be modest (i.e., . log pfu) [ ] . other studies have compared the efficacy of commonly used active agents (ethanol, chlorine, and quaternary ammonium) at different concentrations against human norovirus strains and surrogates (mnv and fcv). data from these studies revealed a complete or relative lack of efficacy of quaternary ammonium against all of the viruses tested [ , , ] . in another study fcv applied on a surface, but not in suspension, was effectively inactivated by a quaternary ammonium-based disinfectant, while cvb , an enterovirus, was resistant [ ] . ethanol has shown minimal efficacy against human norovirus and fcv, but it is active against mnv, with a clear dose-dependent impact [ , [ ] [ ] [ ] . very high concentrations of hypochlorite (≥ ppm) are necessary to inactivate human norovirus and mnv; fcv has been shown to be more susceptible to hypochlorite than human noroviruses [ , , ] . chlorine dioxide (clo ) represents a strong alternative to chlorine, with an increased ability to reduce or inactivate enteric viruses in the environment. clo is very attractive compared to chlorine because it has been shown to be less affected by ph conditions than chlorine and very effective even at low concentrations [ , ] . indeed, several studies have shown that the disinfection efficacy of clo is higher than that of chlorine when tested on enteric viruses, including human rotavirus, enteric adenoviruses, caliciviruses, and enteroviruses [ ] [ ] [ ] . however, commonly active doses of chlorine dioxide against these viruses have been shown to have some, albeit limited, activity against human norovirus [ , ] . taken together, all of these studies have highlighted the fact that human noroviruses are much less sensitive to commonly used disinfectants than surrogate viruses, and one might pay attention to and integrate several parameters when extrapolating data between surrogate viruses and human noroviruses. different peroxyacids, including peracetic, perpropionic, perlactic, and percitric, have been assessed for norovirus inactivation on stainless steel and polyvinyl chloride surfaces. peracetic acid and perpropionic acid have been found to be the most effective. exposure to a solution at mg/ml for min resulted in a -log reduction of the viral titer in all of the conditions tested [ ] . interestingly, peracetic acid airborne disinfectant was reported by our team to be very efficient in inactivating poliovirus applied on stainless steel carriers [ ] . enteric viruses represent an important public health issue both in developed and in developing countries. these nonenveloped viruses have shown a high level of resistance in the environment. they can survive for a long time on animate and inanimate surfaces and be easily transferred to hands when hygiene measures are lacking. the role of fomites has been shown in the transmission of these viruses and the occurrence of outbreaks, especially in closed environments. several factors impact the survival of viruses on fomites, and detailed experimental studies comparing all of the main enteric viruses are scarce. the implication of this strong resistance is the implementation of adequate disinfection strategies in health care centers and the food industry. chemical disinfection is currently the most used in these settings, and great differences have been shown in the effects of various compounds on enteric viruses. nearly constant shedding of diverse enteric viruses by two healthy infants viruses causing gastroenteritis a review of viral gastroenteritis viral gastroenteritis outbreaks in europe survival of human enteric viruses in the environment and food two epidemiologic patterns of norovirus outbreaks: surveillance in england and wales significance of fomites in the spread of respiratory and enteric viral disease the potential spread of infection caused by aerosol contamination of surfaces after flushing a domestic toilet effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season how long do nosocomial pathogens persist on inanimate surfaces? a systematic review survival of enteric viruses on environmental fomites institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission potential role of fomites in the vehicular transmission of human astroviruses effect of relative humidity and air temperature on survival of hepatitis a virus on environmental surfaces the effect of environmental parameters on the survival of airborne infectious agents survival and vehicular spread of human rotaviruses: possible relation to seasonality of outbreaks absolute humidity influences the seasonal persistence and infectivity of human norovirus the relationship between indoor and outdoor temperature, apparent temperature, relative humidity, and absolute humidity detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction prevalence of rotavirus on highrisk fomites in day-care facilities monitoring rotavirus environmental contamination in a pediatric unit using polymerase chain reaction environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit group a rotavirus detection on environmental surfaces in a hospital intensive care unit rotavirus outbreak on a pediatric oncology floor: possible association with toys the role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay spread and prevention of some common viral infections in community facilities and domestic homes a norovirus outbreak at a long-term-care facility: the role of environmental surface contamination a point-source norovirus outbreak caused by exposure to fomites norovirus gii. detection in environmental samples from patient rooms during nosocomial outbreaks environmental viral contamination in a pediatric hospital outpatient waiting area: implications for infection control routine monitoring of adenovirus and norovirus within the health care environment viability of human adenovirus from hospital fomites use of a heminested reverse transcriptase pcr assay for detection of astrovirus in environmental swabs from an outbreak of gastroenteritis in a pediatric primary immunodeficiency unit outbreak of hepatitis a spread by contaminated drinking glasses in a public house comparison of surface sampling methods for virus recovery from fomites evaluation of a new environmental sampling protocol for detection of human norovirus on inanimate surfaces application of pcr-based methods to assess the infectivity of enteric viruses in environmental samples global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis temperature and humidity influences on inactivation kinetics of enteric viruses on surfaces inactivation of feline calicivirus, a norwalk virus surrogate persistence of caliciviruses on environmental surfaces and their transfer to food evaluation of murine norovirus, feline calicivirus, poliovirus, and ms as surrogates for human norovirus in a model of viral persistence in surface water and groundwater survival of norovirus surrogate on various food-contact surfaces tenacity of human norovirus and the surrogates feline calicivirus and murine norovirus during long-term storage on common nonporous food contact surfaces global illness and deaths caused by rotavirus disease in children importance of enteric adenoviruses and in acute gastroenteritis in infants and young children identification and characterization of enteric adenoviruses in infants and children hospitalized for acute gastroenteritis human astroviruses hepatitis a: epidemiology in resource-poor countries non-polio enteroviruses and their association with acute diarrhea in children in india enteroviral pathogenesis of type diabetes: queries and answers survival of enveloped and non-enveloped viruses on inanimate surfaces inactivation of picornaviruses and caliciviruses. . inactivation solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral rna stability of feline caliciviruses in marine water maintained at different temperatures inactivation of caliciviruses surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus viruses contained in droplets applied on warmed surface are rapidly inactivated uv light inactivation of hepatitis a virus, aichi virus, and feline calicivirus on strawberries, green onions, and lettuce inactivation of hepatitis a virus and norovirus surrogate in suspension and on food-contact surfaces using pulsed uv light (pulsed light inactivation of food-borne viruses) ultraviolet-c efficacy against a norovirus surrogate and hepatitis a virus on a stainless steel surface comparative inactivation of enteroviruses and adenovirus by uv light pulsed uv-light inactivation of poliovirus and adenovirus efficacy and mechanisms of murine norovirus inhibition by pulsed-light technology the survival of escherichia coli o on a range of metal surfaces mechanism of copper surface toxicity in escherichia coli o :h and salmonella involves immediate membrane depolarization followed by slower rate of dna destruction which differs from that observed for gram-positive bacteria sustained reduction of microbial burden on common hospital surfaces through introduction of copper copper surfaces reduce the rate of healthcare-acquired infections in the intensive care unit inactivation of norovirus on dry copper alloy surfaces inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity destruction of the capsid and genome of gii. human norovirus occurs during exposure to metal alloys containing copper virus inactivation on hard surfaces or in suspension by chemical disinfectants: systematic review and metaanalysis of norovirus surrogates human norovirus as a foodborne pathogen: challenges and developments comparing human norovirus surrogates: murine norovirus and tulane virus efficacy of commonly used disinfectants for inactivation of human noroviruses and their surrogates comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and gii. norovirus disinfection of human enteric viruses on fomites attachment of noroviruses to stainless steel and their inactivation, using household disinfectants inactivation of coxsackievirus b , feline calicivirus and herpes simplex virus type : unexpected virucidal effect of a disinfectant on a non-enveloped virus applied onto a surface use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer inactivation of murine norovirus by chemical biocides on stainless steel inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide effects of chlorine and chlorine dioxide on human rotavirus infectivity and genome stability investigation on virucidal activity of chlorine dioxide: experimental data on feline calicivirus, hav and coxsackie b measurement of the virolysis of human gii. norovirus in response to disinfectants and sanitisers inactivation of human norovirus using chemical sanitizers study of the virucidal potential of organic peroxyacids against norovirus on food-contact surfaces inactivation of an enterovirus by airborne disinfectants key: cord- - ik jxjy authors: alvarez, mar; zinoviev, kirill; moreno, miguel; lechuga, laura m. title: cantilever biosensors date: - - journal: optical biosensors doi: . /b - - . - sha: doc_id: cord_uid: ik jxjy this chapter describes the application of nano- and micro-electromechanical systems (nems and mems), and more specifically microcantilever structures, as transducers for highly sensitive biosensors. in these devices, named as ‘nanomechanical biosensors,’ a biomolecular interaction produces a change in the mechanical behavior of the transducer (a movement at nanometer scale), which can be measured and analyzed in real time. microcantilevers translate the molecular recognition of biomolecules into a nanomechanical motion that is commonly coupled to an optical read-out system. this chapter discusses the main aspects regarding the physics of microcantilever as well the optical read-out techniques. it reviews the state-of-the-art, and discusses the prospective future directions of this new family of biosensors. nanomechanical sensors are derived from the microfabricated cantilevers used in atomic force microscopy (afm) and are based on the bending or resonance change induced in the cantilever when a biomolecular interaction takes place on one of its surfaces. the cantilever response depends on its mechanical properties, which are determined mainly by their spring constant and resonance frequency. both parameters depend on the cantilever material and its geometry. the increasing number of applications of microcantilevers as biosensors has established these systems as a versatile platform for real-time and in situmeasurements of physical, chemical, and biochemical interactions. further research is banked upon to provide information for increasing the biosensor sensitivity. this chapter describes the application of nano-and micro-electromechanical systems (nems and mems), and more specifically microcantilever structures, as transducers for highly sensitive biosensors. in these devices, named as "nanomechanical biosensors," a biomolecular interaction produces a change in the mechanical behavior of the transducer (a movement at nanometer scale), which can be measured and analyzed in real time. microcantilevers translate the molecular recognition of biomolecules into a nanomechanical motion that is commonly coupled to an optical read-out system. a rectangular beam, clamped at one end, is the most used transducer as it is the simplest mechanical structure that can be easily batch-fabricated at micrometer scale . microcantilevers can be fabricated in arrays of ten to thousands, and for that reason, they are a promising alternative to current biochips as they could permit the parallel, fast, and real-time monitoring of thousands of analytes (proteins, pathogens, dna strands, etc.) without the need for labeling (baller and fritz, ) . nanomechanical sensors are able to detect analytes with picomolar sensitivity and they have the ability for discerning single-base variations in dna strands. recently, microcantilever-based biosensors have arisen as a competitive biosensor alternative for measuring, in a direct way, extremely low forces and masses. when fabricated at the nanoscale (nanocantilevers), the sensitivity goes up and expected limits of detection are in the femtomole-to-attomole range with the possibility for detection at the single-molecule level in real time. in this chapter, the main aspects regarding the physics of microcantilever sensors will be described as well the optical read-out techniques. we will review the state-of-the-art, and we will discuss the prospective future directions of this new family of biosensors. nanomechanical sensors are derived from the microfabricated cantilevers used in atomic force microscopy (afm) and are based on the bending or resonance change induced in the cantilever when, for example, a biomolecular interaction takes place on one of its surfaces. the cantilever response will depend on its mechanical properties, which are determined mainly by their spring constant and resonance frequency. both parameters depend on the cantilever material and its geometry. shows a diagram with both transducer methods and its relation with the properties that could be measured. in the sensors working in the static mode, the bending arises as a consequence of a surface stress change induced by any molecular reaction, which takes places on only one of the cantilever surfaces. the induced surface stress change could be positive or negative, depending on the surface deformation generated (shuttleworth, ) . the cantilever deformation depends mainly on the forces involved in the bioreaction process and is not directly related to the receptor-ligand binding energy. these forces could arise from the bond strength between the receptor and the surface (lineal response) and from the intermolecular interactions between neighboring molecules (non-lineal response). although the factors and phenomena responsible for this change are still not fully understood, forces coming from electrostatic, steric, and van der waals interactions, changes in the surface hydrophobicity or conformational changes of the adsorbed molecules could play an important role (hagan et al., ) . the easiest and most extended model to study the surface stress produced on cantilevers is based on the work by stoney ( ) . this model relates the total surface stress change between the top and the bottom sides (ao- -ao- ) with the cantilever free end displacement, az, the young's modulus, e, the poisson coefficient, v, and the cantilever length, l, and thickness, h, represented by for sensing biomolecular interactions in the static mode, only one surface of the microcantilever must be previously biofunctionalized. this can be a complex task when working with cantilever arrays. in contrast to the static case, the dynamic mode does not require the functionalization of only one cantilever surface, as the resonance frequency depends on the total mass adsorbed on both sides. this mass could produce, at the same time, a change in the cantilever spring constant, affecting the final resonance frequency shift (lu et al., ; gupta et al., ) . in this mode, very high sensitivities can be obtained (in the attogram range), superior to those of other similar mass detectors, such as the quartz crystal microbalance (qcm) (ilic et al., ) . the microcantilever resonator is also characterized by the quality factor (q), which quantifies the energy dissipation and is defined as the ratio between the mechanical energy accumulated and dissipated per vibration cycle. the dissipative mechanisms could be both internal and external; the external damping is the dominant factor when working in air or liquids. the quality factor determines the frequency resolution of the system (lavrik et al., ): so. o operating in liquids, the resonance frequency and the quality factor shift toward much lower values than in air, due to the damping effect of the viscous surround. the quality factor in liquids could be up to -fold lower than in air, reducing the frequency resolution. there are different ways to overcome this limitation: by fabricating cantilevers with higher resonance frequency and quality factors, by measuring the resonance frequency in air under controlled humidity before and after the biochemical recognition, by working with higher order vibration modes, or by using external excitations, among others. for the above reasons, this way of operation is more difficult to implement and most of the cantilever biosensors are based on the static mode. typically, the cantilever dimensions range from tens to hundred of micrometers in length, some tens of micrometers in width, and hundreds of nanometer in thickness. in general, they are made of silicon, silicon nitride, polymers, or piezoelectric materials. the microcantilever properties and sensitivity are determined by the cantilever dimensions and material (young's modulus and poisson coefficient). the selection of the appropriate characteristics depends on the working detection method (static or dynamic), the final application, and the available fabrication technology. in general, smaller spring constants give softer cantilevers, which are more sensitive to bending. making thinner and longer cantilevers could improve the sensitivity, but they are also more fragile and therefore more difficult to handle. in addition, the thermomechanical noise increases. as an example, the standard silicon microfabrication technology allows fabricating micrometer-sized cantilevers with a high length:thickness ratio in a reproducible and inexpensive way. however, the noise arising from the cantilever thermal motion could limit this ratio value . on the other hand, reducing the cantilever length and thickness results in a sensitivity increase for the dynamic cantilever sensors, reducing the sensitivity for the surface stress sensors. polymers, such as su- , have a lower young's modulus than silicon and could be more sensitive for static deflection measurements (even if they do not have a high length:thickness ratio); their fabrication process is relatively inexpensive, fast, and reliable. the final sensitivity could be determined by previously modeling the system to determine the range of dimensions needed for a specific material and working method. modification of the microcantilevers' shape could also improve the sensor sensitivity, but the final sensitivity will also depend on the mechanical variabilities between microcantilevers in one array, the resolution of the detection set-up, and the total capacity for integration. a read-out system capable of monitoring changes with subnanometer resolution is crucial for detecting the nanomechanical motion induced by the biochemical recognition process. in addition to supplying high sensitivity and accuracy, the signal read-out is critical in the realtime measurements acquisition and, in the final biosensor, integration of microsystems. to avoid the influence of external factors, such as non-specific binding or temperature changes, a reference cantilever is typically used. for that reason, and for detecting several analytes at the same time, the read-out schemes have to enable the use of arrays of microcantilevers. there are several techniques suitable for the cantilever response read-out: optical beam deflection, piezoresistivity, piezoelectricity, interferometry, and capacitance are among the most important (lavrik et al., ; ziegler, ) . however, the majority of the biochemical applications carried out with cantilever-based sensors are based on the optical beam deflection method, due to its high sensitivity. a variation of this optical method has been recently developed using an array of a new type of optical waveguide microcantilevers (zinoviev et al., ) . these cantilevers act as a waveguide for conducting light, and it is possible to collect the exit light with a second waveguide or with a photodetector. in this case, the cantilever bending is related to the reduction in intensity of the collected light with respect to the input light. the optical beam deflection method is simple to implement and shows a linear response with sub-angstrom resolution. movement of the cantilever's free end is detected by measuring the reflected laser beam displacement into a position-sensitive photodetector (psd, figure . ). the laser beam displacement over the photodetector, x, is related with the cantilever free end deflection, az, by a simple algebraic equation where l is the cantilever length and d is the photodetectormicrocantilever distance. the read-out implementation of array platforms is technologically challenging, as it requires an array of laser sources with the same number of elements as the cantilever array. the laser's displacement could be measured using an array of photodetectors, adding alignment complications, or using just one photodetector and sequentially switching on and off each laser source to avoid the overlapping of the reflected beams . using a ccd camera and an array of microcantilevers with paddles at its end (yue et al., ) or a scanning laser source to sequentially illuminate each microcantilever are new ways to overcome the problem of overlap. this technique, based on butt-coupled optical waveguides, was proposed as a new alternative to solve some of the limitations of optical beam detection, the most serious of which is integration in array platforms. the use of a cantilever as a waveguide has been demonstrated by different groups (wu and frankena, ; ollier et al., ; budakian and putterman, ; wang et al., ; xu et al., ; zinoviev et al., b; nordin et al., ) . the sub-micron thickness of the cantilevers provides a high sensitivity, while the overall dimensions help to miniaturize the device and make it suitable for further integration in lab-on-chip applications. a schematic view of the optical sensor is shown in figure . . the device can be operated in both the visible and the infrared ranges. the "heart" of the sensor is an optically transparent cantilever of submicron thickness. light from the cantilever is injected into the output waveguide, called the "receptor," separated from the free end of the cantilever by a short gap (see figure . ). both the cantilever and the receptor are total internal reflection waveguides. if the gap is in the order of several microns, the energy transmitted into the receptor changes dramatically with the displacement of the cantilever's free end. the idea is to monitor the changes in the power transmitted into the output waveguide in order to register the deflection of the cantilever caused by any biomolecular interaction occurring on its surface. the device presented by zinoviev et al. ( a) was fabricated using standard silicon technologies. to obtain a straight cantilever free end aligned with the output waveguide, with precision better than ~m, a thermally grown silicon oxide film was used. the input waveguide used for delivering the light into the cantilever should be made out of high temperature low-pressure chemical vapor deposition (lpcvd) silicon nitride because the silica layer cannot work as an optical waveguide on the silicon substrate, due to its lower refractive index. if the input waveguide is fabricated thin enough and utilizes a taper, all the energy can be transmitted into the cantilever, due to the large evanescent field tail of the waveguide mode propagating along the silicon nitride layer. immobilization of bioreceptors on the cantilever surface strongly influences the quality of the bioanalysis to be performed. the efficiency of biomolecule attachment, the accessibility to its targets, and the degree of non-specific binding have to be taken into account. the immobilization process should avoid any change in the biological properties of the receptors, but, at the same time, must keep the chemical and physical properties of the cantilever as uniform as possible in order to generate a large surface stress. the current interest in nanometer-sized cantilever is demanding new immobilization techniques for the manipulation at nanometer scales; newer strategies from synthetic chemistry will become essential for the manipulation of biosensor surfaces and subsequent assembly of receptors. controlled chemical functionality can be accomplished using self-assembled monolayers (sams). the strong affinity of sulphur compounds (thiols, thioethers, and disulfides) for gold and other noble metals make them excellent candidates for nanomechanical biosensing (nuzzo and allara, ; ferretti et al., ) , since the cantilever surface usually has one surface coated with a thin layer of gold ( - nm) to increase reflectance of the laser beam. the immobilization of both dna and proteins on gold surfaces using thiol-sams has become the most widely employed method. the covalent immobilization of proteins on the gold surfaces of the cantilevers can be achieved by a wide variety of chemical procedures, ensuring reproducibility and stability of the protein coating (see figure . ). the main drawback is that some of the functional groups could randomize the orientation of the active sites of the protein. thus, the appropriate immobilization procedure must be chosen in order to avoid inactivation or a decrease in the activity of the protein receptor. several strategies could be employed which have already been useful in many other analytical systems, such as surface plasmon resonance (boozer et al., ) , qcm (mannelli et al., ) , or electrochemical measurements (hianik et al., ) . a very interesting one is to covalently immobilize carboxylate-terminated alkanethiols onto the gold surface, followed by the esterification of the carboxylic groups with -ethyl- -( dimethylaminopropyl)-carbodiimide (edc) and n-hydroxysuccinimide (nhs) (staros et al., ; grabarek and gergely, ; patel et al., ) . another alternative is using a cystamine modified with glutaraldehyde and the subsequent attachment of the protein through an amine group pavlov et al., ) . in addition, we can immobilize biotinylated proteins on avidin monolayers previously attached to the surface using edc or cystamine chemistry. the immobilization procedure developed by park and kim employs the reaction of sulfosuccinimidyl -[ -( -pyridyldithio)pro-pionamido] hexanoate (sulfo-lc-spdp) with the protein nhz-groups to give amide linkages. the subsequent addition of dithiothreitol (dtt) reduces the disulfide to give a thiol, which finally self assembles onto the gold surface (park et al., ) . alternatively, a protein, such as antibody, protein a, or avidin, can be thiolated, with the thiol group subsequently used for immobilization on the metal surface; however, there is a high risk of denaturing the protein and therefore losing its biological function (pyun et al., ) . nucleic acid immobilization is much easier, since chemical modifications can be included in their in vitro synthesis, avoiding the risk of losing its functionality. the direct coupling of dna probes by self assembly of thiol-labeled oligonucleotides is widely used on microcantilever gold surfaces and most of the dna biosensing applications performed with microcantilever technology are based on this strategy (biswal et al., ; lechuga et al., ) . herne and coworkers demonstrated that a mixed-layer with mercaptohexanol enhanced the hybridization rate and minimized the physical adsorption of dna probes on gold surfaces by eliminating the weaker adsorptive contacts between the nucleotide chain and gold. in this way, the majority of the immobilized dna probes are accessible for hybridization with the cantilever biosensors complementary strand. to prevent non-specific binding to the silicon side of the cantilever, a peg-silane coating is used (biswal et al., ) . for silicon optical waveguide microcantilevers, immobilization chemistry offers a multitude of well-established and relatively simple attachment strategies. frequently used silicon modifications, such as aldehyde activation or coating with poly-lysine or nitrocellulose, are typical for the biomolecule attachment (macbeath and schreiber, ) . the highly reactive epoxysilane surface, for example, reacts not only with amino groups, but also with other nucleophilic moieties on protein surfaces like alcohol, thiol, and acid groups, exhibiting a high binding capacity (zhu et al., ) . other methods employ amine-terminal silanes such as aminopropyl-triethoxysilane to provide a reactive amine group at the nitride surface. thus, alkylamine coupling using glutaraldehyde is simple and fast, giving an aldehyde derivative that can form an imine linkage with primary amines on the protein (williams and blanch, ). the direct immobilization via thiol-terminal silanes is also a well-known methodology. using crosslinkers such as edc and nhs, it is possible to control the directional immobilization with more than % of specific binding to the surface (shriver-lake et al., ). the immobilization of different bioreceptors on each cantilever of an array is a complicated task. there are several commercial platforms devoted to the specific functionalization of individual cantilevers. several commercial platforms have been reported using different strategies. for example, the autodrop platform (microdrop) (bietsch et al., ; www.microdrop.com) uses the principle of the ink jet printing technology and employs up to eight dispensers in an area of x x mm. the core of the dispensing head consists of a glass capillary which is surrounded by a tubular piezo actuator. at one end, the capillary forms a nozzle. by applying a voltage pulse, the piezo actuator creates a pressure wave which propagates through the glass into the liquid. this platform has been used for uniform deposition of thiolated dna and others alkenothiols (bietsch et al., ) . the nanojet technology, also from microdrop (www.microdrop.com), forms a liquid jet by means of a dispenser system controlled by time and pressure. provided that the micro valve switches at a very fast rate, volumes down to nl can be dispensed. moreover, a combination of the autodrop and nanojet systems, called "dropjet," is also available, allowing the generation of a large number of drops with well-defined size (www.microdrop.com). cantion has developed the cant{~spot platform, which can be used for delivering reagents onto individual cantilevers as well as onto controlled small areas. its spotter, capable of delivering pl droplets, is connected to a computer-controlled syringe pump for adjusted aspiration and dispensing of reagents (www.cantion.com). although the cantisens | fu- platform from concentris is currently used only for cantilever functionalization, it offers a novel way to immobilize biomolecules on cantilever arrays using capillary techniques; up to four cantilevers can be functionalized simultaneously (www.concentris.ch). finally, a completely different approach under development in several laboratories uses a microfluidics system with an independent flow path for each cantilever in an array (figure . ). with this type of system, immobilization can be performed on each cantilever independently by flowing the appropriate reagents through the individual flow cells. the first chemical sensor based on a macroscopic bimetallic plate was proposed by norton ( ) for hydrogen detection. in the following years, the concept of a device able to provide a direct conversion between the chemical stimulus and the mechanical energy was developed, focusing attention on miniaturized devices (kuhn et al., ; steinberg et al., ) . at the end of s, some integrated miniaturized devices were developed (newell, ; newell et al., ) . however, the idea of fabricating floating structures was given up due to the microelectronics fabrication limitations at that time. as early as , with the appearance of afm (binnig et al., ) and improvements in microelectronic technology, micro-mechanical transducers started to be used routinely and mass production at low cost was possible. atomic force microscopy arose as a surface characterization technique, in air and subsequently in liquid, measuring the cantilever deflection induced by the interaction between a tip placed at its free end and the surface of the sample. expansion of afm techniques showed the microcantilever sensitivity for measuring intermolecular forces between two complementary biological molecules (force spectroscopy), such as the interaction between proteins (avidin~iotin) (lee et al., ) , hybridization between complementary single-stranded dnas (lee et al., ) , or antigen-antibody interactions (dammer et al., ) . the sensitivity of microcantilevers for measuring intermolecular forces, the commercial availability of cantilevers, and their fabrication using standard microelectronic technology resulted, around , in a new type of sensor where the transducer system is based on a silicon microcantilever with a tipless free end (figure . ) (gimzewski et al., ; chen et al., ) . biochemical applications for this type of sensor have been specifically developed for bending-based modes of measurement, with an optical read-out, due to the complexity required for working with the dynamic mode in liquids. the first experiments in solution were focused on ion detection and measurement of changes in surface stress induced by changes in ph or ion concentrations. it was found that the cantilever response depended upon both ph and the ionic strength of the aqueous medium (butt, ) . among other biochemical applications, induction of surface stress by formation of sams datskos and sauers, ; fritz et al., b) or by non-specific adsorption of proteins such as bsa or lipoproteins has been studied (moulin et al., ) . however, the largest growth in cantilever-based biosensor research has occurred after the landmark paper of fritz et al. ( a) , in which sensitive discrimination of single-base variations in dna strands was demonstrated without using fluorescent labels. this paper had a wide impact and marked the beginning of a major research effort in this field. shortly afterwards, microcantilever sensors were also employed to detect diverse biomolecular interactions. several commercial platforms (carrascosa et al., ) based on microcantilever array sensors are now available. the extensive development that cantilever-based biosensing has experienced during the last years has been mainly focused on demonstrating its suitability and sensitivity in a wide range of different fields: genomic, proteomic, environmental control, clinical diagnosis, etc. this section reviews some of the biosensing applications of the microcantilever-optical read-out configurations, as well as the different approaches proposed to increase cantilever biosensor sensitivity. the bending detection method is very sensitive to changes in the surrounding environment, and has been applied to detection of variations in ph or salt concentration using silicon microcantilevers modified and non-modified with sams (butt et al., ; fritz et al., b; . the effect of functionalized cantilevers with sams, such as alkylthiols with different chemical termini or lengths, on surface stress has also been studied datskos and sauers, ; godin et al., ; kohale et al., ) . likewise, different functionalization schemes have shown that cantilevers were able to detect different ions, such as ca + (ji and thundat, ) or cro -in concentrations as low as -~ m (zhang et al., ) , among many others. genomics is one of the fields where the cantilever-based biosensors have generated both interest and controversy. the first publication in this field demonstrated the detection of a single-base mismatch of singlestranded dna chains comprised of nucleotides, with a detection limit of nm. an array of two cantilevers was used, employing one as a reference; the final signal was determined as the difference in bending of the two cantilevers (fritz et al., a) . a subsequent study reported the discrimination of single nucleotide polymorphisms with a single cantilever wu et al., lb) . this opened a discussion about the possibility of detecting the dna hybridization and single nucleotide polymorphisms without a reference cantilever (arntz et al., ; . mckendry et al. ( ) detected nm of target oligonucleotide using an array of eight microcantilevers previously functionalized using microcapillaries containing a ixm solution of thiolated dna probes. thermal denaturation has also been checked (biswal et al., ) , reporting a decrease in the melting temperature with chain length and salt concentration; lower melting temperatures were also reported for surface-grafted dna than for dna in solution. more recently, this technology has been applied for the validation of mrna biomarkers in total cellular rna, with sensitivity in the picomolar range without target amplification (zhang et al., ) . another novel application is the design of synthetic dna motors (shu et al., ) , which generate a microcantilever nanoscale motion via controlled conformational changes (see figure . ). the forces exerted by the conformational change of precise duplex to non-classical i-motif were shown to induce a compressive surface stress of -t- mn/m (which means a single motor force of approximately pn/m). the cantilever direction and amplitude were controlled by buffer ph and the ionic strength, signifying the importance of electrostatic forces in surface stress generation. in the proteomics field, numerous studies have used antibodies as receptors for detecting their complementary proteins. wu et al. ( a) measured two isoforms of prostate-specific antigen (cpsa and fpsa) over a range of concentrations from . ng/ml to ixg/ml, in a background of human serum albumin (hsa) and human plasminogen (hp) at mg/ml (figure . ); these isoforms have proven to be a useful biomarker for early detection of prostate cancer. the limit of detection obtained was at the current limit of elisa ( . ng/ml), with the advantage of being label free. many other applications have been described, such as the detection of nm of streptavidin with biotin functionalized cantilevers (raiteri et al., ) or the real-time recognition of multiple different cardiac biomarker proteins (creatin kinase and myoglobin) (arntz et al., ) . a cantilever-based sensor sensitivity of ~ nm was achieved using single-chain fv (scfv) antibody fragments as receptor molecules, covalently attached to the gold-coated sensor interfaces in directed orientation . the potential of the cantilever-based biosensor for detection conformational changes in proteins has also been explored. conformational change-induced surface stress was studied for the protein calmodulin. the cantilever bent when the calmodulin bound to ca +, in contrast to the cantilevers modified with other proteins, such as hemoglobin and myoglobin, which do not exhibit change conformations upon binding with analytes (yan et al., ) . the ability of cantilever-based sensors for detection of ligand-protein interactions and conformational v x q. "r" applications have expanded to new fields, such as detection of pathogens such as escherichia coli for food quality control (zhang and ji, ) or environmental control. in this latter field, the interactions between the herbicide , -dichlorophenoxyacetic (raiteri et al., ) and the organochlorine insecticide compound dichlorodiphenyltrichloroethane (ddt) with their corresponding monoclonal antibodies have been measured. direct detection of ddt was achieved with a competitive assay, measuring dtt concentrations as low as nm. in addition, the novel development for early osteosarcoma discovery, sensing the interactions between vimentin antibodies and antigens with a single cantilever-based biosensor, proved that cantilever biosensors can provide a suitable platform for life sciences research (milburn et al., ) . also of interest is application of microcantilevers for detection of the feline coronavirus type i (velanki and ji, ) , which demonstrated the feasibility of detecting severe acute respiratory syndrome associated coronavirus (sars-cov). as previously mentioned, the application of resonant mechanical sensors for biochemical recognition in a liquid medium is a great challenge, as a consequence of the cantilever's resonant frequency and quality factor reduction. in spite of this limitation, there are some works that demonstrate detection of biomolecules by measuring the cantilever resonance before and after the binding event. using this methodology, dna hybridization has been sensed using dna strands linked to the cantilever by gold-thiol covalent bonding and gold nanoparticle-modified probes. after hybridization, the gold nanoparticles act as a nucleating agent for the growth of silver particles, which leads to a detectable frequency shift by increasing the cantilever effective mass. this method can detect at least . nm and is able to discriminate a single-base mismatch (suet al., ) . the number of applications performed using the dynamic mode grows when working with pathogens, cells, viruses, etc. that have a large mass. demonstrated the ability to detect both an e. coli antibody monolayer and a single e. coli cell, operating in air and measuring the cantilever thermal noise with the optical bending method. the mass sensitivities for a and ixm long silicon beam were . and . hz/fg, respectively. measurement of growth of pathogens such as aspergillus niger spores and e. coli cells , in a humidity-controlled environment, has also been reported. in these experiments, the resonance frequency shifts as a function of the increasing mass on the cantilever. reducing the cantilever size is a widespread alternative for increasing the frequency resolution, in both air and liquid. with a silicon cantilever - ixm long, - lxm wide, and - nm thick, gupta et al. ( ) established detection of a single vaccinia virus particle with an average mass of . fg (under ambient conditions, and by measuring the cantilever thermal noise). later, they found that the protein attachment increases with cantilever size (figure . ), resulting in an increase in the effective spring constant and a corresponding anomalous increase in frequency for a certain class of cantilevers . the interaction of biotin with streptavidin has been measured in liquids by working with higher cantilever resonance harmonics (ghatkesar et al., ) . as an alternative, the integrated piezoelectric microcantilevers are becoming increasingly popular, showing quality factors one order of magnitude greater than thermal-driven cantilevers, allowing for mass virus detection in the femtogram range ( - fg) in air (johnson et al., ) . a nanomechanical lead zirconium titanate or pzt thin film cantilever, composed of sio /tafpt/pzt/pt/sio on a sin~ supporting layer, was used for detection of different concentrations ( - ng/ml) of prostate-specific antigen (psa) in liquids (hwang et al., ) . for these experiments, the antibody was previously immobilized with calixcrown sams on a gold surface deposited on the cantilever; the subsequent antigen detection was executed in a fabricated poly(dimethylsiloxane) (pdms) flow cell with a txm width channel and a i~l volume reaction chamber, by measuring the resonant frequency with a laser doppler vibrometer (figure . ). currently, there are many different and alternative ways to increase the sensitivity of cantilever-based biosensors, depending on the sensor working mode. for example, production of cantilever surface nanostructures has been demonstrated as a good method for amplifying the bending signal, due to the increased effective surface area (lavrik et al., ; headrick et al., ) . exploitation of polymers for cantilever fabrication results in a cheaper alternative to increase the cantilever static deflection, as the young's moduli of polymers are lower than that of silicon. at present, one of the most widely employed alternative materials for new sensor designs is the su- polymer, due to its favorable properties. a non-vacuum fabrication process to produce arrays of su cantilevers has been reported, demonstrating their application as chemical sensors (ransley et al., ) . zhang and xu ( ) demonstrated the suitability of txm thick polyethylene terephthalate films for microcantilever fabrication using laser micromachining techniques. in this work, dna hybridization experiments showed the capability of detecting base oligonucleotides both experiments were carried out with the optical beam deflection method. another widely employed polymer is pdms. with this polymer, three-dimensional thin structures with different geometries on each side have been fabricated (park et al., ) . in addition to polymer cantilever fabrication, this work demonstrated the mechanical shear stress enhancement of self-organized cardiomyocytes on three-dimensional grooved surfaces compared to that on two-dimensional surfaces (figure . ), revealing quantitative mechanical changes in cells in real time between the relaxated state and the contracted state of cardiomyocytes. other works propose new designs and cantilever configurations fabricated using standard silicon microtechnology to improve sensitivity or solve existing drawbacks. a configuration based on two adjacent cantilevers forming a sensor/reference pair was used to demonstrate the capability of a dna aptamer-protein binding event to generate changes in surface stress (savran et al., ) . this configuration allowed the researchers to directly detect the differential tip deflection between the two cantilevers. for sensors based on an array of microcantilevers and standard optical detection, either with a single or an array of psd, a very low variability figure . esem (environmental scanning electron microscope) image of cardiomyocytes cultured on flat and grooved microcantilever structures. [reproduced with permission from lop publishing limited (park et al., ).] in the initial position of cantilever array is desired. a reduction in the initial angular offset and angle deviation between the cantilevers of an array is achieved by fabricating t-shaped cantilevers . this geometry consists of a sensing cantilever joined to a doubly clamped beam (figure . ) , which allows that all the cantilevers remain flat and parallel to each other in spite of possible non-uniform initial stresses at the anchor region. new designs, such as an array of cantilevers with a common or discrete window or the aforementioned optical waveguide microcantilevers, offer a new and interesting approach for further integration in "lab-on-a-chip" microsystems. figure . shows the device produced by zinoviev et al., which contains cantilever transducers with lengths of several hundreds of microns fabricated in an array of (zinoviev et al., a (zinoviev et al., , . in this work, characterization of the cantilevers' resonance frequency and bending was performed using the optical beam technique for monitoring the waveguide output signal as a function of the cantilever absolute displacement. the sensitivity of both methods, resonant frequency and bending, allows resolution of cantilever displacement to less than . nm. the effect of covering the cantilevers with gold for biosensing applications has also been studied, showing a strong adsorption depending on the cantilever length and thickness as well as the gold layer thickness. in contrast, the higher order modes are getting filtered, which has the advantage of making the cantilevers single mode in the transverse direction. an additional property of the metallized cantilevers is their ability to deform with light absorption, which helps to adjust the initial position of the cantilever free end with respect to the output waveguide. the increasing number of applications of microcantilevers as biosensors has established these systems as a versatile platform for real-time and in situ measurements of physical, chemical, and biochemical interactions. as previously mentioned, one of the main advantages is their ability to detect molecular interactions without any kind of label, reducing the time and cost of sample preparation. another advantage is the low cost of fabrication and mass production of extremely sensitive devices, as well as the flexibility in fabrication techniques to change the size and shape depending on the required application. hence, it is possible to fabricate cantilevers with very low spring constants that are very sensitive for bending detection, or reduce its size (stiffer cantilevers) for mass changes measurements. a disadvantage related with the cantilever dimensions is the mechanicalthermal noise, which could limit the fundamental biosensor detection limit. the microcantilever can be operated in vacuum, gasses, and liquids, although the dynamic resolution is reduced when working in liquids, due to the damping effect over the resonance response. this limitation has rendered the static mode as the most attractive method for biosensing platforms. the main disadvantage related to bending detection is the complex relation between the measured signal and the factors producing it, due to the number of forces acting in the biorecognition processes. although the working principle of mechanical response could be more difficult to understand than in other types of sensors, a new type of information is reported and represents a new alternative for the discrimination of interactions, such as dna single-base mismatch polymorphisms or single-molecule detection, otherwise not possible with other established biosensing techniques. among other advantages, microcantilevers present a reduced sensor area, is compatible with the complementary metal oxide semiconductor (cmos) technology, and can be easily scaled up, being currently fabricated in arrays of tens to thousands of microcantilevers. this could allow the parallel, fast, and real-time monitoring of thousands of analytes (dna strands, proteins, pathogens, cells, etc.) without the need for labels. moreover, the bending and resonance response (frequency, amplitude, q-factor or phase), when simultaneously detected, can supply valuable information. currently, excellent results have been obtained with the optical bending read-out configuration; however, this set-up is limited by environmental optical properties which confound its applications with real samples such as blood. in order to obtain an industrial sensor system, low power consumption and compact portable devices are required. these requirements have led to an increase in the development of integrated detection read-outs with a cmos electric circuitry incorporated . in addition to an integrated read-out, the biotechnology field requires that biosensors be capable of working with small amounts of reagents, with highly reproducible surface functionalization and subsequent recognition processes. for this purpose, an integrated microfluidics system that allows liquid handling in an easy and reproducible approach is needed (bietsch et al., ; yue et al., ; http://www.protiveris.com/ products/pages/sensorcartridge.html). some of the most desired improvements on the cantilever-based biosensors are the increase in the sensitivity and reproducibility, a more in-depth knowledge of the working principles, and a means of integration of these sensors into a portable and miniaturized system. there are different ways to increase the biosensor sensitivity. some are related to optimization of the cantilever material and dimensions, noise reduction, or improvements in the detection methods. the development of a highly sensitive and integrated detection method is one of the most important unresolved matters in this field where new approaches, such as the optical waveguide cantilevers, arise as a great alternative to standard optical beam methods. other techniques based on piezoresistive and piezoelectric microcantilevers allow an easy and more integrated readout and are becoming important during the past several years, although their sensitivities are presently inferior to those obtained with the optical method. other ways to improve sensitivity are focused on the biochemistry, looking for new methods to functionalize the cantilever reproducibly, reducing both the non-specific interactions and the volume of reagents. sams are especially useful for binding assays, allowing surface regeneration for subsequent assays. development of an integrated microfluidic system would be especially valuable for improving the signal reproducibility, reducing the volume required and the total system size. the possibility of working with several cantilevers at the same time, in completely independent microfluidics channels, would allow the detection, in real time, of many different reagents simultaneously, providing this sensor with a greater capability of measurement and higher versatility than other current techniques (such as elisa or dna microarrays). a more fundamental understanding about the cantilever working principles and the factors involved in the cantilever response, both static and dynamic, are needed. this knowledge will provide new useful information for increasing the biosensor sensitivity and for controlling the binding process itself (lu et al., ; reed et al., ; craighead, ; watari et al., ) . proc. natl. acad. sci. usa protein microarray technology: nanomechanical cantilever sensors for microarrays proc. ieee sens mems , th ieee international conference proc. natl acad. sci. usa, micromanufacturing and nanotechnology: nanomechanical cantilever for biological sensors proc. natl acad. sci. usa, gas analyzer, general electric co. us patent , , spie-lntegr proc. spie the authors would like to acknowledge the european union for funding and their partners working within the european project optonanogen (ist- - ). key: cord- -ti qjrn authors: elzaabalawy, assem; meguid, s. a. title: potential of combating transmission of covid- using novel self-cleaning superhydrophobic surfaces: part ii—thermal, chemical, and mechanical durability date: - - journal: int j mech mater des doi: . /s - - -y sha: doc_id: cord_uid: ti qjrn in part i, we identified encapsulation, contamination suppression, and virus elimination as our three governing strategies for developing surfaces to combat the transmission and spread of covid- . we showed that our recent superhydrophobic nanocomposites has the potential of encapsulating and suppressing the virus so as to limit its transmission and spread. in this study, we examine the durability of the newly developed surfaces when subjected to elevated temperature, chemical attack and mechanical damage in the form of abrasion and compressive load. extensive tests were conducted to reveal the effect of these parameters on the surface performance. three aspects of the work were accordingly examined. the first was concerned with controlled thermal stability tests in which the surfaces were subjected to elevated temperatures approaching °c for silicone-based nanocomposites and °c for epoxy-based nanocomposites. the second was concerned with subjecting the surfaces to alkaline and acidic solutions with ph concentrations ranging between and . finally, the third involved surface damage by abrasion tests. our results show clearly that the newly developed superhydrophobic surfaces are capable of resisting the adverse effects of thermal and chemical attacks as well as mechanical abrasion owing to the excellent structural stability and mechanical properties of the constituents of the nanocomposite. moreover, our superhydrophobic monolith demonstrated exceptional regenerative capabilities even after being subjected to damaging compressive stresses of up to mpa. covid- can be transmitted through airborne respiratory droplets, ejected as a result of coughing or sneezing through human contact with contaminated surfaces (yang and wang ; gralinski and menachery ) . recent studies by the national institute of allergy and infectious diseases (usa) have shown that the virus can infect fomites made of metals, polymers and recycled paper for days (van doremalen et al. ) . superhydrophobic surfaces have recently gained popularity in the biomedical sector due to their ability to possess blood repellency characteristics and reduce bacterial/viral adhesion to surfaces and their antifouling properties (falde et al. ; shin et al. ; jaggessar et al. personal protective equipment (ppe) that have high contact angles (ca) and low sliding angles (sa) can significantly reduce a virus's carryover potential and provide antimicrobial properties (katoh et al. ; tomšič et al. ; yeerken et al. ) . in part i, we demonstrated the potential in creating superhydrophobic coatings and monoliths that are capable to combating the transmission and spread of covid- . however, one of the main challenges that restricts the widespread utilization of superhydrophobic coatings in real applications is their durability (ellinas et al. ; jeevahan et al. ) . specifically, thermal and chemical stability as well as mechanical strength. elevated temperatures can lead to the thermal degradation of hydrocarbon coatings (cha et al. ) or thermal desorption of the hydrophobic material (saleema and farzaneh ) , which result in the loss of surface superhydrophobicity. additionally, some polymeric superhydrophobic surfaces cannot withstand elevated temperatures and are not recommended to operate above their glass transition temperature (ellinas et al. ) . similarly, chemical attack due to exposure to acidic or alkaline solutions can result in severe deterioration of the superhydrophobicity of the treated surfaces (baratidarband et al. ) . for instance, highly alkaline or acidic solutions can result in dissolving the hydrophobic functional groups (zhao et al. ; ishizaki et al. ) and cause chemical desorption of the hydrophobic material (cui et al. ) . mechanical wear is considered one of the main performance degradation mechanisms of superhydrophobic surfaces (milionis et al. ) . indeed, numerous studies and tests have been conducted to evaluate the influence of abrasion tests on the mechanical durability of these surfaces (ellinas et al. ; mortazavi and khonsari ) . superhydrophobic surfaces generally suffer from weak mechanical durability due to the fragility of their nano/microhierarchical surface structure (mortazavi and khonsari ; zhang et al. ) . additionally, polymeric materials involved in fabricating superhydrophobic surfaces usually suffer from poor mechanical stability and robustness (ellinas et al. ). in our previous work (elzaabalawy et al. ) , we demonstrated that high external compressive loads can also lead to permanent plastic deformation of the surface hierarchical structure and the embedment of the hydrophobic nanoparticles, which ultimately cause a complete loss of superhydrophobicity. covid- spreads mainly through droplet transmission or via contacting fomites. we believe that surfaces coated with or created from a superhydrophobic nanocomposite has the potential of combating virus transmission and spread through a -step strategy discussed in part i. the superhydrophobic nanocomposites acquire their protection characteristics through chemical modification as well as a hierarchical nano/ microstructure of the modified surface. however, typical surfaces are subjected to elevated temperatures, chemical attacks and mechanical damage during service. as a result, the surface repellency characteristics may be altered and the effectiveness and the potential of the surface to encapsulate and suppress the virus might be lost. for nanocomposite superhydrophobic surfaces, utilizing different polymeric materials can be used to vary the flexibility and durability of the developed surfaces. for instance, silicone polymers can be used when flexibility is sought, and epoxy can be employed to meet the requirements of harsh mechanical environments. the durability of the nanocomposite can also be enhanced and tailored by selecting constituent materials that possess high thermal and chemical stability. moreover, superhydrophobic monoliths with regenerative capabilities can be employed in severe service conditions to accommodate mechanical damage. since these monoliths acquire water repellency features throughout the entire bulk of the material, surfaces can be regenerated by removing the damaged layer and exposing a new superhydrophobic layer. in this case, superhydrophobicity can be maintained as long as the monolith's thickness can accommodate a layer removal. the aim of part ii is to assess the ability of the superhydrophobic coating and monoliths discussed in part i to resist thermal degradation, chemical attacks, and mechanical damage. three aspects of the work were accordingly examined. the first was concerned with thermal stability tests in which the superhydrophobic surfaces were subjected to elevated temperatures approaching °c for silicone-based nanocomposites and °c for epoxy-based nanocomposites. the second was concerned with subjecting the surfaces to alkaline and acidic environments with ph values ranging between and . finally, the mechanical durability of the surface of epoxy-based nanocomposite coating was assessed using abrasion tests, while the regenerative capability of the silicone-based nanocomposite monoliths was evaluated using compressive loading. given below is a summary of the approach adopted and the outcomes of our research. in this research program, superhydrophobic coatings and monoliths were developed using epoxy and silicone nanocomposites. we demonstrated the ability of these coatings to modify the surface characteristics of different substrate materials, as shown in fig. . we have also succeeded in fabricating a bulk that is entirely superhydrophobic. in order to assess the thermal, chemical, and mechanical stability of these surfaces, multiple tests were conducted. since superhydrophobic surfaces are characterized by ca above °and sa below °, the superhydrophobicity in these tests was assessed by measuring the contact and sliding angles. contact angles were measured using an oca ec contact angle analyzer (dataphysics instruments, fig. ), while sliding angles were measured using an in-house apparatus with controlled surface tilting until droplets roll off is achieved. as organic polymers, epoxy thermosets will degrade under thermo-oxidative conditions, and this degradation typically initiates at the surface. it is therefore necessary to conduct thermal stability tests for the superhydrophobic surface to ensure that water repellency is maintained after being subjected to surges of elevated temperatures. unlike organic polymers, silicone is characterized by its higher thermal resistance due to the high stability of its siloxane backbone. in order to examine the thermal durability, coated samples and monoliths were placed in an oven for h. tests were conducted for different temperatures and the maximum temperature was selected based on the polymer's auto-ignition temperature. upon completing the test, samples were left to cool down, and then the contact and sliding angles were re-evaluated to assess their superhydrophobicity characteristics. for epoxy-based nanocomposites, thermal durability tests were conducted for temperatures between and °c in °c increments. as depicted in fig. , no variation was observed to the values of ca (* °) or sa (* °) for all of the tested temperatures. these results verify that the coated samples maintained their superhydrophobicity and the siloxane-modification process (part i) succeeded in enhancing the water repellency of epoxy without compromising its durability. silicone-based nanocomposites were tested for temperatures between °c and °c in °c increments. as shown in fig. , the coated samples and monoliths maintained high ca (* °for monoliths and * °for coatings) and low sa ( °f or monoliths and °for coatings) throughout the entire temperature range. these results further demonstrate the capability of the strong siloxane backbone within the polymer chemical structure to withstand elevated temperatures and maintain the superhydrophobicity of the nanocomposite. chemical attacks or short-term immersion in corrosive solutions can result in severe degradation of the surface characteristics. to assess the chemical stability of the developed superhydrophobic nanocomposites, samples were immersed in acidic and alkaline solutions for h at room temperature in a fume hood, as depicted in fig. . solutions were prepared using sulphuric acid or sodium hydroxide, which were diluted using distilled water to achieve ph values between and . following the immersion, water repellency of the samples was again evaluated by measuring the contact and sliding angles. following the immersion tests, samples were observed to check their reflective appearance in the fig. thermal durability test results for silicone-based a coating and b monolith solution, which indicates a stable cassie-baxter wetting state, and to be completely dry upon removal. furthermore, following the immersion, the ca and sa were measured for the epoxy-based and silicone-based nanocomposites, as depicted in figs. and , respectively. the measurements revealed that the nanocomposites are capable of maintaining their excellent superhydrophobicity for the entire range of ph values tested. accordingly, these nanocomposites can operate and effectively maintain their performance in harsh service conditions that are subjected to acidic or alkaline chemical attacks. abrasion is considered the most widely test used to evaluate the mechanical properties of superhydrophobic coatings (milionis et al. ) . since siliconebased coatings are sought to provide flexibility rather than mechanical strength, abrasion tests were only conducted to evaluate the strength and durability of epoxy-based nanocomposite coatings. as schematically shown in fig. , the samples were placed face down on silicon carbide coarse sandpaper ( grit) under a calibrated weight pressure of * kpa. abrasion cycles ( cm of linear displacement) were performed in alternating directions. following each interval of cycles, debris were removed, and the contact and sliding angles were measured. the contact and sliding angles measurements following abrasion cycle intervals are demonstrated in fig. . after abrasion cycles, the coating was only able to maintain its high contact angle ([ °), while the sliding angle increased above °. in order to examine the degradation caused to the nano/microstructure of the surface, sem images were obtained, figure the results indicate that excessive abrasion cycles induce damage to the surface's hierarchical structure (fig. b ) compared to the original (fig. a) . however, the nanocomposite coating was able to superbly sustain its superhydrophobicity after abrasion cycles, which is attributed to the excellent adhesion strength and mechanical properties of epoxy polymers. although the epoxy-based nanocomposite coating demonstrated excellent mechanical durability characteristics, service conditions can sometimes be harsher than a coating can handle. in those situations, a silicone-based nanocomposite monolith that is entirely superhydrophobic is an excellent replacement. under harsh mechanical environment, the superhydrophobic surface's hierarchical structure would be damaged. to obviate this damage, the surface can overcome that damage by the regeneration of a fresh superhydrophobic layer under controlled abrasion. in order to demonstrate these regenerative capabilities, monoliths were exposed to compressive stresses ranging between and mpa with the aid of a uniaxial electrohydraulic servo-controlled testing machine (instron ). samples with a square crosssection ( mm mm) were placed between two flat metallic plates, and then the loading was applied at a displacement rate of mm/min until the desired load was obtained. the compressive load was then maintained for min before the samples were unloaded. subsequently, ca and sa were measured after unloading and after samples were regenerated. the test set up is depicted in fig. . the compression test results (fig. ) revealed that the monolith can withstand stresses up to mpa without affecting its superhydrophobicity, in which ca and sa are maintained at * °and °respectively. for increased compressive stresses ( and mpa), gradual decrease in the ca and increase in sa were noticed. at a compressive stress of mpa, ca reached * °and droplets were pinned to the surface and did not roll off even when applying a finite inclination angle. as shown in fig. , sem images indicated that this deterioration was caused by the plastic deformation in the surface's hierarchical structure as a result of the high compressive stresses. remarkably, the surface's superhydrophobicity was fully restored upon the removal of the damaged layer through abrasion, and the respective ca and sa were fully recovered to * °and °, as depicted in fig. . as observed, the regeneration process was successful for all values of destructive stresses ( - mpa). in this work, we examined the durability of the newly developed superhydrophobic surfaces proposed in part i to potentially combat the transmission and spread of covid- . thermal degradation, chemical attack and mechanical abrasion tests were conducted, and the superhydrophobic surface characteristics were re-tested and re-evaluated. our extensive test results revealed that the developed epoxy-based and siliconebased nanocomposites can maintain their superhydrophobicity after being subjected for h to elevated temperatures of °c and °c, respectively. the developed surfaces also preserved their characteristics after being immersed in alkaline and acidic solutions (ph values - ) for h. in order to assess the mechanical durability of epoxy-based nanocomposites, samples were subjected to abrasion tests, and they were capable of maintaining their superhydrophobicity for abrasion cycles. moreover, the regeneration capabilities of the silicone-based nanocomposites were demonstrated by subjecting the monoliths to destructive compressive loads (up to mpa), and then the high ca and low sa were easily restored through abrasion. these results verify the ability of these surfaces to survive harsh service conditions and their potential use to combat the transmission and spread of infected surfaces. it is important to note that we have neither examined the combined effect of these parameters upon the surface characteristics of the developed superhydrophobic surfaces nor the prolonged exposure to these conditions (aging), which are considered beyond the scope of the current study. science and engineering of superhydrophobic surfaces: review of corrosion resistancechemical and mechanical stability thermal stability of superhydrophobic, nanostructured surfaces fabrication and corrosion resistance of a hydrophobic micro-arc oxidation coating on az mg alloy durable superhydrophobic and superamphiphobic polymeric surfaces and their applications: a review sem images of the monolith showing its regenerative capability a prior to compression, b after being subjected to a quasistatic compressive stress of mpa, and c after regeneration multifunctional silica-silicone nanocomposite with regenerative superhydrophobic capabilities superhydrophobic materials for biomedical applications return of the coronavirus: -ncov corrosion resistance and chemical stability of super-hydrophobic film deposited on magnesium alloy az by microwave plasma-enhanced chemical vapor deposition bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants superhydrophobic surfaces: a review on fundamentals, applications, and challenges potential risk of virus carryover by fabrics of personal protective gowns recent advances in the mechanical durability of superhydrophobic materials on the degradation of superhydrophobic surfaces: a review thermal effect on superhydrophobic performance of stearic acid modified zno nanotowers bioinspired extreme wetting surfaces for biomedical applications sol-gel coating of cellulose fibres with antimicrobial and repellent properties aerosol and surface stability of sars-cov- as compared with sars-cov- covid- : a new challenge for human beings chemical stable, superhydrophobic and self-cleaning fabrics prepared by two-step coating of a polytetrafluoroethylene membrane and silica nanoparticles superhydrophobic surfaces for the reduction of bacterial adhesion controllable nanoscale engineering of vertically aligned mos ultrathin nanosheets by nitrogen doping of d graphene hydrogel for improved electrocatalytic hydrogen evolution key: cord- -tk v hoj authors: nan title: environmental and safety issues with nanoparticles date: - - journal: nanoparticle technology handbook doi: . /b - - - - . - sha: doc_id: cord_uid: tk v hoj nan since nanoparticles have superior surface activity and can be applied to the production of particles with various functions, they are extremely important for the future development of sophisticated material technologies. on the other hand, this superior activity of nanoparticles is a cause of trouble from the perspective of safety, and does not always have a positive influence on the environment. attention must also be paid to impact on health. nevertheless, all technologies have negative aspects, and overcoming these kinds of problems, we will be able to utilize the superior characteristics of nanoparticles for practical purposes. to achieve this goal, it is necessary to fully understand the influence of nanoparticles on the environment and the relevant safety issues. this chapter evaluates the relationship between nanoparticles and the environment, and also describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment will be clarified from the viewpoint of what kind of influence nanoparticles generated either artificially or naturally have on the environment. the influence on the indoor environment, where nanoparticles are produced, will also be clarified. the safety of nanoparticles will be clearly described from the perspective of the trouble caused by the superior surface activity of nanoparticles; the effect of the compositional characteristics of nanoparticles, and also the influence on health. a method for assessing the influence of nanoparticles using quantum dots is also explained. in the final section, methods for removing nanoparticles from gas and liquid are described as technology to control the influence of nanoparticles on the environment. in our atmospheric environment, particles ranging from several nanometers to several tenth micron orders are suspended. they are emitted into the atmosphere at the rate of . billion tons every year. emission sources are classified as either natural or artificial. natural particles occupy % of total particles, consisting mainly of salt particles (ϳ billion ton) from the sea and soil particles (ϳ . billion ton) from the land. on the other hand, the latter particles are brought about by human activities. although occupying only % of the total emitted particles, their size is mostly of submicron order and because they contain hazardous chemical components such as nitrates, sulfates, hydrocarbons, heavy metals, etc. in high concentration, their effects on the ecosystem are serious. fig. . . shows an overview of the size and concentration ranges of various aerosol particles. as it can be seen, the number concentration of atmospheric aerosol which we inhale every day ranges from several thousand particles per cm in clean area to several hundred thousands in dusty areas, and the size range lies between nm and several tens of micrometer. fig. . . shows mass-based size distribution of atmospheric aerosol particles. since the size distribution in the nanosize range appears only when the sources of particle generation exist, the size distribution is usually bimodal with peaks in the size range of a few to micron and submicron. the former peak consists of naturally generated coarse particles such as soil dust, sea salt spray, and so on. on the contrary, the latter contains plenty of artificially generated particles, some of which grow from molecules (in most cases vapor state) exhausted by human activities through chemical reaction, condensation, and coagulation. particle growth rarely leads to particles larger than m unless high concentration of vapors or particulate matters which cause the above-mentioned growth mechanisms exist in the atmosphere. as it can be seen from the differences in the particle generation process, fine particles generated from molecules or nanoparticles are much more complicated in their chemical component than the coarse particles, and sometimes have serious adverse health effects. such fine particles are called pm . , which is defined for particles less than . m including nanosized particles. recent epidemiologic investigation reports that the concentration of pm . showed a positive correlation to the mortality due to pulmonary diseases [ ] . various research techniques are used in order to understand the process of particle growth and to trace back to the source of pollution. an example is shown in fig. . . where a characteristic function of sulfur dioxide is shown taking into account all possible factors related to particle growth. where f is the characteristic function that expresses particle size, particle concentration, particle composition, and so on [ , ] . particulate materials in water are present in the form of colloids. these colloid particles are classified into inorganic colloids. examples of the former are oxides of aluminum, silicon and other substances, and typical examples of the latter are substances such as humic acid and fulvic acid. while the structure and molecular weight of particles vary depending on the area of water, it is known that what are usually present in water are comparatively small colloids (particles smaller than nm). the number concentration of colloid particles in ground water, or a typical water area environment, ranges from to (number / m ) and varies significantly depending on the geochemical conditions of the aquifer. it is known that in moving water, colloid particles sometimes act as a medium in conjunction with water and in some cases move faster than water. homogeneous porous layer such as a sand layer, and most of the colloid particles are trapped. the mechanism of partical trap in this layer is explained by the sand filtration theory. c and d are a gravel layer and a rock bed, respectively, and both have high water permeability with large gaps and cracks. particles can also pass through easily. safety and movement characteristics of colloid particles have a significant influence on the movement of materials such as ionized molecules in aquatic environments. since fine particles such as nanoparticles in particular are highly stable as colloid particles, it will be very important in the future to understand their influence. at the same time, these characteristics are considered to have a high potential to be developed for further application of nanoparticles. in most cases, nanoparticles in exhaust gases are studied from the viewpoint of the influence of total particulate matters on the environment. the term "nanoparticles" is used only in a few cases, "fine particles" is usually used for investigation. since nanoparticles are part of fine particles, this section will be described from this perspective. major sources of combustion exhaust gases are stationary large-scale combustors and diesel engines for stationary and portable use. for stationary combustors, fuels such as coal, oil, and gas are used. lighter fuels have a lower rate of particulate emission, but have a higher fine particle content including nanoparticles. fig. . . [ ] shows the frequency distribution in combustion of coal and heavy oil. fig. . . a and b are the distributions on a number and mass basis, respectively. as these figures clearly show, the total weight of particles of a size of m or smaller is extremely low, while their total number is, on the contrary, very large. it is clear that, while the total quantity of particulate material is far larger in coal combustion than in oil combustion, the difference is less when it comes to particles m or less in diameter, including nanoparticles. most of the particles contained in pulverized coal combustion exhaust gases are considered to be formed as particulate materials directly from ash content, which is originally contained in coal and also includes some unburned carbon. particularly, almost all large-size particles are considered to be this type of particle. on the other hand, fine particles include two types. one type is formed in the process by which low boiling point metal contained in coal ash is evaporated and vaporized in a high-temperature combustion field and then becomes particles in the exhaust gas cooling process. the other includes carbon particles formed in the gas phase, or so-called soot, which is generated due to the delay in oxygen supply for combustion of evaporated volatile matter in the initial stage. fig. . . [ ] shows the relationship between the trace metal content in coal ash and the particle diameter. aluminum with a high boiling point has a constant concentration regardless of the particle diameter. however it is obvious that in the case of metals with a lower boiling point, the smaller the particle diameter, the larger the content. with regard to particles with sizes m or smaller in the nano domain, it has been clarified that the generated amount is increased rapidly by reducing combustion air supply or by weakening the oxidation atmosphere in the volatile matter combustion area, for example, when air supply from a burner is reduced in twostage combustion. this also demonstrates the significant contribution of carbon particles formed in the gas phase. also in the case of ash from heavy oil combustion, there are large particles of a carbon residue type generated from sprayed liquid particles and particles formed in the gas phase as well. as in the case of coal, trace metal contained in heavy oil with a low boiling point is concentrated into fine particles and discharged. also in the case of the combustion of liquefied natural gas, carbon particles formed in the gas phase are generated, albeit in trace amounts. in contrast, only in the case of diesel engines, fuel is injected into the high-temperature and highpressure atmosphere produced by compressing only air to induce spontaneous ignition, and combustion continues with a heterogeneous mixture of fuel and air in the combustion chamber. therefore, particulate materials mainly consisting of unburnt carbon are generated due to incomplete combustion. fig. . . [ ] shows changes in the diameter of particles according to changes in the diesel engine load. it is obvious that the overall concentration of particles increases with the increase in the load rate of the engine. according to observations using sem, fine particles in diesel engine exhaust gases have also been found to comprise fine primary particles of a size several tens of nanometers, and coarse particles with carbon hydride condensed on the surface of secondary aggregates of primary particles. influence of particle diameter on trace element contents. the volume of industrial and domestic wastewater is increasing significantly year by year with the change in the lifestyle based on mass consumption and mass disposal brought about by the dramatic development of the economy and industry. effective advanced wastewater treatment is required because wastewater contains a variety of constituents such as particles, organic materials, and emulsion depending on the resource. inorganic nanoparticles are not generally stabilized in the liquid because they form aggregates of some sort more or less. for wastewater reclamation and reuse, these nanoparticles can be removed from the liquid by the advanced treatment processes such as membrane filtration following biological treatment processes. organic materials such as macromolecules are regarded as soft nanoparticles judging from their sizes, in contrast with hard inorganic particles. chemical mechanical polishing (cmp) is one of the fastest growing processes in semiconductor industry, and it has become an integral part of the state-of-the-art fabrication line for the multilayer wiring board of large-scale integrated circuit (lsi). besides the semiconductor devices, cmp is widely applied to the magnetic head, the magnetic memory, and the imaging devices. the process is primarily used for polishing the device side of a semiconductor wafer through the mechanical downforce of slurry abrasive in combination with chemical oxidation of wafer surface. in general, colloidal silica is used as abrasive slurry to planarize the oxide wafer surface. particles in slurry are highly charged to avoid aggregations between particles or between particles and wafer surfaces. during the process, large volumes of ultrapure water are consumed to clean the surface of the wafer, which generates large quantity of cmp wastewater typically having high solid content resulting from slurry abrasive particles of sio , al o , or ceo , depending on the nature of the cmp application. the quantity of cmp wastewater generated is expected to increase proportionally with the growing needs of the cmp processes. as a result, the treatment and reuse of cmp wastewater has become increasingly necessary. the cmp wastewater has been generally treated with the conventional chemical coagulation-sedimentation process, producing large quantity of sludge. currently, a membrane filtration process coupled with chemical pretreatment is used to separate the nanoscale particles from the cmp wastewater to reclaim the water [ , ] . wastewater of nanosized metal colloid, which is hard to be removed by coagulation-sedimentation process, is discharged in such diverse fields as metalworking factory, electronic components factory, and pigment-manufacturing factory [ ] . it is reported that various trace elements of heavy metal are contained in wastewater discharged from a pulp production plant [ ] . a spent emulsion, which contains nanosized copper colloid, is discharged from plants manufacturing copper cables for electrical industry, and the treatment for purification of effluents is examined by the integrated membrane system based on ultrafiltration (uf) and nanofiltration (nf) [ ] . a glass company generates the wastewater containing fine clay and glass particles from the grinding process of glass surfaces during production of crt glass used for tvs and monitors. separation of fine clay and glass particles by microfiltration (mf)/uf is examined in order to treat glass industry wastewater for reuse in the manufacturing process [ ] . the colored substances are free from regulatory constraint of water quality so far because they are not considered hazardous substances. however, water color is being recently used as a standard for the judgment of the purity in water because the removal of color becomes important for wastewater reclamation and reuse. dye works are scattered across the country as the industry with local tradition. the dyehouse effluent is discharged in large quantity, and it has extremely complex composition because it contains not only dye but also dyeing aid and finishing agent. in general, dye cannot be removed by standard biological treatment because of its low environmental biodegradability. dye wastewater is treated by coagulation-sedimentation and activated sludge processes, and nanoparticles produced in the course of the treatment are released into the environment [ ] . the color is often imparted by organic substances, predominantly humic substances. aquatic humic substances including humic and fulvic acids are a term referring to a broad class of naturally occurring mixture of organic compounds, ubiquitous in surface waters, ground waters, and soil pore waters. they are a complex mixture of heterogeneous organic materials in terms of elemental composition, chemical functionality, and molecular size distribution since humic substances can be derived from any organic materials, including plant and animal debris, microfauna, biowaste, pesticide, and others. the molecular weights of humic acids range from several thousands to several tens of thousands daltons, and those of fulvic acids range from several tens of thousands to several hundred thousands daltons. because of this versatility, humic substances are known to significantly affect the behavior of some pollutants in natural environments, such as trace metal speciation and toxicity, solubilization and adsorption of hydrophobic organic compounds, and disinfection by-product formation [ ] . melanoidins are natural polymeric compounds of dark brown color, and they are closely related to humic substances. they are produced by a set of consecutive and parallel nonenzymatic reactions taking place between amino compounds and carbohydrates during a maillard reaction [ ] . they are contained in the molasses wastewater from alcohol distillery, sugar processing and refinery industry, and glutamate processing industry. such wastewater containing melanoidins has frequently caused a coloration problem of water environment, and thus the suitable decolorization treatment is required in many fermentation and sugar industries using molasses. treatments by flocculation, ozonation, and electrolysis are promising in color removal [ ] . food-processing wastewater usually contains a variety of organic materials in varying degree of concentration. in cheese-making in the dairy products industry, only ϳ % of the initial milk volume becomes product, cheese, and the other % becomes by-product, liquid cheese whey. since cheese whey is a protein-and lactose-rich by-product of cheese production, its cost-effective utilization is becoming increasingly important. recent developments in membrane technology have provided exciting new opportunities for large-scale protein and lactose fractionation in whey treatment [ ] . in textile industry, typically it takes over l of water to process just kg of textile material. not only the washing water must be treated to recover important by-products such as lanolin, but bleaching and dyeing chemicals must also be removed before discharge back to the rivers [ ] . surfactants are a primary constituent of the detergent used in the household routinely, and also they are widely used in industry and agriculture because they have several functions such as washing, emulsification, and dispersion. the surfactants are usually present in the solution in the form of the micelle, and large amounts of surfactant wastewater are discharged in the rivers [ ] . pesticides whose molecular weight ranged from to da (ϳ nm) have been used in great quantities not only for agricultural use but also in golf links and resort. therefore, the wastewater and effluent treatments have become an important issue, and pesticide separation by nf membranes is found to be very efficient [ ] . the potential reclamation of high-quality water produced by the advanced treatment of the secondary effluent of the municipal sewage has come a long way in recent years. the sewage contains various components such as virus [ ] , pharmaceutical substances [ ] , and endocrine disrupting compounds derived from zoonotic excretory substances [ ] . the advanced treatment of such chemical contaminants at low level becomes increasingly important. as mentioned above, the removal of nanoparticles contained in wastewater is stringently required to recycle the reclaimed wastewater in a wide variety of industries such as chemical industry, textile industry, pulp and papermaking industry, food-processing industry, dairy products industry, and pharmaceutical industry. also for domestic wastewater, the reuse of the reclaimed wastewater for nonpotable purposes is becoming more and more important, and this is expected to raise awareness of the behaviors of nanoparticles contained in wastewater in order to upgrade the water treatment processes. in recent urbanized lifestyles people tend to spend more time in enclosed buildings or residences than outdoors. therefore, it is of great importance to characterize indoor particles and correlate between indoor and outdoor ones from the viewpoint of evaluating the influence of indoor air quality (iaq) on human health. as shown in table . . , indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. . . . secondary particle formation by gas phase ozonolysis for particle formation resulting from chemical reaction via ozone, the reaction of terpenes is very common in indoor environments as well as atmospheric ones. terpenes are emitted from fragrance-containing vegetable oils such as pine oil and citrus oil, and wooden materials including woody furniture [ ] . meanwhile, sources of emission of ozone are air cleaners, air-conditioners, laser printers using corona discharge, and infiltration of outdoor air. terpenes are generic terms of unsaturated organic compounds that are composed of isoprene as unit (e.g. -pinene and limonene). these compounds used for household applications readily react with ozone because they have one or more double bonds. it has been proposed that, as shown in fig. . . , the reaction mainly proceeds to form less volatile pinonic acid via pinonaldehyde of intermediate [ ] . furthermore, the acid-catalyzed reaction allows the products to convert into higher molecular weight compounds by the polymerization via carbonyl groups in the aldehydes and the aldol condensation [ ] . it has been reported that the resultant generated particles have a size distribution with a peak diameter of about nm, and that the products by terpenes ozonolysis irritate human airways [ ] . nanoparticles are also generated from air humidifiers or negative air-ion generators in which water is atomized. in general, humidifiers are mainly categorized into vaporization type and atomization type [ ] . the former does not entrain impurities in water when it is fed into indoor spaces of interest. meanwhile, the latter has the drawback that nvrs are suspended in spaces to be humidified by feeding water via spraying and sonication. the nvrs in tap water include colloidal particles and soluble fraction such as silicates, sulfates, carbonates, and chlorides. the size of nvr particles, d p _ r can be estimated from the following equation: where dp_ m is the droplet size, c the mass fraction of nvr in the droplets, m the droplet density, and r the nvr particle density, respectively. assuming that m-sized droplets ( m ϭ , kg/m , r ϭ , kg/m ) are formed by an ultrasonic nebulizer, and the mass fraction of nvr in city water is ppm ( Ϫ ), the nvr particle size, d p_r is estimated to be nm. recently, a wide variety of negative ion generators using the lenard effect, corona discharge, uv/photoelectron emission and electrospray have been commercialized and attention has been focused on features such as air purification and physiological activation [ ] . among them there are the ion generators that atomize water based on the lenard effect and electrospray form the nvr particles as by-product in addition to ion products if the supplied water contains nonvolatile impurities. fig. . . shows an example of electrical mobility distribution for ions generated by the electrospray method (positive in this case) [ ] . this method atomizes liquid fed to a tip of a capillary electrode to form fine droplets with large amounts of charge by applying high voltage between the tip and the downstream counter electrode. when water in the generated droplets evaporates and their surface charge density attains the charge limit called "rayleigh limit", this phenomenon induces their self-fragmentation followed by the formation of a high concentration of cluster ions. in this figure, the high-mobility peak on the right side corresponds to the cluster ions. these ions are nm in size assuming that they are singly charged. meanwhile, another peak on the figure results from nvr in water and then its height increases with the increase in the fraction of tap water in the fed liquid. the electrical-mobility-equivalent size of nvr particles measured by differential mobility analyzer (dma) -condensation nucleus counter (cnc) method ranges from to nm and their concentration is on the order of particles/cm . comparing the forementioned electrical mobility distribution with the particle size one, the nvr nanoparticles are estimated to hold about charges. accompanying the recent proliferation of computers, the use of inkjet printers and electrophotographic machines such as laser printer and photocopier is becoming common in homes as well as offices. it has been reported that these devices emit various sorts of pollutants. the eco-friendlinessoriented standards such as blue angle standard [ ] regulate the maximum permissible limits of benzene, styrene, total volatile organic compounds (tvoc), ozone and particles. as the regulation of particles is based on the emitted mass per hour, mainly the relatively coarser particles such as toner and dust adhering to paper have been targeted. however, some reports have revealed that nanoparticles are emitted from inkjet printers or laser printers [ ] . fig. . . depicts the size distribution of particles emitted from a laser printer measured by a scanning mobility particle sizer (smps). as seen in the figure, nanoparticles with a peak diameter of around nm are generated in printing mode, whereas the emission in the case of feeding paper without printing is about one third of the normal printing mode. furthermore, these particles were dried by passing them through a diffusion dryer because they are thought to originate from the nucleation of water vapor emitted from papers in the fixation process. as a result, it was found that most particles formed in the paper feed mode evaporated and then vanished, while particles in the printing mode contained nonvolatile components as well as water. from these results it is anticipated that the particles are derived from styrene remaining in the toner even though their composition has still not been identified. meanwhile, it is thought that during ink discharge ink-jet printers emit not only the main ink droplets but also their satellites (about m) to result in nanometer-sized nvrs during printing. one of the most significant source of indoor nanoparticles relevant to combustion is cooking such as frying and sautéing [ ] . some reports said that over % of the particles by number were in the ultrafine fraction range during cooking with bimodal peaks at and nm, attaining the number concentration on the order of particles / m and the emission rate of particles / h. owing to lifestyles in asian countries, cigarette smoke, incense, and mosquito coils also contribute to indoor nanoparticle levels [ ] . it was reported that especially in the indian subcontinent the combustion of biofuels such as straw and dried cattle manure used for cooking could have a significant impact on climate change in the south asian region [ ] . sidestream cigarette smoke also contains nanoparticles, having a concentration distribution with the main peak between . and . m [ ] . in addition, it was found that nanoparticles a peak size of nm formed by the nucleation of vapor fraction in filtered sidestream smoke immediately after burning when the dilution of smoke by air was insufficient [ ] . attention should be paid to air cleaners when a high concentration of cigarette smoke has to be treated by the cleaners using a single unit air filter. airborne virus particles or virions are typically in the - nm size range, and are a good example of nanoparticle bioaerosols. smaller viruses typically contain one subunit, which consists of an outer protein capsid, internal nucleic acid (e.g. dna and rna), and other internal proteins. corona virus that causes sars and influenza are good examples of them. the viruses most often are transmitted through direct contact with an infected person, such as by shaking hands, hugging or kissing, while sometimes it is spread by nasal droplets. however, it is still unknown how these virus particles behave in the case of airborne infection. recently, the studies that attempt to elucidate the behavior have been progressing [ ] . this section describes the sources of nanoparticle generation in industrial processes by categorizing them into specific processes where a cleanroom is used and other general ones. the sources of emission of unwanted nanoparticles in general workplaces are categorized as fumes from hot processes (e.g., smelting, refining, and welding) and from (incomplete) combustion processes. favorable conditions required for the generation of nanoparticles are found in workplaces where there is ( ) presence of vaporizable material, ( ) sufficiently high temperature to produce enough vapor, followed by condensation to form an independent aerosol, and ( ) rapid cooling and a large temperature gradient. there have been so many studies on occupational exposure to fine particles in the field of public health. in general, high spikes of nanoparticle concentration are observed during active operations, followed by a gradual decay after the operation, primarily because of coagulation, evaporation, dilution, and/or deposition. the fraction of the total number of nanoparticles generally decreases, whereas that of the number of submicrometer particles increases with time and distance from the point of emission. in order to accurately estimate exposure, the effects of spatial and temporal changes will need to be evaluated. therefore, it is important to identify the time required for the concentration to decline to the normal or background levels. as an example of reports on grinding processes, fig. . . shows the case where a steel substrate was ground upon using a high-speed grinder [ ] . from the figure the distribution of concentration of generated particles has a distinct bimodality, one with the finer peak at around nm and the coarser one at around m. the former results from within the grinder motor and the volatilization or combustion of amenable ground substrate and/or grinding materials, the latter from the mechanical abrasion and attrition. however, the resultant total concentration on the order of particles/cm is not so high. . . . industrial processes with cleanrooms cleanrooms and associated controlled environments (e.g., in the case of an iso class cleanroom, the maximum permissible airborne particle concentration is less than particles/m for particles with the size of . m or larger, while the airborne particle concentration in ordinary indoor environments is on the order of particles/m or higher) are usually adopted to avoid particle contamination in industrial processes where precision products such as engineered nanoparticles, semiconductors, and other electronic or optical devices are fabricated because the deposition of particles onto product surfaces causes their yield reduction and quality deterioration. the emission sources in cleanroom environments are tabulated in table . . . since some of the listed emission sources emit trace amounts of nanoparticles, these nanoparticles are not regarded as particulate contaminant but as chemical or molecular one. in this section, these nanometer-sized solid substances formed on solid surfaces by chemical reaction are also included. size distribution of nanoparticles generated when a steel plate was ground with a high-speed grinder. ( ) air exhaled by humans emissions from human bodies are a minor contribution in ordinary indoor situations because airborne particle concentration in such places is quite high, whereas the emission cannot be seen as negligible in cleanroom environments. the major human emissions are thought to be atmospheric dust deposited on clothes and skin fragments, and most of these particles are submicrometer in size. meanwhile, particles in exhaled air are composed of fine liquid droplets from spittle ( . % of water), and then evaporate to form nanoparticles of nvr. in fig. . . an example of size distributions of particles in exhaled air before and after smoking is shown [ ] . when measuring particles in exhaled air, the air was introduced into a measuring device after drying them by passing them through a diffusion dryer. the size distribution of particles in exhaled air before smoking (n db ϭ ) has a bimodality, one with the peak size of . m, and the other of nm or smaller. the former peak comes from atmospheric aerosols as it decreases with the increase in number of deep breaths in a clean booth (n db ). the latter originates from nvr particles of spittle droplets. incidentally, since smoking induces the rapid increase in number concentration of particles . m or larger by times or more and for nanoparticles by about double, special attention should be paid to the management of personnel's clothes such as face mask when they enter a cleanroom after smoking. ( ) emission from ionizers ionizers are commonly used in cleanrooms to eliminate electrostatic charge on substrates for precision electronic devices. the most popular ionizer is a corona-discharge type. corona-discharge type ionizers are categorized into the following three groups; ac, dc and pulsed-dc types. the issues of emission of contaminants such as ozone, no x and particles have been pointed out [ ] . these issues are also applicable to air cleaners using a corona discharger. among these problems is that the particle emission has a potential for particle contamination onto product surfaces and eventually decline in product yield. the particle emission, which has been studied since the s, is caused by foreign particle deposition onto electrodes, electrode erosion, and gas-to-particle conversion. the issue of electrode erosion can be solved by the improvement of electrode materials, whereas for the issue of gas-to-particle conversion, the airborne molecular contamination (amc) control to be ionized has to be made. it was reported that fundamentals ch. environmental and safety issues with nanoparticles corona-discharge ionizer (e.g. gas-to-particle conversion of low-molecular-weight cyclosiloxane) boron-containing particles from borosilicate glass fibers of hepa filter haze by chemical reaction on solid surfaces (precipitation of ammonium salt and silica) watermark on wafer surfaces at drying leakage from thin film and nanoparticles processing equipment silicon-containing compounds that precipitated on the electrodes result from the gas-to-particle conversion of low-molecular-weight cyclosiloxane (lmcs) from silicone sealant via corona discharge [ ] . ( ) boron-containing particles from hepa filter with borosilicate glass fibers the use of hepa and ulpa filters made of borosilicate glass fibers prevails in the most cleanrooms. it has been known that owing to chemical reaction in equation ( . . ) bf vapor is formed from glass fiber filters by passing hf gas leaking from wet cleaning equipment through the filters. boron, which is a dopant element for semiconductors, has been thought to be a contaminant that might cause failure in semiconductor devices if it comes from the surroundings. in addition, it has been revealed that trace amounts of boron in the form of boric acid (h bo ) are also formed from the fibers via the reaction with moisture in the surrounding air (equation ( . . )). fig. . . depicts the change in volatilized boron mass from various filters in terms of airborne boron concentration. especially, at the initial stage just after the initiation of ventilation, the volatilized boron mass increases with increase in relative humidity [ ] . boric acid, which is solid at room temperature, is surmised to form in the particulate form. however, its existence was identified only by chemical analysis because it is present only in trace amounts. haze might form on glass surfaces of lenses and mirrors for optical instruments if they are exposed for a long time to cleanroom environments where amcs are not controlled properly. the haze is more likely to bring about the insufficient light delivery onto a surface to be exposed in photolithographic processes. one of the reasons is that ammonium sulfate ((nh ) so ) is formed, which then precipitates on the glass surfaces via chemical reaction of sulfur dioxide (so ) with ammonia or amines. for another reason, hexamethyldisilazane (hmds) used as additive in resist coating or lmcs from silicone sealant is adsorbed, and then decomposed to form silica precipitates on glass surfaces by photochemical reaction during laser irradiation, followed by the unwanted decline in laser penetration [ ] . as another example a report said that tiny projections, which are also known as "haze", with a size of . m or smaller were formed on silicon wafer surfaces owing to the adsorption of organosilicate compounds in thin film formation processes with cvd. it is similarly caused by the precipitation of sio [ ] . ( ) watermarks on solid surfaces during drying when a silicon wafer surface is cleaned with deionized water and then dried in air, a watermark is formed on it via the mechanisms demonstrated in fig. . . . oxygen in air is dissolved and diffused into water droplets or adsorbed water on a wafer surface, followed by the formation of silicate compounds via silanol reaction. the watermark on a wafer surface is detected in the form of nanometer-sized particles by an electron microscope [ ] . ( ) leakage from nanoparticle production processes in regard to the risks to processing equipments by nanoparticle leakage from production processes, the vdi report in germany [ ] has been described in detail. the production of engineered nanoparticles can be generally categorized into two approaches. one is a "top-down" approach that is initiated with a bulk material and then breaks it into finer pieces using some form of energy such as etching, ball milling, sputtering, and laser ablation. the other approach is to synthesize materials from the atomic or molecular level by growth and assembly to form the desired nanoparticles. processes included in this "bottom-up" category are sol-gel, chemical vapor deposition, flame synthesis laser pyrolysis, and so on. most of these processes are performed in a closed reaction chamber installed in a cleanroom or associated controlled environment. human exposure to these engineered particles does not take place during synthesis unless there is an unexpected system failure (e.g. rupture of a seal). human exposure is more likely to occur after the manufacturing when opening the reaction chamber, drying the products, or in the post-process handling of the products. the release of nanoparticles during production chamber cleaning operations is another critical point. cleaning typically involves using water or some solvent. brushes, sponges, or tissues used in the cleaning will carry nanoparticles into the waste stream. disposal of the waste and wastewater may become a source of nanoparticle release into the environment. further, conditioning of nanoparticles such as compression, coating, and composition to form final products may also result in the release to the environment and resultant exposure although very few studies have been carried out on this subject. recent studies [ ] to evaluate the aerosol discharge during the handling of carbon nanotubes showed that the generation of nanoparticles occurred under vacuum to remove spilled nanotube materials or vigorous mechanical agitation. however, they reported that the concentrations were very low. in addition, measurements of nanoparticle levels during final packaging of carbon black, which is a typical engineered nanoparticle material, showed that there was no increase in nearby air [ ] . study on the safety of nanoparticles has started only recently, and no sufficiently systemized results have been obtained. what should be noted in particular is that the possibility of radial troubles caused by particulate matters are considered to increase by the decrease of particle diameter in nanoparticles. one typical example is the problem of dust explosion, caused by the high surface reactivity of fine particles. in other words, since nanoparticles are extremely fine particles, dust explosion is more likely to occur. explosion is more likely to occur because fine particles are different in their composition, in that low boiling point metal can be easily condensed, as described in section . . however, of particular note here is that, since all particles do not necessarily exist independently in the form of a single particle, the possibility of dust explosion does not simply increase as particles become finer. fine particles with sizes of m or smaller such as nanoparticles have an extremely high agglomeration propensity and secondary particles can be easily generated. therefore, in some cases they conversely behave like large particles. these are the points to be taken into consideration when studying the problems caused by nanoparticles. as shown in fig. . . [ ] , the effect of the particle diameter on dust explosion tends to be that the smaller the particle diameter of the dust, the lower the minimum explosion concentration. in other words, explosion can be induced under conditions of lower concentration of particles in air as the particle diameter becomes smaller. due to the difficulty of conducting experiments to suspend particles with the same size in a uniform concentration, this result was obtained from particles far larger than nanoparticles; however, it has been clarified qualitatively that the smaller the particle diameter, the higher the possibility of dust explosion. from the perspective of composition, the possibility of explosion increase if materials that react easily with oxygen at low temperature are condensed into particles of small diameter. therefore, with regard to the effect of particle diameter on dust explosion, more careful attention needs to be paid in the case of combined materials than in the case of uniform materials, as assumed in fig. . . . as described before, however, since nanoparticles are considered to exist often as agglomerates, it is necessary from the perspective of the particle diameter to take into consideration the diameter of not only primary particles but also of particles after agglomeration. to address the safety of nanoparticles, it will be important in the future to elucidate their behavior in detail including these factors. the terms 'nanoparticles' and 'nanomaterial' have been used for particles of which one representative dimension, for example, diameter of particles on cross-sectional diameter of fibers has at least nm or less. some people hold that the majority of such fine particles are exhaled without depositing in the respirator tract, and that therefore the particles may not cause pulmonary diseases. however, the properties of nanoparticles are known to be different from the bulk material they are derived from. in cases where the biological effects of bulk materials have been reported, nanosized particles of these materials may be expected to have stronger dose response for the health effects. every effort must be made to clarify the uncertainty on the risks of these nanomaterials [ ] . at the present time, there is no regulation or standard for assessing the biological effects of nanomaterials, and therefore there is a paucity of toxicological data concerning nanomaterials. much more systematic and strategic studies are needed to enable risk assessments for human health [ ] [ ] [ ] [ ] [ ] . as regards risk assessment and risk management of nanomaterials, the characterization and identification of anticipated risks should be first determined for chemical substances or foods. conventional assessment methods are applicable for water-soluble particles. for insoluble nanoparticles, the assessment of potential health hazards should be made based on their properties or toxicity and dose-response relationship. the risk is a product of hazard and exposure; even if a nanoparticle has a hazard, the risk is lower when the possibility of exposure to the nanoparticle is small [ ] . influence of particle diameter on lower limit of particle concentration for explosion. ( ) exposure routes for nanoparticles nanoparticles can either be deliberately introduced into the body for medical purposes (drug delivery systems) or absorbed involuntarily from the environment (inhalation of nanoparticle-containing dust in the air). a distinction should also be drawn between nanoparticles manufactured for industrial application and those unintentionally generated and released in the environment, such as welding fumes or diesel exhaust particles (dep). in the fields of environmental science and toxicology, numerous studies on the potential health hazards caused by ultrafine particles have been conducted. practically, there are several definitions of nanoparticles or ultrafine particles, however, findings regarding biological effects of the ultrafine particles are useful as a starting point for estimating the effects of nanoparticles on human health. human and animals contact with nanoparticles through various routes: nanoparticles can be inhaled in the air, swallowed in the water, ingested in food, and absorbed via the skin in cosmetics. for successful risk assessment, it is important to determine how nanomaterials or nanoparticles are used, such as composites, surface coating, or powders. coatings or powders have the potential to release a part of their nanomaterials into the environment. workers who come into contact with nanomaterials have the possibility of exposure to nanoparticles at the workplace. consumers of products using nanotechnology can also be exposed to them. attention needs to be paid to the environments and ecosystems in which nanoparticles and nanomaterials are released. nanoparticles in the products may change their size, quantity, and composition during their life-cycle of manufacturing, use, transportation, and disposal. ( ) respiratory uptake of nanoparticles inhalation is the main route of exposure to nanoparticles. particles inhaled with the air through the mouth and nose pass through the throat (nasopharynx and oropharynx) and tracheobronchial tree before reaching the alveolar region where oxygen moves from the alveoli to the blood and carbon dioxide moves from the blood to the alveoli. how deeply particles can penetrate and where they become deposited on each respiratory airway such as the nasal cavity, tracheobronchial tree, and the alveoli depend on their size under the various deposition mechanisms: inertial impaction, gravitational sedimentation and diffusion, etc. the respiratory airway includes the anterior nasal passage, posterior nasal passage, pharynx, larynx, trachea, main bronchi, bronchi, bronchioles, terminal bronchioles, alveolar duct, and alveoli, as shown in fig. . . . in the human lungs, the trachea divides asymmetrically into the right main bronchus that enters the right lung where it divides into three lobes, that is, an upper, a middle, and a lower, and the left main bronchus that enters the left lung where it divides into two lobes, that is, an upper and a lower. the trachea divides into two branches, dividing progressively to the terminal alveolus. to quantitatively assess the pulmonary particle deposition needs human lung morphology models, respiratory physiology based models of the entire lung airway system and aerosol deposition models based on many experimental findings. in , the icrp task group on lung dynamic (icrp: the international commission of radiological protection) published their revised lung model [ ] . the deposition, clearance, and translocation of particles in each of the compartments were described. while the model has been widely used in the nuclear field, it is applicable to conventional aerosols as well as radioactive aerosol. in the nuclear field, aerosols including radon progeny that used to be nanoparticles have been studied. fig. . . shows the deposition fractions of inhaled particles per adult nasal respiration of . m /h in each region re-calculated for the nasopharynx, tracheobronchial, and the pulmonary (alveolar) region based on the model. inhaled aerosol particles deposit on different regions depending on their size; for example, nanoparticles larger than nm deposit mostly in the alveoli and those less than nm deposit in the nasal cavity. how deeply particles penetrate into the lung depends on their size. nanoparticles can reach pulmonary region in the lung and deposit more intensively and this, therefore, has become one of the reasons for concern about the effects of nanoparticles on human health. however, deposition of nanoparticle chain-like agglomeration and fibrous particles such as carbon nanotubes cannot be estimated by this model. most inhaled nanoparticles are deposited on the surface of the respiratory tract. generally, if insoluble particles are deposited in the ciliated airspaces which are lined with a mucous layer, they are transported to the digestive tract with the mucous flow by mucociliary movements. particles deposited on nonciliated bronchioles and alveoli are phagocytosed by macrophages, which is a kind of white blood cell. as a result, their residence time is longer, however, they are usually transported to the ciliated upper part of the respiratory tract. removal of deposited particles described above is called clearance. when the amount of deposited particles is below a certain value, no health effect is produced. in relation to a macrophage response to particles, crystalline silica is hazardous, whereas titanium dioxide is not. pneumoconiosis is a well-known lung disease that is caused by exposure to dust particles of several micrometers in diameter. silicosis is a typical form of pneumoconiosis resulting from exposure to crystalline silica dust and characterized by a progressive fibrosis of the lungs. the macrophage-mediated clearance (phagocytes) was effective for micron and submicron particles. it has been reported that only % of deposited nanoparticles were removed by the clearance mechanism [ ] . it has been suggested that the remaining nanoparticles may pass through the alveolar walls, penetrate into the blood or lymphatic circulation, and be transported to other organs. many studies have shown that the smaller the particle size the greater the mobility or they pass easily through the alveolar wall and enter into the bloodstream. it is further presumed that the mechanism of health effects of nanoparticles on cardiovascular system other than the respiratory tract is similar to that in the airborne ultrafine particles from dep. in japan, two reference values, the 'administrative control levels' (acl) and the 'occupational exposure limits' (oels), are used for the regulation of hazardous chemicals as well as dust (particle matters). oels are recommended and revised every year by the japan society for occupational health. oel (oel-mean) for mean concentration of a chemical substance is defined as the reference value to the mean exposure concentration at or below which adverse health effects caused by the substance do not appear in most workers working for h a day, h a week under a moderate workload [ ] . the 'threshold limit value' (tlv) has the same definition (but may not be the same values as oel of the same substance) provided by the american conference of governmental industrial hygienists (acgih). acl is an index to determine the control class to judge the propriety of the working environment control based on the results for working environment measurement, which have been implemented for the unit work area in accordance with the working environment measurement standard. the results of working environment measurement are evaluated by classifying the working environments concerned into three control classes (control class i, ii, and iii). these classes are used as the standards to classify the level of the working environment concerned. among those subject to working environment measurement, these standards apply to workplace where dust, lead, organic solvent, and specified chemical substances are used. article of the industrial safety and health law stipulates that certain workplaces in which harmful substances are involved or harmful work operations are performed shall be the subject to working environment measurement. a % cut-off size of the dust particle is set at m for both standards and is far larger than nanosized particles. oel or acl [ ] for particulate matters are usually based on mass concentration, that is, milligram per cubic meter. therefore, if the particulate matters have broad size distribution, the contribution of nanoparticles is not large to in terms of mass of particles. evidence from a number of toxicological studies on insoluble particles indicates that the primary determinant of the health effect of particles depends on the surface area of particles deposited [ , ] . on the basis of the results from a number of in vitro studies of insoluble nanoparticles, a hypothetical cellular interaction has been proposed [ ] : inflammation and oxidative stress can be mediated by several primary pathways: ( ) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; ( ) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; ( ) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or ( ) intracellular distribution of insoluble nanoparticles to mitochondria generates oxidative stress. in the workplace, the concentration of nanoparticles may be at a high level and most of the nanoparticles become agglomerates, while nanoparticles will form single nanoparticle at low levels in the general environment. it is a matter of debate whether agglomerates of nanoparticles react as a larger particle or a single nanoparticle in the lung or other organs. if insoluble particles are retained in the lung for a longer time without enough clearance mechanisms, they can cause pulmonary inflammation or pneumoconiosis. it is of interest that nanoparticles deposited in the lung can move into the blood vessel through alveolar epithelium and they can damage vessels or produce blood clots [ , ] . in a recent study, nanoparticles deposited in the nose may move directly to the brain via the olfactory bulb [ ] . ( ) biological effects of fullerene the biological effects of fullerene have being investigated intensively. in rats dosed orally with radioisotope-labeled c fullerenes, most were excreted in the feces and some were found in the urine. a small amount of them can be absorbed via the gastrointestinal tract. in contrast, in the same study, % of the same labeled fullerenes administered intravenously were retained after week, with most found in the liver [ ] . ld s (acute toxicity) by intraperitoneal injection in mice and rats were . and . g/kg, respectively. the dose of . g/kg orally in rats did not result in death. the reproductive translocation of fullerenes was also observed in mice. fullerenes have shown mutagenic activity in ames tests. fullerenes have shown no skin irritation or allergic reactions [ ] . on the other hand, fullerenes are being tested for possible medical use. fullerenes are basically hydrophobic but water-soluble derivatives have been synthesized to be used as drugs or its carrier. the derivative can be anticipated as drugs, for example, anti-aids drug. it has been stated that the toxicity of fullerenes changes due to slight structural changes including chemical modification [ ] . ( ) biological effects of carbon nanotubes carbon nanotubes are chemically stable and are similar in form and size to asbestos; these characteristics have given rise to concern that carbon nanotubes may have the potential to cause pulmonary diseases such as lung cancer and mesothelioma similar to asbestos. a few data are available concerning the biological effects of carbon nanotubes. the biological effects of carbon nanotubes are being researched. epithelioid granulomas and interstitial inflammation are induced in mice and rats following exposure to single-walled carbon nanotubes [ ] [ ] [ ] . untreated carbon nanotubes contain the nanoparticles of transition metals such as iron and nickel, which are used as catalysts in forming carbon nanotubes. these nickel-containing carbon nanotubes have been reported to be toxic [ ] . the concentration of airborne asbestos fibers is expressed as a number concentration, that is, fibres per cubic centimeter or fiber per liter. when fiber concentrations are determined by phase contrast light microscopy, the fibers with a diameter of less than m, a length longer than m, and a lengthto-diameter ratio (aspect ratio) greater than are counted [ ] . asbestos fibers having nanosized diameter were often observed in analyses of environmental samples using electron microscopy. international agency for research on cancer (iarc) rated asbestos as a known human carcinogen (group ) [ ] and the concentration of chrysotile asbestos is expressed as a risk level of . fiber/cm [ ] . health effects of vitreous fibers and other asbestos substitutes have been assessed to determine their oels or their carcinogenicity in humans. the health effects of carbon nanotubes are being intensively investigated now. the oel for carbon black respirable dust is mg/m and these for activated charcoal and graphite are . mg/m in each [ ] . in the ref. [ ] , while rats and mice inhaled carbon black with a particle diameter of ϳ nm at a concentration of - mg/m did not produce any specific changes, particles (agglomerate of small particles) of ϳ nm at a concentration of - mg/m produced early pulmonary changes. micronsized titanium dioxide particles are thought to have almost no toxicity and often used as a negative control substance. the oel for titanium dioxide is mg/m for respirable fraction [ ] . however, the results of a series of studies by oberdörster et al. [ , , , ] on submicron-and nanosized titanium dioxide suggested that as size decreases, inflammatory effects are intensified, and normally nontoxic substances may assume hazardous characteristics. fig. . . shows a part of the results by oberdörster et al. in which rats and mice were exposed to anatase titanium dioxide particles [ ] . their results have been frequently cited in the discussion of whether the health effects of fine particles should be based on its mass or its surface area. in fig. . . , percentages of neutrophils in lung lavage of rats are shown as indicators of inflammation after intratracheal instillation of different mass doses of and nm tio particles. the steeper dose response of nanosized tio particles is observed than for submicron tio particles when the dose is expressed as mass (fig. . . a ). if the same dose-response relationship as in fig. . . a is indicated as particle surface area (fig. . . b), the particle surface area seems to be a more appropriate dosimetric for comparing effects of different-sized particles of the same chemical structure. zinc oxide is a white powder and used in pigments. the oel is mg/m for its respirable fraction [ ] , and the value for zinc oxide fume which causes metal fume fever is under consideration. nanoparticles of transition metals and rare earth elements and their oxides will be used widely. since many of these metals and their oxides have biological effects, particular attention should be given to them. nickel compounds are rated as a human carcinogen (group ) by iarc. in particular, nickel oxide is particularly insoluble among the nickel compounds and remains longer in the lung. nanosized nickel oxide particles have greater toxicity to the lung than larger particles [ , ] . pulmonary inflammatory responses induced by nanosized cobalt particles have been reported [ ] [ ] [ ] . biological effects of nanosized particles of other transient metals such as iron and manganese have received attention [ ] . rare earth elements are a general term of chemical elements consisting of scandium (sc), yttrium (y), and a lanthanide series of elements from lanthanum (la) to lutetium (lu). these elements have been used in magnetic alloy, fluorescent and hydrogen storage alloy. particularly cerium oxide nanoparticles are frequently used as a fuel additive and are incorporated in cosmetics formulation. the potential biological and environmental effects of these elements have not sufficiently been investigated. it has been demonstrated that ld s for these elements in oral and intravenous administration are in a range from several dozens to several thousands milligrams per kilograms, indicating that none of these elements has high toxicity. in the results of studies in which the biopersistence and the distribution of rare earth compounds in the body were investigated, for example, the compounds deposited in bones and teeth, and organs including lung, liver, spleen and kidney following intratracheal, oral, intravenous, and intraperitoneal administration. although the compounds deposited predominantly in the liver other than bones, it has also been reported that the distribution of the compounds in the lung and spleen increased when the dose was increased [ ] . the demand for indium compounds has been sharply increasing. the compounds have been used in the materials for transparent electrodes for flat panel displays. in japan, the cases of pulmonary interstitial pneumonia and pulmonary fibrosis have been reported in workers engaged in cutting and grinding of sintered indium-tin oxide (ito) and potentially having inhaled the dusts released from ito [ , ] . the biological effects of indium arsenic compounds and indium phosphorus compounds also have been investigated. the acgih has proposed a value of . mg / m for their tlv, and the value has been applied tentatively in japan. we have been experiencing amazing progress of the technology on the processing for the nanometer-sized materials. the applications cover even biomedical engineering in addition to information technology, material, environmental science, and energy production [ ] [ ] [ ] [ ] [ ] . as the result, many kinds of new materials have been designed, fabricated, and discarded. from now on, this movement will be accelerated and even more new functional materials will be distributed in the world. here, we should not forget the safety of those materials in the process of the production, usage, and discard. without this safety assessment, we will go into the same problems as that of asbestos just we are facing now. first of all, we have to conduct experiments to reveal the minimal concentration for emergence of the toxicity. in other words, we have to fix the standard value for the threshold concentration for each material first of all. if we do not fix it, we should not use the material at any concentration, which means any engineering process could not be carried out. the applications in the various fields have started all over the world, and the safety assessment is urgently needed. in this article, we introduce the methods of the safety assessment of the semiconductor nanoparticles and describe the safety and the threshold depending on the surface treatment. the production process and the surface treatment play one of the most important roles for the safety of the nanoparticles. cd and se semiconductor nanoparticles coated with zns are one of the most widely used for the strong intensity of the fluorescent activity. cd oxide and se compounds once dispersed in the tri-octhil-phosphine oxide (topo) heated up to Њc generate nanoparticles by self-assembly. then zns enhances the stability of the structure and raises up the fluorescent intensity than without the coated one. nanoparticles, thus manufactured, as materials for a novel memory in the field of intelligence technology (it), and as super-micro devices for laser in the field of optics, have been developed all over the world [ ] [ ] [ ] [ ] [ ] . these nanoparticles cannot be dissolved into water, but dissolved into organic solvents like toluene. therefore, for biological and medical applications, various technologies for surface-conjugations to make them hydrophilic [ , ] have been developed. for example, nanoparticles covered with topo are hydrophobic because an alkyl group on them is hydrophobic. therefore, a technology for replacing this alkyl group with hydrophilic carbonic acid (making the whole particles soluble in water) has been developed [ ] . nanoparticles, thus surface-treated, can be dissolved into water, like sodium salt or potassium salt. with this method, various kinds of materials have been surface-conjugated. the mtt assay method is a way to evaluate the hazard assessment of nanoparticles, in which the activation metabolism in a mitochondrium in a cell is measured and the influence of nanoparticles on the prolification of the cell is qualitified. the mtt is a kind of tetrazolium, whose molecular formula is c h brn s. taken into a cell, it is decomposed by a dehydrogenase enzyme in a mitochondrium into a pigment called 'hormazan'. the measurement of the fluorescence intensity of the pigment shows the prolification of the cell [ ] [ ] [ ] . fig. . . . shows the hazard assessment of vero cells and kidney cells of the african green monkey against cdse/zns nanoparticles. the horizontal axis shows the concentrations of the nanoparticles and the vertical axis the fluorescence intensity of the hormazan at nm, that is, the metabolism of the cells. the figure indicates that cyto-toxicity is not observed for cells when the concentration is less than . m. this result means that this concentration is the threshold of the cell toxicity. likewise, cyto-toxicity is not observed for the hela cells and for the human primary cells. further, in order to find out how the sizes of nanoparticles influence the cyto-toxicity, the cyto-toxicities were evaluated with three kinds of quantum dots; one whose fluorescence wavelength is nm, red, one with nm, yellow, and the other with nm, green. the following results were obtained. the largest quantum dots whose fluorescence wavelength is nm show a tendency to give cyto-toxicity. cyto-toxicity is observed at concentrations more than m [ ] . another method to evaluate cyto-toxicity is the flow cytometry [ ] . the mtt assay alone cannot tell whether the toxicity observed is lethal to the cells or just restrains the prolification of them. in the flow cytometry, the nuclei of dead cells are dyed with propidium iodide (pi) after the nanoparticles are taken in and the ratios of the dead cells are measured. fig. . . shows the lethal cyto-toxicity of mua conjugated nanoparticles ( nm, green) against vero cells. the vertical axis indicates the numbers of the cells, and the horizontal axes show the fluorescence intensities and the cyto-toxicities. these experiments also show that dead cells cannot be observed at concentrations less than . m even though nanoparticles are taken in, as was shown in the mtt assay. however, at concentrations more than m, the nanoparticles taken in cause damage to more than the half of the cells. that is, the cyto-toxicity of mua quantum dots against cells is lethal [ ] . nanoparticles have been surface-conjugated for applications for various uses. some surface-conjugations cause more grave cyto-toxicity than others. therefore, relations between surface-conjugations and their safety for cells have to be considered. in order to find out the relations, the safety evaluations of nanoparticles surface-conjugated with two materials were made; one is with mua (quantum dots-cooh) and the other is with glycerol (quantum dots-oh), and their purified and unpurified particles. fig. . . shows that the purification reduces the cyto-toxicity for the quantum dots-oh, and that the toxicity remains the same after the purification for the quantum dots-cooh. mua itself, a material with which particles are conjugated, has cyto-toxicity. this experiment shows that toxicity against cells is connected not only with particles themselves but also with kinds of surfaceconjugations and degrees of purification. it is shown in the above experiments that the toxicity of nanoparticles is lethal to cells. next, we found out by the comet assay method whether the toxicity is derived from damaged dna. the method is a way to evaluate quantitative damage of dna by electrophoresis. the fragmented dna seeps out of their cells by treating cells, whose dna has been fragmented, with agarose-gel to break their cell membrane, and then electrophoresing them. it looks like the "tales of comets". cells, whose dna has not been fragmented, have their nuclei keeping their spherical shape after electrophoresis, and the tales of comets cannot be observed [ ] [ ] [ ] [ ] . fig. . . shows the results of the experiments with quantum dots conjugated with cooh (both purified and unpurified) to wtk- cells, a human lymphoblast mutation strain [ ] . the vertical axis shows the lengths of the tails, and the horizontal axis the concentrations of the quantum dots. the concentration of the nanoparticles is m. the results are as follows: the unpurified quantum dots with cooh caused damage to dna. on the other hand, the electrophoresis with the purified quantum dots does not show dna damage. this is probably because the dna damage has been repaired during the longer cultivation time. conducted with quantum dots conjugated with mua, and quantum dots-nh with an amino group. dna damage was not observed in either case. the results indicate that as far as particles themselves do not break down, the cyto-toxicity of quantum dots is derived from the chemical properties of the materials covering the quantum dots. the safety evaluation of nanoparticles has not been conducted sufficiently. as indicated above, the procedure for surface-conjugation could apply not only to cd/se nanoparticles but also to other nanoparticles. today, various kinds of techniques for surfaceconjugations have been available in academic papers, proceedings, and on the internet. some of them are widely known and others are patented. those techniques are all shared among the human race. even at this moment, the human beings are making breakthroughs in various fields and developing different kinds of technologies. sharing these technologies will lead to still more speedy developments of yet more advanced technologies. to achieve it, these technologies should be so structured that different fields, for example, bioimaging and biotechnology, structured on their own, can be linked to each other. such structured knowledge will play an essential part in merging different fields. in order to prevent nanoparticles release from a system so as to maintain environmental safety, the removal technique of nanoparticles must be established. in this section, separation techniques of particles from exhausted or suspended gas and liquid are described focusing on particles less than nm. generally, as shown in fig. . . , all particle separators for a dispersed system employ either one of three basic forms of particle separation. on the left hand side of the figure lie the separation methods in which particles are collected only by force field (electrostatic force, centrifugal force, gravity force, etc.), and the representative separator is electrostatic precipitator (esp). if some obstacles (collectors) are placed into the particle laden stream, particle separation is facilitated because particles are collected on obstacles with a smaller deviation from the fluid flow by the force exerting on the particles compared to the case without obstacles. typical collectors of this form are air filter, deep bed filter, etc. on the right hand side of the figure lie separators that collect particles utilizing only sieving effect of obstacles without any force field. in this case, geometrical size of channel between the obstacles must be smaller than that of particles. membrane filter, fabric filter, etc. belong to this group. when we apply the above collection forms to nanoparticles, the major collection mechanisms are brownian diffusion and electrostatic force for particles in gas, while sieving effect and interception/adhesion forces for those in liquid. as mentioned above, most airborne particles are collected by separators utilizing various kinds of forces such as gravity, centrifugal force, electrostatic forces, inertia, brownian diffusion force, and so on. therefore, the migration velocities or displacement of a particle per second due to the individual forces gives the basis for the comparison of removal efficiencies due to each force. in fig. . . , migration velocities of particles due to various forces are depicted against particle diameter at normal temperature and pressure for particle density of g/cm [ ] . as seen from the figure, the velocities due to gravity, centrifugal force, and inertia monotonically decrease with decreasing particle diameter, suggesting that the removal of nanoparticles with these forces is difficult. on the contrary, the velocities due to brownian diffusion and electrostatic forces increase with decreasing particle diameter for particles less than nm. this suggests that brownian diffusion and electrostatic forces are most effective in collecting nanoparticles. fig. . . summarizes typical conventional dust collectors. among them, the effective collectors for nanoparticles are esp and fabric/air filter. however, for the case of esp, which relies on only electrostatic force, nanoparticles (Ͻ nm) fail to carry even one electron resulting in low collection efficiency. in this case electrically charged filters are effective because we can expect the combined effects of electrostatic forces and brownian diffusion. among charged filters, so-called electret filter, which consists of permanently polarized fibers, is the most favorable filter because of its charge stability. particle penetration data of electret filter are plotted against particle diameter in fig. . . and compared with that of uncharged filter with the same structure. for the three combinations of charged states of fiber and particle there exist the most penetrating particle diameters. for the uncharged fiber, collection efficiency of uncharged particle has a minimum at nm and increases with decreasing particle diameter, showing that nm is the most penetrating particle size. for the charged fiber, particle collection efficiency is very high even for uncharged particle, and the efficiency for charged nanoparticles is extremely high because of strong coulombic force between fiber and particle. the experimental data plotted in fig. . . are qualitatively in good agreement with the theoretical prediction following the particle size dependency on particle migration velocity (shown in fig. . . ). however, as particle size becomes smaller and comparable with the size of a molecule, particles may rebound on a collector surface, and the adhesion probability of particles drops, resulting in a decrease in collection efficiency. fig. . . is an example of experimental data that confirm the particle rebound [ ] . the figure shows the penetration of nanoparticles through a grounded circular tube. the solid curve is the theoretical line derived by assuming that particles are deposited from a laminar flow in a tube by brownian diffusion. it is evident that experimental penetration deviates from the theoretical line for particles less than nm. this means that molecular behavior begins to appear when the particle size becomes as small as nm, and as a result, the collection efficiency is reduced. it should be noted that considerable amounts of nanosized particles are contained in diesel exhaust particles (dep), possibly penetrating through the honeycomb type (tubular channel) diesel particulate filters (dpf). there are two types of methods that differ in the way the nanoparticles in liquid are collected. the first group, called membrane filtration, constrains the particles by a membrane, and the liquid is allowed to flow freely through the membrane. in the second group of ultracentrifugation, the liquid is constrained in a rotating vessel, and the particles move freely within the liquid by an external field of acceleration caused by ultracentrifugal field. these methods have been quite extensively used in separation of macromolecules and molecules from liquid, and they are recently becoming important also in separation of nanoparticles from liquid. in pressure-driven membrane filtration processes, the pressure gradient across the membrane would force solvent and smaller species through the pores of the membrane, while the larger molecules/particles would be retained. membrane filtration processes are usually classified into three general categories according to the size of separating components, as shown in fig. . . . microfiltration (mf) is designed to retain suspended particles in the range of nm- m. ultrafiltration (uf), on the other hand, retains macromolecules or nanoparticles in the range of - nm (nominal molecular weight cut-off (nmwco) ranging from , to , , da). nanofiltration (nf) is a relatively new process that uses charged membranes, and it covers molecular sizes ranging from . to nm (nmwco ranging from to , ). it is useful in that it can separate dissociated forms of a compound from the undissociated form. one of the major factors limiting the use of membrane filtration is membrane fouling, resulting in a dramatic decline in flux with time of operation. to account for the membrane fouling, the resistancein-series model is frequently employed. the resistance model becomes ( . . ) where u is the permeate flux, v the filtrate volume per unit membrane area, the filtration time, p the applied transmembrane pressure, the viscosity of the permeate, r t the total resistance, r bm the resistance of the membrane per se plus the clogging of the membrane pores, r c the resistance of the filter cake, and r cp the resistance of the concentration polarization layer. significance of each resistance in membrane filtration is as follows. the membrane, even in the absence of any suspended particle, has a natural flow resistance. during membrane filtration, particles become attached to the pore channel of the membrane thereby reducing the flow channel dimension, or pores become blocked off completely. the last two effects lead to resistances that are due to adsorption and pore blocking. the blocking filtration model introduced by hermans and bredée [ ] , and grace [ ] is most commonly used as an interpretation of such phenomena. the clogging of the membrane pores is strongly influenced by such solution environment as ph and the ionic strength. the permeate flux of bovine serum albumin (bsa) (pi . , molecular weight , , stokes-einstein diameter . nm) solution by permeable mf membrane (nominal pore size . m) is lowest at around the isoelectric point [ ] . as the bsa molecule carries no net charge at the isoelectric point, the molecule is in its most compact state at that point. the bsa molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. as a result of this, the flow resistance increases markedly at around the isoelectric point. in dead-end membrane filtration, which has a feed and permeate stream, each with the same mass flow rate, the resistance of the filter cake plays a major part in the filtration resistance. therefore, the cake filtration theory can be applied, and thus the permeate flux u is described by ( . . ) where m is the ratio of wet to dry cake masses, s the mass fraction of solids in the solution, av the average specific filtration resistance, the density of the permeate, and v m the fictitious filtrate volume per unit membrane area, equivalent to the flow resistance of the membrane [ ] . for fine particle suspensions, colloidal forces which arise from interaction between the suspended particles control the nature of the filter cake. the average specific filtration resistance av and the average porosity av of the filter cake are strongly affected by the solution properties, including ph and electrolyte strength. for instance, in mf of suspensions of the titanium dioxide (pi . , the original mean specific surface area size nm), av goes through a minimum, and av is much larger near the isoelectric point [ ] , as shown in fig. . . . the titanium dioxide particles are destabilized around the isoelectric point where the van der waals attraction is more dominant. consequently, the particle tends to come together, that is, to flocculate, and the very porous flocs are then formed. thus, it is speculated that the filter cake formed from such porous flocs has often loose and wet structures. on the other hand, the filter cake becomes compact and dry when the particle carries the charge. since the most loose filter cake forms around the isoelectric ph, the filter cake is most permeable. it is interesting to note that the results in protein uf had a distinctly different behavior. in protein uf of bsa solution, the filter cake is in its most compact state around the isoelectric point [ ] , as shown in fig. . . . since the bsa molecules are hydrophilic colloids, their stability in the solution would appear to be influenced not only by the presence of a surface charge on the protein but also by hydration of the surface layers of the protein. the bsa molecules, because of hydrated layers surrounding them, are not destabilized by such consideration as depression of the electrical double layer. thus, the bsa molecules have water bound to them even around the isoelectric point. the hydrophilic bsa molecules maintain a dispersed state in the solution due to hydration of the surface layers of the protein even around the isoelectric point. when a bsa molecule acquires a charge, the filter cake becomes loose and wet due to electrostatic repulsion between the charged bsa molecules. this contrasts to the compact filter cake around the isoelectric point. the average specific filtration resistance av has a definite maximum around the isoelectric point since a compact filter cake provides a large hydraulic flow resistance. most membrane filtration processes are operated in the cross-flow mode, in which the feed is moved tangentially to the membrane surface so that the filter cake is continuously sheared off. during membrane filtration, particles in the feed are brought to the upstream surface of the membrane by convective transport, and this results in a higher local concentration of the rejected particles at the membrane surface as compared to the bulk solution which is referred to as concentration polarization [ ] . the particle concentration in the solution adjacent to the membrane varies from the value at the membrane surface, c m , to that in the bulk feed solution, c b , over a distance equal to the concentration boundary layer thickness . the resulting concentration gradient causes the particles to be transported back into the bulk solution due to diffusional effects. at steady state, the rate of convective transport of particle toward the membrane is balanced by the rate of particle transport through the membrane plus the rate of the diffusive back transport of particle. thus, the permeate flux u is given by ( . . ) where cp is the particle concentration in the permeate, k (ϭd/ ) the mass transfer coefficient, and d the diffusion coefficient. the osmotic pressure model assumes that the deviation from pure water flux occurs solely due to the osmotic pressure difference across the membrane, and thus the permeate flux u is given by ( . . ) where is the osmotic pressure, which is a function of the concentration. equation ( . . ) means that the effective driving force across the clean membrane is pϪ{ (c m )Ϫ (c p )}. replacing pϪ{ (c m )Ϫ (c p )} by the hydraulic pressure at the membrane surface, p m , equation ( . . ) reduces to the cake filtration equation. to minimize the effects of cake buildup and concentration polarization, membrane filtration is usually conducted using the cross-flow geometry in which the feed flow is parallel to the membrane and perpendicular to the filtrate flow [ ] . however, especially in mf the energy requirements associated with pumping the feed (plus any recirculation flow) along the membrane surface are typically very high. thus, there have been some innovations in recent years with cakeless filtration. the rotating disk module in which the membrane disk is stationary is suited for large-scale operation [ ] . it is possible to enhance the permeate flux by using the vibrating modules [ , ] . in the rotating cylinder device with the membrane on the inner rotating cylinder, counter-rotating taylor vortices within the annular gap are available [ , ] . dean vortices that twist and spiral in the direction of flow inside a highly curved channel, similar to vortices in rotary modules can result in enhanced flux [ ] . these vortices, or flow instabilities, induce turbulence into the system. periodic removal of the formed filter cake is also effective for enhancing the permeate flux. recently, several methods have been investigated: back washing using the filtrate or air pressurization [ ] , periodic rotation of the cylindrical membrane [ ] , pulsatile flow [ ] , high-frequency transmembrane pressure pulsing with a frequency around . - hz [ ] . dead-end upward filtration, where the filtrate flow is in the opposite direction to gravity, and dead-end inclined filtration, where the membrane is inclined, can reduce the cake formation onto the membrane in uf of nanoparticulate suspension and protein solutions. in upward uf of silica sol (mean diameter . nm) and bsa solution, a sustained permeate flux is achieved, as shown in fig. . . [ , ] , because the filter cake overlying the membrane is exfoliated continuously under the gravitational force acting on the particles comprising the filter cake. another approach for enhancing the permeate flux is to employ external force fields. electrofiltration, in which an applied electric field is used to drive charged particles away from the membrane surface, has been developed. in electrofiltration, the accumulation of the particles on the membrane surface is limited by the imposed electrophoretic force. in addition, the permeate flux through the filter cake is dramatically enhanced due to electroosmosis as a secondary electrokinetic phenomenon. this method can be applied to a broad combination ranging from mf of particulate suspension such as bentonite [ ] to uf of protein solution. fig. . . shows the reciprocal permeate flux (d /dv) versus the permeate volume per unit membrane area, v, for various values of the strength of the dc electric field, e [ ] . the steady permeate flux increases noticeably with the magnitude of the imposed field strength. also, a higher electric field strength causes the permeate flux to equilibrate more rapidly. a method has been developed for removing humic substances by hybrid uf combined with both flocculation and adsorption treatments, as shown in fig. . . [ ] . flocculation by use of polyaluminum chloride (pacl) is particularly effective for the removal of humic acids, which constitute the relatively high molecular weight fractions of humic substances, whereas adsorption by use of powdered activated carbon (pac) is able to remove fulvic acids of relatively low molecular weight effectively, which cannot be fully flocculated by pacl. hybrid uf in combination with flocculation and adsorption treatments exhibits high permeate quality because the flocs and pac are easily retained by the uf membrane. in ultracentrifugal sedimentation, ultracentrifugal force field of several tens of thousands of revolutions per minute is applied to a rotor. in recent years, ultracentrifugal sedimentation is employed for concentrating dilute protein solutions and for separating proteins and other large biological molecules from low-molecular-weight solutes or from much larger particles. fig. . . shows the results for ultracentrifugal sedimentation of an aqueous solution of the mixtures of bsa and egg white lysozyme (pi . , mw , ) measured using schlieren optics in an analytical ultracentrifuge [ ] . the angular acceleration of the rotor is , rad/s. the symbol r i and r i in the figure represent the distances from the center of rotation to the sedimentation boundary at time and , respectively. the electrical nature of macromolecules plays a significant role in determining the sedimentation behavior in ultracentrifugation of binary protein mixtures. in the ph range where both protein molecules were electropositive, the molecules sediment independently due to the electrostatic repulsive force acting between bsa and lysozyme molecules. denpun kagaku filtr. sep institute for quality assurance and certification: basic criteria for the award of the environmental label (printer ral-uz ) proc. air cleaning contam proc. air cleaning contam proc. air cleaning contam chemical contamination in semiconductor processing environments and its countermeasures the japan society of industrial machinery manufactures: report on behavior control of individual sort of contaminants - report on introduction of advanced technologies to environmental equipment industry industrial application of nanomaterials -chance and risks. future technologies, division of vdi technologiezentrum nanoscience and nanotechnologies: isbn recommendation of occupational exposure limits who: environmental health series . world health organization summaries & evaluations -nickel and nickel compounds rapid colorimetric assay for cellular growth and survival ouyou earozorugaku crossflow filtration: theory and practice encyclopedia of fluid mechanics: slurry flow technology filtr. sep key: cord- -n o ih authors: barker, j.; stevens, d.; bloomfield, s.f. title: spread and prevention of some common viral infections in community facilities and domestic homes date: - - journal: j appl microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: n o ih nan nearly one thousand different types of viruses are known to infect humans and it is estimated that they account for approximately % of all human infections (horsfall ) . viruses are spread easily through closed environments such as the home, schools, workplaces, transport systems, etc. although many of the respiratory and gastrointestinal infections caused by viruses can be asymptomatic or relatively mild and self-limiting (coughs and colds, etc.), they still represent a signi®cant economic burden. increasing numbers of people who have reduced immunity to infection, for whom the consequences of infection can be much more serious, are now cared for at home. at risk groups include not only the immunocompromised but also the elderly, neonates, pregnant women, hospital patients discharged into the community, individuals using immu- nosuppressive drugs and also those using invasive systems (indwelling catheters) or inhalation systems or devices. otherwise healthy family members with asthma or allergies also have increased susceptibility to infection. in the uk it is estimated that one in six people in the community belong to an`at risk' group (bloom®eld ) . world health organisation estimates suggest that, by , there will be more than million people over years old in the world, two-thirds of them in developing countries (anon. ) . viruses are probably the most common cause of infectious disease acquired within indoor environments. close personal contact within the home and community settings, such as daycare centres and schools, makes them ideal places for the spread of viral infections. infected individuals can shed up to virus particles per ml of faeces with the possibility of transfer of the virus by contaminated hands to surfaces in the bathroom or toilet. viruses that cause tonsillitis, colds, croup, bronchiolitis, in¯uenza, pneumonia and other respiratory tract infections can be spread in aerosolized droplets. aerosols produced by coughing, sneezing and talking can be inhaled directly by a susceptible host or may settle onto surfaces. touching hands or fomites, such as eating utensils, towels or doorknobs, inadvertently contaminated with fresh secretions or vomit, etc. from an infected person and then transferring the virus from the hands to the eyes, nose or mouth, are further routes of spread. infants are especially vulnerable to such infections because they frequently place objects, such as toys, into their mouths. transfer of viruses to food during handling and preparation via hands and food contact surfaces is an important route of spread of viral gastroenteritis. amongst health care professionals there is growing awareness that improved standards of hand, surface and air hygiene in community settings could do much to prevent the spread of viral infections within these environments. the purpose of this paper is to review the evidence base for this assumption. since viral infections are not easily treated, prevention of infection is still the main route of control. assessment of the impact of hygiene is made dif®cult by the general lack of quantitative epidemiological data and, even where evidence for cross-contamination as a causative factor in an outbreak exists, it is always circumstantial. a further problem in assessing whether contamination found on hands or other surfaces might represent a hazard is that the infectious dose can vary signi®cantly according to the pathogenicity of the organism and the immune status of the host. thus the case for practising good hygiene in these settings rests largely on evidence showing that cross-contamination can occur in these environments coupled with laboratory data demonstrating the ef®cacy of hygiene procedures in minimizing microbiological contamination. in developed countries it is estimated that ± % of infectious gastroenteritis cases are attributable to viruses (thompson ) . surveillance data from the uk show that reported outbreaks of viral intestinal infection have increased rapidly over the last years; epidemiological data for and (evans et al. ) show that rotavirus, astrovirus, norwalk-like viruses (nlvs; also known as small round structured viruses) and other caliciviruses were responsible for % of all reported outbreaks of infectious intestinal disease (iid). other data indicate that nlvs and rotavirus are the commonest pathogens causing outbreaks of gastroenteritis in homes for the elderly (djuretic et al. ; ryan et al. ; dedman et al. ) . over the period ± a uk study involving some participants was carried out to evaluate rates of iid in the community and presenting to general practice which has given valuable insights into the epidemiology of viral infections in the community (wheeler et al. ) . the study indicated that as many as one in ®ve people in the general uk population develop iid each year with an estimated á million cases occurring annually. it has long been recognized that, since cases and outbreaks related to viral agents are often unreported, the impact of viral intestinal infections may be much greater than national surveillance suggests. the ®ndings of the community study con®rmed the validity of this assumption. wheeler and co-workers estimated that for every one case of rotavirus and nlv reported to national surveillance a further cases of rotavirus and cases of nlv occur in the community. uk surveillance covering ± showed nlv as a signi®cant cause of epidemic gastroenteritis in community residential and nursing homes, accounting for % of all reported general gastroenteritis outbreaks (evans et al. ) . the rate of reported nlv infection reaches a peak in children under years and again in the elderly. foodborne outbreaks can arise from contaminated raw food such as shell®sh and also through secondary contamination from food handlers carrying the virus. foods implicated in outbreaks are mainly those eaten raw, or those not cooked after handling, e.g. salads, cold meats and fruit. worldwide, rotavirus is probably the most important viral pathogen causing diarrhoeal disease in infants, infecting virtually all children aged ± years (parashar et al. a ). however, a recent study by pang et al. ( ) of children between months and years of age with acute gastroenteritis, has shown that human caliciviruses are found as commonly as rotaviruses. in developing countries rotavirus accounts for approximately % of all diarrhoeal episodes and % of all diarrhoea-associated deaths of children under years of age, resulting in an estimated childhood deaths each year. it is second only to upper respiratory infections in infants under years old as a major cause of death in the developing world (glass et al. ) . each year in the us, á million children under years old are affected by rotavirus diarrhoea, resulting in visits to the doctor and hospitalizations (parashar et al. b) . a large proportion of hospital admissions due to gastroenteritis in children under years old were caused by rotavirus in both the uk and hong kong chan et al. ) . in older children % of hospital admissions for acute diarrhoea were associated with rotavirus (lewis et al. ) . a study by isaacs et al. ( ) showed that % of children under years old who visited their doctor with diarrhoea had a rotavirus infection. indications are that hospital admissions only represent a small percentage of rotavirus infections; the majority will be treated by general practitioners. rotavirus infections are highly seasonal, peaking in the winter months (brandt et al. ; ryan et al. ; dedman et al. ) . it has been suggested that low humidity and people spending more time indoors contribute to the spread of rotavirus infections (anon. ) . such conditions may make it possible for the virus to be spread by the airborne route through environmental contamination (brandt et al. ) . a study in the us revealed that rotaviruses infected one or more members in % of families, including % of children and % of adults (rodriguez et al. ) . within infected families rotavirus infection was found in % of children and % of adults. some adults acquired rotavirus infections a few days after their children's illnesses, suggesting that the children rather than the parents brought infection into the home. rodriguez et al. ( ) also found rotavirus infection in % of adult family contacts of children hospitalized with gastroenteritis. in a community study in new zealand, in families with an index case of rotavirus infection, children were more frequently infected than adults. once a family member became infected there was a high probability of crossinfection (grimwood et al. ). among children with diarrhoea attending daycare centres, lew et al. ( ) detected astroviruses and adenoviruses. astrovirus was signi®cantly more common in children with diarrhoea than those without diarrhoea. enteric adenoviruses were detected in an equal percentage in children with and without diarrhoea. children can excrete astrovirus before the onset of diarrhoea and up to d after the diarrhoea has stopped (mitchell et al. ) . although astrovirus primarily infects the young, the elderly can also be affected, with reported outbreaks in care homes and hospital wards for the elderly (gray et al. ; lewis et al. ) . enteric adenoviruses (generally serotypes and ) are also associated with outbreaks of gastroenteritis in schools, paediatric hospital wards and nursing homes (lebaron et al. ). they may be second to rotavirus as a cause of gastroenteritis in young children (blacklow and greenberg ) . globally, hepatitis a virus (hav) is the most common cause of hepatitis in man (melnick ) . contaminated water or food, particularly ®lter-feeding shell®sh, frequently transmit hepatitis a but other foods are occasionally implicated (raw milk, dairy products and cold meats). the virus is excreted in high numbers in faeces and is spread from person to person primarily by the faecal±oral route. when personal hygiene is not observed, food handlers may unintentionally transfer the virus to food during the incubation period of the disease (sundkvist et al. ) . outbreaks of viral hepatitis occur in institutions such as daycare centres, hospitals, nurseries and schools (bern et al. ; dickinson ) . these outbreaks may lead to secondary cases in the general community (hadler et al. ) . infections caused by in¯uenza viruses, rhinoviruses, coronaviruses and respiratory syncytial viruses (rsvs) are a major health burden. estimates suggest that adults suffer two to ®ve colds per year and infants and preschool children have about four to eight colds per year (sperber ) . although such infections are often regarded as trivial, taking into account lost days from work and school, hospital admissions and mortality rates in infants and the elderly, the health and economic costs are considerable. although the common cold can be caused by a number of viruses, rhinoviruses and coronaviruses predominate. rhinoviruses are responsible for outbreaks of the common cold in the general community such as schools, daycare centres and hospitals (denny et al. ; krilov et al. ; kellner et al. ) . rhinoviruses and coronaviruses have been found to cause a greater disease burden in elderly people living at home, compared with in¯uenza virus or rsv (nicholson et al. ) . in¯uenza affects all age groups, but it is the elderly and persons with underlying health problems who are at particular risk from complications of in¯uenza and are more likely to require hospitalization. respiratory syncytial virus infections occur all over the world and outbreaks are common in the cold season in temperate climates and in the rainy season in tropical climates. respiratory syncytial virus is a major cause of respiratory illness in young children, affecting about % of children by the age of years (crowcroft et al. ; simoes ) . school-aged children often carry rsv to their homes and spread infection to younger siblings. attack rates within families are high, with about % of family members, including adults, becoming infected. in most family outbreaks although more than % of infections are symptomatic they are not usually severe (berglund ; hall et al. a) . infants admitted to hospital with rsv bronchiolitis or pneumonia tend to shed the virus abundantly and for prolonged periods allowing ample opportunity for spread (hall et al. b) . in adults rsv infection generally results in a`common cold' type illness although it can sometimes produce a`¯u-like' syndrome indistinguishable from in¯uenza. antibodies resulting from an early childhood rsv infection do not prevent further rsv infections later in life. respiratory syncytial virus is known to cause a high incidence of pneumonia and death in the elderly. in england and wales it is estimated that rsv causes ± % more deaths than in¯uenza, causing about deaths each winter (nicholson ) . parain¯uenza viruses (pivs) are a further major group of respiratory pathogens. they cause severe colds, croup, bronchitis and pneumonia in children and adults and in infants the virus can cause life-threatening disease (hall ) . infection is probably spread by aerosols in addition to direct contact with contaminated surfaces (hall et al. ) . brady et al. ( ) noted that the persistence of piv on hospital surfaces contaminated with patients' secretions was a potential source of transmission. cross infection from an infected person to a new host depends on a number of factors, including the number of virus particles shed by the infected person, their stability in the environment, in aerosols or on surfaces and the potential for spread within a closed environment (valenti ) . viruses that increase¯uid secretions or irritate the respiratory epithelium induce coughing and sneezing, which in turn increases the shedding and transmission of the virus. although diarrhoea eliminates organisms from the gut, it increases the potential for contamination of the environment and spread of the virus infection. the more particles shed the greater their survival and the greater the chance of reaching a new host. equally important, the likelihood of cross infection depends on the number of particles that reach the new host, the immune status of that host and the route by which they become infected. virus particles can be shed in large numbers in various body¯uids from an infected person or a carrier, including blood, faeces, saliva, urine and nasal secretions. the nonenveloped viruses have greater resistance to drying and thus spread more easily than enveloped viruses, which are less stable in the environment. laboratory studies show that rotavirus, adenovirus, poliovirus, herpes simplex virus and hav can survive for signi®cant periods on dry surfaces (nerurkar et al. ; abad et al. ) . although few studies have been carried out in domestic homes, studies in children's daycare centres (lew et al. ; keswick et al. b; butz et al. ) and in hospitals (samadi et al. ; akhter et al. ) show that viruses can survive on surfaces and that virus transfer and survival on hands play a part in the transmission of infections. bellamy et al. ( ) investigated the domestic environment for the presence of viruses and body¯uids that may contain viruses. haemoglobin was found on % of surfaces (taps, washbasins, toilet bowls and seats), indicating the presence of blood and possible contamination with bloodborne viruses. amylase (an indicator of saliva, sweat and urine) was found on % of surfaces, which were frequently handled or in contact with urine. this highlights that surfaces may remain soiled for some time and may not be thoroughly cleaned. bellamy et al. ( ) also detected enteroviral rna in three of environmental samples (tap handle, telephone handpiece and toilet bowl). a previous study has shown that enteric viruses can survive on environmental surfaces for up to d (abad et al. ) . virus transmission in childcare facilities was studied using modi®ed cauli¯ower virus dna as an environmental marker (jiang et al. ) . the viral dna, introduced through treated toy balls, spread within a few hours of handling. although the marker treated objects were removed after d, the viral dna continued circulating in the facilities for up to weeks. hand contact with contaminated surfaces played an important part in transmission. the markers were also detected in the children's homes, on the hands of family members and environmental surfaces, including toys. transmission of viruses in a household setting has been recently studied, using bacteriophage /x as a model virus with resistance properties similar to polio-or parvoviruses (rheinbaben et al. ) . contaminated door handles and skin surfaces were found to be ef®cient vectors of contamination. at least persons could be contaminated one after another by touching a contaminated door handle. successive transmission from one person to another could be followed up to the sixth contact person. transfer from contaminated door handles to other surfaces was also con®rmed under everyday life conditions in a¯at shared by four students. studies focusing on home and community settings are providing a better understanding of how infectious disease is spread in these environments. such studies suggest that the airborne route is by no means the sole route of transmission of respiratory infections caused by rhinovirus and rsv and that iid, particularly that of viral origin, can arise from various sources of which food is only one. evans et al. ( ) reported that, whereas of outbreaks of infection attributed to salmonella were`mainly foodborne' and regarded as`mainly person to person', for reported outbreaks of nlv infection were attributed to personto-person transfer and only were reported as foodborne. although epidemiological and surveillance studies provide vital information on modes of infection transmission during outbreaks, they give only a limited picture of how sporadic person-to-person transmission actually occurs within community and home environments. although such data are limited, it is generally acknowledged that person-to-person transmission is associated not only with poor hand hygiene but also airborne or surface-to-surface transmission. what cannot be deduced from current data is the relative importance of these different modes of person-to-person transmission and how it may differ for different viral agents and different communities (home, daycare, etc.). in particular, it is dif®cult to assess the importance of environmental contamination as a source of secondary cases, but recent data show that this can be a signi®cant factor, particularly in the transmission of nlvs (anon. ) . although direct evidence is lacking, outbreaks in hotels and cruise ships in which recurrent waves of infection occurred in successive cohorts of guests strongly suggests transmission of nlv via environmental sites and surfaces (ho et al. ; gellert et al. ; mcevoy et al. ; cheesbrough et al. ) . these and other studies which can be used to assess the routes of transmission of viral infections in community and home settings are discussed in the following sections. . . rotavirus. rotavirus is shed in large numbers from an infected person, with faeces often containing > particles per gram. children and adults can be asymptomatic excreters of rotavirus (ansari et al. a ) and rotavirus excretion can persist for up to d after diarrhoea has stopped in symptomatic patients (pickering et al. ). more recently, a hospital study showed that % of the immunocompetent children excreted rotavirus particles for more than d and as long as d after the onset of diarrhoea (richardson et al. ) . rotavirus can survive on human hands and transfer of infectious virus to animate and non-porous inanimate surfaces has been demonstrated (sattar et al. ; ansari et al. ). ward et al. ( ) examined the transfer of rotavirus from contaminated surfaces to the mouth and from surfaces to hands to the mouth. all of the volunteers who licked rotavirus-contaminated plates became infected whereas, of those individuals touching the virus-contaminated plates with their ®ngers and then their mouths, only about half became infected. a number of studies in child daycare centres have shown that rotavirus can be widely disseminated when outbreaks occur. in one such centre faecal contamination of hands and the environment was demonstrated during an outbreak of rotavirus diarrhoea (keswick et al. a) . other studies in daycare centres have shown that ± % of surfaces sampled can be contaminated with rotavirus. in particular, hand contact surfaces (e.g. refrigerator handle, toilet handles, telephone receivers and toys) and moist surfaces such as sinks, water fountains and water-play tables were contaminated with the virus (keswick et al. a; wilde et al. ; butz et al. ) . soule et al. ( ) found that there was an increase in the number of environmental surfaces contaminated with rotavirus in a hospital paediatric unit when there was an increase in the number of children suffering from rotavirus gastroenteritis. of the surfaces in direct contact with children (thermometers, play mats and toys) rotavirus was detected in % of samples compared with % for surfaces without direct contact (telephones, door handles and washbasins). these ®ndings were similar to those of akhter et al. ( ) who showed that widespread rotavirus contamination of a paediatric ward and playroom correlated with the presence of patients infected with rotavirus. in a treatment centre in bangladesh, handwashings from % of the attendants of patients with diarrhoea (children under years) were positive for rotavirus antigens (samadi et al. ) . rotavirus was also found in handwashings of % of attendants of patients with non-rotavirus diarrhoea, indicating that they may have come into contact with other attendants and patients in adjacent beds. this highlights the potential for contaminated hands to spread the infection. projectile vomiting associated with nlvs is probably a major source of cross-infection because it is estimated that ´ particles are distributed as an aerosol into the environment during a vomiting attack (caul ) . a recent report (anon. ) showed how aerosols produced by vomiting can be inhaled or can contaminate hands or work surfaces, with the potential for subsequent transfer to foods or direct hand-to-mouth transfer. the importance of airborne transmission was demonstrated in a recent outbreak in a restaurant where no food source was detected but an analysis of the attack rate showed an inverse correlation with the distance from a person who had vomited (marks et al. ) . the potential for secondary transmission via environmental surfaces in semiclosed communities was demonstrated following a wedding reception, where an outbreak of nlv gastroenteritis affected % of guests (patterson et al. ) . the previous day, a kitchen assistant had vomited in a sink that was subsequently used for preparing vegetables eaten by the wedding guests. further evidence for transmission of nlv via aerosols and environmental surfaces comes from reports of recurrent waves of nlv gastroenteritis occuring in successive cohorts of guests on a cruise ship over a -year period (ho et al. ) . it was found that the risk of gastroenteritis amongst passengers who shared toilet facilities was twice that of those who had a private bathroom. nosocomial spread is also a major concern. norwalk-like virus gastroenteritis in an elderly care unit in a hospital spread rapidly within and between wards, affecting both patients and staff. analysis of risk exposure showed areas where patients had vomited to be the most signi®cant factor for the spread of nlvs to staff (chadwick and mccann ) . a study by cheesbrough et al. ( ) showed that carpets can also harbour nlvs and serve as reservoirs of infection. two carpet ®tters became ill after removing a carpet from a hospital ward d after the last case in a nlv outbreak. routine vacuuming every day since the outbreak had not removed the virus. during an outbreak of vomiting and diarrhoea due to nlv in a long-stay ward for the mentally ill, environmental samples were collected on the affected ward of which ( %) were positive by reverse transcriptase-polymerase chain reaction. positive swabs were from lockers, curtains and commodes and were con®ned to the immediate environment of the affected patients (green et al. ) . most recently the potential for environmental spread of nlvs was demonstrated in a prolonged hotel outbreak in successive cohorts of guests (cheesbrough et al. ) . environmental sampling demonstrated widespread dissemination of the virus on hand contact and other surfaces. from the patterns of infection it was concluded that, although infectious aerosols were probably the main route of dissemination of infection within a particular cohort of guests, contact with contaminated fomites was the most likely factor responsible for maintaining the outbreak by forming the link between successive cohorts. as with other enteric viruses hav is shed from an infected person in large numbers and is able to survive on environmental surfaces (mbithi et al. ) and be readily transferred to hands (mbithi et al. ) . fomites are potential risk factors in the spread of the virus, especially in hospital wards, daycare centres or restaurants (cliver ) . a recent outbreak of hav was associated with a public house whose barman had chronic diarrhoea and had served drinks while incubating hepatitis a himself (sundkvist et al. ) . fomite transmission by contamination of glasses was the likely route of spread. assessments of community outbreaks of hav have shown that persons involved in nappy-changing in daycare centres often handle food and that this is a potential risk for transmission (hadler and mcfarland ) . hepatitis a virus may be acquired from children who are excreting hav, the majority of whom are asymptomatic (fox et al. ) . a signi®cant percentage ( ± %) of susceptible household contacts of index cases with acute hav infection are at risk of acquiring acute hav infection from the index case (minuk et al. ) . a higher rate of hav infection amongst children ( ± %) compared with the parents ( %) suggests that play activity among children is a signi®cant factor for hav transmission in households. it is generally accepted that respiratory viruses, such as those which cause the common cold and¯u, are spread from person to person by aerosol transmission due to sneezing and coughing. nevertheless, there is growing evidence that a signi®cant proportion of¯u and particularly cold viruses are spread via hands and surfaces such as handkerchiefs and tissues, tap and door handles, telephones or other surfaces touched by an infected person (eccles ; goldman ). cross infection can occur either by handshaking or by touching the contaminated surface. rubbing either the nasal mucosa or the eyes with virus-contaminated hands can cause infection. sattar et al. ( ) have shown that rhinoviruses can survive on environmental surfaces for several hours. infectious viruses have been recovered from naturally contaminated objects in the surroundings of persons with rhinovirus colds (reed ) . clean hands can readily pick up the virus by touching or handling such objects (ansari et al. b) . as much as % of infectious rhinovirus on contaminated hands has been shown to transfer to a recipient's ®ngers after contact of only s (gwaltney et al. ) . after handling contaminated coffee cup handles and other objects, more than % of subjects developed an infection (gwaltney and hendley ) . hendley et al. ( ) and reed ( ) have demonstrated that rhinoviruses can survive for several hours on the hands and selfinoculation by rubbing of the nasal mucosa or conjunctivae via virus-contaminated ®ngers can lead to infection. in¯uenza virus can be shed before the onset of symptoms and for up to d after onset and individuals with in¯uenza can be infectious before they develop symptoms and for up to a week afterwards. both in¯uenza a and b virus have been shown to survive on hard surfaces such as stainless steel and plastic for ± h and on absorbent surfaces such as cloth, paper and tissues for up to h (bean et al. ) . it was shown that in¯uenza a virus could be transferred from contaminated surfaces to hands for up to h after the surface was inoculated. epidemiological evidence supports the laboratory data because an in¯uenza outbreak in a nursing home suggested that the virus was spread by staff, through hands contaminated directly with body¯uids, or by touching contaminated fomites (morens and rash ) . there is similar evidence for the environmental survival and spread of piv and rsv. ansari et al. ( b) demonstrated the transfer of piv from stainless steel surfaces to clean ®ngers, which suggests that fomites have a role as a reservoir for the spread of the virus. further, piv could be recovered from non-absorbent surfaces for as long as h when the surface remained moist. however, brady et al. ( ) found that, when material containing piv was spread and allowed to dry, virus was only recoverable for up to h. these workers also showed that piv persisted on the skin for at least h after contamination, which reinforces the need to perform vigorous handwashing before and after contact with patients and their environment. likewise, hall et al. ( ) showed that rsv was recovered from hands touching surfaces contaminated with fresh secretions from rsv-infected infants. evidence showing that direct and indirect contact is a key factor in transmission of rsv infection is further reviewed by goldmann ). humans are the only known reservoir of herpes simplex virus (hsv). the virus is most commonly spread by oral secretions and can be shed by persons with or without symptoms. herpes simplex virus can be recovered from the skin for up to h after inoculation of the hands with the virus (bardell ) . the virus was more readily transmitted from moist drops than from drops which had been allowed to dry, although touching dried virus-containing droplets on the skin with a moistened ®nger resulted in transmission of the virus. infectious hsv has also been recovered from environmental surfaces such as doorknobs and toilet seats, although it is not clear what role fomites play in the spread of herpes viruses (larson and bryson ; bardell ; bardell ) . a recent study demonstrated the rapid and broad contamination of the environment with varicella-zoster virus (vzv) when a family member acquired the disease (asano et al. ) . eight days after onset of the index case vzv dna was detected in both samples from the patient and on the surfaces of an air-conditioning ®lter, a table, television channel push-buttons and a door handle. the virus was also detected on the hands of the parents and children. two siblings developed the disease d after onset of the index case. the problem in assessing whether contamination in the environment might be a hazard is that the infectious dose can vary signi®cantly according to the immune status of the individual. it is clear that increasingly the variability in immune status of individuals is becoming a signi®cant factor in community and domestic settings as well as in the hospital environment. although some viruses survive relatively poorly in the environment, the low infectious dose of many viral pathogens, even for individuals regarded as healthy', suggests that, where body¯uids naturally contaminate objects with a high viral load, the virus can persist in suf®cient numbers to act as sources of infection for several hours, weeks or even months (sattar and springthorpe ) . the infective dose for nlvs may be as low as ± particles, indicating that both aerosol and surface contamination could be a route of transfer of infection (caul ) . likewise, the infective dose for rotavirus may be as few as particles and person-to-person transmission probably perpetuates endemic disease (ward et al. ) . a minimal infective dose of less than plaque-forming units has been demonstrated for poliovirus (minor et al. ) . respiratory viruses also have low infective doses. for rhinoviruses, the infective dose via the nasal route may be less than tcid (couch ) , i.e. the tissue culture infective dose infecting % of the cells. conversely, pivs have an infective dose via the intranasal route of tcid (smith et al. ) . although studies about the survival characteristics of viruses represent an important component in understanding the infection potential and the preventive role of hygiene, much of our knowledge comes from reports of infection outbreaks where hygiene procedures have been defective or from case control studies. fifteen such reports have been examined in which viral contamination was directly implicated or for which viral agents were likely to have been the cause of the infections. the effects of the hygiene intervention procedures are summarized in table and relate to daycare and other community centres where the concentration of people and activity provides the most cost-effective setting for evaluation of the impact of hygiene procedures. although opportunities for cross-contamination and cross-infection may occur less frequently in the home it could be argued that, since the ratio of homes to daycare centres is very large, the impact of these environments on the overall infection rates across a community may not be so dissimilar, even though daycare centres bring more people together. none of the investigations cited relate speci®cally to the home but fornasini et al. ( ) and osterholm et al. ( ) report studies of disease transmission from daycare centres to the home where it is transferred among family members. in eight of the studies only the impact of handwashing was evaluated. in a -week handwashing education programme in child daycare centres, black et al. ( ) showed that the incidence of diarrhoea in children was rarely washed hands associated with higher incidence of infection st. sauver et al. ( ) signi®cantly reduced compared with two control centres. kilgore et al. ( ) studied the prevalence of neonatal rotavirus infection in bangladesh. they found an increased risk for neonatal rotavirus infection among infants whose mothers reported no handwashing during care of the neonate. in a study carried out during the cold and¯u season at two daycare centres, fewer colds were reported in the test group of ± -year-olds using proper and frequent handwashing techniques than in the control group. in the test centre the proportion of colds remained fairly constant at á % whilst in the control group the proportion of colds increased from á % to á % (niffenegger ) . carter et al. ( ) demonstrated that families who used an iodine-based hand disinfectant, known to kill rhinoviruses, had lower rates of infection than families using an inactive handwash. to decrease respiratory infections in senior daycare centres, staff were educated on viral transmission and the value of handwashing (falsey et al. ). in the intervention year, the infection rate among those attending the centres was signi®cantly lower than in the previous years, with an almost % decrease in the infection rate. roberts et al. ( a) carried out a randomized controlled trial of the effect of infection control measures on the frequency of upper respiratory infection in childcare. the intervention measures were training of childcare staff about transmission of infection, handwashing and aseptic nose-wiping technique. when compliance with infection control practice was high, the incidence of colds was reduced by %. a similar study by these workers also examined the effects of infection control measures on the frequency of diarrhoeal episodes in childcare using a randomized controlled trial (roberts et al. b) . they found that, for those centres in which children's compliance with handwashing was high, diarrhoeal episodes were reduced by %. in the us, an outbreak of aseptic meningitis due to echovirus was reported amongst parents with children attending a childcare centre. it was found that more frequent handwashing among the teachers compared with the parents of young children was associated with signi®cantly lower rates of infection (helfand et al. ) . in six of the studies handwashing combined with environmental decontamination or other control measures was considered, whilst one study which related to nlv infection highlighted only the importance of environmental disinfection. in a preschool daycare centre, respiratory and gastrointestinal infections decreased following implementation of measures which included reinforcing existing handwashing procedures and education of staff and families on issues of infection control including environmental surface cleaning and disinfection and disinfection of toys (krilov et al. ) . uhari and mottonen ( ) transmission of infections in child daycare centres. it was evident that most of the infections that did occur were viral. the programme included increased handwashing, cleaning of the daycare centres and regular washing of toys. both the children and staff had signi®cantly fewer infections that those in control centres. st. sauver et al. ( ) studied hygienic practices and the prevalence of respiratory illness in children attending daycare homes. never or rarely washing hands by both children and carers was associated with a higher frequency of respiratory illness in both family and group daycare homes. using shared cloth towels rather than individual paper towels and washing of sleeping mats less than once a week were also associated with a higher frequency of upper respiratory infection. isaacs et al. ( ) reported a sevenfold reduction in the incidence of rsv in a hospital when patients and staff were educated about the importance of handwashing and infected babies were segregated. before intervention á % of children under years old developed nosocomial rsv, whilst after intervention only á % developed infection. following implementation of a hygiene intervention programme that included handwashing education, use of gloves, disposable nappy pads and an alcohol-based hand rinse the incidence of enteric illness was lowered in intervention child daycare homes as compared with control homes (butz et al. ) . as stated previously, contamination of the environment may be considerable during an outbreak of nlv. following outbreaks of viral gastroenteritis on consecutive cruises, a ship was cleared and disinfected at the end of the fourth cruise in order to interrupt transmission of nlv (mcevoy et al. ) . fewer than cases presented in each of the ®fth and sixth cruises compared with cases during the fourth cruise. control measures included cleaning and disinfection of cabins, crew and staff quarters and communal bathrooms and steam cleaning of soft furnishings. hygiene measures were also introduced into the kitchen. the contamination of soft furnishing in areas where individuals have vomited presents a dif®cult cleaning problem and steam cleaning has been recommended by mcevoy et al. ( ) . during a hospital gastroenteritis outbreak caused by nlv, the attack rate among patients decreased in several wards following the implementation of environmental hygiene procedures (chadwick and mccann ) . infection control measures implemented included cleaning and chemical disinfection (ward¯oors, toilet areas, toilet seats, taps and spillages of vomit and faeces) to reduce environmental contamination. hypochlorite solution ( %) was used for disinfection of places contaminated with vomit or faeces and á % hypochlorite for general disinfection of ward oors and toilet areas. since the studies highlighted in table involved the implementation of infection control programmes involving several steps, it is dif®cult to attribute the effectiveness of the hygiene procedures to one speci®c aspect because they were inherently multifaceted. indeed, the results do not prove that the various interventions had a direct effect in decreasing infection rates. it could be argued that the reduced rates were due to variations in individual susceptibility to infection or to a lower incidence of the pathogen amongst the case control groups. nevertheless, overall, there is convincing circumstantial evidence to suggest that improved standards of hygiene can have a signi®cant impact in reducing the rates of respiratory, intestinal and other viral infections in childcare facilities, domestic homes, hospitals and adult care centres and the circulation of infections between these communities. the data reviewed show how improved standards of education and integrated hygiene measures, including hand and environmental hygiene, could have a signi®cant impact in reducing infectious diseases within community and home environments. in recent years the concept of risk assessment or hazard analysis critical control point (haccp) has successfully controlled microbial risks in food and other manufacturing environments. traditionally, the public has tended to regard good hygiene as creating an environment free of germs. to devise a hygiene policy that has real public health bene®ts, it is now accepted that a risk-based approach should also be adopted (bloom®eld and scott ; jones ; scott ) . a risk assessment approach to hygiene starts from the premise that homes and other settings always contain potentially harmful microbes (people, pets, food, etc.) and that good hygiene is not about eradication but about targeting measures in the places and at the times that matter, in order to limit risks of exposure. for both the hands and for environmental surfaces hygiene can be achieved by physical removal of organisms from the surface. alternatively, organisms can be inactivated in situ by a disinfection process or a combination of both physical removal and disinfection. in many situations such as the hands, and cooking and eating utensils, appropriate risk reduction can be achieved using detergent and hot water. however, since, in this situation, hygiene is achieved by removal of the microbes from the surface, if it is to be effective it must be applied in conjunction with a thorough rinsing process with clean water and must take account of the strength of attachment of the microbes to the surface (eginton et al. ) . stevens and holah ( ) showed that wiping of contaminated abraded surfaces using a sponge followed by a -s rinse produced a -log reduction in bacterial contamination of stainless steel surfaces but only a ± á -log reduction on enamelled steel, mineral resin and polycarbonate surfaces. scanning electron microscope studies showed that bacteria were typically retained in surface imperfections, such that surfaces which sustained the most extensive damage retained higher numbers of bacteria. studies by schurmann and eggers ( ) showed that enteric viruses may be more strongly bound to the skin surface and that the inclusion of an abrasive substance, such as sand or aluminium hydroxide, in the handwash preparation is advisable to achieve effective virus removal. recent studies of the transmission of viruses in a household setting using bacteriophage /x as a model showed that virus spread was not prevented by the usual standards of hand hygiene as practised in the household (rheinbaben et al. ) . the virus was reisolated after h from the hands of all persons tested even after normal use and cleaning of the hands. biocides that have activity against both enveloped and non-enveloped viruses include chlorine-and iodine±releasing agents, peracids and ozone (rotter ; sattar and springthorpe ) . biocide effectiveness depends on the nature of the virus, the surface carrier, the presence of interfering substances such as organic soil and hard water salts and the contact time. although these compounds can be used for disinfection of environmental surfaces they are generally too toxic and irritant for use on the skin. in achieving decontamination of hands, although handrub and handwash products currently available may have good activity against bacterial pathogens, activity against viral contamination is variable and depends on the type of virus. rotter ( ) suggested that, although alcoholic handrubs are effective against enveloped viruses such as in¯uenza, piv, herpes and rsv, activity against non-enveloped viruses such as rotaviruses, rhinovirus, poliovirus, adenovirus, nlv and hepatitis virus is limited unless extended contact times (up to min) are used. similarly, agents such as triclosan and chorhexidine have some activity against enveloped virus but are not considered effective against nonenveloped viruses. it is well established that viruses are shed in large numbers and can survive for long periods on surfaces or fomites commonly found in many environments and this emphasizes the possible role of surfaces in the transmission of viruses. faeces can contain up to virus particles per gram and vomit up to per millilitre so the potential for hand and environmental contamination is considerable. viral shedding may begin before the onset of symptoms and may continue for several days or even weeks after the symptoms have ceased. virus transfer from surfaces to hands, ®ngers and food has been demonstrated. other studies have shown a high rate of spread once a viral infection is introduced into a family home or institution. improved handwashing and surface hygiene procedures have been shown to interrupt the transmission of viral infections via hands, surfaces or fomites. although the importance of hygiene and most particularly handwashing cannot be over-emphasized as a means of reducing infections it can be dif®cult to enforce even in healthcare facilities where staff should be aware of the infection risks. studies have shown that handwashing compliance amongst healthcare workers is variable (daniels and rees ; mcguckin et al. ; nishimura et al. ; pittet et al. ) . in a department of surgery, clinicians washed their hands between examinations in only % of cases (daniels and rees ) . in continuous videocamera surveillance of an intensive care unit personnel complied with handwashing in % of entries, whereas visitors of patients complied in % of entries (nishimura et al. ) . a recent survey of beliefs and attitudes towards hygiene in domestic homes showed that over % recognized handwashing as a key preventative measure in ensuring food safety and % believed that surface cleaning was also important. however, the respondents admitted that they would not carry out these procedures as frequently as they thought they should (mathias ) . the importance of hands in the transmission of virus infections is well recognized and many of the studies cited in this review relate speci®cally to handwashing interventions. increasingly however, there is evidence that cross contamination via surfaces is a signi®cant contributory factor. most particularly hand contact with contaminated surfaces is likely to be the cause of such cross contamination. sattar and springthorpe ( ) and lieberman ( ) emphasized that, if hygiene programmes are to be effective, hand hygiene education must be integrated with education about the importance of surface and air hygiene in prevention of infection transmission. to motivate changes in attitude to hygiene it will be necessary to gain acceptance that homes and other community settings will always contain potentially harmful microbes and that good hygiene is not about eradication but about targeting the correct measures at the times that matter, in order to reduce infection risks. there is a need for wider understanding of the potential for contaminated surfaces to act as unidenti®ed vectors of pathogens, in the recontamination of hands, during the infection transmission cycle. the epidemiological evidence to date shows that raising awareness about the importance of key procedures, such as through handwashing and surface hygiene (particularly hand and food contact surfaces), will have a considerable impact in the control and prevention of infectious organisms. survival of enteric viruses on environmental fomites viral contamination of environmental surfaces on a general paediatric ward and playroom in a major referral centre in riyadh outbreaks of viral gastroenteritis associated with srsvs a working party of the public health laboratory service salmonella committee. the prevention of human transmission of gastro-intestinal infections, infestations and bacterial intoxications the world health report life in the st century, a vision for all geneva: world health organisation rotavirus survival on human hands and transfer of infectious virus to animate and non-porous inanimate surfaces survival and vehicular spread of human rotaviruses Ð possible relation to seasonality of outbreaks potential role of hands in the spread of respiratory viral infections Ð studies with human parain¯uenza virus and rhinovirus spread of varicella-zoster virus dna to family members and environments from siblings with varicella in a household hand-to-hand transmission of herpes simplex virus type survival of herpes simplex virus type on some frequently touched objects in the home and public buildings survival of herpes simplex virus type in saliva and tap water contaminating some common objects detection of viruses and body¯uids which may contain viruses in the domestic environment respiratory syncytical virus infections in families the magnitude of the global problem of diarrhoeal disease: a ten year update handwashing to prevent diarrhoea in day-care centres viral gastroenteritis how hygienic should our homes be? professional care of cross-contamination and infection in the domestic environment and the role of chemical disinfectants survival and disinfection of parain¯uenza viruses on environmental surfaces rotavirus gastroenteritis and weather prevalence of rotavirus on high-risk fomites in day care facilities occurrence of infectious symptoms in children in day care homes rhinovirus inactivation by aqueous iodine in vitro and on skin small round structured viruses Ð airborne transmission and hospital control transmission of a small round structured virus by vomiting during a hospital outbreak of gastroenteritis rotavirus infection in hong kong: epidemiology and estimates of disease burden possible prolonged environmental survival of small round structured viruses widespread environmental contamination with norwalk-like viruses (nlv) detected in a prolonged hotel outbreak of gastroenteritis rhinoviruses respiratory syncytical virus: an underestimated cause of respiratory infection, with prospects for a vaccine handwashing: simple but effective surveillance of small round structured virus infection in england and wales acute respiratory infections in day care hepatitis a Ð new information on an old virus infectious intestinal disease in elderly people international scienti®c forum on home hygiene. spread of common colds and in¯uenza quanti®cation of the ease of removal of bacteria from surfaces evaluation of a handwashing intervention to reduce respiratory illness rates in senior day-care centers trimethoprim-resistant escherichia coli in households of children attending day care centres an outbreak of gastro-enteritis caused by small round structured virus in a geriatric convalescent facility transmission of viral respiratory infections in the home an outbreak of gastroenteritis in a home for the elderly associated with astrovirus type and humans calicivirus the role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay spread of rotavirus with families: a communitybased study transmission of experimental rhinovirus infection by contaminated surfaces handto-hand transmission of rhinovirus colds hepatitis in day care centers: epidemiology and prevention hepatitis a in daycare centers Ð a community-wide assessment parain¯uenza viruses respiratory syncytical virus infections in infants: quantitation and duration of shedding possible transmission by fomites of respiratory syncytial virus respiratory syncytial virus infections within families ) echovirus infection and aseptic meningitis in parents of children attending a child-care centre transmission of rhinovirus colds by self inoculation viral gastroenteritis aboard a cruise ship general principles and historical aspects childhood gastroenteritis: a population study handwashing and cohorting in prevention of hospital acquired infections with respiratory syncytial virus pathogen transmission in child care settings studied by using a cauli¯ower virus dna as a surrogate marker application of haccp to identifying hygiene risks in the home contribution of rhinoviruses to respiratory viral infections in childhood: a prospective study on a mentally hospitalized infant population prevalence of rotavirus in children in day-care centres survival and detection of rotaviruses on environmental surfaces in day care centres neonatal rotavirus infection in bangladesh: strain characterization and risk factors for nosocomial infection impact of an infection control program in a specialized preschool the association of rhinoviruses with lower respiratory tract diseases in hospitalized patients fomites and herpes simples virus: the toilet seat revisited viral agents of gastroenteritis: public health importance and outbreak management astrovirus and adenovirus associated with diarrhoea in children in day care settings outbreaks of astrovirus type and rotavirus gastroenteritis in a geriatric inpatient population a year's experience of the rotavirus syndrome and its association with respiratory illness rotavirus and other viral causes of gastroenteritis evidence for airborne transmission of norwalk-like virus (nlv) in a hotel restaurant the use of consumer knowledge, beliefs and attitudes in the development of a local authority strategy for domestic food hygiene education survival of hepatitis a virus on human hands and its transfer on contact with animate and inanimate surfaces effect of relative humidity and air temperature on survival of hepatitis a virus on environmental surfaces an outbreak of viral gastroenteritis on a cruise ship patient education model for increasing handwashing compliance history and epidemiology of hepatitis a virus human infective dose determinations for oral poliovirus type vaccine in infants the risks of transmission of acute hepatitis a and b virus infection in an urban centre outbreaks of astrovirus gastroenterititis in day care centres lessons from a nursing home outbreak of in¯uenza a survival of herpes simplex in water specimens collected from hot tubs in spa facilities and on plastic surfaces impact of in¯uenza and respiratory syncytial virus on mortality in england and wales from acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, populations based study of disease burden proper handwashing promotes wellness in child care handwashing before entering the intensive care unit: what we learned from continuous video-camera surveillance infectious diseases and child day care human caliciviruses in acute gastro-enteritis of young children in the community hospitalizations associated with rotavirus diarrhoea in the us, through : surveillance based on the new icd- -cm rotavirus speci®c diagnostic code outbreak of small round structured virus gastroenteritis arose after kitchen assistant vomited asymptomatic excretion of rotavirus before and after rotavirus diarrhoea in children in day care centres compliance with handwashing in a teaching hospital an investigation on the possible transmission of rhinovirus colds through indirect contact transmission of viruses via contact in a household setting: experiments using bacteriophage /x as a model virus extended excretion of rotavirus after severe diarrhoea in young children effect of infection control measures on the frequency of upper respiratory infection in child care: a randomised effect of infection control measures on the frequency of diarrhoeal episodes in child are: a randomised longitudinal study of rotavirus infection and gastroenteritis in families served by a paediatric medical practice: clinical and epidemiological observations common exposure outbreak of gastroenteritis due to type rotavirus with high secondary attack rates within families handwashing and hand disinfection hospital admissions attributable to rotavirus infection in england and wales outbreaks of infectious intestinal disease in residential institutions in england and wales detection of rotavirus in handwashings of attendants of children with diarrhoea chemical disinfection to interrupt the transfer of rhinovirus type from environmental surfaces to hands institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission viricidal activity of biocides an experimental study on the epidemiology of enteroviruses Ð water and soap washing of poliovirus contaminated hands, its effectiveness and kinetics hygiene issues in the home respiratory syncytial virus infection protective effect of antibody to parain¯uenza type virus monitoring rotavirus environmental contamination in a paediatric unit using polymerase chain reaction the common cold hygienic practices and acute respiratory illness in family and group day care homes the effect of wiping and spraywash temperature on bacterial retention on abraded domestic sink surfaces outbreak of hepatitis a spread by contaminated drinking glasses in a public house infectious diarrhoea in children: controlling transmission in the child care setting an open randomized controlled trail of infection prevention in child day-care centres selected viruses of nosocomial importance prevention of surfaceto-human transmission of rotavirus by treatment with disinfectant spray human rotavirus studies in volunteers: determination of infectious dose and serological response to infection study of infectious intestinal disease in england: rates in the community, presenting to general practice and reported to national surveillance detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction key: cord- - b t r authors: bordatchev, evgueni v.; cvijanovic, srdjan j.; tutunea-fatan, remus o. title: preliminary experimental analysis of the surface topography formation during laser polishing h tooling steel using statistical characteristics of the surface amplitude distribution date: - - journal: procedia manufacturing doi: . /j.promfg. . . sha: doc_id: cord_uid: b t r abstract surface finish is one of the most important quality characteristics of fabricated components. to complement that, laser polishing (lp) is one of the advanced manufacturing surface finishing techniques that has been recently developed and successfully employed for improving surface quality without deteriorating the overall structural form through surface smoothing by melting and redistributing a thin layer of molten material. this paper proposes a statistical digital twin of the lp process and demonstrates the applicability of amplitude distribution statistical characteristics in the experimental analysis of surface topography formation during lp process. initially, the thermodynamic transformation of the initial surface topography is considered by means of technical cybernetics and machine learning approaches to describe two of the most critical lp process components, namely: thermodynamic melting and solidification of both solid material and surface topography. to exemplify the effective application of statistical amplitude distribution characteristics, lp experiments were conducted with two different laser powers ( w and w) on flat and ground initial surfaces and resulting surface topographies were measured. several amplitude distribution characteristics, such as roughness average value, averaged transverse profile as a w-shape, averaged transverse roughness profile, and probability distribution function were calculated. after that, actual molten material area, volume redistribution and final surface quality were comparatively analyzed. it was shown that the proportion between two components of the lp thermodynamic transformation and surface topography is critically dependent on laser power. as such, during low-power conditions (< w), surface quality is predominantly determined by the thermodynamic transformation of initial surface topography and therefore only this component can be used for statistically reliable lp process modelling and digital identification. in summary, amplitude distribution characteristics have several advantages in building a comprehensive understanding of the molten material redistributing along and across lp line. according to industry axioms, functional components fabricated from various ferrous and non-ferrous materials require superior surface qualities. laser polishing (lp) is an advanced surface finishing technique that has been continuously developed and enhanced over the past two decades [ ] [ ] [ ] [ ] [ ] [ ] . lp is productive to the level of several seconds/cm and improves surface quality by smoothing, melting and redistributing a thin layer of molten material. this happens without affecting the form precision and accuracy of the polished component. lp does not involve material removal or addition. lp does not require a physical contact between tool and workpiece and is typically regarded as an environmentally friendly process. however, lp performance depends on the physical-mechanical properties of the workpiece material, lp process parameters as related to the delivery of the laser energy (optics and motion), complex thermodynamics of lasermaterial interactions, as well as several other process/system details. according to industry axioms, functional components fabricated from various ferrous and non-ferrous materials require superior surface qualities. laser polishing (lp) is an advanced surface finishing technique that has been continuously developed and enhanced over the past two decades [ ] [ ] [ ] [ ] [ ] [ ] . lp is productive to the level of several seconds/cm and improves surface quality by smoothing, melting and redistributing a thin layer of molten material. this happens without affecting the form precision and accuracy of the polished component. lp does not involve material removal or addition. lp does not require a physical contact between tool and workpiece and is typically regarded as an environmentally friendly process. however, lp performance depends on the physical-mechanical properties of the workpiece material, lp process parameters as related to the delivery of the laser energy (optics and motion), complex thermodynamics of lasermaterial interactions, as well as several other process/system details. according to industry axioms, functional components fabricated from various ferrous and non-ferrous materials require superior surface qualities. laser polishing (lp) is an advanced surface finishing technique that has been continuously developed and enhanced over the past two decades [ ] [ ] [ ] [ ] [ ] [ ] . lp is productive to the level of several seconds/cm and improves surface quality by smoothing, melting and redistributing a thin layer of molten material. this happens without affecting the form precision and accuracy of the polished component. lp does not involve material removal or addition. lp does not require a physical contact between tool and workpiece and is typically regarded as an environmentally friendly process. however, lp performance depends on the physical-mechanical properties of the workpiece material, lp process parameters as related to the delivery of the laser energy (optics and motion), complex thermodynamics of lasermaterial interactions, as well as several other process/system details. according to industry axioms, functional components fabricated from various ferrous and non-ferrous materials require superior surface qualities. laser polishing (lp) is an advanced surface finishing technique that has been continuously developed and enhanced over the past two decades [ ] [ ] [ ] [ ] [ ] [ ] . lp is productive to the level of several seconds/cm and improves surface quality by smoothing, melting and redistributing a thin layer of molten material. this happens without affecting the form precision and accuracy of the polished component. lp does not involve material removal or addition. lp does not require a physical contact between tool and workpiece and is typically regarded as an environmentally friendly process. however, lp performance depends on the physical-mechanical properties of the workpiece material, lp process parameters as related to the delivery of the laser energy (optics and motion), complex thermodynamics of lasermaterial interactions, as well as several other process/system details. th sme north american manufacturing research conference, namrc (cancelled due to covid- ) the main functional goal of lp is related to the flattening of the initial surface to a desired polished topography. during lp process, cnc-controlled laser beam travels over the surface and produces a localized pool of the molten material. molten pool travels along with the laser spot and because of that, a thin top layer of material is continuously melting while being redistributed. the newly formed surface topography rapidly solidifies improving surface quality up to % [ ] . the current study plans to demonstrate that the resulting quality of the lp surface is affected by two interrelated thermodynamic processes induced by the moving laser source, namely melting, redistribution and solidification of both solid body and surface topography. for this purpose, a statistical twin of the lp process was developed, and several lp experiments were performed on initially flat and ground surfaces. following that, amplitude distribution statistical characteristics were calculated and analyzed to identify contributions from laser melting of solid body and surface topography. as shown in the past [ ] , final lp surface topography is comprised of two distorted material components: laser modification of both solid body and top surface topography. in addition to these two components, lp surface incorporates two distinct classes of surface structures: i) ripples and undercuts produced by the dynamics of the melt/solidification front, and ii) bulges, step structures, and martensite needles produced by plastic deformation and changes in the microstructure. the two material components affected by laser energy can be regarded as the result of two lp regimes that are essentially dependent on the thickness of the molten layer, namely: shallow surface melting (ssm) and surface over melting (som) [ ] . ssm regime was defined as having a molten layer thickness smaller than peak-to-valley distance such that molten peaks will flow down to valleys under the action of the capillary pressure. ssm can be understood as a capillary regime [ ] in which dominating surface tension and viscosity smoothens high spatial frequencies of the surface topography. by contrast, som regime is present when a molten layer thickness is greater than the peak-to-valley distance. the presence of som implies that the initial surface topography will disappear completely. the two regimes coexist during lp but they are present in different ratios [ , , ] and the dominating one will essentially define the mechanism underlying the final surface quality. preliminary statistical modelling, identification and spectral analysis of the laser micropolishing (lµp) process were previously conducted. according to [ ] , lµp can be considered as a single-input/ single-output dynamic system. in a different approach, lp process can also be regarded as a thermodynamic formation of the polished surface topography hlp(x,y). previous lp studies [ , , , ] have demonstrated that the thermodynamics of the laser-material interactions simultaneously affects both solid body and initial surface topography. this enables the modelling of lp process from two perspectives: i) thermodynamic distortion of the flat solid body, and ii) laser smoothing of the initial surface topography hini(x,y) into hlp(x,y). therefore, the digital twin of the lp process can be defined as a thermodynamic transfer function wlp(•) having horig(x,y) as a combined two-fold input, namely solid material and its surface topography. in this case, topography hlp(x,y) is considered as an output that combines the two aspects mentioned above (thermodynamic and statistical) that would concurrently roughen and polish horig(x,y). the superposition of these two simultaneous processes will define the final quality of the surface. as such, lp parameter optimization will consist in the minimization of the thermodynamic material distortion as well as the maximization of laser smoothing effect as applied on the initial surface topography hini(x,y). a schematic representation of the statistical digital twin of lp is shown in fig. . where wlp(jω) is the transfer function of lp process; sflat(ω), sini(ω) and slp(ω) are the autospectrums of flat, initial and polished surface profiles, respectively; ω is the spatial frequency, and j = √- is the imaginary unit. this technical cybernetic description allows modelling and understanding lp process as a linear thermodynamic system transforming statistical characteristics of initial into lp surface (e.g. probability density functions f(x)) and laser beam interactions with flat solid body and non-uniform initial surface. lp experiments with identical process parameters are to be applied on two different initial surface (flat and ground) with an underlying assumption that the distortion of the flat surface topography hini(x,y) into hlp(x,y) underlies the thermodynamic component of surface formation wlp(•), whereas the redistribution of amplitudes of hini(x,y) into hlp(x,y) will drive the statistical component of the lp process. initial surface of a flat sample with sa = . - . μm was obtained via single point diamond cutting. ground initial surface topography incorporates a variety of spatial frequencies having a profile average roughness ra = . … . μm. the lp system shown in fig. a is a complex opto-electromechanical cnc-controlled system and consisting of a highprecision -axis motion system and a nm, w continuous wave (cw) laser from ipg photonics (model ylr- ). the motion system is controlled by a cnc aerotech controller with a positional resolution of . fig. . a) lp system and b) surface analysis methodology. envelope, the laser beam was directed to a -axis galvanometric scanning head, manufactured by scanlab and with an f-theta objective having a focal length of mm. two sets of lp experiments with the same parameters were performed by varying laser power and travel speed (fig. ) in order to investigate two different lp regimes (ssm vs som) as well as contributions of melting solid body and initial surface topography on the final lp topography. the analysis of the surface topography presented in fig. b involved the following steps: a) measure initial surface geometry hini(x,y) between alignment marks, b) perform lp experiments on the previously measured initial surface, c) measure lp surface geometry hlp(x,y), and d) calculate statistical characteristics of amplitude distribution for both hini(x,y) and hlp(x,y), a process to be followed by their comparison. the sample surfaces before and after lp experiments were measured using a wyko nt white light interferometer having an Å height measurement resolution and . μm/pixel resolution. an autostitching technique was used to expand the limited field of view. furthermore, measured and digitized hini(x) and hlp(x) were used for calculating specific statistical parameters and characteristics of the transverse profile amplitude distribution along y axis, such as roughness average value ra, averaged transverse x-x cross-section hx(y), averaged roughness transverse ra profile ra(y), density histogram px(y), and material ratio function mr(y). the effect of laser power on surface topography formation and its thermodynamic and statistical components was studied by comparing initial and lp longitudinal y-y cross-sections hini(x) and hlp(x) of ground and flat samples with different lp powers ( w and w). the post-polished surface topography in fig. suggests clear differences between initial and lp profiles of ground and flat surfaces. two main observations can made based on the results presented in fig. :  lp with w of applied power is associated with ssm, capillary and laser micropolishing regimes as lp process acts as a moving average of the ground surface topography. more specifically, lp process melts peaks to fill cavities. this will consequently smoothen the surface profile and reduce initial surface roughness since ra drops from . um to . um for the ground surface (see fig. a ). this regime will minimally affect the surface waviness, and it is characterized by a very low molten depth that cannot be measured accurately. ssm is associated with a strong statistical correlation between initial and lp surfaces and very high damping of profile amplitudes in the high areal frequency range. this represents the statistical component of the transfer function of the lp process wlp(•).  lp with w of applied laser power worsens the quality of the initially flat surface. more specifically, its ra increased by % from . µm to . µm (see fig. b ) so-called "surface bulging" phenomenon. this mean value is also laser power dependent since it increased from to . µm for ground surface and to . µm for flat solid body, respectively. the bulging effect can be seen as a compound/composite effect since its characteristic bulging height was determined by upward movements of the bulk material as well as contributions from melting peaks. when lp is performed at w of applied laser power (see fig. ), som regime and thermo-capillary effects are present. in this case, lp process acts as an amplitude modulator of the ground surface topography. the initially flat surface topography was completely eroded since ra increased from . µm to . µm. the amplitude distribution of frequency components has also changed significantly. as expected [ , ] , the bulging effect was more prominent for w, rather than w of power. unlike in the low power case, ra for lp ground and flat samples are comparable: . µm vs . µm (see figs. a and b), thus suggesting that molten material from bulk solid body and surface topography are significantly blending with each other. although ra has still improved considerably from . µm to . µm (see fig. a ), the thermodynamic mechanism involved in lp surface formation was completely different in a sense that this time, solid body elevation seems to play a predominant role on the overall process of bulge formation. as a next step in the current analysis, the overall formation of lp line topography was analyzed in detail on both flat and ground surfaces polished by averaging transversal x-x crosssections along laser travel trajectory (see fig. ). these crosssections represent the result of the molten material redistribution during lp and overall form quality of the lp line. as it was shown before [ ] , typically, the averaged transversal x-x cross-section is characterized by a specific "w"-shape. the central bulge is always present after lp and its height increases with higher laser power levels: . µm for w vs . µm for w (ground sample case). the lateral pocket depths also increase from . µm for w to . µm for w. one other important characteristic of the averaged transversal x-x cross-section is an areal material distribution under the "w"-shape. this distribution consists of a centrally raised area (bulging) coupled with laterally lower regions (pocket). the direct comparison of ra(y) profiles on fig. indicates clearly that laser melting of solid body with w does not significantly affect the surface quality of lp ground geometry while w will significantly alter it. this implies that the ratio between the two components of the lp thermodynamic transformation is critically dependent on the applied laser power. additional aspects of the material redistribution during lp can be also explored using a classical probability density function f(y) [ , ] for analysis of surface topography amplitudes distributions. experimental analysis and comparison of f(y) for lp with w and w are shown in fig. . for the initial flat surface, fini,flat(y) is very similar to a dirac delta function with a very sharp amplitude of . after applying w lp to initial flat surface, fini,flat, w(y) becomes wider with reduced maximum amplitude. this describes the thermodynamic transformation of solid material, which increases average surface waviness and roughness and produces a bulging effect. fini,ground, w(y) for lp with w has an opposite signature, where gaussian distribution of the initial ground surface is transformed into a narrowed shape with higher maximum amplitude. this represents the combination of both thermodynamic transformations during lpa) bulging induced by melting-and-solidification of solid material and resulted in increasing the waviness and b) surface smoothing induced by melting-and-solidification of initial surface topography and resulted in surface quality improvement. same observations mentioned above can be applied for lp with w however producing bigger bulging effect, larger amplitude of the surface waviness, and in overall less surface quality. this is because lp simultaneously and continuously melts, redistributes and resolidifies solid material within a laser-material interaction volume. the present study advocates towards the use of advanced statistical amplitude distribution characteristics in the analysis of the lp process. in this context, the emphasis was placed on the experimental characterization of two major components of thermodynamic transformation of the initial surface. the current body of work warrants several important conclusions:  lp is a complex physical-thermo-dynamic process, with two major thermodynamic components: i) melting, redistribution and solidification of both solid material and ii) initial surface topography.  concept of the statistical digital twin of the lp process has been introduced and explored in this study as the transformation of the statistical characteristics of the initial and lp surfaces, such as a probability density function.  thermodynamic transformation of solid material generally leads to bulging and increased surface waviness.  thermodynamic transformation of the initial surface generally leads to smoothing as well as desirable results in surface quality improvement.  final lp surface is formed by a complex, volumetric, and non-linear combination of the two aforementioned components. their superposition will define the final surface quality.  the two thermodynamic components are critically dependent on the applied laser power. more specifically, low power conditions imply that surface quality is predominantly formed by the thermodynamic transformation of initial surface topography and therefore only this component can be used for statistically reliable lp process modelling and its digital identification.  a superior understanding of all aspects associated with thermodynamic melting, redistribution and solidification of workpiece material requires further in-depth analysis and extensive modelling and experimental investigations.  future extension of this work will focus on the variation of other process parameters (such as travel speed) in an attempt to further understand the mechanisms underlying the lp process. polieren von werkzeugstählen mit laserstrahlung editor: tailored light : laser allication technology laser polishing pulsed laser polishing of micro-milled ti al v samples improving surface finish in pulsed laser micro polishing using thermocapillary flow performance of laser polishing in finishing of metallic surfaces processand material-induced surface structures during laser polishing laser polishing of tool steel with co laser and high-power diode laser impact of initial surface parameters on the final quality of laser micro-polished surfaces effect of initial surface topography during laser polishing process: statistical analysis handbook of surface and nanometrology engineering applications of correlation and spectral analysis this study is the result of collaboration between the national research council of canada (london, ontario) and western university (london, ontario). partial financial support was also provided by the natural sciences and engineering research council (nserc) of canada.⁑ these authors contributed equally to this work key: cord- -oj jjjm authors: miroshnichenko, igor; sheremet, mikhail title: numerical simulation of heat transfer in an enclosure with time-periodic heat generation using finite-difference method date: - - journal: computational science - iccs doi: . / - - - - _ sha: doc_id: cord_uid: oj jjjm this paper reports a numerical investigation of highly coupled system of partial differential equations, simulating the fluid flow and heat transfer in a large-scale enclosure with time-periodic heat generation. the bottom wall of the enclosure is insulated, and heat exchange with the environment is modeled at other external boundaries. the heater with time-periodic heat generation is located at the bottom of the enclosure. the internal surfaces of both the heater and walls are assumed to be gray. air is the working fluid and the rayleigh number is ( ). to solve the governing equations with dimensionless vorticity – stream function – temperature variables, the finite difference method has been used. the developed model has been validated through a comparison with data of other authors. the effect of surface emissivity and periodic heat generation on nusselt numbers and both stream function and temperature distributions has been investigated. the results showed that the influence of the thermal radiation on total thermal transmission increases with surface emissivity of walls and heater surfaces. the present numerical method can be applied in several engineering problems, such as designing passive cooling systems and the simulation of heat transfer in building constructions. many industrial processes are associated with convective heat transfer in large-scale enclosures. one significant application from an energy-saving point of view is an effective optimization of energy consumption for heating buildings. the main and relevant topics for construction and design of buildings are to provide both energy efficiency and thermal comforts for inhabitants. the efficient optimization and thorough design of buildings require modern experimental and numerical approaches. in specialized literature one can find a large number of articles relating to convective heat transfer inside a large-scale enclosure due to its applications in buildings [ ] [ ] [ ] . several studies on free convection inside enclosures have been focused on attic space, where the free convection mechanism is sensitive. a comprehensive review of thermal transmission in attic-shaped spaces has been presented by saha and khan [ ] . their findings have indicated that most works have been performed in the laminar and transition regimes. at the same time, as practice shows, the airflow in the real attic must be fully turbulent [ , ] . therefore, numerical analysis of hydrodynamics and thermal transmission inside attic (or other large areas) should be carried out in this regime. the review of das et al. [ ] has summarized the studies on natural convection in different (non-square) shapes of enclosures and enclosures with wavy and curved walls. a number of works showed that change of both the angle and aspect ratio of the parallelogrammic and triangular cavities had a big effect on the flow fields. the influence of the different parameters such as the darcy number, prandtl number, rayleigh number, volume fraction of the nanoparticles and irreversibility distribution ratios has also been analyzed. these results provide helpful insight into possible strategies to enhance convective heat transfer inside complicated enclosures. analysis of the effect of a room heater (location, size and power) on heat transfer and air flow is high important problem. experimental and numerical simulation of turbulent modes of natural convection in a large-scale cavity with a small heat source has been carried out by zhang et al. [ ] . the central plane velocity fields above the plate have been measured using piv method and temperature measurements have been obtained using thermocouples. the numerical modeling was conducted by fluent software using various models of turbulence. the analysis of inertia and buoyancy force discussed in this paper helps to better understand the nature of flow inside the enclosure. it should be noted that radiative heat transfer plays an important role in thermal transmission inside enclosures. as practice shows, radiation can have a big effect both on hydrodynamic characteristics and heat transfer even at relatively low temperature differences [ ] [ ] [ ] [ ] . a detailed review of works in the field of turbulent free convection without and with radiation in rectangular cavities was conducted by miroshnichenko and sheremet [ ] . inclination angle and shape of the cavities, thermal boundary and initial conditions, various radiative properties and heat source location have also been investigated. sharma et al. [ ] have investigated the interaction effects between turbulent thermogravitational convective heat transport and thermal radiation in bottom heated cavities. the aspect ratio in this study varies from . to and ra based on cavity width varies from to . they have found a correlation using aspect ratio and rayleigh number for determining the mean convective nusselt number. kogawa et al. [ ] have studied the radiation impact on turbulent modes of free convective energy transport inside closed volume. they have analyzed four different radiation models (surface radiation, non-radiation, gas radiation and combined radiation) to understand the radiation influence of the gas and from the borders. their results have shown that the effect of gas radiation on radiative energy transport is insignificant, while the surface radiation effect is dominant. to accurately predict convective-radiative heat transfer, various numerical approaches, namely, finite difference and finite volume methods, finite element and lattice boltzmann methods are used [ ] [ ] [ ] . the impact of non-uniform and uniform heating of inclined walls on free convection in an isosceles triangular cavity has been studied by basak et al. [ ] using the finite element method. they have considered two various cases of thermal boundary conditions, viz., non-uniformly and uniformly heating of two inclined walls. the main goal of that study was to analyze the flow and temperature fields with detailed analysis of heat transfer estimates for free convection in triangular cavity. their findings indicated that geometry does not have much effect on flow structure at small prandtl numbers. to the best of our knowledge, investigation of the impact of time periodic heat generation on convective flows in an enclosure having internal heating has not received due attention. the aim of this work is to simulate unsteady turbulent energy transport in a large-scale enclosure with a heater using the finite difference method. analysis of other heat transfer modes (radiation and conduction inside the heater and solid walls) noticeably affects natural convection and essentially complicates the mathematical model. moreover, we conducted a comprehensive study of the effect of time-periodic heat generation inside the enclosure. the geometry of the problem as shown in fig. is a large-scale enclosure bounded by massive walls of finite conductivity with a local heat-generating element. the heat-generating element is located on the bottom wall and it has time-periodic heat generation. time-periodic heat generation was determined as q ¼ q v À sin f Á t ð Þ ð Þ . the solid walls of the enclosure are of finite thermal conductivity k w and finite thickness l. the external border of the lower wall is adiabatic. no-slip conditions are accepted for cavity walls. the heat exchange with the outside is simulated at external surfaces. all internal walls of the large-scale enclosure are diffuse, opaque and gray emitters. the considered air flow is turbulent in nature. the properties of air are supposed to be permanent except for the density where the boussinesq approximation is considered. taking into account the above assumptions the reynolds-averaged navier-stokes equations can be written in the following form [ , ] : where g k defines the generation or dissipation of turbulent kinetic energy due to buoyancy, p k describes the production of k and present terms are expressed as here x , x are the physical coordinates; t is the temperature; u , u are the velocity components in the projection on the x and x axes, respectively; e is the dissipation rate of the kinetic energy of turbulence; a w is the coefficient of thermal diffusivity of the solid wall material; m is the coefficient of kinematic viscosity; m t is the coefficient of turbulent viscosity; g is the gravitational acceleration; k is the kinetic energy of turbulence; a t is the coefficient of turbulent thermal diffusivity; b is the temperature coefficient of volume expansion; t is the time; l is the characteristic size of the cavity (fig. ) . the kolmogorov-prandtl formula m t ¼ c l k e was used to calculate turbulent viscosity, q v is the volume density of heat flux. the governing eqs. ( )-( ) can be written in a slightly different form, which eliminates the need to search for a pressure field. the form of equations, where the variables are the vorticity (x) and stream function (w), allows us to reduce the number of differential equations, thereby lead to a decrease in the time needed for calculations. in the present paper, a numerical study of the convective-radiative thermal transmission has been carried out in dimensionless variables. the scales of temperature, velocity, distance, time, kinetic energy of turbulence, dissipation rate of kinetic energy of turbulence, stream function and vorticity are chosen as , respectively. with the aim of detailed study of the both energy and momentum transport near the solid walls, a non-uniform grid has been introduced using a special algebraic coordinate transformation [ , ] : where j is a compaction parameter and a, b are the geometrical characteristics. derivatives of the first and second orders in spatial coordinates are: the air flow and heat transfer were determined by subsequent characteristics: the prandtl number (pr), the ostrogradsky number (os) and the rayleigh number (ra). these parameters are: it should be noted that the volumetric heat generation from the heater is described by the ostrogradsky number. taking into account the algebraic coordinate transformation noted above boundary and initial conditions are considered in the following form: at s = psi n; g; ð Þ¼x n; g; ð Þ¼k n; g; ð Þ¼e n; g; ð Þ¼ ; h n; g; ð Þ¼ : at s > at the boundary η = : @h @g ¼ ; at the boundaries n = and n ¼ þ l=l: @n @x @h @n ¼ bi Á h (the heat exchange with an external environment is simulated); at the boundary g ¼ þ l=l: @g @y @h @g ¼ bi Á h (the heat exchange with an outside is simulated); at the heater surface: @h hs @ n ¼ k air k hs @h air @ n À n rad q rad ; at the internal surfaces of the walls-air boundary, parallel to the axis on: at the internal surfaces of the walls-air boundary, parallel to the axis oη: air @n @x @h w @n ¼ @n @x @h air @n À n rad q rad : the boundary conditions for the turbulent characteristics were presented in detail previously in [ ] . it is necessary to understand the impact of radiative mechanism of energy transfer on the hydrodynamics and thermal transmission in the analyzed domain. the non-dimensional net radiative thermal flux q rad is defined as [ ] : here bi ¼ hl=k w is the biot number, r is the stefan-boltzmann constant, k air is the air thermal conductivity, n rad ¼ rt hs l k air t hs À t e ð Þ ½ is the radiation number, f k-i is the view factor from kth unit to the ith unit of the chamber, a i;j ¼ a i a j is the thermal diffusivity ratio, k i;j ¼ k i k j is the thermal conductivity ratio, f ¼ t e =t hs is the temperature parameter, q rad;k is the net radiative thermal flux (dimensionless), r k is the dimensionless radiosity of the kth unit of a chamber,ẽ is the surface emissivity. to solve the set of governing equations the finite-difference method is used. the system ( )-( ) is a combination of elliptic and parabolic equations. so, the secondorder accurate central differences scheme was used to describe the elliptical equation. the successive over relaxation method was used to solve the difference equation. to solve the parabolic equations the locally one-dimensional scheme was adopted. the diffusion terms in parabolic equations were discretized using central differences scheme of the second-order accuracy, whereas accurate upwind difference scheme was applied to discretize the convective terms. first-order scheme was employed for the transient term. the resulting systems of linear equations were worked out by tridiagonal matrix algorithm (thomas method). next, we consider in detail the solution of elliptic equations for the stream function. taking into account the mesh transformation, a spatio-temporal uniform grid was constructed: denote f n i ; g j ; s n À Á ¼ f n i;j , to approximate the derivatives of the first and second orders, the central differences are used: the approximation relation for the time derivative is as follows: @f @s % f n þ i;j À f n i;j s : poisson's equation for the stream function taking into account the coordinate transformation is defined as: one approach to solving elliptic equations is to use central differences to approximate second-order derivatives. as a result, we obtain the following discrete equation: here k is a number of iterations. denote further, the resulting system of linear algebraic equations is solved by successive over relaxation method: the relaxation parameterwas chosen experimentally from the results of many numerical experiments. a thorough criterion for stream function variable is used to receive converged solutions at each time step. the convergence condition \ À must be satisfied by variable w k ij at any grid point (i, j), here k is a given iteration parameter. this difference scheme is unconditionally stable and has an approximation order o h n þ h g . in order to verify the numerical algorithm, the developed computational code has been validated successfully using various numerical results. in the case of natural convection inside the differentially heated enclosure, the developed computational code has been validated successfully using the numerical results (variation of the average nusselt number) of dixit and babu [ ] , zhuo and zhong [ ] and le quere [ ] ( table ) . the numerical simulation has been carried out on coarser and finer meshes, in order to check the grid independence. these computational tests with various grids allowed choosing the suitable grid size selection without compromising both accuracy and cpu time. the mesh sensitivity investigation was examined forẽ ¼ , ra = . three different meshes of  ,  and  were applied to verify the grid independence. the fluid flow rate, average convective nusselt number at the heater surface and average heater temperature are shown in table . the obtained results have shown that the  grid provided a good accuracy. for example, the maximum difference in terms of the average convective nusselt number between case of  points and case of  points is less than . %. in this connection, the non-uniform  grid has been selected for analysis. numerical simulation is reported for the following values of key parameters: pr = . , ra = , h/l = . , f = . , os = , n rad = . , ẽ . the main attention was paid to the impact of both surface emissivity and time-periodic heat generation on distributions of both integral parameters (average radiative and convective nusselt numbers at the heater surface) and local parameters (streamlines and isotherms). the geometry (fig. ) is selected with an eye to simulate thermal transmission and air motion within the room with a heater. the present results are received using one intel core i processor of . ghz with gb memory ram. figure shows the distribution of isolines of the stream function and temperature as a function of the surface emissivity (ẽ ¼ : a,ẽ ¼ : b) at f = . p. two lowintensity symmetric convective cells determining clockwise (right) and counterclockwise (left) circulations are formed inside the large-scale enclosure. the formation of the present vortex structures is caused by cooling of the analyzed area due to the heat exchange with the environment (since the ambient temperature is lower than the initial temperature inside the enclosure), as well as the influence of the heater which located on the bottom wall of the enclosure. the presence of an upward flow of warm air in the central part of the cavity reflects the formation of a thermal plume. the bottom solid wall is a zone least affected by the environment, this is due to heat dissipation from the heat source. with an increase in the surface emissivity, the intensity of convective flow decreases, which is confirmed by the characteristic decrease in the maximum value of the stream function in the core of the convective cell w j jẽ ¼ : max ¼ : \ w j jẽ ¼ : max ¼ : . just to clarify again, thermal transmission is caused by both the effect of time-periodic heat generation from the heater and the cooling of the enclosure owing to the heat exchange with an environment. the radiation significantly affects the distribution of temperature inside the cavity. in general, an increment of surface emissivity allows increasing the mean temperature inside the air-filled enclosure. the impact of the surface emissivity of heater and walls surfaces, as well as dimensionless time on the average convective and radiative nusselt numbers (at the surface of the heater) has been analyzed numerically in fig. . it should be noted that if the boundary conditions are not isothermal (for example, volumetric heat generation from the heater) then achieving the steady state conditions is extremely difficult. timeperiodic heat generation was determined as q ¼ q v À sin f s ð Þ ð Þ . when surface emissivity values increase, the temperature gradient at the heater surface is reduced and natural convection is also weakened. so, with an increase inẽ, a characteristic decrease in the intensity of convective heat transfer has been observed (at s = , the average convective nusselt number decreases by . % when changing ofẽ from . to . ), while a growth of the average radiative nusselt number with increasing surface emissivity values has been observed (at s = , a change ofẽ from . to . leads to an increase in the average radiative nusselt number up to . times). although profiles are time-periodic, the peaks and valleys are increasing with time. this is due to the fact that heat conduction equation inside the heater has an internal heat generation term. more detailed influence of the values of surface emissivity on the temperature profiles at the middle cross-sect. = . is depicted in fig. . a rise of surface emissivity of internal surfaces affects a slight decreasing temperature directly within the heater. this fact also leads to a growth of the mean temperature within the large-scale enclosure. it should be noted that the increase in surface emissivity is manifested in a noticeable decrease in temperature inside the heater. in order to study the influence of time-periodic heat generation on energy transport, the convective heat exchange from the heater was estimated by the average convective nusselt number. figure presents the average convective nusselt number for various surface emissivities at the heater-air boundary. the results have indicated that there is no time point which characterizes the stationary distribution of heat transfer coefficients. this is due to time-periodic heat generation inside the local heater. it is worth noting that a decrease in parameter f leads to growth of period of oscillation. numerical simulation of convective-radiative heat transfer in a large-scale enclosure with heat-conducting walls of finite thickness in the presence of local energy source with timeperiodic heat generation has been carried out. to generate the systems of linear equations using vorticity and stream function, the finite difference technique has been employed. the developed computational code has been validated through a comparison with data of other authors. as a result of the research, various distributions of integral and local parameters characterizing the hydrodynamic and thermal transmission in the enclosure have been obtained. a role of the natural convection in the total energy transport decreases with an increase in the surface emissivity of both solid walls and heater. it has been shown that in a large-scale enclosures with one heat-generating element under conditions of convective heat exchange with external environment the heat removal from the heatgeneration element can be increased even with small growth ofẽ. the surface radiation has a significant effect on the total heat exchange and can reach more than % of the total heat flux, particularly if the heater and wall surfaces have high emissivity. evaluation of the eddy viscosity turbulence models for the simulation of convection-radiation coupled heat transfer in indoor environment experimental study of radiation and free convection in an enclosure with a radiant ceiling heating system numerical simulation of a vertical solar collector integrated in a building frame: radiation and turbulent natural convection coupling a review of natural convection and heat transfer in attic-shaped space turbulent natural convection in an air-filled isosceles triangular enclosure numerical analysis of turbulent natural convection heat transfer inside a triangular-shaped enclosure utilizing computational fluid dynamic code studies on natural convection within enclosures of various (non-square) shapes -a review piv measurement and simulation of turbulent thermal free convection over a small heat source in a large enclosed cavity interaction effects between surface radiation and turbulent natural convection in square and rectangular enclosures a dimensionless solution to radiation and turbulent natural convection in square and rectangular enclosures interaction effects between laminar natural convection and surface radiation in tilted square and shallow enclosures numerical analysis of spatial unsteady regimes of conjugate convective-radiative heat transfer in a closed volume with an energy source turbulent natural convection heat transfer in rectangular enclosures using experimental and numerical approaches: a review conjugate turbulent natural convection with surface radiation in air-filled rectangular enclosures influence of radiation effect on turbulent natural convection in cubic cavity at normal temperature atmospheric gas numerical study of natural convection-surface radiation coupling in air-filled square cavities interaction between natural convection and surface thermal radiation in tilted slender cavities transient combined natural convection and radiation in a double space cavity with conducting walls finite element analysis of natural convection flow in a isosceles triangular enclosure due to uniform and non-uniform heating at the side walls effect of thermal conductivity and emissivity of solid walls on time-dependent turbulent conjugate convective-radiative heat transfer numerical simulation of turbulent natural convection combined with surface thermal radiation in a square cavity simulation of high rayleigh number natural convection in a square cavity using the lattice boltzmann method les-based filter-matrix lattice boltzmann model for simulating turbulent natural convection in a square cavity accurate solutions to the square thermally driven cavity at high rayleigh number acknowledgements. this work was supported by the russian science foundation (project no. - - ). key: cord- - g kmpdi authors: makino, hisao; emi, hitoshi; yamaguchi, akimasa; iritani, eiji; namiki, norikazu; myojo, toshihiko; yamamoto, kenji title: environmental and safety issues with nanoparticles date: - - journal: nanoparticle technology handbook doi: . /b - - . - sha: doc_id: cord_uid: g kmpdi this chapter evaluates the relationship between nanoparticles and the environment, and describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment is clarified from the viewpoint of the kind of influence nanoparticles generated either artificially or naturally have on the environment, such as in atmosphere, groundwater, wastewaters, and exhaust gases. indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. the influence of nanoparticles on the indoor environment is discussed in the chapter. it describes the sources of nanoparticle generation in general industrial processes such as grinding processes, and in cleanroom or controlled environment industrial processes, such as exhaled air, ionizers, and haze by chemical reaction on solid surfaces. the chapter discusses safety issues related to nanoparticles such as possibility of dust explosion, health risks and biological effects of nanoparticle materials such as carbon nanotubes, fullerenes, nanosized metal oxides, and carbon black. the chapter also discusses methods for removing nanoparticles from gas and liquid as technology to control the influence of nanoparticles on the environment. since nanoparticles have superior surface activity and can be applied to the production of particles with various functions, they are extremely important for the future development of sophisticated material technologies. on the other hand, this superior activity of nanoparticles is a cause of trouble from the perspective of safety, and does not always have a positive influence on the environment. attention must also be paid to impact on health. nevertheless, all technologies have negative aspects, and overcoming these kinds of problems, we will be able to utilize the superior characteristics of nanoparticles for practical purposes. to achieve this goal, it is necessary to fully understand the influence of nanoparticles on the environment and the relevant safety issues. this chapter evaluates the relationship between nanoparticles and the environment, and also describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment will be clarified from the viewpoint of what kind of influence nanoparticles generated either artificially or naturally have on the environment. the influence on the indoor environment, where nanoparticles are produced, will also be clarified. the safety of nanoparticles will be clearly described from the perspective of the trouble caused by the superior surface activity of nanoparticles; the effect of the compositional characteristics of nanoparticles, and also the influence on health. a method for assessing the influence of nanoparticles using quantum dots is also explained. in the final section, methods for removing nanoparticles from gas and liquid are described as technology to control the influence of nanoparticles on the environment. in our atmospheric environment, particles ranging from several nanometers to several tenth micron orders are suspended. they are emitted into the atmosphere at the rate of . billion tons every year. emission sources are classified as either natural or artificial. natural particles occupy % of total particles, consisting mainly of salt particles (ϳ billion ton) from the sea and soil particles (ϳ . billion ton) from the land. on the other hand, the latter particles are brought about by human activities. although occupying only % of the total emitted particles, their size is mostly of submicron order and because they contain hazardous chemical components such as nitrates, sulfates, hydrocarbons, heavy metals, etc. in high concentration, their effects on the ecosystem are serious. fig. . . shows an overview of the size and concentration ranges of various aerosol particles. as it can be seen, the number concentration of atmospheric aerosol which we inhale every day ranges from several thousand particles per cm in clean area to several hundred thousands in dusty areas, and the size range lies between nm and several tens of micrometer. fig. . . shows mass-based size distribution of atmospheric aerosol particles. since the size distribution in the nanosize range appears only when the sources of particle generation exist, the size distribution is usually bimodal with peaks in the size range of a few to micron and submicron. the former peak consists of naturally generated coarse particles such as soil dust, sea salt spray, and so on. on the contrary, the latter contains plenty of artificially generated particles, some of which grow from molecules (in most cases vapor state) exhausted by human activities through chemical reaction, condensation, and coagulation. particle growth rarely leads to particles larger than m unless high concentration of vapors or particulate matters which cause the above-mentioned growth mechanisms exist in the atmosphere. as it can be seen from the differences in the particle generation process, fine particles generated from molecules or nanoparticles are much more complicated in their chemical component than the coarse particles, and sometimes have serious adverse health effects. such fine particles are called pm . , which is defined for particles less than . m including nanosized particles. recent epidemiologic investigation reports that the concentration of pm . showed a positive correlation to the mortality due to pulmonary diseases [ ] . various research techniques are used in order to understand the process of particle growth and to trace back to the source of pollution. an example is shown in fig. . . where a characteristic function of sulfur dioxide is shown taking into account all possible factors related to particle growth. where f is the characteristic function that expresses particle size, particle concentration, particle composition, and so on [ , ] . particulate materials in water are present in the form of colloids. these colloid particles are classified into inorganic colloids. examples of the former are oxides of aluminum, silicon and other substances, and typical examples of the latter are substances such as humic acid and fulvic acid. while the structure and molecular weight of particles vary depending on the area of water, it is known that what are usually present in water are comparatively small colloids (particles smaller than nm). the number concentration of colloid particles in ground water, or a typical water area environment, ranges from to (number / m ) and varies significantly depending on the geochemical conditions of the aquifer. it is known that in moving water, colloid particles sometimes act as a medium in conjunction with water and in some cases move faster than water. homogeneous porous layer such as a sand layer, and most of the colloid particles are trapped. the mechanism of partical trap in this layer is explained by the sand filtration theory. c and d are a gravel layer and a rock bed, respectively, and both have high water permeability with large gaps and cracks. particles can also pass through easily. safety and movement characteristics of colloid particles have a significant influence on the movement of materials such as ionized molecules in aquatic environments. since fine particles such as nanoparticles in particular are highly stable as colloid particles, it will be very important in the future to understand their influence. at the same time, these characteristics are considered to have a high potential to be developed for further application of nanoparticles. in most cases, nanoparticles in exhaust gases are studied from the viewpoint of the influence of total particulate matters on the environment. the term "nanoparticles" is used only in a few cases, "fine particles" is usually used for investigation. since nanoparticles are part of fine particles, this section will be described from this perspective. major sources of combustion exhaust gases are stationary large-scale combustors and diesel engines for stationary and portable use. for stationary combustors, fuels such as coal, oil, and gas are used. lighter fuels have a lower rate of particulate emission, but have a higher fine particle content including nanoparticles. fig. . . [ ] shows the frequency distribution in combustion of coal and heavy oil. fig. . . a and b are the distributions on a number and mass basis, respectively. as these figures clearly show, the total weight of particles of a size of m or smaller is extremely low, while their total number is, on the contrary, very large. it is clear that, while the total quantity of particulate material is far larger in coal combustion than in oil combustion, the difference is less when it comes to particles m or less in diameter, including nanoparticles. most of the particles contained in pulverized coal combustion exhaust gases are considered to be formed as particulate materials directly from ash content, which is originally contained in coal and also includes some unburned carbon. particularly, almost all large-size particles are considered to be this type of particle. on the other hand, fine particles include two types. one type is formed in the process by which low boiling point metal contained in coal ash is evaporated and vaporized in a high-temperature combustion field and then becomes particles in the exhaust gas cooling process. the other includes carbon particles formed in the gas phase, or so-called soot, which is generated due to the delay in oxygen supply for combustion of evaporated volatile matter in the initial stage. fig. . . [ ] shows the relationship between the trace metal content in coal ash and the particle diameter. aluminum with a high boiling point has a constant concentration regardless of the particle diameter. however it is obvious that in the case of metals with a lower boiling point, the smaller the particle diameter, the larger the content. with regard to particles with sizes m or smaller in the nano domain, it has been clarified that the generated amount is increased rapidly by reducing combustion air supply or by weakening the oxidation atmosphere in the volatile matter combustion area, for example, when air supply from a burner is reduced in twostage combustion. this also demonstrates the significant contribution of carbon particles formed in the gas phase. also in the case of ash from heavy oil combustion, there are large particles of a carbon residue type generated from sprayed liquid particles and particles formed in the gas phase as well. as in the case of coal, trace metal contained in heavy oil with a low boiling point is concentrated into fine particles and discharged. also in the case of the combustion of liquefied natural gas, carbon particles formed in the gas phase are generated, albeit in trace amounts. in contrast, only in the case of diesel engines, fuel is injected into the high-temperature and highpressure atmosphere produced by compressing only air to induce spontaneous ignition, and combustion continues with a heterogeneous mixture of fuel and air in the combustion chamber. therefore, particulate materials mainly consisting of unburnt carbon are generated due to incomplete combustion. fig. . . [ ] shows changes in the diameter of particles according to changes in the diesel engine load. it is obvious that the overall concentration of particles increases with the increase in the load rate of the engine. according to observations using sem, fine particles in diesel engine exhaust gases have also been found to comprise fine primary particles of a size several tens of nanometers, and coarse particles with carbon hydride condensed on the surface of secondary aggregates of primary particles. influence of particle diameter on trace element contents. the volume of industrial and domestic wastewater is increasing significantly year by year with the change in the lifestyle based on mass consumption and mass disposal brought about by the dramatic development of the economy and industry. effective advanced wastewater treatment is required because wastewater contains a variety of constituents such as particles, organic materials, and emulsion depending on the resource. inorganic nanoparticles are not generally stabilized in the liquid because they form aggregates of some sort more or less. for wastewater reclamation and reuse, these nanoparticles can be removed from the liquid by the advanced treatment processes such as membrane filtration following biological treatment processes. organic materials such as macromolecules are regarded as soft nanoparticles judging from their sizes, in contrast with hard inorganic particles. chemical mechanical polishing (cmp) is one of the fastest growing processes in semiconductor industry, and it has become an integral part of the state-of-the-art fabrication line for the multilayer wiring board of large-scale integrated circuit (lsi). besides the semiconductor devices, cmp is widely applied to the magnetic head, the magnetic memory, and the imaging devices. the process is primarily used for polishing the device side of a semiconductor wafer through the mechanical downforce of slurry abrasive in combination with chemical oxidation of wafer surface. in general, colloidal silica is used as abrasive slurry to planarize the oxide wafer surface. particles in slurry are highly charged to avoid aggregations between particles or between particles and wafer surfaces. during the process, large volumes of ultrapure water are consumed to clean the surface of the wafer, which generates large quantity of cmp wastewater typically having high solid content resulting from slurry abrasive particles of sio , al o , or ceo , depending on the nature of the cmp application. the quantity of cmp wastewater generated is expected to increase proportionally with the growing needs of the cmp processes. as a result, the treatment and reuse of cmp wastewater has become increasingly necessary. the cmp wastewater has been generally treated with the conventional chemical coagulation-sedimentation process, producing large quantity of sludge. currently, a membrane filtration process coupled with chemical pretreatment is used to separate the nanoscale particles from the cmp wastewater to reclaim the water [ , ] . wastewater of nanosized metal colloid, which is hard to be removed by coagulation-sedimentation process, is discharged in such diverse fields as metalworking factory, electronic components factory, and pigment-manufacturing factory [ ] . it is reported that various trace elements of heavy metal are contained in wastewater discharged from a pulp production plant [ ] . a spent emulsion, which contains nanosized copper colloid, is discharged from plants manufacturing copper cables for electrical industry, and the treatment for purification of effluents is examined by the integrated membrane system based on ultrafiltration (uf) and nanofiltration (nf) [ ] . a glass company generates the wastewater containing fine clay and glass particles from the grinding process of glass surfaces during production of crt glass used for tvs and monitors. separation of fine clay and glass particles by microfiltration (mf)/uf is examined in order to treat glass industry wastewater for reuse in the manufacturing process [ ] . the colored substances are free from regulatory constraint of water quality so far because they are not considered hazardous substances. however, water color is being recently used as a standard for the judgment of the purity in water because the removal of color becomes important for wastewater reclamation and reuse. dye works are scattered across the country as the industry with local tradition. the dyehouse effluent is discharged in large quantity, and it has extremely complex composition because it contains not only dye but also dyeing aid and finishing agent. in general, dye cannot be removed by standard biological treatment because of its low environmental biodegradability. dye wastewater is treated by coagulation-sedimentation and activated sludge processes, and nanoparticles produced in the course of the treatment are released into the environment [ ] . the color is often imparted by organic substances, predominantly humic substances. aquatic humic substances including humic and fulvic acids are a term referring to a broad class of naturally occurring mixture of organic compounds, ubiquitous in surface waters, ground waters, and soil pore waters. they are a complex mixture of heterogeneous organic materials in terms of elemental composition, chemical functionality, and molecular size distribution since humic substances can be derived from any organic materials, including plant and animal debris, microfauna, biowaste, pesticide, and others. the molecular weights of humic acids range from several thousands to several tens of thousands daltons, and those of fulvic acids range from several tens of thousands to several hundred thousands daltons. because of this versatility, humic substances are known to significantly affect the behavior of some pollutants in natural environments, such as trace metal speciation and toxicity, solubilization and adsorption of hydrophobic organic compounds, and disinfection by-product formation [ ] . melanoidins are natural polymeric compounds of dark brown color, and they are closely related to humic substances. they are produced by a set of consecutive and parallel nonenzymatic reactions taking place between amino compounds and carbohydrates during a maillard reaction [ ] . they are contained in the molasses wastewater from alcohol distillery, sugar processing and refinery industry, and glutamate processing industry. such wastewater containing melanoidins has frequently caused a coloration problem of water environment, and thus the suitable decolorization treatment is required in many fermentation and sugar industries using molasses. treatments by flocculation, ozonation, and electrolysis are promising in color removal [ ] . food-processing wastewater usually contains a variety of organic materials in varying degree of concentration. in cheese-making in the dairy products industry, only ϳ % of the initial milk volume becomes product, cheese, and the other % becomes by-product, liquid cheese whey. since cheese whey is a protein-and lactose-rich by-product of cheese production, its cost-effective utilization is becoming increasingly important. recent developments in membrane technology have provided exciting new opportunities for large-scale protein and lactose fractionation in whey treatment [ ] . in textile industry, typically it takes over l of water to process just kg of textile material. not only the washing water must be treated to recover important by-products such as lanolin, but bleaching and dyeing chemicals must also be removed before discharge back to the rivers [ ] . surfactants are a primary constituent of the detergent used in the household routinely, and also they are widely used in industry and agriculture because they have several functions such as washing, emulsification, and dispersion. the surfactants are usually present in the solution in the form of the micelle, and large amounts of surfactant wastewater are discharged in the rivers [ ] . pesticides whose molecular weight ranged from to da (ϳ nm) have been used in great quantities not only for agricultural use but also in golf links and resort. therefore, the wastewater and effluent treatments have become an important issue, and pesticide separation by nf membranes is found to be very efficient [ ] . the potential reclamation of high-quality water produced by the advanced treatment of the secondary effluent of the municipal sewage has come a long way in recent years. the sewage contains various components such as virus [ ] , pharmaceutical substances [ ] , and endocrine disrupting compounds derived from zoonotic excretory substances [ ] . the advanced treatment of such chemical contaminants at low level becomes increasingly important. as mentioned above, the removal of nanoparticles contained in wastewater is stringently required to recycle the reclaimed wastewater in a wide variety of industries such as chemical industry, textile industry, pulp and papermaking industry, food-processing industry, dairy products industry, and pharmaceutical industry. also for domestic wastewater, the reuse of the reclaimed wastewater for nonpotable purposes is becoming more and more important, and this is expected to raise awareness of the behaviors of nanoparticles contained in wastewater in order to upgrade the water treatment processes. in recent urbanized lifestyles people tend to spend more time in enclosed buildings or residences than outdoors. therefore, it is of great importance to characterize indoor particles and correlate between indoor and outdoor ones from the viewpoint of evaluating the influence of indoor air quality (iaq) on human health. as shown in table . . , indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. . . . secondary particle formation by gas phase ozonolysis for particle formation resulting from chemical reaction via ozone, the reaction of terpenes is very common in indoor environments as well as atmospheric ones. terpenes are emitted from fragrance-containing vegetable oils such as pine oil and citrus oil, and wooden materials including woody furniture [ ] . meanwhile, sources of emission of ozone are air cleaners, air-conditioners, laser printers using corona discharge, and infiltration of outdoor air. terpenes are generic terms of unsaturated organic compounds that are composed of isoprene as unit (e.g. -pinene and limonene). these compounds used for household applications readily react with ozone because they have one or more double bonds. it has been proposed that, as shown in fig. . . , the reaction mainly proceeds to form less volatile pinonic acid via pinonaldehyde of intermediate [ ] . furthermore, the acid-catalyzed reaction allows the products to convert into higher molecular weight compounds by the polymerization via carbonyl groups in the aldehydes and the aldol condensation [ ] . it has been reported that the resultant generated particles have a size distribution with a peak diameter of about nm, and that the products by terpenes ozonolysis irritate human airways [ ] . nanoparticles are also generated from air humidifiers or negative air-ion generators in which water is atomized. in general, humidifiers are mainly categorized into vaporization type and atomization type [ ] . the former does not entrain impurities in water when it is fed into indoor spaces of interest. meanwhile, the latter has the drawback that nvrs are suspended in spaces to be humidified by feeding water via spraying and sonication. the nvrs in tap water include colloidal particles and soluble fraction such as silicates, sulfates, carbonates, and chlorides. the size of nvr particles, d p _ r can be estimated from the following equation: where dp_ m is the droplet size, c the mass fraction of nvr in the droplets, m the droplet density, and r the nvr particle density, respectively. assuming that m-sized droplets ( m ϭ , kg/m , r ϭ , kg/m ) are formed by an ultrasonic nebulizer, and the mass fraction of nvr in city water is ppm ( Ϫ ), the nvr particle size, d p_r is estimated to be nm. recently, a wide variety of negative ion generators using the lenard effect, corona discharge, uv/photoelectron emission and electrospray have been commercialized and attention has been focused on features such as air purification and physiological activation [ ] . among them there are the ion generators that atomize water based on the lenard effect and electrospray form the nvr particles as by-product in addition to ion products if the supplied water contains nonvolatile impurities. fig. . . shows an example of electrical mobility distribution for ions generated by the electrospray method (positive in this case) [ ] . this method atomizes liquid fed to a tip of a capillary electrode to form fine droplets with large amounts of charge by applying high voltage between the tip and the downstream counter electrode. when water in the generated droplets evaporates and their surface charge density attains the charge limit called "rayleigh limit", this phenomenon induces their self-fragmentation followed by the formation of a high concentration of cluster ions. in this figure, the high-mobility peak on the right side corresponds to the cluster ions. these ions are nm in size assuming that they are singly charged. meanwhile, another peak on the figure results from nvr in water and then its height increases with the increase in the fraction of tap water in the fed liquid. the electrical-mobility-equivalent size of nvr particles measured by differential mobility analyzer (dma) -condensation nucleus counter (cnc) method ranges from to nm and their concentration is on the order of particles/cm . comparing the forementioned electrical mobility distribution with the particle size one, the nvr nanoparticles are estimated to hold about charges. accompanying the recent proliferation of computers, the use of inkjet printers and electrophotographic machines such as laser printer and photocopier is becoming common in homes as well as offices. it has been reported that these devices emit various sorts of pollutants. the eco-friendlinessoriented standards such as blue angle standard [ ] regulate the maximum permissible limits of benzene, styrene, total volatile organic compounds (tvoc), ozone and particles. as the regulation of particles is based on the emitted mass per hour, mainly the relatively coarser particles such as toner and dust adhering to paper have been targeted. however, some reports have revealed that nanoparticles are emitted from inkjet printers or laser printers [ ] . fig. . . depicts the size distribution of particles emitted from a laser printer measured by a scanning mobility particle sizer (smps). as seen in the figure, nanoparticles with a peak diameter of around nm are generated in printing mode, whereas the emission in the case of feeding paper without printing is about one third of the normal printing mode. furthermore, these particles were dried by passing them through a diffusion dryer because they are thought to originate from the nucleation of water vapor emitted from papers in the fixation process. as a result, it was found that most particles formed in the paper feed mode evaporated and then vanished, while particles in the printing mode contained nonvolatile components as well as water. from these results it is anticipated that the particles are derived from styrene remaining in the toner even though their composition has still not been identified. meanwhile, it is thought that during ink discharge ink-jet printers emit not only the main ink droplets but also their satellites (about m) to result in nanometer-sized nvrs during printing. one of the most significant source of indoor nanoparticles relevant to combustion is cooking such as frying and sautéing [ ] . some reports said that over % of the particles by number were in the ultrafine fraction range during cooking with bimodal peaks at and nm, attaining the number concentration on the order of particles / m and the emission rate of particles / h. owing to lifestyles in asian countries, cigarette smoke, incense, and mosquito coils also contribute to indoor nanoparticle levels [ ] . it was reported that especially in the indian subcontinent the combustion of biofuels such as straw and dried cattle manure used for cooking could have a significant impact on climate change in the south asian region [ ] . sidestream cigarette smoke also contains nanoparticles, having a concentration distribution with the main peak between . and . m [ ] . in addition, it was found that nanoparticles a peak size of nm formed by the nucleation of vapor fraction in filtered sidestream smoke immediately after burning when the dilution of smoke by air was insufficient [ ] . attention should be paid to air cleaners when a high concentration of cigarette smoke has to be treated by the cleaners using a single unit air filter. airborne virus particles or virions are typically in the - nm size range, and are a good example of nanoparticle bioaerosols. smaller viruses typically contain one subunit, which consists of an outer protein capsid, internal nucleic acid (e.g. dna and rna), and other internal proteins. corona virus that causes sars and influenza are good examples of them. the viruses most often are transmitted through direct contact with an infected person, such as by shaking hands, hugging or kissing, while sometimes it is spread by nasal droplets. however, it is still unknown how these virus particles behave in the case of airborne infection. recently, the studies that attempt to elucidate the behavior have been progressing [ ] . this section describes the sources of nanoparticle generation in industrial processes by categorizing them into specific processes where a cleanroom is used and other general ones. the sources of emission of unwanted nanoparticles in general workplaces are categorized as fumes from hot processes (e.g., smelting, refining, and welding) and from (incomplete) combustion processes. favorable conditions required for the generation of nanoparticles are found in workplaces where there is ( ) presence of vaporizable material, ( ) sufficiently high temperature to produce enough vapor, followed by condensation to form an independent aerosol, and ( ) rapid cooling and a large temperature gradient. there have been so many studies on occupational exposure to fine particles in the field of public health. in general, high spikes of nanoparticle concentration are observed during active operations, followed by a gradual decay after the operation, primarily because of coagulation, evaporation, dilution, and/or deposition. the fraction of the total number of nanoparticles generally decreases, whereas that of the number of submicrometer particles increases with time and distance from the point of emission. in order to accurately estimate exposure, the effects of spatial and temporal changes will need to be evaluated. therefore, it is important to identify the time required for the concentration to decline to the normal or background levels. as an example of reports on grinding processes, fig. . . shows the case where a steel substrate was ground upon using a high-speed grinder [ ] . from the figure the distribution of concentration of generated particles has a distinct bimodality, one with the finer peak at around nm and the coarser one at around m. the former results from within the grinder motor and the volatilization or combustion of amenable ground substrate and/or grinding materials, the latter from the mechanical abrasion and attrition. however, the resultant total concentration on the order of particles/cm is not so high. cleanrooms and associated controlled environments (e.g., in the case of an iso class cleanroom, the maximum permissible airborne particle concentration is less than particles/m for particles with the size of . m or larger, while the airborne particle concentration in ordinary indoor environments is on the order of particles/m or higher) are usually adopted to avoid particle contamination in industrial processes where precision products such as engineered nanoparticles, semiconductors, and other electronic or optical devices are fabricated because the deposition of particles onto product surfaces causes their yield reduction and quality deterioration. the emission sources in cleanroom environments are tabulated in table . . . since some of the listed emission sources emit trace amounts of nanoparticles, these nanoparticles are not regarded as particulate contaminant but as chemical or molecular one. in this section, these nanometer-sized solid substances formed on solid surfaces by chemical reaction are also included. size distribution of nanoparticles generated when a steel plate was ground with a high-speed grinder. ( ) air exhaled by humans emissions from human bodies are a minor contribution in ordinary indoor situations because airborne particle concentration in such places is quite high, whereas the emission cannot be seen as negligible in cleanroom environments. the major human emissions are thought to be atmospheric dust deposited on clothes and skin fragments, and most of these particles are submicrometer in size. meanwhile, particles in exhaled air are composed of fine liquid droplets from spittle ( . % of water), and then evaporate to form nanoparticles of nvr. in fig. . . an example of size distributions of particles in exhaled air before and after smoking is shown [ ] . when measuring particles in exhaled air, the air was introduced into a measuring device after drying them by passing them through a diffusion dryer. the size distribution of particles in exhaled air before smoking (n db ϭ ) has a bimodality, one with the peak size of . m, and the other of nm or smaller. the former peak comes from atmospheric aerosols as it decreases with the increase in number of deep breaths in a clean booth (n db ). the latter originates from nvr particles of spittle droplets. incidentally, since smoking induces the rapid increase in number concentration of particles . m or larger by times or more and for nanoparticles by about double, special attention should be paid to the management of personnel's clothes such as face mask when they enter a cleanroom after smoking. ( ) emission from ionizers ionizers are commonly used in cleanrooms to eliminate electrostatic charge on substrates for precision electronic devices. the most popular ionizer is a corona-discharge type. corona-discharge type ionizers are categorized into the following three groups; ac, dc and pulsed-dc types. the issues of emission of contaminants such as ozone, no x and particles have been pointed out [ ] . these issues are also applicable to air cleaners using a corona discharger. among these problems is that the particle emission has a potential for particle contamination onto product surfaces and eventually decline in product yield. the particle emission, which has been studied since the s, is caused by foreign particle deposition onto electrodes, electrode erosion, and gas-to-particle conversion. the issue of electrode erosion can be solved by the improvement of electrode materials, whereas for the issue of gas-to-particle conversion, the airborne molecular contamination (amc) control to be ionized has to be made. it was reported that exhaled air of human corona-discharge ionizer (e.g. gas-to-particle conversion of low-molecular-weight cyclosiloxane) boron-containing particles from borosilicate glass fibers of hepa filter haze by chemical reaction on solid surfaces (precipitation of ammonium salt and silica) watermark on wafer surfaces at drying leakage from thin film and nanoparticles processing equipment silicon-containing compounds that precipitated on the electrodes result from the gas-to-particle conversion of low-molecular-weight cyclosiloxane (lmcs) from silicone sealant via corona discharge [ ] . ( ) boron-containing particles from hepa filter with borosilicate glass fibers the use of hepa and ulpa filters made of borosilicate glass fibers prevails in the most cleanrooms. it has been known that owing to chemical reaction in equation ( . . ) bf vapor is formed from glass fiber filters by passing hf gas leaking from wet cleaning equipment through the filters. boron, which is a dopant element for semiconductors, has been thought to be a contaminant that might cause failure in semiconductor devices if it comes from the surroundings. in addition, it has been revealed that trace amounts of boron in the form of boric acid (h bo ) are also formed from the fibers via the reaction with moisture in the surrounding air (equation ( . . )). fig. . . depicts the change in volatilized boron mass from various filters in terms of airborne boron concentration. especially, at the initial stage just after the initiation of ventilation, the volatilized boron mass increases with increase in relative humidity [ ] . boric acid, which is solid at room temperature, is surmised to form in the particulate form. however, its existence was identified only by chemical analysis because it is present only in trace amounts. haze might form on glass surfaces of lenses and mirrors for optical instruments if they are exposed for a long time to cleanroom environments where amcs are not controlled properly. the haze is more likely to bring about the insufficient light delivery onto a surface to be exposed in photolithographic processes. one of the reasons is that ammonium sulfate ((nh ) so ) is formed, which then precipitates on the glass surfaces via chemical reaction of sulfur dioxide (so ) with ammonia or amines. for another reason, hexamethyldisilazane (hmds) used as additive in resist coating or lmcs from silicone sealant is adsorbed, and then decomposed to form silica precipitates on glass surfaces by photochemical reaction during laser irradiation, followed by the unwanted decline in laser penetration [ ] . as another example a report said that tiny projections, which are also known as "haze", with a size of . m or smaller were formed on silicon wafer surfaces owing to the adsorption of organosilicate compounds in thin film formation processes with cvd. it is similarly caused by the precipitation of sio [ ] . ( ) watermarks on solid surfaces during drying when a silicon wafer surface is cleaned with deionized water and then dried in air, a watermark is formed on it via the mechanisms demonstrated in fig. . . . oxygen in air is dissolved and diffused into water droplets or adsorbed water on a wafer surface, followed by the formation of silicate compounds via silanol reaction. the watermark on a wafer surface is detected in the form of nanometer-sized particles by an electron microscope [ ] . ( ) leakage from nanoparticle production processes in regard to the risks to processing equipments by nanoparticle leakage from production processes, the vdi report in germany [ ] has been described in detail. the production of engineered nanoparticles can be generally categorized into two approaches. one is a "top-down" approach that is initiated with a bulk material and then breaks it into finer pieces using some form of energy such as etching, ball milling, sputtering, and laser ablation. the other approach is to synthesize materials from the atomic or molecular level by growth and assembly to form the desired nanoparticles. processes included in this "bottom-up" category are sol-gel, chemical vapor deposition, flame synthesis laser pyrolysis, and so on. most of these processes are performed in a closed reaction chamber installed in a cleanroom or associated controlled environment. human exposure to these engineered particles does not take place during synthesis unless there is an unexpected system failure (e.g. rupture of a seal). human exposure is more likely to occur after the manufacturing when opening the reaction chamber, drying the products, or in the post-process handling of the products. the release of nanoparticles during production chamber cleaning operations is another critical point. cleaning typically involves using water or some solvent. brushes, sponges, or tissues used in the cleaning will carry nanoparticles into the waste stream. disposal of the waste and wastewater may become a source of nanoparticle release into the environment. further, conditioning of nanoparticles such as compression, coating, and composition to form final products may also result in the release to the environment and resultant exposure although very few studies have been carried out on this subject. recent studies [ ] to evaluate the aerosol discharge during the handling of carbon nanotubes showed that the generation of nanoparticles occurred under vacuum to remove spilled nanotube materials or vigorous mechanical agitation. however, they reported that the concentrations were very low. in addition, measurements of nanoparticle levels during final packaging of carbon black, which is a typical engineered nanoparticle material, showed that there was no increase in nearby air [ ] . study on the safety of nanoparticles has started only recently, and no sufficiently systemized results have been obtained. what should be noted in particular is that the possibility of radial troubles caused by particulate matters are considered to increase by the decrease of particle diameter in nanoparticles. one typical example is the problem of dust explosion, caused by the high surface reactivity of fine particles. in other words, since nanoparticles are extremely fine particles, dust explosion is more likely to occur. explosion is more likely to occur because fine particles are different in their composition, in that low boiling point metal can be easily condensed, as described in section . . however, of particular note here is that, since all particles do not necessarily exist independently in the form of a single particle, the possibility of dust explosion does not simply increase as particles become finer. fine particles with sizes of m or smaller such as nanoparticles have an extremely high agglomeration propensity and secondary particles can be easily generated. therefore, in some cases they conversely behave like large particles. these are the points to be taken into consideration when studying the problems caused by nanoparticles. as shown in fig. . . [ ] , the effect of the particle diameter on dust explosion tends to be that the smaller the particle diameter of the dust, the lower the minimum explosion concentration. in other words, explosion can be induced under conditions of lower concentration of particles in air as the particle diameter becomes smaller. due to the difficulty of conducting experiments to suspend particles with the same size in a uniform concentration, this result was obtained from particles far larger than nanoparticles; however, it has been clarified qualitatively that the smaller the particle diameter, the higher the possibility of dust explosion. from the perspective of composition, the possibility of explosion increase if materials that react easily with oxygen at low temperature are condensed into particles of small diameter. therefore, with regard to the effect of particle diameter on dust explosion, more careful attention needs to be paid in the case of combined materials than in the case of uniform materials, as assumed in fig. . . . as described before, however, since nanoparticles are considered to exist often as agglomerates, it is necessary from the perspective of the particle diameter to take into consideration the diameter of not only primary particles but also of particles after agglomeration. to address the safety of nanoparticles, it will be important in the future to elucidate their behavior in detail including these factors. the terms 'nanoparticles' and 'nanomaterial' have been used for particles of which one representative dimension, for example, diameter of particles on cross-sectional diameter of fibers has at least nm or less. some people hold that the majority of such fine particles are exhaled without depositing in the respirator tract, and that therefore the particles may not cause pulmonary diseases. however, the properties of nanoparticles are known to be different from the bulk material they are derived from. in cases where the biological effects of bulk materials have been reported, nanosized particles of these materials may be expected to have stronger dose response for the health effects. every effort must be made to clarify the uncertainty on the risks of these nanomaterials [ ] . at the present time, there is no regulation or standard for assessing the biological effects of nanomaterials, and therefore there is a paucity of toxicological data concerning nanomaterials. much more systematic and strategic studies are needed to enable risk assessments for human health [ ] [ ] [ ] [ ] [ ] . as regards risk assessment and risk management of nanomaterials, the characterization and identification of anticipated risks should be first determined for chemical substances or foods. conventional assessment methods are applicable for water-soluble particles. for insoluble nanoparticles, the assessment of potential health hazards should be made based on their properties or toxicity and dose-response relationship. the risk is a product of hazard and exposure; even if a nanoparticle has a hazard, the risk is lower when the possibility of exposure to the nanoparticle is small [ ] . influence of particle diameter on lower limit of particle concentration for explosion. ( ) exposure routes for nanoparticles nanoparticles can either be deliberately introduced into the body for medical purposes (drug delivery systems) or absorbed involuntarily from the environment (inhalation of nanoparticle-containing dust in the air). a distinction should also be drawn between nanoparticles manufactured for industrial application and those unintentionally generated and released in the environment, such as welding fumes or diesel exhaust particles (dep). in the fields of environmental science and toxicology, numerous studies on the potential health hazards caused by ultrafine particles have been conducted. practically, there are several definitions of nanoparticles or ultrafine particles, however, findings regarding biological effects of the ultrafine particles are useful as a starting point for estimating the effects of nanoparticles on human health. human and animals contact with nanoparticles through various routes: nanoparticles can be inhaled in the air, swallowed in the water, ingested in food, and absorbed via the skin in cosmetics. for successful risk assessment, it is important to determine how nanomaterials or nanoparticles are used, such as composites, surface coating, or powders. coatings or powders have the potential to release a part of their nanomaterials into the environment. workers who come into contact with nanomaterials have the possibility of exposure to nanoparticles at the workplace. consumers of products using nanotechnology can also be exposed to them. attention needs to be paid to the environments and ecosystems in which nanoparticles and nanomaterials are released. nanoparticles in the products may change their size, quantity, and composition during their life-cycle of manufacturing, use, transportation, and disposal. ( ) respiratory uptake of nanoparticles inhalation is the main route of exposure to nanoparticles. particles inhaled with the air through the mouth and nose pass through the throat (nasopharynx and oropharynx) and tracheobronchial tree before reaching the alveolar region where oxygen moves from the alveoli to the blood and carbon dioxide moves from the blood to the alveoli. how deeply particles can penetrate and where they become deposited on each respiratory airway such as the nasal cavity, tracheobronchial tree, and the alveoli depend on their size under the various deposition mechanisms: inertial impaction, gravitational sedimentation and diffusion, etc. the respiratory airway includes the anterior nasal passage, posterior nasal passage, pharynx, larynx, trachea, main bronchi, bronchi, bronchioles, terminal bronchioles, alveolar duct, and alveoli, as shown in fig. . . . in the human lungs, the trachea divides asymmetrically into the right main bronchus that enters the right lung where it divides into three lobes, that is, an upper, a middle, and a lower, and the left main bronchus that enters the left lung where it divides into two lobes, that is, an upper and a lower. the trachea divides into two branches, dividing progressively to the terminal alveolus. to quantitatively assess the pulmonary particle deposition needs human lung morphology models, respiratory physiology based models of the entire lung airway system and aerosol deposition models based on many experimental findings. in , the icrp task group on lung dynamic (icrp: the international commission of radiological protection) published their revised lung model [ ] . the deposition, clearance, and translocation of particles in each of the compartments were described. while the model has been widely used in the nuclear field, it is applicable to conventional aerosols as well as radioactive aerosol. in the nuclear field, aerosols including radon progeny that used to be nanoparticles have been studied. fig. . . shows the deposition fractions of inhaled particles per adult nasal respiration of . m /h in each region re-calculated for the nasopharynx, tracheobronchial, and the pulmonary (alveolar) region based on the model. inhaled aerosol particles deposit on different regions depending on their size; for example, nanoparticles larger than nm deposit mostly in the alveoli and those less than nm deposit in the nasal cavity. how deeply particles penetrate into the lung depends on their size. nanoparticles can reach pulmonary region in the lung and deposit more intensively and this, therefore, has become one of the reasons for concern about the effects of nanoparticles on human health. however, deposition of nanoparticle chain-like agglomeration and fibrous particles such as carbon nanotubes cannot be estimated by this model. most inhaled nanoparticles are deposited on the surface of the respiratory tract. generally, if insoluble particles are deposited in the ciliated airspaces which are lined with a mucous layer, they are transported to the digestive tract with the mucous flow by mucociliary movements. particles deposited on nonciliated bronchioles and alveoli are phagocytosed by macrophages, which is a kind of white blood cell. as a result, their residence time is longer, however, they are usually transported to the ciliated upper part of the respiratory tract. removal of deposited particles described above is called clearance. when the amount of deposited particles is below a certain value, no health effect is produced. in relation to a macrophage response to particles, crystalline silica is hazardous, whereas titanium dioxide is not. pneumoconiosis is a well-known lung disease that is caused by exposure to dust particles of several micrometers in diameter. silicosis is a typical form of pneumoconiosis resulting from exposure to crystalline silica dust and characterized by a progressive fibrosis of the lungs. the macrophage-mediated clearance (phagocytes) was effective for micron and submicron particles. it has been reported that only % of deposited nanoparticles were removed by the clearance mechanism [ ] . it has been suggested that the remaining nanoparticles may pass through the alveolar walls, penetrate into the blood or lymphatic circulation, and be transported to other organs. many studies have shown that the smaller the particle size the greater the mobility or they pass easily through the alveolar wall and enter into the bloodstream. it is further presumed that the mechanism of health effects of nanoparticles on cardiovascular system other than the respiratory tract is similar to that in the airborne ultrafine particles from dep. in japan, two reference values, the 'administrative control levels' (acl) and the 'occupational exposure limits' (oels), are used for the regulation of hazardous chemicals as well as dust (particle matters). oels are recommended and revised every year by the japan society for occupational health. oel (oel-mean) for mean concentration of a chemical substance is defined as the reference value to the mean exposure concentration at or below which adverse health effects caused by the substance do not appear in most workers working for h a day, h a week under a moderate workload [ ] . the 'threshold limit value' (tlv) has the same definition (but may not be the same values as oel of the same substance) provided by the american conference of governmental industrial hygienists (acgih). acl is an index to determine the control class to judge the propriety of the working environment control based on the results for working environment measurement, which have been implemented for the unit work area in accordance with the working environment measurement standard. the results of working environment measurement are evaluated by classifying the working environments concerned into three control classes (control class i, ii, and iii). these classes are used as the standards to classify the level of the working environment concerned. among those subject to working environment measurement, these standards apply to workplace where dust, lead, organic solvent, and specified chemical substances are used. article of the industrial safety and health law stipulates that certain workplaces in which harmful substances are involved or harmful work operations are performed shall be the subject to working environment measurement. a % cut-off size of the dust particle is set at m for both standards and is far larger than nanosized particles. oel or acl [ ] for particulate matters are usually based on mass concentration, that is, milligram per cubic meter. therefore, if the particulate matters have broad size distribution, the contribution of nanoparticles is not large to in terms of mass of particles. evidence from a number of toxicological studies on insoluble particles indicates that the primary determinant of the health effect of particles depends on the surface area of particles deposited [ , ] . on the basis of the results from a number of in vitro studies of insoluble nanoparticles, a hypothetical cellular interaction has been proposed [ ] : inflammation and oxidative stress can be mediated by several primary pathways: ( ) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; ( ) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; ( ) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or ( ) intracellular distribution of insoluble nanoparticles to mitochondria generates oxidative stress. in the workplace, the concentration of nanoparticles may be at a high level and most of the nanoparticles become agglomerates, while nanoparticles will form single nanoparticle at low levels in the general environment. it is a matter of debate whether agglomerates of nanoparticles react as a larger particle or a single nanoparticle in the lung or other organs. if insoluble particles are retained in the lung for a longer time without enough clearance mechanisms, they can cause pulmonary inflammation or pneumoconiosis. it is of interest that nanoparticles deposited in the lung can move into the blood vessel through alveolar epithelium and they can damage vessels or produce blood clots [ , ] . in a recent study, nanoparticles deposited in the nose may move directly to the brain via the olfactory bulb [ ] . ( ) biological effects of fullerene the biological effects of fullerene have being investigated intensively. in rats dosed orally with radioisotope-labeled c fullerenes, most were excreted in the feces and some were found in the urine. a small amount of them can be absorbed via the gastrointestinal tract. in contrast, in the same study, % of the same labeled fullerenes administered intravenously were retained after week, with most found in the liver [ ] . ld s (acute toxicity) by intraperitoneal injection in mice and rats were . and . g/kg, respectively. the dose of . g/kg orally in rats did not result in death. the reproductive translocation of fullerenes was also observed in mice. fullerenes have shown mutagenic activity in ames tests. fullerenes have shown no skin irritation or allergic reactions [ ] . on the other hand, fullerenes are being tested for possible medical use. fullerenes are basically hydrophobic but water-soluble derivatives have been synthesized to be used as drugs or its carrier. the derivative can be anticipated as drugs, for example, anti-aids drug. it has been stated that the toxicity of fullerenes changes due to slight structural changes including chemical modification [ ] . ( ) biological effects of carbon nanotubes carbon nanotubes are chemically stable and are similar in form and size to asbestos; these characteristics have given rise to concern that carbon nanotubes may have the potential to cause pulmonary diseases such as lung cancer and mesothelioma similar to asbestos. a few data are available concerning the biological effects of carbon nanotubes. the biological effects of carbon nanotubes are being researched. epithelioid granulomas and interstitial inflammation are induced in mice and rats following exposure to single-walled carbon nanotubes [ ] [ ] [ ] . untreated carbon nanotubes contain the nanoparticles of transition metals such as iron and nickel, which are used as catalysts in forming carbon nanotubes. these nickel-containing carbon nanotubes have been reported to be toxic [ ] . the concentration of airborne asbestos fibers is expressed as a number concentration, that is, fibres per cubic centimeter or fiber per liter. when fiber concentrations are determined by phase contrast light microscopy, the fibers with a diameter of less than m, a length longer than m, and a lengthto-diameter ratio (aspect ratio) greater than are counted [ ] . asbestos fibers having nanosized diameter were often observed in analyses of environmental samples using electron microscopy. international agency for research on cancer (iarc) rated asbestos as a known human carcinogen (group ) [ ] and the concentration of chrysotile asbestos is expressed as a risk level of . fiber/cm [ ] . health effects of vitreous fibers and other asbestos substitutes have been assessed to determine their oels or their carcinogenicity in humans. the health effects of carbon nanotubes are being intensively investigated now. the oel for carbon black respirable dust is mg/m and these for activated charcoal and graphite are . mg/m in each [ ] . in the ref. [ ] , while rats and mice inhaled carbon black with a particle diameter of ϳ nm at a concentration of - mg/m did not produce any specific changes, particles (agglomerate of small particles) of ϳ nm at a concentration of - mg/m produced early pulmonary changes. micronsized titanium dioxide particles are thought to have almost no toxicity and often used as a negative control substance. the oel for titanium dioxide is mg/m for respirable fraction [ ] . however, the results of a series of studies by oberdörster et al. [ , , , ] on submicron-and nanosized titanium dioxide suggested that as size decreases, inflammatory effects are intensified, and normally nontoxic substances may assume hazardous characteristics. fig. . . shows a part of the results by oberdörster et al. in which rats and mice were exposed to anatase titanium dioxide particles [ ] . their results have been frequently cited in the discussion of whether the health effects of fine particles should be based on its mass or its surface area. in fig. . . , percentages of neutrophils in lung lavage of rats are shown as indicators of inflammation after intratracheal instillation of different mass doses of and nm tio particles. the steeper dose response of nanosized tio particles is observed than for submicron tio particles when the dose is expressed as mass (fig. . . a ). if the same dose-response relationship as in fig. . . a is indicated as particle surface area (fig. . . b), the particle surface area seems to be a more appropriate dosimetric for comparing effects of different-sized particles of the same chemical structure. zinc oxide is a white powder and used in pigments. the oel is mg/m for its respirable fraction [ ] , and the value for zinc oxide fume which causes metal fume fever is under consideration. nanoparticles of transition metals and rare earth elements and their oxides will be used widely. since many of these metals and their oxides have biological effects, particular attention should be given to them. nickel compounds are rated as a human carcinogen (group ) by iarc. in particular, nickel oxide is particularly insoluble among the nickel compounds and remains longer in the lung. nanosized nickel oxide particles have greater toxicity to the lung than larger particles [ , ] . pulmonary inflammatory responses induced by nanosized cobalt particles have been reported [ ] [ ] [ ] . biological effects of nanosized particles of other transient metals such as iron and manganese have received attention [ ] . rare earth elements are a general term of chemical elements consisting of scandium (sc), yttrium (y), and a lanthanide series of elements from lanthanum (la) to lutetium (lu). these elements have been used in magnetic alloy, fluorescent and hydrogen storage alloy. particularly cerium oxide nanoparticles are frequently used as a fuel additive and are incorporated in cosmetics formulation. the potential biological and environmental effects of these elements have not sufficiently been investigated. it has been demonstrated that ld s for these elements in oral and intravenous administration are in a range from several dozens to several thousands milligrams per kilograms, indicating that none of these elements has high toxicity. in the results of studies in which the biopersistence and the distribution of rare earth compounds in the body were investigated, for example, the compounds deposited in bones and teeth, and organs including lung, liver, spleen and kidney following intratracheal, oral, intravenous, and intraperitoneal administration. although the compounds deposited predominantly in the liver other than bones, it has also been reported that the distribution of the compounds in the lung and spleen increased when the dose was increased [ ] . the demand for indium compounds has been sharply increasing. the compounds have been used in the materials for transparent electrodes for flat panel displays. in japan, the cases of pulmonary interstitial pneumonia and pulmonary fibrosis have been reported in workers engaged in cutting and grinding of sintered indium-tin oxide (ito) and potentially having inhaled the dusts released from ito [ , ] . the biological effects of indium arsenic compounds and indium phosphorus compounds also have been investigated. the acgih has proposed a value of . mg / m for their tlv, and the value has been applied tentatively in japan. we have been experiencing amazing progress of the technology on the processing for the nanometer-sized materials. the applications cover even biomedical engineering in addition to information technology, material, environmental science, and energy production [ ] [ ] [ ] [ ] [ ] . as the result, many kinds of new materials have been designed, fabricated, and discarded. from now on, this movement will be accelerated and even more new functional materials will be distributed in the world. here, we should not forget the safety of those materials in the process of the production, usage, and discard. without this safety assessment, we will go into the same problems as that of asbestos just we are facing now. first of all, we have to conduct experiments to reveal the minimal concentration for emergence of the toxicity. in other words, we have to fix the standard value for the threshold concentration for each material first of all. if we do not fix it, we should not use the material at any concentration, which means any engineering process could not be carried out. the applications in the various fields have started all over the world, and the safety assessment is urgently needed. in this article, we introduce the methods of the safety assessment of the semiconductor nanoparticles and describe the safety and the threshold depending on the surface treatment. the production process and the surface treatment play one of the most important roles for the safety of the nanoparticles. cd and se semiconductor nanoparticles coated with zns are one of the most widely used for the strong intensity of the fluorescent activity. cd oxide and se compounds once dispersed in the tri-octhil-phosphine oxide (topo) heated up to Њc generate nanoparticles by self-assembly. then zns enhances the stability of the structure and raises up the fluorescent intensity than without the coated one. nanoparticles, thus manufactured, as materials for a novel memory in the field of intelligence technology (it), and as super-micro devices for laser in the field of optics, have been developed all over the world [ ] [ ] [ ] [ ] [ ] . these nanoparticles cannot be dissolved into water, but dissolved into organic solvents like toluene. therefore, for biological and medical applications, various technologies for surface-conjugations to make them hydrophilic [ , ] have been developed. for example, nanoparticles covered with topo are hydrophobic because an alkyl group on them is hydrophobic. therefore, a technology for replacing this alkyl group with hydrophilic carbonic acid (making the whole particles soluble in water) has been developed [ ] . nanoparticles, thus surface-treated, can be dissolved into water, like sodium salt or potassium salt. with this method, various kinds of materials have been surface-conjugated. the mtt assay method is a way to evaluate the hazard assessment of nanoparticles, in which the activation metabolism in a mitochondrium in a cell is measured and the influence of nanoparticles on the prolification of the cell is qualitified. the mtt is a kind of tetrazolium, whose molecular formula is c h brn s. taken into a cell, it is decomposed by a dehydrogenase enzyme in a mitochondrium into a pigment called 'hormazan'. the measurement of the fluorescence intensity of the pigment shows the prolification of the cell [ ] [ ] [ ] . fig. . . . shows the hazard assessment of vero cells and kidney cells of the african green monkey against cdse/zns nanoparticles. the horizontal axis shows the concentrations of the nanoparticles and the vertical axis the fluorescence intensity of the hormazan at nm, that is, the metabolism of the cells. the figure indicates that cyto-toxicity is not observed for cells when the concentration is less than . m. this result means that this concentration is the threshold of the cell toxicity. likewise, cyto-toxicity is not observed for the hela cells and for the human primary cells. further, in order to find out how the sizes of nanoparticles influence the cyto-toxicity, the cyto-toxicities were evaluated with three kinds of quantum dots; one whose fluorescence wavelength is nm, red, one with nm, yellow, and the other with nm, green. the following results were obtained. the largest quantum dots whose fluorescence wavelength is nm show a tendency to give cyto-toxicity. cyto-toxicity is observed at concentrations more than m [ ] . another method to evaluate cyto-toxicity is the flow cytometry [ ] . the mtt assay alone cannot tell whether the toxicity observed is lethal to the cells or just restrains the prolification of them. in the flow cytometry, the nuclei of dead cells are dyed with propidium iodide (pi) after the nanoparticles are taken in and the ratios of the dead cells are measured. fig. . . shows the lethal cyto-toxicity of mua conjugated nanoparticles ( nm, green) against vero cells. the vertical axis indicates the numbers of the cells, and the horizontal axes show the fluorescence intensities and the cyto-toxicities. these experiments also show that dead cells cannot be observed at concentrations less than . m even though nanoparticles are taken in, as was shown in the mtt assay. however, at concentrations more than m, the nanoparticles taken in cause damage to more than the half of the cells. that is, the cyto-toxicity of mua quantum dots against cells is lethal [ ] . nanoparticles have been surface-conjugated for applications for various uses. some surface-conjugations cause more grave cyto-toxicity than others. therefore, relations between surface-conjugations and their safety for cells have to be considered. in order to find out the relations, the safety evaluations of nanoparticles surface-conjugated with two materials were made; one is with mua (quantum dots-cooh) and the other is with glycerol (quantum dots-oh), and their purified and unpurified particles. fig. . . shows that the purification reduces the cyto-toxicity for the quantum dots-oh, and that the toxicity remains the same after the purification for the quantum dots-cooh. mua itself, a material with which particles are conjugated, has cyto-toxicity. this experiment shows that toxicity against cells is connected not only with particles themselves but also with kinds of surfaceconjugations and degrees of purification. it is shown in the above experiments that the toxicity of nanoparticles is lethal to cells. next, we found out by the comet assay method whether the toxicity is derived from damaged dna. the method is a way to evaluate quantitative damage of dna by electrophoresis. the fragmented dna seeps out of their cells by treating cells, whose dna has been fragmented, with agarose-gel to break their cell membrane, and then electrophoresing them. it looks like the "tales of comets". cells, whose dna has not been fragmented, have their nuclei keeping their spherical shape after electrophoresis, and the tales of comets cannot be observed [ ] [ ] [ ] [ ] . fig. . . shows the results of the experiments with quantum dots conjugated with cooh (both purified and unpurified) to wtk- cells, a human lymphoblast mutation strain [ ] . the vertical axis shows the lengths of the tails, and the horizontal axis the concentrations of the quantum dots. the concentration of the nanoparticles is m. the results are as follows: the unpurified quantum dots with cooh caused damage to dna. on the other hand, the electrophoresis with the purified quantum dots does not show dna damage. this is probably because the dna damage has been repaired during the longer cultivation time. conducted with quantum dots conjugated with mua, and quantum dots-nh with an amino group. dna damage was not observed in either case. the results indicate that as far as particles themselves do not break down, the cyto-toxicity of quantum dots is derived from the chemical properties of the materials covering the quantum dots. the safety evaluation of nanoparticles has not been conducted sufficiently. as indicated above, the procedure for surface-conjugation could apply not only to cd/se nanoparticles but also to other nanoparticles. today, various kinds of techniques for surfaceconjugations have been available in academic papers, proceedings, and on the internet. some of them are widely known and others are patented. those techniques are all shared among the human race. even at this moment, the human beings are making breakthroughs in various fields and developing different kinds of technologies. sharing these technologies will lead to still more speedy developments of yet more advanced technologies. to achieve it, these technologies should be so structured that different fields, for example, bioimaging and biotechnology, structured on their own, can be linked to each other. such structured knowledge will play an essential part in merging different fields. in order to prevent nanoparticles release from a system so as to maintain environmental safety, the removal technique of nanoparticles must be established. in this section, separation techniques of particles from exhausted or suspended gas and liquid are described focusing on particles less than nm. generally, as shown in fig. . . , all particle separators for a dispersed system employ either one of three basic forms of particle separation. on the left hand side of the figure lie the separation methods in which particles are collected only by force field (electrostatic force, centrifugal force, gravity force, etc.), and the representative separator is electrostatic precipitator (esp). if some obstacles (collectors) are placed into the particle laden stream, particle separation is facilitated because particles are collected on obstacles with a smaller deviation from the fluid flow by the force exerting on the particles compared to the case without obstacles. typical collectors of this form are air filter, deep bed filter, etc. on the right hand side of the figure lie separators that collect particles utilizing only sieving effect of obstacles without any force field. in this case, geometrical size of channel between the obstacles must be smaller than that of particles. membrane filter, fabric filter, etc. belong to this group. when we apply the above collection forms to nanoparticles, the major collection mechanisms are brownian diffusion and electrostatic force for particles in gas, while sieving effect and interception/adhesion forces for those in liquid. as mentioned above, most airborne particles are collected by separators utilizing various kinds of forces such as gravity, centrifugal force, electrostatic forces, inertia, brownian diffusion force, and so on. therefore, the migration velocities or displacement of a particle per second due to the individual forces gives the basis for the comparison of removal efficiencies due to each force. in fig. . . , migration velocities of particles due to various forces are depicted against particle diameter at normal temperature and pressure for particle density of g/cm [ ] . as seen from the figure, the velocities due to gravity, centrifugal force, and inertia monotonically decrease with decreasing particle diameter, suggesting that the removal of nanoparticles with these forces is difficult. on the contrary, the velocities due to brownian diffusion and electrostatic forces increase with decreasing particle diameter for particles less than nm. this suggests that brownian diffusion and electrostatic forces are most effective in collecting nanoparticles. fig. . . summarizes typical conventional dust collectors. among them, the effective collectors for nanoparticles are esp and fabric/air filter. however, for the case of esp, which relies on only electrostatic force, nanoparticles (Ͻ nm) fail to carry even one electron resulting in low collection efficiency. in this case electrically charged filters are effective because we can expect the combined effects of electrostatic forces and brownian diffusion. among charged filters, so-called electret filter, which consists of permanently polarized fibers, is the most favorable filter because of its charge stability. particle penetration data of electret filter are plotted against particle diameter in fig. . . and compared with that of uncharged filter with the same structure. for the three combinations of charged states of fiber and particle there exist the most penetrating particle diameters. for the uncharged fiber, collection efficiency of uncharged particle has a minimum at nm and increases with decreasing particle diameter, showing that nm is the most penetrating particle size. for the charged fiber, particle collection efficiency is very high even for uncharged particle, and the efficiency for charged nanoparticles is extremely high because of strong coulombic force between fiber and particle. the experimental data plotted in fig. . . are qualitatively in good agreement with the theoretical prediction following the particle size dependency on particle migration velocity (shown in fig. . . ). however, as particle size becomes smaller and comparable with the size of a molecule, particles may rebound on a collector surface, and the adhesion probability of particles drops, resulting in a decrease in collection efficiency. fig. . . is an example of experimental data that confirm the particle rebound [ ] . the figure shows the penetration of nanoparticles through a grounded circular tube. the solid curve is the theoretical line derived by assuming that particles are deposited from a laminar flow in a tube by brownian diffusion. it is evident that experimental penetration deviates from the theoretical line for particles less than nm. this means that molecular behavior begins to appear when the particle size becomes as small as nm, and as a result, the collection efficiency is reduced. it should be noted that considerable amounts of nanosized particles are contained in diesel exhaust particles (dep), possibly penetrating through the honeycomb type (tubular channel) diesel particulate filters (dpf). there are two types of methods that differ in the way the nanoparticles in liquid are collected. the first group, called membrane filtration, constrains the particles by a membrane, and the liquid is allowed to flow freely through the membrane. in the second group of ultracentrifugation, the liquid is constrained in a rotating vessel, and the particles move freely within the liquid by an external field of acceleration caused by ultracentrifugal field. these methods have been quite extensively used in separation of macromolecules and molecules from liquid, and they are recently becoming important also in separation of nanoparticles from liquid. in pressure-driven membrane filtration processes, the pressure gradient across the membrane would force solvent and smaller species through the pores of the membrane, while the larger molecules/particles would be retained. membrane filtration processes are usually classified into three general categories according to the size of separating components, as shown in fig. . . . microfiltration (mf) is designed to retain suspended particles in the range of nm- m. ultrafiltration (uf), on the other hand, retains macromolecules or nanoparticles in the range of - nm (nominal molecular weight cut-off (nmwco) ranging from , to , , da). nanofiltration (nf) is a relatively new process that uses charged membranes, and it covers molecular sizes ranging from . to nm (nmwco ranging from to , ). it is useful in that it can separate dissociated forms of a compound from the undissociated form. one of the major factors limiting the use of membrane filtration is membrane fouling, resulting in a dramatic decline in flux with time of operation. to account for the membrane fouling, the resistancein-series model is frequently employed. the resistance model becomes ( . . ) where u is the permeate flux, v the filtrate volume per unit membrane area, the filtration time, p the applied transmembrane pressure, the viscosity of the permeate, r t the total resistance, r bm the resistance of the membrane per se plus the clogging of the membrane pores, r c the resistance of the filter cake, and r cp the resistance of the concentration polarization layer. significance of each resistance in membrane filtration is as follows. the membrane, even in the absence of any suspended particle, has a natural flow resistance. during membrane filtration, particles become attached to the pore channel of the membrane thereby reducing the flow channel dimension, or pores become blocked off completely. the last two effects lead to resistances that are due to adsorption and pore blocking. the blocking filtration model introduced by hermans and bredée [ ] , and grace [ ] is most commonly used as an interpretation of such phenomena. the clogging of the membrane pores is strongly influenced by such solution environment as ph and the ionic strength. the permeate flux of bovine serum albumin (bsa) (pi . , molecular weight , , stokes-einstein diameter . nm) solution by permeable mf membrane (nominal pore size . m) is lowest at around the isoelectric point [ ] . as the bsa molecule carries no net charge at the isoelectric point, the molecule is in its most compact state at that point. the bsa molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. as a result of this, the flow resistance increases markedly at around the isoelectric point. in dead-end membrane filtration, which has a feed and permeate stream, each with the same mass flow rate, the resistance of the filter cake plays a major part in the filtration resistance. therefore, the cake filtration theory can be applied, and thus the permeate flux u is described by ( . . ) where m is the ratio of wet to dry cake masses, s the mass fraction of solids in the solution, av the average specific filtration resistance, the density of the permeate, and v m the fictitious filtrate volume per unit membrane area, equivalent to the flow resistance of the membrane [ ] . for fine particle suspensions, colloidal forces which arise from interaction between the suspended particles control the nature of the filter cake. the average specific filtration resistance av and the average porosity av of the filter cake are strongly affected by the solution properties, including ph and electrolyte strength. for instance, in mf of suspensions of the titanium dioxide (pi . , the original mean specific surface area size nm), av goes through a minimum, and av is much larger near the isoelectric point [ ] , as shown in fig. . . . the titanium dioxide particles are destabilized around the isoelectric point where the van der waals attraction is more dominant. consequently, the particle tends to come together, that is, to flocculate, and the very porous flocs are then formed. thus, it is speculated that the filter cake formed from such porous flocs has often loose and wet structures. on the other hand, the filter cake becomes compact and dry when the particle carries the charge. since the most loose filter cake forms around the isoelectric ph, the filter cake is most permeable. it is interesting to note that the results in protein uf had a distinctly different behavior. in protein uf of bsa solution, the filter cake is in its most compact state around the isoelectric point [ ] , as shown in fig. . . . since the bsa molecules are hydrophilic colloids, their stability in the solution would appear to be influenced not only by the presence of a surface charge on the protein but also by hydration of the surface layers of the protein. the bsa molecules, because of hydrated layers surrounding them, are not destabilized by such consideration as depression of the electrical double layer. thus, the bsa molecules have water bound to them even around the isoelectric point. the hydrophilic bsa molecules maintain a dispersed state in the solution due to hydration of the surface layers of the protein even around the isoelectric point. when a bsa molecule acquires a charge, the filter cake becomes loose and wet due to electrostatic repulsion between the charged bsa molecules. this contrasts to the compact filter cake around the isoelectric point. the average specific filtration resistance av has a definite maximum around the isoelectric point since a compact filter cake provides a large hydraulic flow resistance. most membrane filtration processes are operated in the cross-flow mode, in which the feed is moved tangentially to the membrane surface so that the filter cake is continuously sheared off. during membrane filtration, particles in the feed are brought to the upstream surface of the membrane by convective transport, and this results in a higher local concentration of the rejected particles at the membrane surface as compared to the bulk solution which is referred to as concentration polarization [ ] . the particle concentration in the solution adjacent to the membrane varies from the value at the membrane surface, c m , to that in the bulk feed solution, c b , over a distance equal to the concentration boundary layer thickness . the resulting concentration gradient causes the particles to be transported back into the bulk solution due to diffusional effects. at steady state, the rate of convective transport of particle toward the membrane is balanced by the rate of particle transport through the membrane plus the rate of the diffusive back transport of particle. thus, the permeate flux u is given by ( . . ) where cp is the particle concentration in the permeate, k (ϭd/ ) the mass transfer coefficient, and d the diffusion coefficient. the osmotic pressure model assumes that the deviation from pure water flux occurs solely due to the osmotic pressure difference across the membrane, and thus the permeate flux u is given by ( . . ) where is the osmotic pressure, which is a function of the concentration. equation ( . . ) means that the effective driving force across the clean membrane is pϪ{ (c m )Ϫ (c p )}. replacing pϪ{ (c m )Ϫ (c p )} by the hydraulic pressure at the membrane surface, p m , equation ( . . ) reduces to the cake filtration equation. to minimize the effects of cake buildup and concentration polarization, membrane filtration is usually conducted using the cross-flow geometry in which the feed flow is parallel to the membrane and perpendicular to the filtrate flow [ ] . however, especially in mf the energy requirements associated with pumping the feed (plus any recirculation flow) along the membrane surface are typically very high. thus, there have been some innovations in recent years with cakeless filtration. the rotating disk module in which the membrane disk is stationary is suited for large-scale operation [ ] . it is possible to enhance the permeate flux by using the vibrating modules [ , ] . in the rotating cylinder device with the membrane on the inner rotating cylinder, counter-rotating taylor vortices within the annular gap are available [ , ] . dean vortices that twist and spiral in the direction of flow inside a highly curved channel, similar to vortices in rotary modules can result in enhanced flux [ ] . these vortices, or flow instabilities, induce turbulence into the system. periodic removal of the formed filter cake is also effective for enhancing the permeate flux. recently, several methods have been investigated: back washing using the filtrate or air pressurization [ ] , periodic rotation of the cylindrical membrane [ ] , pulsatile flow [ ] , high-frequency transmembrane pressure pulsing with a frequency around . - hz [ ] . dead-end upward filtration, where the filtrate flow is in the opposite direction to gravity, and dead-end inclined filtration, where the membrane is inclined, can reduce the cake formation onto the membrane in uf of nanoparticulate suspension and protein solutions. in upward uf of silica sol (mean diameter . nm) and bsa solution, a sustained permeate flux is achieved, as shown in fig. . . [ , ] , because the filter cake overlying the membrane is exfoliated continuously under the gravitational force acting on the particles comprising the filter cake. another approach for enhancing the permeate flux is to employ external force fields. electrofiltration, in which an applied electric field is used to drive charged particles away from the membrane surface, has been developed. in electrofiltration, the accumulation of the particles on the membrane surface is limited by the imposed electrophoretic force. in addition, the permeate flux through the filter cake is dramatically enhanced due to electroosmosis as a secondary electrokinetic phenomenon. this method can be applied to a broad combination ranging from mf of particulate suspension such as bentonite [ ] to uf of protein solution. fig. . . shows the reciprocal permeate flux (d /dv) versus the permeate volume per unit membrane area, v, for various values of the strength of the dc electric field, e [ ] . the steady permeate flux increases noticeably with the magnitude of the imposed field strength. also, a higher electric field strength causes the permeate flux to equilibrate more rapidly. a method has been developed for removing humic substances by hybrid uf combined with both flocculation and adsorption treatments, as shown in fig. . . [ ] . flocculation by use of polyaluminum chloride (pacl) is particularly effective for the removal of humic acids, which constitute the relatively high molecular weight fractions of humic substances, whereas adsorption by use of powdered activated carbon (pac) is able to remove fulvic acids of relatively low molecular weight effectively, which cannot be fully flocculated by pacl. hybrid uf in combination with flocculation and adsorption treatments exhibits high permeate quality because the flocs and pac are easily retained by the uf membrane. in ultracentrifugal sedimentation, ultracentrifugal force field of several tens of thousands of revolutions per minute is applied to a rotor. in recent years, ultracentrifugal sedimentation is employed for concentrating dilute protein solutions and for separating proteins and other large biological molecules from low-molecular-weight solutes or from much larger particles. fig. . . shows the results for ultracentrifugal sedimentation of an aqueous solution of the mixtures of bsa and egg white lysozyme (pi . , mw , ) measured using schlieren optics in an analytical ultracentrifuge [ ] . the angular acceleration of the rotor is , rad/s. the symbol r i and r i in the figure represent the distances from the center of rotation to the sedimentation boundary at time and , respectively. the electrical nature of macromolecules plays a significant role in determining the sedimentation behavior in ultracentrifugation of binary protein mixtures. in the ph range where both protein molecules were electropositive, the molecules sediment independently due to the electrostatic repulsive force acting between bsa and lysozyme molecules. denpun kagaku filtr. sep institute for quality assurance and certification: basic criteria for the award of the environmental label (printer ral-uz ) proc. air cleaning contam proc. air cleaning contam proc. air cleaning contam chemical contamination in semiconductor processing environments and its countermeasures the japan society of industrial machinery manufactures: report on behavior control of individual sort of contaminants - report on introduction of advanced technologies to environmental equipment industry industrial application of nanomaterials -chance and risks. future technologies, division of vdi technologiezentrum nanoscience and nanotechnologies: isbn recommendation of occupational exposure limits who: environmental health series . world health organization summaries & evaluations -nickel and nickel compounds rapid colorimetric assay for cellular growth and survival ouyou earozorugaku crossflow filtration: theory and practice encyclopedia of fluid mechanics: slurry flow technology filtr. sep key: cord- -xbw k m authors: castano, nicolas; cordts, seth; jalil, myra kurosu; zhang, kevin; koppaka, saisneha; bick, alison; paul, rajorshi; tang, sindy ky title: fomite transmission and disinfection strategies for sars-cov- and related viruses date: - - journal: nan doi: nan sha: doc_id: cord_uid: xbw k m contaminated objects or surfaces, referred to as fomites, play a critical role in the spread of viruses, including sars-cov- , the virus responsible for the covid- pandemic. the long persistence of viruses (hours to days) on surfaces calls for an urgent need for surface disinfection strategies to intercept virus transmission and the spread of the disease. elucidating the physicochemical processes and surface science underlying the adsorption and transfer of virus between surfaces, as well as their inactivation, are important in understanding how the disease is transmitted, and in developing effective interception strategies. this review aims to summarize the current knowledge and underlying physicochemical processes of virus transmission, in particular via fomites, and common disinfection approaches. gaps in knowledge and needs for further research are also identified. the review focuses on sars-cov- , but will supplement the discussions with related viruses. membrane. the lipid bilayer is susceptible to chemical disruption, for example, by surfactants. disruption of the lipid envelope could render the virus inactive. in addition to the lipid layer, the m and e proteins could be targets for the inactivation or weakening of sars-cov- due to their critical roles in viral envelope assembly and replication. while enveloped viruses are more susceptible to inactivation than non-enveloped viruses, they possess the ability to adapt the envelope molecular profile to evade immune systems. , while the exact size of sars-cov- has not been reported, the approximate diameter of the closely related sars-cov- is - nm, with spikes that extend ~ nm out (total diameter of ~ - nm). the isoelectric point (pi) of viruses is important in determining their adsorption characteristics. based on the protein composition, the pi of the m and n proteins of other coronaviruses have been computed theoretically to be ~ . - . . [ ] [ ] [ ] the pi of the m and n proteins on sars-cov- are likely to be within the same range. although the overall isoelectric points of coronaviruses have not been reported, they are expected to be largely influenced by the isoelectric properties of m and n proteins, , and can be further approximated by accounting for the dissociation constants of all amino acids of the virus. sars-cov- requires biosafety level (bsl) facilities to handle. to facilitate the investigation of its infectivity, transmission, and disinfection, it is useful to identify surrogates with similar structures to sars-cov- but with reduced risk of human infection. the first class of surrogates involves the use of natural viruses with low infectivity in humans. table shows these surrogate viruses, host cells, and bsl levels reported thus far. , a second class of surrogates is pseudotyped viruses. they are derived from parent viruses such as the murine leukemia virus (mlv), human immunodeficiency virus (hiv), and herpes simplex virus (hsv). the genome of the parents are modified for safer use in bsl labs. the synthesis of pseudotyped viruses is highly adaptable and allows for the incorporation of various kinds of envelope glycoproteins. , for example, sars-cov- s glycoprotein has been incorporated into a lentiviral pseudotyped virion system to determine the potential drug targets for the virus. a third class of surrogates involves artificial capsids that emulate the viral architecture. for example, peptide capsids have been constructed using capsid proteins to serve as nonpathogenic viral surrogates. they have been used to study aspects of viral infectivity, applied as antimicrobial agents to disrupt bacterial lipid bilayer membranes, and programmed to carry specific genetic cargo and deliver into the cytoplasm of human cells. understanding the transmission routes of viruses is crucial to the development of effective control measures. three primary transmission routes have been found to contribute to the spread of respiratory viruses (e.g., sars-cov- and - , measles, hcov, rhinovirus, and influenza virus) ( figure a ): ) direct contact between individuals, ) indirect contact via contaminated objects (fomites), ) airborne transmission via droplets and aerosols. direct contact involves the transmission of the virus through physical contact between an infected host and a susceptible individual. direct contact is a potent transmission route since the viral load can be large, and the virus spends a shorter amount of time outside of a host compared with other routes of transmission. for mers, sars-cov- , and sars-cov- , direct contact is considered a major transmission route. , indirect contact involves the transmission of the virus through a contaminated intermediate object called a fomite. fomites are inanimate objects or surfaces that can become contaminated by the physical contact with either another infected fomite or skin, or by settling airborne particles. fomite transmission can occur when an individual touches a contaminated fomite, and then touches their facial membranes ( figure b ). numerous studies have implicated fomites as a significant virus transmission route in a range of environments. , although the transfer efficiency of sars-cov- from fomites to other fomites or skin is not well characterized, the transfer efficiency of a number of viruses has been investigated, and will be detailed in section . respiratory viruses can also become airborne and spread via particles generated by sneezing, coughing, talking, or exhaling. the particles generated can be classified into droplets or aerosols based on a cut-off diameter of μm. these airborne droplets and aerosols can cause infection through inhalation into the respiratory tract, or by settling onto fomites. due to their large size, droplets typically fall within - m of the source within seconds (figure ). , [ ] [ ] [ ] aerosols are smaller and can remain suspended in the air for minutes to hours (figure ) , depending on the environmental conditions. , such prolonged suspension could increase the distance the virus travels from the source, and the number of individuals and fomites exposed to the virus. detailed experimental and theoretical approaches have estimated that more violent events such as sneezing can deposit droplets and aerosols up to - m away from the source. , following the initial respiratory event, nearby air currents (e.g., from ventilation or wind) can re-suspend aerosols and extend the range over which aerosols can travel. although the degree to which sars-cov- can be transmitted by aerosol remains unclear, early evidence from a study in wuhan hospitals reported that the virus was detected in aerosol samples from areas open to the general public, ~ - meters from the source. airborne droplets and aerosols can also deposit onto and contaminate fomites. a study on sars-cov- infected patients in isolation rooms showed contamination of high-contact surfaces such as doorknobs and bedrails, as well as air outlet fans which indicated virus transfer from aerosols to a surface. while the transmission routes discussed above are generally accepted as the main transmission routes of respiratory viruses, sewage and dust-borne transmission have also been implicated as possible routes. sars-cov- and sars-cov- have both been detected in sewage, suggesting the possibility of transmission via the fecal-oral route, or via aerosolization of sewage caused by flushing. , furthermore, dust-borne transmission has been proposed as a possible mechanism in the spread of avian influenza in chickens. the relative importance of each transmission route in the spread of sars-cov- and most respiratory viruses remains an open question. a trend that is generally accepted, though, is that the risk of infection increases for persons who are in close proximity to an infected individual for extended periods of time. for example, the probability of transmission of sars-cov- between family members and close contacts and among passengers and workers on cruise ships were much higher than that among the general population. additionally, a ferret model showed high transmission efficiencies of sars-cov- among ferrets living in close quarters, while ferrets separated by a permeable partition were infected less efficiently. because of the uncertainty or unavailability of quantitative data, it is difficult to draw conclusions about how each transmission route contributes to the increased risk of infection in close proximity. furthermore, there is often a confounding effect between transmission routes. for example, persons in sufficiently close proximity for droplet-based transmission are likely exposed to a great intensity of virus-laden aerosols simultaneously. in a model of influenza infection assessing the relative contributions of each transmission route to infection risk within a household, fomite transmission was estimated as a major, if not the dominant, transmission route. although the relative importance of each transmission route remains poorly understood, there has been a growing consensus that contaminated fomites play a critical role in the spread of viruses. in order to determine the viability of a virus on surfaces and in aerosols, it is crucial that the methods of collecting virus particles are effective in representing the virus titer from the environmental sample accurately. additionally, the methods used to analyze the collected samples must have high specificity and sensitivity. in this section, we summarize the current collection and analysis methods, as well as opportunities for improvement. the most common methods of collecting aerosolized virus particles use a gelatin filter because it is highly efficient in collecting virus particles without affecting viral infectivity. gelatin filters can also be dissolved easily for harvesting, culturing, and quantifying live virus particles. in a recent study, the stability of sars-cov- and sars-cov- in aerosols was measured by generating an aerosol and feeding the aerosol into a goldberg drum containing a gelatin filter. alternative methods for collecting aerosolized viruses have been covered in a previous review. briefly, these methods use solid impactors (e.g., andersen sampler, slit sampler, and cyclone sampler), liquid impactors (e.g., all-glass impingers, swirling aerosol collector), filters (e.g., gelatin, cellulose, polycarbonate, ptfe, or cotton), or electrostatic precipitators. the downside of these approaches is the significant time delay (minutes to hours) between particle collection and virus quantification. in an effort to develop real-time methods for virus aerosol collection and detection, microfluidicsbased assays have been developed. briefly, these methods include microfluidic optical immunosensing of latex agglutination, , aerosol detection by impingement onto a microfluidic droplet detector, and label-free virus capture using carbon nanotubes and detection by raman spectroscopy. however, these approaches are still in development and are not yet validated for wide-spread use. no study has evaluated the efficiency of virus collection from an exhaustive list of surfaces. in a recent study examining commonly used collection methods, the most effective method for recovering ms bacteriophages from nonporous fomites used polyester-tipped swabs pre-wetted in either one-quarter-strength ringer's solution or saline solution. this method recovered a median fraction of . for infective ms , and . for ms rna from stainless steel and pvc surfaces. the two prevailing methods for detecting viral rna and viable virus particles are reverse transcriptase polymer chain reactions (rt-pcr) and plaque assays, respectively. rt-pcr assays are used to determine both the presence of viral rna and the number of copies of viral rna. while various forms of rt-pcr exist (e.g. rrt-pcr, qrt-pcr, and lamp-pcr), they all follow a general principle of amplifying specific viral rna for detection and quantification. rt-pcr assays are well characterized, straight-forward to perform, and do not require cell culture. their limitation is the inability to determine virus infectivity. plaque assays involve culturing cells that are susceptible to virus infection in a titration of the collected virus samples, monitoring the cytopathic effects, and counting plaque forming units (pfu). several recent preprint publications have used vero e cells to quantify the presence of infective sars-cov- and sars-cov- . , - while plaque assays are the most popular method for determining infectivity, they have several limitations, including: ) the propensity to human error, ) the long duration of the assay (possibly exceeding a week) due to the time required for observable cytopathic effects and/or pfus, ) the lack of a reliable host cell model for some viruses, and ) the absence of plaque formation in some viruses. to address some of these limitations, alternative approaches have been developed. one alternative is the % tissue culture infectious dose (tcid ), an endpoint dilution assay that quantifies the infectious titer required to produce cytopathic effects in % of a tissue culture. however, the tcid assay is also susceptible to some of the limitations of the plaque assay (e.g., long durations before cytopathic effects are detectable). to improve the detection limit of plaque assays, integrated cell culture followed by quantitative pcr (icc-qpcr) has been used. the number of infective virus particles is enhanced for detection by first culturing the virus particles with host cells. the virus is then extracted for rt-pcr to quantify the amount of viral rna. samples that had infective virus particles produced a larger end-point value during rt-pcr than samples that did not have viable virus. icc-qpcr has been used for many virus strains. , [ ] [ ] [ ] [ ] an alternative method to detect viable viruses without cell culture is to pretreat the collected samples with proteinase k and rnase before performing rt-pcr. if the virus envelope is damaged (i.e., the virus is non-infective), proteinase k and rnase will break down the capsid and any rna that is not in a stable viral envelope. however, this method still requires optimization and broader validation. , other detection methods exist but have not been fully developed or validated for coronavirus. for example, elisa and immunofluorescence assays that take - minutes have been developed for influenza virus detection. while these tests have high specificity ( . % from bivariate random-effects regression), they have modest sensitivity ( . %) only. as can be seen, most current methods take a few hours to measure virus rna concentration and days to measure virus infectivity. additionally, most methods require instrumentation and/or cell culture that may be inaccessible. there is a critical need for the rapid detection (< hours) of viable infective virus not only from patient samples, but also from aerosols, droplets, and fomites to better understand the infection risk via these transmission routes. mathematical models of infection risk can be useful to estimate the relative importance of transmission routes and to evaluate the effectiveness of preventative measures. here we discuss several existing models of an individual's risk of infection from their immediate environment. a more detailed review can be found elsewhere. large-scale models at the community level are beyond the scope of this review, but can be found in prior work. , models of infection risk often consist of two tasks: estimating the viral load, or dose, received, and estimating the infection risk based on the dose received. infection risk models can be classified as deterministic, where an individual is infected if the dose exceeds a critical value, or stochastic, where an individual's probability of infection is a function of the dose received. typically, stochastic models are more biologically relevant. to estimate the dose received, various strategies exist to model the transmission routes of the virus from the surroundings to the individual. in the case of aerosol transmission, a poisson distribution is often used to describe the distribution of virus particles in the air. this distribution can be used to estimate the dose an individual receives through aerosol inhalation. for the wells-riley model (discussed later), the airborne "dose" is formulated in terms of a hypothetical unit called a quantum of infection. , the wells-riley "dose" includes parameters such as the quanta generation rate, room ventilation rate, and exposure time to describe aerosol transmission and can be further modified to account for complexities such as uneven mixing. in the case of fomite transmission, some models estimate the surface concentration through deposition from an airborne source, while others directly prescribe a distribution on a surface based on experimental measurements. the rate of virus transferred between two surfaces is often formulated as the product of contact frequency, transfer efficiency, surface concentration, and contact area in eq. , where Ṅ is the rate of virus transferred to surface , , is the frequency of contact between surfaces and , , is the transfer efficiency from surface to , is the virus concentration on surface , and , is the contact area between surface and . the virus can be transferred serially between surfaces before finally transferring to an individual's facial membranes, giving the final dose received through fomite transmission. more complex models integrate multiple transmission routes and phenomena together, such as a markov chain model of fomite and aerosol transmission that include a gradual loss of virus viability. to estimate the infection risk based on the dose received, two popular models have emerged: the wells-riley model and the dose-response model. the wells-riley curve is an exponential curve and is based on a hypothetical "dose" unit called a quantum of infection as described above. , a basic form of the wells-riley curve is shown in eq. , where is the probability of infection, is the number of infectors, is the quanta generation rate, is the pulmonary ventilation rate of the susceptible individual, is the exposure time interval, and is the room ventilation rate with clean air. while the wells-riley model is convenient to use, its formulation based on the quantum of infection limits its application to aerosol transmission only. still, it remains a useful model and has been applied to various cases including sars-cov- . the dose-response model was adapted for respiratory viruses from models of toxicity. the dose input is a physical quantity of the virus, and can be extended to multiple situations including aerosol transmission, fomite transmission, and the efficacy of surface disinfection strategies. a basic form of an exponential dose-response curve is shown in eq. , where is the probability of infection, is the number of infectors, is the number of airborne pathogens released per infector per unit time, is the pulmonary ventilation rate of the susceptible individual, is the deposition fraction of pathogens in the alveolar region, is the exposure time interval, and is the room ventilation rate with clean air. extensions and alternative forms of the dose-response curve can be found elsewhere. , some studies have begun to apply these models of individual infection risk to a larger system such as a household. for example, interactions between multiple healthy individuals, infected individuals, and objects in a household can be modelled and the dose-response model is then applied to each individual. , ultimately, the choice of the model depends on the application, and models of infection risks have emphasized the effectiveness of promising interventions including fomite disinfection. , the ability of a virus to transfer between and persist on different surfaces, including skin, plays a crucial role in the overall infectivity of a virus by means of fomite transmission. understanding the adsorption and transfer rates between skin and fomites is critical for modeling the spread of viruses. , furthermore, understanding virus persistence on different surfaces under different environmental factors can inform decision making for disinfection protocols. in this section, we will review the factors affecting virus adsorption, transfer, and persistence on different surfaces, and then discuss surfaces that are at high risk of contamination. the adsorption of virus on fomites and their subsequent transfer to other surfaces is a multi-factor problem that depends on the properties of the virus, the fomite, and the environment. the physical description of virus adsorption borrows from formulations of colloid adsorption, treating virus particles as soft colloidal spheres and using gibbs free energy to model the interactions between virus particles and the adsorbing surface. like colloid adsorption onto surfaces, virus adsorption onto fomites is primarily driven by electrostatic, hydrophobic, and van der waals interactions ( figure ). the relative contribution of these interactions is modulated by environment ph and ionic strength. , , classical models of virus adsorption adopt the derjaguin-landau-verwey-overbeek (dlvo) theory for colloid adsorption onto surfaces. it accounts for electrostatic and van der waals interactions between viruses and surfaces. [ ] [ ] [ ] however, the extended-dlvo (xdlvo) model, which considers hydrophobic interactions, was found to agree more with experimental observations of virus adsorption. [ ] [ ] [ ] xdlvo is expressed in terms of gibbs free energy of interaction, shown in eq. , where electrostatic or double-layer (dl), hydrophobic (hyd), and van der waals (vdw) contributions are summed. entropy changes ( ) are usually ignored. a negative favors adsorption. , detailed formulations for each component of the total free energy for a spherical virus particle adsorbing onto a flat plate can be found in prior work. electrostatic forces drive long-range adsorption dynamics dictated by the radius of the virus's electrical double-layer (debye length) and the charge of the absorbing surface. , all viruses, including sars-cov- , express unique protein markers on their surfaces. these markers consist of weakly acidic or basic polypeptides and amino acids ionizing residues that give viruses characteristic isoelectric points (pi) (also see section . ). , the net charge of a virus is thus determined by the ph of its environment. the net charge on a virus causes the formation of an electrical double-layer that extends from the stern layer, the first layer of immobile charges attached to the surface of the virus particles, and across the gouy diffuse layer, the region of charge imbalance that results in an electrical potential. [ ] [ ] [ ] in addition to ph, the ionic strength of the surrounding medium is another important parameter affecting electrostatic interaction. at high ionic concentrations (> mm nano ) , electrostatic screening stunts the zeta potential at the charge slipping plane and weakens the effects of surface charge for both attractive and repulsive interactions. in the absence of electrostatic interaction, if adsorption occurs, it is typically attributed to hydrophobic interactions. , the hydrophobic effect causes an attractive force between a virus and adsorbing surface. the effect is due to electron-donor and -acceptor, i.e., lewis acid-base, interfacial interactions. under hydrophobic interactions, there is a tendency of apolar species such as molecular chains or particles to aggregate, providing an energetically favorable mechanism of adsorption due to the minimization of interfacial area between the virus and the adsorbing material. in the absence of electrostatic interactions, hydrophobic effects dominate because they are apolar by nature. the energy of hydrophobic interactions largely depends on the prevalence of hydrophobic groups on a virus particle's surface. greater virus hydrophobicity has been shown to correlate with higher rates of adsorption regardless of ionic strength. , , the presence of chaotropic (i.e., scn -, ci ccoo -) or anti-chaotropic (i.e., no -, so -, f -) agents can promote or hinder, respectively, hydrophobic adsorption. hydrophobic interactions are considered to have a short-range effect compared with electrostatics. van der waals forces are considered to be of secondary importance. their relative contribution, as with electrostatic and hydrophobic interaction, is a function of virus and environmental properties. for example, van der waals forces may play a significant role in the adsorption of viruses that carry a neutral charge in their environment. furthermore, materials known to generate large van der waals potentials are also more likely to adsorb viruses. the contribution of van der waals forces to adsorption can be quantified by lifshitz theory, which predicts that materials with higher dielectric susceptibility produce higher van der waals potentials. by this reasoning, metals have better adsorbing effectiveness than most organic substances. in general, the effectiveness of materials to absorb viruses follows: metals > sulfides > transition metal oxides > sio > organics. this theory suggests that high ionic strength or a fluid ph equal to virus pi is necessary for adsorption to most organics. under these conditions, the debye length is shortened and viruses are able to get sufficiently near to organic surfaces to absorb by van der waals interactions. , , there exist some gaps in the comprehensive understanding of the physicochemical mechanisms in virus adsorption onto fomites. while xdlvo theory on virus adsorption can begin to explain the observations of many virus strains, including sars-cov- , readily adsorbing to a variety of non-porous surfaces (e.g., steel, glass, plastic), , , , , the observed virus adsorption onto porous fomite surfaces (e.g., cardboard, cloth) is not well described by xdlvo. some studies have indicated the need to account for steric effects and surface roughness. , other studies emphasized the pitfalls of modeling viruses as soft colloids with homogeneous charge distributions. unlike a soft colloid or even a virus-like particle (vlp) engineered with viral structural proteins, the pi of true viruses depends on the complex physicochemical structure of the outer surface and the genetic material packed within the capsid. , , , the state of understanding in physicochemical mechanisms of virus adsorption in aqueous environments is fairly advanced, but there is still significant room for research in elucidating the mechanisms of dry contact transfer. although a number of studies have quantified the rates of transfer between dry surfaces, including skin (also see section . ), the precise physicochemical basis for virus transfer in dry conditions has been unaddressed. the tendency of a virus to transfer between fomites is likely determined by differences in adsorption energies between the two surfaces. in the case of porous materials, lower rates of transfer are likely due to viruses entrapped in their matrix due to increased surface area for attachment. , to our knowledge, no work has examined the physicochemical interactions, adsorption, and transfer kinetics of sars-cov- on different surfaces especially in dry conditions. despite a lack of data on the transfer efficiency of coronaviruses, numerous studies have examined the transfer efficiency for other viruses. an overall mean transfer efficiency of % ± % was found between fingerpads (either washed or unwashed prior to inoculation with a virus) and glass for three types of non-enveloped bacteriophages (ms , φx , and fr). the efficiency was calculated by measuring the viral pfu of the surface before and after contact. in this study, prior handwashing was found to reduce the transfer efficiencies only slightly. the reduction due to washing was greater in fingerpad-to-glass transmission than glass-to-fingerpad. this result is likely because of changes in the skin moisture or ph due to handwashing before inoculation with a virus. a similar transfer efficiency was found for ms from fingertips to glass and to acrylic (~ %), but this value increased to . % in humid conditions. transfer efficiency of psd- phage from hand to mouth was found to be . %, representing a skin to skin pathway. studies have shown that viruses adsorbed on surfaces can maintain high rates of survival and infection potential. exactly how long viruses retain their viability on a surface is highly variable and dependent on: ) surface porosity, ) environmental factors, and ) virus envelope characteristics. , first, nonporous surfaces, compared with porous surfaces, are more effective in receiving and transferring viruses, and are typically better at preserving virus viability because they do not draw moisture away from adsorbed viruses. however, if a porous material is inoculated, it is capable of harboring most strains of viruses (especially at low temperatures e.g., ℃), and can remain contagious despite the lower rates of transfer to skin. sars-cov- has demonstrated an ability to contaminate a wide range of porous and nonporous fomites. table shows the persistence of sars-cov- and other coronaviruses on various surfaces. to our knowledge, no studies have evaluated the persistence of sars-cov- or sars-cov- on skin. however, parainfluenza was shown to be % viable within minutes, while rhinovirus was shown to be . % and % viable on skin after and hours, respectively. the percent of viability was determined by comparing pfu before inoculation and at various times points. we note that no work has explicitly investigated the physicochemical reasons why some surfaces support longer virus persistence. as viruses can be inactivated by desiccation, improved persistence is likely due to the ability of a surface to maintain a moist microenvironment. second, environmental variables such as temperature, humidity, and resident microfauna can influence virus adsorption and viability. in general, increased temperature and moderate humidity levels have adverse effects on the persistence and viability of coronaviruses and other viruses. in a study on the viability of dried sars-cov- on smooth plastic surfaces, the virus was found to be viable for over days at - °c with - % relative humidity (rh). however, virus viability decreased significantly (> log reduction) at °c with > % rh. in another study using phi as a surrogate, the virus survived best at high (> %) and low (< %) rhs. they also found that rh is a more significant factor in virus survivability than absolute humidity (ah). in addition to temperature and humidity, the presence of other microbes can also influence the survival of viruses. though the presence of microbes can reduce the rate of desiccation of the viral particles enhancing their persistence and viability, microbial proteases and fungal enzymes can be harmful to their existence. , third, viral persistence on fomites also depends on the type and the strain of the virus. in general, non-enveloped enteric viruses (e.g., adenovirus, rotavirus) can persist on fomites longer than enveloped viruses (e.g., coronaviruses). the lack of a lipid membrane in non-enveloped viruses make them less susceptible to inactivation than enveloped viruses, where the disintegration of the lipid envelope (e.g., by common disinfectants; see details in section ) causes the loss of the viral envelope proteins involved in virus adsorption and cell penetration thereby rendering them inactive. additionally, non-enveloped viruses are less susceptible to desiccation than their enveloped counterparts because of their lack of lipid membrane envelopes. these characteristics make them easier to spread and persist on surfaces over long periods of time compared with enveloped viruses. in principle, all surfaces or objects can be considered potential fomites and are at risk of contamination. , in practice, knowledge of which objects are at high-risk of contamination could guide the design of optimal disinfection strategies. for a given object, the risk of contamination can depend on the interaction between the virus and the material, the frequency at which the object is contacted, the object's distance from an infected individual, and the environmental conditions. first, the combination of virus composition and surface properties can influence the likelihood of contamination (see details in section . ). second, objects that are frequently handled or are in high contact with individuals are at higher risk of contamination. in a hospital setting, contamination has been detected on numerous high-contact surfaces, including door handles, bed rails, tables, call/control panels, other near-patient surfaces, office equipment, and even sterile packaging. , a study of the isolation rooms of sars-cov- infected patients in singapore showed contamination of a similar list of high-contact surfaces. while the floor of the isolation room and the shoes worn by individuals entering and exiting the room tested positive for sars-cov- , the floor immediately outside tested negative, suggesting contamination by footwear is low. third, an object's proximity to an infected individual affects its risk of contamination. an object can be contaminated from a distance due to deposition of droplets or aerosols onto its surface. the risk of contamination by droplets or aerosols decreases when the object is further away from infected individuals, as viral shedding by coughing, sneezing, or exhaling can potentially deposit droplets and aerosols onto fomites as far as - m away. , in the aforementioned singapore study, all air samples taken from the isolation room tested negative while the air outlet fans tested positive, suggesting that sars-cov- is not detectably aerosolized in these conditions but is still able to transfer from air to a potential fomite. a study in wuhan hospitals found that the highest concentrations of sars-cov- in the air were, surprisingly, not in patient rooms but in toilet facilities. . even aerosol generation from personal protective equipment (ppe) removal can create fomites. doffing ppe has the potential to aerosolize the virus and transfer it to other ppe in changing rooms. fourth, the environmental conditions can affect an object's risk of contamination. air currents could potentially determine the flight path of droplets and aerosols, as proposed in a case study of a guangzhou restaurant where the sars-cov- infection pattern aligned with the air conditioning currents. the amount of foot traffic and the degree of connectivity between rooms could also affect where high sars-cov concentrations may be found. we note a limitation to many of these studies is the use of rt-pcr to identify viral rna. the presence of viral rna is not indicative of viability, and viral culture is needed to determine infective virus . the above factors can be used to help identify and predict surfaces at high-risk of contamination. to further quantify the role of these surfaces as fomites, surface viral concentrations need to be measured, and contact frequencies can be derived from observational studies. such quantifications can be used as input parameters in modeling infection risk and designing optimal disinfection strategies. for example, a model of disinfecting strategies for methicillin-resistant staphylococcus aureus (mrsa) predicted once-daily whole room cleaning to be less efficient than frequent targeting of high contact surfaces in preventing indirect contact transmission. strategies to intercept fomite transmission revolve around inactivating the virus, improving personal hygiene, or using ppe. here, we discuss different mechanisms of virus inactivation on surfaces and hands, focusing on strategies that have been shown to inactivate sars-cov- and other enveloped viruses. we will not discuss ppe as it has been discussed elsewhere. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in order for a virus to be infective, it must fuse with a host cell, insert its genome into the cell, and replicate. these processes require an enveloped virus to have an intact envelope and nucleocapsid. to inactivate a virus, at least one of these components needs to be disrupted. it is important to understand the mechanisms of virus inactivation based on virus composition, structure and function in order to: ) understand the efficacy of disinfectants on viruses, ) predict the response of a new strain of virus to a disinfectant, ) identify common sites on proteins, envelopes or genomes that are vulnerable to disinfectant treatment that are shared by many viruses, and ) enhance the design of antiviral agents and therapies that target specific viral components. one method of predicting virus inactivation mechanisms is a composition-based method. it takes into account the reaction rate constants between disinfectants and specific nucleotides and amino acids found in viral structures (e.g., proteins, nucleic acids, and envelope lipids) ( table ). the relative contribution of a viral structure to inactivation can be predicted by summing the relative abundance of each nucleotide or amino acid multiplied by the respective rate constants for a given disinfectant. , a limitation of the composition-based method is that it does not account for the complex interactions between adjacent monomers. to our knowledge, no model exists that accounts for viral structure as well as composition. this section summarizes current disinfection strategies and their effectiveness for sars-cov- and related viruses (figure ) . uv irradiation is a widely-used method of surface disinfection. here, we discuss the inactivation of sars-cov- by uvc and solar irradiation. uvc irradiation ( - nm, typically nm is used) damages nucleic acid bases in genetic material, and to a lesser extent, proteins in virus capsids. , uvc irradiation induces dimerization of adjacent uracil bases in rna, forming pyrimidine dimers that disrupt the rna structure, which inhibits the viral replication process and inactivates the virus. , exposure of sars-cov- to a uvc light source ( nm, µw/cm ) held cm above the virus resulted in a ~ . log tcid /ml reduction in virus titer within minutes. after this point, virus inactivation plateaued, until viral activity became undetectable at minutes. exposure to other uv wavelengths (e.g., uva) was found to be insufficient to inactivate the virus. uvc irradiation as a disinfection strategy poses a few challenges. the time required to inactivate sars-cov- using uvc ( nm, µw/cm ), ~ minutes, is significantly longer than the time required using chemical disinfectants ( s to min). , this time to inactivate only applies to regions of an object directly exposed to uvc irradiation. disinfectant effectiveness reduces significantly in shadowed regions. additionally, uvc radiation may pose health risks, including skin cancer and ocular damage to exposed individuals. nonetheless, uvc-based disinfection can be valuable for use in applications where the irradiation can be shielded from humans, and have been used in, for example, empty buses and other vehicles. the inactivation of viruses by solar irradiation has also been studied, especially in the context of the disinfection of water. the range of uv wavelengths in sunlight that reach the surface of the earth is between and nm, as uvc is typically completely blocked by the atmosphere. , the antiviral properties of sunlight primarily come from uvb light, which can also form pyrimidine dimers, but these mechanisms are not as well studied as the mechanisms of uvc-based disinfection. additionally, the solar spectrum, especially in the uv wavelengths, can vary significantly depending on environmental factors, the time of day, and the season. such factors can lead to large variations in the efficiency of virus inactivation by sunlight. a wide variety of chemical disinfectants are currently available to combat the spread of sars-cov- (table ). the effectiveness varies depending on the virus inactivation mechanism. in general, there are modes of inactivation by disinfectants: ) disruption of the lipid layer of the envelope (e.g., ethanol and detergents), , ) modification of important protein sites on the envelope or capsid (e.g., chlorine and glutaraldehyde), , , , , and ) reaction with the nucleotides and amino acids in the genetic material, leading to the degradation of the nucleic acids (e.g., chlorine). , chemical disinfectants are typically evaluated with suspension and carrier tests. suspension tests combine a known titer of a virus in solution with a disinfectant and evaluate virus titer after a period of time that depends on the disinfectant manufacturer's directions of use. however, suspension tests are considered less challenging for the disinfectant under scrutiny, and may not reflect the practical usage of disinfectants to clean contaminated surfaces. quantitative carrier tests are performed by allowing an aliquot of virus solution to dry on a surface before applying the disinfectant. this test is conducted under conditions that are more relevant to practical use of disinfectants, and is, therefore, a more appropriate measure of disinfectant effectiveness. chemical disinfectants have been evaluated for their ability to inactivate various types of coronavirus. , for sars-cov- , % and % household bleach, % ethanol, . % povidoneiodine, . % chloroxylenol, . % chlorhexidine, and . % benzalkonium chloride have been found to reduce an initial viral load of ~ . log tcid /ml to undetectable levels at room temperature within minutes in suspension tests. for other coronaviruses (e.g., sars-cov- , mers-cov, and mhv), %, %, % and % ethanol; - % -propanol; a mixture of % -propanol and % -propanol; . % sodium hypochlorite; and %, % and . % povidone iodine were found to reduce viral activity by at least log within seconds in suspension tests. carrier tests performed on stainless steel disks showed > log reduction within minute for hcov- e, mhv, and tgev exposed to % ethanol and for hcov- e exposed to . - . % sodium hypochlorite and % glutaraldehyde. in another study, hydrogen peroxide vapor inactivated tgev in a carrier test by a reduction of . - . log , but it took - hours to do so. prior carrier tests have been performed primarily on stainless steel, and to our knowledge, no carrier tests have been conducted for sars-cov- on any material. results using stainless steel carriers may not reflect disinfectant effectiveness on other fomites with different surface properties (e.g., surface chemistry, wettability, porosity, roughness). the dependence of disinfectant effectiveness on surface properties remains an open question. plasma is an ionized gas made up of charged and uncharged particles (i.e., ions and electrons, and molecules and atoms, respectively), reactive species, and uv photons. inactivates sars-cov- . uv light may be applicable to surfaces sensitive to heat or chemicals. uv irradiation is less efficient at low temperatures (e.g., < °c ) and is not suitable for all environments. incompatible with photosensitive materials. as heavy particles. cold atmospheric plasma (cap) is a low-temperature, non-thermal plasma that is produced by a variety of methods using gases such as helium, argon, nitrogen, heliox and/or air. two common methods for producing cap are dielectric barrier discharge and atmospheric pressure plasma jet. cap has been considered for disinfection applications in dentistry and oncology and in food processing. , the antimicrobial properties of cap are attributed to reactive oxygen and nitrogen species generated in the non-thermal plasma. [ ] [ ] [ ] the detailed inactivation mechanisms are still under investigation. however, it is believed that the reactive species damage genetic material and proteins. in one study, singlet oxygen in plasma was implicated in the inactivation of bacteriophages through multiple mechanisms involving reactions with amino acids and dna nucleotide oxidation and crosslinking, but the primary mechanism was thought to be singlet oxygen-induced crosslinking of capsid proteins. cap has been shown to inactivate potato virus y in water, in a study aimed to decontaminate water supply systems. cap was found to inactivate potato virus y in minute, compared with minutes using mg/l of hydrogen peroxide. plasma-activated water (paw) is another form of plasma-based disinfection. water is treated with non-thermal plasma to produce paw, which is more acidic and contains more reactive oxygen and nitrogen species than regular water rendering it antimicrobial. paw has been proposed as an antiviral agent. for example, in a study testing paw for its potential application in producing an inactivated vaccine for newcastle disease (nd), the enveloped virus responsible for avian nd was found to be fully inactivated in paw. paw has also been proposed for its potential application in the disinfection of fresh produce. while promising in these studies, the effectiveness of cap or paw on inactivating coronaviruses remains to be demonstrated. heat treatment is a well-known method for disinfecting surfaces. at temperatures exceeding ~ °c, viral capsid proteins are denatured and rna is damaged. sars-cov- has been shown to become inactivated within minutes at °c, with a reduction from an initial concentration of ~ . log tcid /ml to undetectable levels. sufficiently high temperatures should be used. moderately high temperatures ( - °c) only cause minor damage to the protein capsid, and fail to inactivate some viruses. autoclave is a common method of sterilizing equipment using heat treatment in a laboratory or clinical environment. autoclaves produce steam at high temperatures (~ °c) in a pressurized chamber. at this temperature, most microbes, including viruses, are inactivated. the surface being sterilized is exposed to the high temperature and pressure environment for a varying amount of time, depending on the material and size of the object. liquids are usually sterilized for - minutes, while objects made of glass and plastics require ~ minutes of sterilization. in one experiment, avian coronavirus and avian pneumovirus carried by cotton swabs were inactivated after heat treatment using an autoclave for minutes. in the same study, heating the same viruses in a microwave oven for seconds was also found to be sufficient for inactivation. engineering self-disinfecting surfaces is an emerging avenue of research for preventing infection transmission by fomites. while certain materials like copper and silver have long been known to possess intrinsic antimicrobial properties, various types of surface modification and functionalization can also give rise to antimicrobial properties against bacteria and viruses. , only a limited number of works have focused on virus-specific self-disinfection. this section highlights some of these studies. readers are referred to a recent review for details. copper and silver alloys are known viricidal agents that inactivate viruses through multiple modes of action. the primary mechanism involves direct interaction between metal ions and microbial proteins, or indirect interaction through the formation of radicals that are damaging to dna and lipid membranes. , copper has been shown to retain its effectiveness across a range of humidities and temperatures, while silver had drastically reduced antimicrobial effectiveness at low humidities (~ % rh). pure copper and alloys with - % copper were found to be most effective in inactivating viruses. abrasion and removal of the outer oxide layer caused a slight decrease in effectiveness. in one study, x pfu/cm of non-enveloped murine norovirus was inactivated in under hours at room temperature. inactivation of norovirus by copper was found to be up to x faster in dry conditions compared to wet, but the mechanisms underlying such differences were unclear. in another study, copper yielded a near -log reduction in enveloped influenza a virus particle count after hours. table includes the effectiveness of copper on some coronaviruses in lowering viability periods. some studies examining the clinical effectiveness of copper surfaces have shown notable improvement towards infection control benchmarks with % less bacteria when compared with control plastic surfaces on icu beds. photocatalytic action has been shown to be highly effective in inactivating microbes by damaging dna and lipid membranes via the photocatalyzed formation of hydroxyl radicals in the presence of photoactive oxides. , numerous enveloped and non-enveloped viruses have been shown to become inactivated by photocatalytic disinfection. titanium dioxide (tio ) is a popular photocatalytic material due to its long lifetime, effectiveness over a wide range of microbes, and environmental friendliness. tio has the potential to provide antiviral protection to a range of materials. for example, cotton fabrics have been impregnated with tio via magnesium nanoparticle carriers. tio impregnated into resin, fiberglass, and pvc have also been used to coat various surfaces in hospitals, schools, and other public places. despite the potentials that self-disinfecting surfaces present, widespread adoption of selfdisinfecting surfaces, especially in hospitals, has been limited by three main obstacles. the first is a lack of clinical trials showing their efficacy in practice. second, the costs associated with upgrading or retrofitting equipment discourages hospitals from taking initiatives to introduce selfdisinfecting surfaces, though the savings from decreasing nosocomial infections could offset this cost. third, characterization of effectiveness over repeated cycles has not yet been quantified. frequent handwashing can lower the incidence of transfer from fomites to facial membranes via contact. considering the frequency that adults touch their faces ( times per hour) and the risk of infection that is associated with face touching, handwashing is a critically important personal hygiene habit. in a hospital setting, the who recommends critical moments for healthcare workers to wash hands: ) before contact with a patient, ) before a cleaning procedure, ) after exposure to bodily fluids, ) after contact with a patient, and ) after contact with fomites surrounding patients. although virus transfer to hands is only mildly reduced after recent handwashing, , handwashing is effective in reducing the spread of a virus from hands. , however, handwashing is only as effective as the frequency, the effectiveness of the antiseptic, and thoroughness. the cdc recommends washing for a minimum of seconds. this recommendation was based on a few empirical studies, [ ] [ ] [ ] including one that investigated handwashing practices such as wash time ( s vs. s) and effect of soiled hands on infectivity reduction. to evaluate the effectiveness of a handwashing, a fingerpad method is typically used. , here a virus is inoculated on pre-cleaned fingerpads, allowed to dry, then subjected to exposure to an antiseptic by static contact with the fingerpad. , the astm specifies that an effective handwashing antiseptic must yield a minimum reduction of log ( . %) in virus titer from the initial inoculation titer. however, this standard does not specify a minimum contact time between the fingerpad and the antiseptic. another potential drawback of these standard tests is that they may not be representative of in vivo handwashing behavior of healthcare workers or the general public. viruses present a unique challenge for handwashing in that their structure and ability to survive on skin may evade inactivation by handwashing methods customized for bacterial disinfection. alcohol and isopropanol-based antiseptics ( - % ethanol) are the most effective non-hazardous antiseptic, especially against enveloped viruses. other who-recommended antiviral antiseptics (from the most to the least effective) are iodophors ( . - %), chlorhexidine ( . - %), and chloroxylenol ( . - %), all of which are less effective than alcohol. in regard to hand sanitizers, sars-cov- has been confirmed to be the most susceptible to ethanol and isopropanol using suspension tests with part virus at tcid /ml, part media, and parts by volume of an ethanol-or isopropanol-based who-recommended antiseptic formulation. a > log sars-cov- reduction was achieved in seconds using ethanol and isopropanol formulations at % and % concentrations, respectively, and using dilutions as low as %. , . methods of applying chemical disinfectants chemical disinfection remains one of the most commonly used methods for virus disinfection. the effectiveness of chemical disinfection depends on the disinfectant contact time, the surface properties of the fomite, and other environmental factors. as such, how the chemical disinfectant is applied has a significant impact on the disinfection effectiveness. this section discusses two common methods of disinfectant application: wiping, and spraying. for chemical disinfectants to work properly, they must be directly applied to the target surface. the most straightforward and conventional method of applying a chemical disinfectant to a surface is to use a manual wipe. manual wiping utilizes both physical removal of viruses (which may not kill the viruses), and chemical activity of the disinfectant. the chemical disinfectant can be applied immediately before wiping, or the wipe can be packaged and pre-wetted with the disinfectant. the wipe process typically takes seconds to complete. despite its convenience, manual wiping is limited by human error and cross-contamination between surfaces. , if the proper protocols are followed, manual wiping can effectively disinfect surfaces contaminated with norovirus, , adenovirus polyomavirus, and numerous bacteria including staphylococcus aureus and clostridium difficile. however, the effectiveness of manual wiping depends on multiple factors including the type of wipe, the type of disinfectant, the target pathogen, the wiping technique (e.g., area covered, pressure applied), and the ratio of disinfectant volume to target surface area. insight into these factors, such as the effectiveness of microfiber wipes, the number of wipe passes over a surface, and adsorption of disinfectants to wipe material, could serve to optimize wipe protocols. the key limitations of manual wiping arise from human error in the wiping process, and cross-contamination of pathogens. multiple studies reported that only ~ % of near-patient surfaces in hospitals were cleaned according to policy. if wipes are re-used between surfaces, there is a risk of transferring pathogens between surfaces. these limitations could potentially have serious consequences especially in a hospital setting, and highlight the need for an automated and effective disinfection strategy. while spray disinfectants are commonly used to disinfect surfaces, their effectiveness has not been well characterized. to our knowledge, no comprehensive model has characterized spray disinfection efficiency taking into account aerosol physics, virus heterogeneity, and surface characteristics. nevertheless, given that disinfection effectiveness depends on disinfectant contact time (and thus disinfectant volume, if the evaporation of disinfectant is fast), the spray characteristics (e.g., spray droplet size and density) and disinfectant droplet deposition on surfaces are critical factors that must be considered in any attempt to evaluate the effectiveness of spray disinfection ( figure ). atomization is a general term referring to the disintegration of a liquid stream into droplets. table summarizes a few common commercial atomizers. atomizers that have been used for applying surface disinfectants (and pesticides) include electrostatic atomizers, hydraulic atomizers, pressure atomizers, spill return atomizers, and ultrasonic atomizers. in particular, spill return atomizers, being able to produce fine sprays, have been shown to be applicable for disinfecting healthcare surfaces. for classroom, healthcare, and general disinfection purposes, handheld electrostatic sprayers also exist with adjustable spray parameters. , in the agriculture industry, electrostatic spray systems have been mounted on unmanned aerial vehicles for spraying pesticides on crops. , drop size and drop-size distribution are critical parameters determining spray disinfection efficiency. table summarizes several considerations in the choice of droplet size for spray disinfection. the covid- pandemic has revealed major gaps in our scientific knowledge, not only in the biology of how the virus infects humans, but also the role of physicochemical processes and surface science in the transmission and inactivation of the virus. box lists some of the open questions we have identified. addressing these questions will allow us to devise more effective strategies to combat the spread of the disease. for example, quantitative models predicting the locations of high-risk areas within a building and high-risk objects within those areas can inform the prioritization for disinfection. the identification of surfaces with high contamination risk also presents an opportunity for self-cleaning communal surfaces such as water faucets or door handles. a better understanding of disinfectant effectiveness on different surfaces and their potential side effects allows one to choose the optimal disinfection strategy for specific applications. while our review is by no means exhaustive, we hope that it can provide the basis for researchers in the physical sciences interested in covid- to take on some of the open research challenges, so that as a community we can be better prepared for the next pandemic. identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus a nucleocapsid protein the m protein of sars-cov: basic structural and immunological properties the coronavirus nucleocapsid protein virus isoelectric point determination using single-particle chemical force microscopy the impact of capsid proteins on virus removal and inactivation during water treatment processes who. laboratory biosafety' ' guidance related to the novel coronavirus world health organization production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level setting retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov antimicrobial peptide capsids of de novo design survival of surrogate coronaviruses in water effects of air temperature and relative humidity on coronavirus survival on surfaces inactivation of surrogate coronaviruses on hard surfaces by health care germicides transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells stability of porcine epidemic diarrhea virus on fomite materials at different temperatures. veterinary sciences severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain canine coronavirus inactivation with physical and chemical agents action of disinfectants on canine coronavirus replication in vitro survival of the enveloped virus phi in droplets as a function of relative humidity, absolute humidity, and temperature effect of testing method on apparent activities of antiviral disinfectants and antiseptics surface cleaning and disinfection: efficacy assessment of four chlorine types using escherichia coli and the ebola surrogate phi virus transfer at the skin-liquid interface viruses at solid-water interfaces: a systematic assessment of interactions driving adsorption turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid- the role of particle size in aerosolised pathogen transmission: a review face touching: a frequent habit that has implications for hand hygiene early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia factors determining human-to-human transmissibility of zoonotic pathogens via contact on air-borne infection* study ii. droplets and droplet nuclei how far droplets can move in indoor environments--revisiting the wells evaporation-falling curve violent expiratory events: on coughing and sneezing review and comparison between the wells-riley and dose-response approaches to risk assessment of infectious respiratory diseases modeling infectious disease dynamics in the complex landscape of global health the mathematics of infectious diseases an analytical framework for relating dose, risk, and incidence: an application to occupational tuberculosis infection airborne contagion and air hygiene. an ecological study of droplet infections. airborne contagion and air hygiene. an ecological study of droplet infections airborne spread of measles in a suburban elementary school exploring surface cleaning strategies in hospital to prevent contact transmission of methicillin-resistant staphylococcus aureus modeling surface disinfection needs to meet microbial risk reduction targets an integrated model of infection risk in a health-care environment a probabilistic transmission dynamic model to assess indoor airborne infection risks a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection modeling of human viruses on hands and risk of infection in an office workplace using micro-activity data quantifying the routes of transmission for pandemic influenza fomite-mediated transmission as a sufficient pathway: a comparative analysis across three viral pathogens electrostatic forces control nonspecific virus attachmentto lettuce applied and theoretical aspects of virus adsorption to surfaces physical chemistry of virus adsorption and degradation on inorganic surfaces: its relation to wastewater treatment environmental protection technology acid-base interfacial interactions in aqueous media virus deposition onto polyelectrolyte-coated surfaces: a study with bacteriophage ms attachment of bacteriophages ms and Φx onto kaolinite and montmorillonite: extended-dlvo interactions virus' (ms , phix , and aichi) attachment on sand measured by atomic force microscopy and their transport through sand columns isoelectric point is an inadequate descriptor of ms , phi x and prd phages adhesion on abiotic surfaces isoelectric points of viruses the influence of ionic strength on the interaction of viruses with charged surfaces under environmental conditions a gouy-chapman-stern model of the double layer at a (metal)/(ionic liquid) interface emergence of a stern layer from the incorporation of hydration interactions into the gouy-chapman model of the electrical double layer forces dictating colloidal interactions between viruses and soil non-dlvo adhesion of f-specific rna bacteriophages to abiotic surfaces: importance of surface roughness, hydrophobic and electrostatic interactions impact of internal rna on aggregation and electrokinetics of viruses: comparison between ms phage and corresponding virus-like particles effect of ionic composition of suspending solution on virus adsorption by a soil column detection of air and surface contamination by severe acute respiratory syndrome coronavirus (sars-cov- ) in hospital rooms of infected patients persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents impact of chemical and structural anisotropy on the electrophoretic mobility of spherical soft multilayer particles: the case of bacteriophage ms quantitative nanoscale electrostatics of viruses issues concerning survival of viruses on surfaces transfer efficiency of bacteria and viruses from porous and nonporous fomites to fingers under different relative humidity conditions virus transfer between fingerpads and fomites comparative surface-to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage electrostatic forces control nonspecific virus attachment to lettuce survival of influenza viruses on environmental surfaces potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus and rhinovirus the effects of temperature and relative humidity on the viability of the sars coronavirus virus survival in the environment with special attention to survival in sewage droplets and other environmental media of fecal or respiratory origin formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system aerosol and surface stability of hcov- (sars-cov- ) compared to sars-cov- stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions survival of human coronaviruses e and oc in suspension and after drying onsurfaces: a possible source ofhospital-acquired infections human coronavirus e remains infectious on common touch surface materials antimicrobial surfaces and their potential in reducing the role of the inanimate environment in the incidence of hospital-acquired infections covid- outbreak associated with air conditioning in restaurant novel coronavirus (covid- ) pandemic: built environment considerations to reduce transmission a quantitative approach to defining "high-touch" surfaces in hospitals covid- ) outbreak: what the department of endoscopy should know decontamination and reuse of n respirators with hydrogen peroxide vapor to address worldwide personal protective equipment shortages during the sars-cov- (covid- ) pandemic evaluation of an ethanol-based spray disinfectant for decontamination of cover gowns prior to removal survival and disinfection of an enveloped surrogate virus on tyvek suits used for health care personal protective equipment disinfecting personal protective equipment with pulsed xenon ultraviolet as a risk mitigation strategy for health care workers evaluation of five decontamination methods for filtering facepiece respirators can n respirators be reused after disinfection? how many times? disinfection of reusable elastomeric respirators by health care workers: a feasibility study and development of standard operating procedures rapid evidence summary on sars-cov- survivorship and disinfection, and a reusable ppe protocol using a double-hit process assessment of n respirator decontamination and re-use for sars-cov- virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity virus disinfection mechanisms: the role of virus composition, structure, and function mechanisms of methods for hepatitis c virus inactivation capsid functions of inactivated human picornaviruses and feline calicivirus uv disinfection of adenoviruses: molecular indications of dna damage efficiency molecular detection, quantification and characterization of human polyomavirus jc from waste water in rio de janeiro, brazil framework for using quantitative pcr as a nonculture based method to estimate virus infectivity mechanisms of inactivation of hepatitis a virus by chlorine antiseptics and disinfectants: activity, action, and resistance formaldehyde and glutaraldehyde in the fixation of chromatin for electron microscopy antimicrobial activity, uses and mechanism of action of glutaraldehyde quantitative pcr for determining the infectivity of bacteriophage ms upon inactivation by heat, uv-b radiation, and singlet oxygen: advantages and limitations of an enzymatic treatment to reduce false-positive results sunlight-mediated inactivation of health-relevant microorganisms in water: a review of mechanisms and modeling approaches inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov ultraviolet radiation from welding and possible risk of skin and ocular malignancy protection against a wide uv wavelength range by bragg reflection from polycrystalline colloidal photonic crystals full inactivation of alphaviruses in single particle and crystallized forms astm. standard practice to assess the activity of microbicides against viruses in suspension: active standard astm e a disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides stability and inactivation of sars coronavirus virucidal activity of whorecommended formulations against enveloped viruses including zika, ebola and emerging coronaviruses efficacy of various disinfectants against sars coronavirus the antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for sars coronavirus rapid and effective virucidal activity of povidone-iodine products against middle east respiratory syndrome coronavirus (mers-cov) and modified vaccinia virus ankara (mva) chemical disinfection of nonporous inanimate surfaces experimentally contaminated with four human pathogenic viruses evaluating the virucidal efficacy of hydrogen peroxide vapour using uvc light-emitting diodes at wavelengths of to nanometers to inactivate foodborne pathogens and pasteurize sliced cheese safety data sheet disinfectants for use against sars spectrum chemical. material safety data sheet safety data sheet: chlorhexidine solution % cold atmospheric plasma: methods of production and application in dentistry and oncology cold atmospheric plasma as a novel method for inactivation of potato virus y in water samples bactericidal effect of various non-thermal plasma agents and the influence of experimental conditions in microbial inactivation: a review preparation of the inactivated newcastle disease vaccine by plasma activated water and evaluation of its protection efficacy mechanism of virus inactivation by cold atmospheric-pressure plasma and plasma-activated water plasma activated water (paw): chemistry, physico-chemical properties, applications in food and agriculture guide to autoclaving plastics and glass microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction controlling hospital-acquired infection: focus on the role of the environment and new technologies for decontamination a critical evaluation of current protocols for self-sterilizing surfaces designed to reduce viral nosocomial infections self-disinfecting surfaces and infection control silver coordination polymers for prevention of implant infection: thiol interaction, impact on respiratory chain enzymes, and hydroxyl radical induction the iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity effects of temperature and humidity on the efficacy of methicillin-resistant staphylococcus aureus challenged antimicrobial materials containing silver and copper inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity inactivation of norovirus on dry copper alloy surfaces inactivation of influenza a virus on copper versus stainless steel surfaces self-disinfecting copper beds sustain terminal cleaning and disinfection effects throughout patient care photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity understanding the antimicrobial mechanism of tio₂-based nanocomposite films in a pathogenic bacterium fabrication of visible light-induced antibacterial and self-cleaning cotton fabrics using manganese doped tio nanoparticles virus and bacteria self-shielding system self-disinfecting surfaces: review of current methodologies and future prospects hygienic hand antiseptics: should they not have activity and label claims against viruses? am guidelines on hand hygiene in health care; patient safety; world health organization epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs show me the science -how to field trial of a low cost method to evaluate hand cleanliness outbreaks where food workers have been implicated in the spread of foodborne disease quantifying the effect of hand wash duration, soap use, ground beef debris, and drying methods on the removal of enterobacter aerogenes on hands alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions guideline for hand hygiene in health-care settings methods to evaluate the microbicidal activities of hand-rub and hand-wash agents comparative in vivo efficiencies of handwashing agents against hepatitis a virus (hm- ) and poliovirus type (sabin) some principles of virucidal testing determining the virus-eliminating effectiveness of hygienic handwash and handrub agents using the fingerpads of adults; e ; standard who guidelines' ' on hand hygiene in health care: a summary the crucial role of wiping in decontamination of high-touch environmental surfaces: review of current status and directions for the future evaluating hygienic cleaning in health care settings: what you do not know can harm your patients limitations of the efficacy of surface disinfection in the healthcare setting effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces evaluation of the virucidal efficacy of disinfectant wipes with a test method simulating practical conditions best practice in healthcare environment decontamination a laboratory evaluation of the decontamination properties of microfibre cloths three swipes and you're out: how many swipes are needed to decontaminate plastic with disposable wipes? adsorption of available chlorine and quaternary by cotton and wool fabrics from disinfecting solutions efficacy of disinfectant-impregnated wipes used for surface disinfection in hospitals: a review fine sprays for disinfection within healthcare the dynamics of aerocolloidal systems: international reviews in aerosol physics and chemistry; revised ry. ry's technologies & applications electrostatic spray nozzle a capacitive type of electrostatic spraying nozzle spray systems co. automatic & air atomizing spray nozzles what determines the drop size in sprays? rotary atomizers for spray dryers twin-fluid atomization of viscous liquids: the effect of atomizer construction on breakup process, spray stability and droplet size ultrasonic atomization sonics & materials, inc. vibra-cell ultrasonic liquid processors ultrasonic atomization: effect of liquid phase properties we acknowledge support from the national science foundation (award # ). key: cord- - d i ix authors: querido, micaela machado; aguiar, lívia; neves, paula; pereira, cristiana costa; teixeira, joão paulo title: self-disinfecting surfaces and infection control date: - - journal: colloids surf b biointerfaces doi: . /j.colsurfb. . . sha: doc_id: cord_uid: d i ix according to world health organization, every year in the european union, million patients acquire a healthcare associated infection. even though some microorganisms represent no threat to healthy people, hospitals harbor different levels of immunocompetent individuals, namely patients receiving immunosuppressors, with previous infections, or those with extremes of age (young children and elderly), requiring the implementation of effective control measures. public spaces have also been found an important source of infectious disease outbreaks due to poor or none infection control measures applied. in both places, surfaces play a major role on microorganisms’ propagation, yet they are very often neglected, with very few guidelines about efficient cleaning measures and microbiological assessment available. to overcome surface contamination problems, new strategies are being designed to limit the microorganisms’ ability to survive over surfaces and materials. surface modification and/or functionalization to prevent contamination is a hot-topic of research and several different approaches have been developed lately. surfaces with anti-adhesive properties, with incorporated antimicrobial substances or modified with biological active metals are some of the strategies recently proposed. this review intends to summarize the problems associated with contaminated surfaces and their importance on infection spreading, and to present some of the strategies developed to prevent this public health problem, namely some already being commercialized. microorganisms' spreading, and consequent infection propagation is a serious concern worldwide, yet there are still very few guidelines or legislation for infection propagation control in public spaces. the exception is made for healthcare facilities with the existence of few guidelines and orientations for infection prevention and control, nevertheless hospital acquired infection (hai) remains a tremendous problem. one of the main causes for the high number of hai reported by the world health organization (who) is related to the ability of the microorganisms to survive for long periods in hostile environments such as dry surfaces [ , ] . rooms occupied with infected patients often have their surfaces contaminated with pathogens leading to the contamination of hands and gloves of medical staff, and consequent transfer of these microorganisms to patients or onto other surfaces. this cross-contamination mechanism has already been recognized and lead to the implementation of hygiene procedures for hands when contacting with patients. still, it is more likely for a patient to get an infection when staying in a room previously occupied by an infected patient. thus, infected patients are the primary source of surface and material contamination. visitors and/or asymptomatic carriers also contribute to the spread of microorganisms, especially in situations where there is an apparent sense of safety [ ] . as said before, there are guidelines for surface cleaning and disinfection in hospitals, however, those recommendations are not sufficient to solve such a problem and the incorrect following of the disinfection instructions can even cause greater contamination problems [ ] . this review considered english-language articles retrieved from pubmed database literature searches, bibliographies from published articles, and infection-control books and chapters, in a total of references published between and , considering the following criteria: the most recent studies performed on microbiological analysis on different surfaces reporting samplings performed on food contact surfaces, public spaces and hospital surfaces, where microorganisms occur naturally. surfaces studies, the most recent and relevant works developed on surface modification or functionalization were selected and two main types of surfaces were considered for this review -anti-adhesive surfaces and antimicrobial surfaces, along with their subtypes. finally, products already commercially available with enough reliable manufacturers' information about the product and its adequacy to the topics discussed in this review were also included. contamination spreading by contact can occur either by direct contact with an infected patient, or indirectly through a contaminated object or surface [ ] . surfaces represent an important way of transmission of diseases since they can act as a reservoir of microorganisms that may spread to whoever contacts with the surface. especially in crowded spots as public transports or public spaces and places susceptible of the presence of microorganisms like healthcare facilities, surfaces may represent a serious source of infection spreading [ ] . table describes the microorganisms detected in hospital, food handling, and other environmental surfaces. it is estimated that each year, in the united states alone, . million episodes of foodborne illness occur resulting nearly , hospitalizations and over deaths. the microorganisms most commonly associated with this phenomenon are norovirus, salmonella spp., clostridium perfringens and campylobacter [ ] . food contamination can result from several factors and occur at different points of the preparation process. from farming to the moment of consuming, food goes through several steps where contamination by microorganisms can occur. temperature and storage conditions are important factors to prevent food spoilage however, there are other factors that can cause microbiological contamination [ ] . food-contact surfaces' contamination can cause food contamination with pathogens during processing or packaging. bacteria are naturally present in plants and animals and it is easy for them to attach to a food contact surface during handling. due to adhesion mechanisms bacteria can remain on the surfaces and contaminate other foods [ ] . chopping boards, knifes and preparation tables are just some of the food preparation instruments found contaminated with bacteria [ ] . saad et al. [ ] evaluated hygiene conditions on food preparation facilities by analysing food-contact surfaces and the results were quite table examples of microorganisms isolated from different surfaces and some of their associated pathologies. adverse, suggesting fecal contamination. coliform bacteria were found in dining tables ( %), food trays ( %), cooking pots ( %) and kitchen faucets ( %). in addition, e. coli was identified in some cooking pots. in this study, surfaces with a count of total mesophilic aerobes of > cfu/cm or > cfu/cm for e. coli and coliforms were considered to fail hygiene criteria, according to the commission of the european communities guidelines [ ] for hygiene conditions in fresh meat processing facilities. although most countries have guidelines about hygiene measures and good practice on food processing facilities not always the cleaning procedures are sufficient to prevent microorganisms propagation [ ] . most of the times the cleaning cloths used in cleaning can become contaminated if soaked with disinfectants in the wrong dilution (common fault in food handling facilities), contributing to microorganisms' spread to previously uncontaminated surfaces [ ] . in the food industry, the occurrence of biofilm formation is also a common event due to inefficient cleaning and disinfection. the remaining microorganisms can survive on the surfaces, especially if food residues remain. this will promote the development and multiplication of bacteria with consequent biofilm formation that is extremely hard to eliminate, becoming even more difficult to disinfect the surface [ ] . schools and day-care centers are hot spots of infection spreading. lack of hygiene habits, common among very young children, is one of the main causes of contamination between kids, leading to the contamination of other children, staff, parents or people in the community, either by direct contact or by contaminating surfaces. [ ] . ibfelt et al. [ ] evaluated the presence of bacteria and viruses in day-care centers in denmark and different surfaces from toilets, kitchens and playrooms were analyzed. the results proved contamination by several bacteria and virus. despite the main bacteria present were non-pathogenic, several surfaces revealed the presence of coliform and nasopharyngeal bacteria. respiratory viruses were mainly present on toys but also on pillows and playroom tables. gastrointestinal viruses although less prevalent were found in some surfaces, mainly on playroom pillows and toilet surfaces. a study by jerković-mujkić et al. [ ] analyzed potential contamination in public telephones and the results showed high presence of microorganisms on the surfaces. staphylococcus epidermidis ( . %) and bacillus subtilis ( %) were the most common bacteria identified, however more species were isolated. mukherjee et al. [ ] evaluated the presence of microorganisms on different surfaces of a fitness center. the samples were collected from skin-contact surfaces as floor mats and exercise instruments, regularly shared by many people. in total, species were identified with the presence of some pathogenic or potential pathogenic bacteria like salmonella, staphylococcus or klebsiella. staphylococci were present in the majority of the surfaces being one the most prevalent species, namely s. aureus, s. epidermidis and staphyloccocus saprophyticus. kandel et al. [ ] developed a study with stupefying results. they compared microbiological contamination of toilet surfaces with buttons on hospital elevators, and the buttons showed higher colonization ( %) than the toilets ( %). the most common bacteria on both surfaces were staphylococcus and streptococcus, however other bacterial species were founded, as wells as fungi. otter and french [ ] studied the presence of microorganisms in hand-touch surface in public transport system. the samples were collected from several surfaces in trains, stations and buses of london. the presence of bacteria was confirmed, and most sites presented a median bacterial concentration of cfu cm − . even though there are no guidelines for acceptable values for bacteria's presence on surfaces of public spaces this value is higher than the recommended for foodprocessing surfaces [ ] and hospital hand-contact surfaces [ , ] . it has been recognized that hospitals' contaminated surfaces and medical equipment contribute to the contamination of patients and medical staff [ , ] . several factors contribute to this occurrence but the mains reason is associated with the high prevalence of microorganisms in healthcare facilities. pathogens with the ability to cause high rates of infection come from different sources such as patients' endogenous flora ( - %), hands of medical staff and assistants ( - %), antibiotic-driven changes in patients' flora ( - %) and other, including contamination of the environment ( %) [ ] . carling et al. [ , ] have shown that in some cases less than % of hospital surfaces can be considered clean after terminal disinfection procedures. however, for specific surfaces the rate can be even more alarming with less than % of bedpan cleaners, bathroom hand-holds, light switches, and doorknobs being cleaned [ ] . contact between hands and gloves of medical staff with contaminated surfaces leads to pathogens' spreading to other surfaces and people [ ] . particularly if they are immunocompromised patients, the risk of developing an infection is very high [ ] . stiefel et al. [ ] found out that the level of contamination of gloves on medical staff after contact with patients' skin sites was actually quite similar ( % vs %) to the level of contamination after contacting with surfaces on methicillin-resistant staphylocococcus aureus (mrsa) carriers' rooms, suggesting that environmental surfaces may pose serious risks. an identical study from guerrero et al. [ ] found similar results for c. difficile contamination, proving that the risk of hand contamination after contact with patients' skin was the same than when contacting with rooms' highly-touched surfaces ( % vs %). this issue is also related to the compliance of hospital workers on performing disinfection protocols. a recent study from stahmeyer et al. [ ] showed that only % of healthcare workers washed their hands before contact with a patient, and % washed their hands after contact with the patient. also, this study found out that . % of healthcare workers' hand hygiene procedure lasted less than s (average . s), when the recommendations is that it should last around s. another study evaluating disinfection compliance by healthcare workers showed that only % of the enquired radiologists affirmed to disinfect their workstation daily, % admitted never to have disinfected their workstation, and % said to have never received any recommendation on how to perform the disinfection [ ] . several studies have proved that surfaces in rooms of patients infected with important pathogens are frequently contaminated, and that a person admitted to a room previously occupied by an infected patient has an increased likelihood of developing colonization or infection with that pathogen [ , ] . a study performed in portugal by geadas farias et al. [ ] revealed hospital surfaces' contamination with different microorganisms, mostly pseudomonas and staphylococcus. several surfaces were contaminated but some of those with higher microbiological presence were surfaces associated with water distribution system as sinks, taps, showers and drains. viana et al. [ ] performed an analysis on hospital mattresses, and resistant bacteria were found on about % of the evaluated mattresses. a. baumannii was the main species found, being present in . % of the mattresses, but also other pathogens were collected as k. pneumoniae and p. aeruginosa. this study also identified the microorganisms found on mattresses and showed that in % of the cases the microorganisms were not related to the current patient admitted in the room but to the previous one, concluding that even after cleaning microorganisms still remain on the mattresses being a possible source of contamination to other patients. despite most studies evaluating the effectiveness of room disinfection have focused on some specific surfaces considered dirty (sink, toilets, etc.), all spots must be considered since contamination can be present on unsuspected surfaces [ ] . actually, white et al. [ ] found out in an hospital that microbiologically the sinks and floors were cleaner than hand touch sites as chairs, beds and cardiac monitors buttons. shek et al. [ ] developed a study where hospital curtains of burn and plastic surgery units were microbiologically tested. the results proved a considerable presence of bacteria on the curtains, even multi resistant bacteria as mrsa. levin et al. [ ] found that in situations of poor infection control practices it is possible for patients to become a source for unresolved contamination of portable radiographic equipment with pathogenic bacteria, namely resistant bacteria. lestari et al. [ ] performed a similar study but evaluating ecg lead wires, and among the lead wires analyzed only were non-contaminated with some type of microorganism. coagulase negative staphylococci and aerobic spore forming bacteria, two important types of pathogens, were present in % and % of the wires respectively. the ineffective cleaning associated with the ability of microorganisms to survive on surfaces under hostile conditions, for long periods of time, is a serious cause of pathogens spreading to people and to other surfaces [ ] . the presence of biofilms, the substances used in the cleaning process, and the method and frequency of cleaning, all interfere in the grade to which microorganisms resist to the cleaning and disinfection procedures. badly executed cleaning procedures may bring bigger problems than not cleaning at all. the transmission of microorganisms from a contaminated surface to a clean one can occur if the cleaning method is not correct. also, most detergents do not have antimicrobial activity they only act in the microorganisms' removal, leading to the contamination of the cleaning cloths with viable microorganisms, and its consequent spread to non-contaminated surfaces [ ] . the frequency of cleaning is also an important factor, since surfaces such as toilets, sinks, and other known as dirty surfaces tend be cleaned frequently [ ] . however, other surfaces thought safe but still highly touched are often neglected, being poorly cleaned. for example, according to the practical guidelines for infection control in health care facilities [ ] , mattresses without plastic covers must be steam cleaned if contaminated with body fluids. however, if body fluid contamination does not occur the mattresses are not so often cleaned, allowing microorganisms to accumulate with time and use, as proved by viana et al. [ ] . also geadas farias et al. [ ] have found that surfaces usually not considered dirty but highly touched as light switches or nursing trays were contaminated with several microorganisms. a very recent study by johani et al. [ ] revealed the presence of biofilms in many surfaces of intensive care units despite regular disinfection procedures. several highly touched surfaces were analyzed and in %, the presence of biofilm was proved by scanning electron microscopy. in many situations the lack of adequate training of the environmental cleaning services or of the medical staff ultimately promotes the use of inappropriate detergents or germicides promoting the survival of microorganisms on the surfaces, even after cleaning [ ] . among the most used disinfectants are substances based on hypochlorite, alcohols, aldehydes, phenols and quaternary ammonium compounds [ ] . however, not all of these substances are efficient against some specific types of microorganisms, namely spore forming pathogens like c. difficile [ ] . some disinfectants may cause resistance on the microorganisms if not used within specific parameters [ ] . there are several guidelines [ , , ] for infection control that suggest cleaning and disinfection methodologies to be applied in healthcare facilities. these guidelines not only suggest how to disinfect medical equipment but also how to clean environmental surfaces as floors, furniture or toilet seats. however, different guidelines present different opinions on the frequency of cleaning or advisable disinfectants/detergents for each situation since there is no standardization for the methods and cleaning agents to be used. in addition, there is a lack of guidelines suggesting effective standardized methods to assess environmental cleaning. visual evaluation is still the most used method to determine a surface cleanness, however even if a surface is apparently clean it can contain a high microbial load [ ] . according to the commission of the european communities [ ] guidelines for hygiene conditions in fresh meat processing facilities, food-contact surfaces with > cfu/cm for total viable count and > cfu/cm for enterobacteriaceae are considered unacceptable. there are no established limit values for microorganisms on surfaces for public spaces, and there is poor control on cleaning performance. as depicted in section . surfaces in these places are often contaminated showing values higher than those allowed on healthcare facilities ( . - cfus/cm ). more regulation should be made to assess the cleaning and disinfection efficacy and compliance on such places, namely on spaces with more susceptible people as schools, day-care centers or elderly homes. more guidelines about how to clean and how often would be a good option, as well as a higher microbiological assessment on surfaces, that is rarely or never done in public spaces. for healthcare facilities, there is some controversy about the acceptable number of microorganisms on surfaces since there are no established thresholds, only tentative suggestions from researchers. for some authors, clean hand-contact surfaces in hospitals must have less than cfus/cm , with an increased risk of infection for values above that [ ] . other researchers have proposed the maximum value of . cfus/cm as the limit level of contamination in clean hospital surfaces [ , ] . for some specific microorganisms, considered indicator organisms, the limit value must be even lower. the presence of such microorganisms (for example s. aureus, c. difficile, vancomycin-resistant enterococci or acinetobacter spp.) is considered indicative of the need for cleaning even at levels of cfus/cm [ ] . some reference limit-values either from governmental entities or from research studies are listed in table . the limit of < cfus/cm for indicator microorganisms makes sense since it has been shown that an infectious dose of cfus/cm was sufficient to cause c. difficile infection on mice [ ] . also, it was observed that very low doses of norovirus have the capacity to cause infection [ ] . these results show that microbiological contamination on surfaces poses a real risk of infection, since the environmental infectious dose can be very low for some microorganisms. there is still the need for reaching a consensus and creating general guidelines for healthcare facilities. establishing clear limit values and implementing frequent microbiological assessment protocols are some of the steps that should be in the horizon. to avoid contamination and consequent infection propagation on surfaces, several alternative strategies have been developed recently. application of aerosols or uv light on contaminated surfaces are good examples of no-touch strategies, and they seem to be a good option to be applied in hospital environment [ ] . however, these approaches still present some limitations. uv light, in hospital facilities, can only be applied in empty rooms, therefore it cannot be used for a daily cleaning routine. also, it only disinfects areas directly reached by the uv light, so many objects and surfaces remain contaminated after this disinfection approach. aerosols are efficient but take a long time to decontaminate the surfaces compared to conventional cleaning or uv light. besides that, these strategies are also exclusively used in empty rooms. this factor increases the necessary time to do the final disinfection of the room and perform the beds turnover in hospitals [ ] . cold plasma technology is another disinfection strategy that has recently been applied on surfaces. plasma is the fourth state of mater and it can be generated from gases that became ionized with lots of ions, electrons and free radicals that will provide good electric conductivity. plasma is known to have high levels of energy and it can change molecules composition making them highly ionized and with many free radicals moving randomly in all directions. for example, when a molecule of hydrogen peroxide is exposed to this technology the double bond on the molecule is broken, creating reactive oxygen species like hydroxyl radicals that in need to seek equilibrium will bind to surrounding microorganisms destroying them by oxidation [ , ] this technology has already been tested in food industry to decontaminate vegetables [ ] and meat [ ] with good results obtained and also to decontaminate surfaces as packaging materials. puligundla et al. [ ] have tested the elimination of different bacteria such as e. coli, salmonella typhimurium and s. aureus, from several materials often used on food packages such as glass, polypropylene, polyethylene, nylon and paper foil. the results were quite promising with several log reductions after to minutes of treatment. even though the results of this emerging technology are quite encouraging as a possible strategy to disinfect surfaces, more studies are needed to assure its safety and profitability to disinfect larger surfaces. surface modification/functionalization is another common procedure to obtain anti-adhesive or antimicrobial properties on different materials. this approach has several advantages over conventional disinfection techniques. for example, antimicrobial surfaces are in constant process of activity oppositely to no-touch technologies or conventional cleaning. this way the antimicrobial charge on the surfaces is reduced immediately after contact preventing its propagation and consequent contamination of surrounding surfaces or people [ , ] . other advantage is the possibility of these surfaces to be present in populated environments since they cause no harm to people with no need to remove people from the rooms to perform the disinfection. in addition, modified surfaces are not restricted to external surfaces. medical devices such as urinary catheters, central venous catheters, prosthesis or contact lenses are some of the surfaces that have been functionalized to obtain better compatibility properties and lower infection risk when inserted on the human body [ ] [ ] [ ] [ ] . self-disinfecting surfaces are an emerging topic with more and more products coming up. some of the most recent strategies developed to obtain surfaces with anti-adhesive and antimicrobial properties are discussed below. several natural surfaces have suffered diverse evolutionary processes that turned them resistant to microorganisms' colonization. natural and bio-mimicked surfaces of insects' wings, sharks' skin, and lotus' leaves exhibit antibiofouling properties by preventing different cells, particles or microorganisms from attaching to their surface [ ] . considering this, some of these natural anti-adhesive or self-cleaning surfaces have been investigated for their potential application on micro/nanostructured surfaces development. however it is also possible to give anti-adhesive properties to a surface by modifying the material's chemistry, namely using self-assembled monolayers or polymer brushes immobilized on the surface (fig. ) ( [ ] ; [ , ] ). there are several ways to produce materials with anti-fouling properties acting at the level of surface modification with chemical structures. functional groups exhibited by the material's surface interact with those in the microorganism' cells determining the kinetics of microbial adhesion. for instance, surfaces highly negatively charged, i.e. polyanionic surfaces have the ability to repulse the bacteria with polyanionic glycocalice (most of gram-positive bacteria) through electrostatic interactions. however, gram-negative bacteria have policationic glycocalices, so this surface modification mechanism is only effective against some bacterial species and sometimes only against specific strain types [ ] . zwitterionic materials, such as carboxybetaine, sulfobetaine, and phosphobetaine also have anti-fouling properties. zwitterionic polymers have an equal number of anionic and cationic groups present in their repeating unit. this fact gives these polymers ultra-hydrophilicity and consequently great hydration ability [ , ] . studies concerning surface modification with zwitterionic polymers have proved to be a promising strategy to reduce microorganisms' adhesion [ , ] . very recently, a study using a tyramine-conjugated sulfobetaine polymer, grafted on polyurethane showed a great reduction in s. aureus adhesion to the surface [ ] . coating with polymeric brushes can also prevent microorganisms' adhesion. apart from avoiding direct contact between microorganisms and the surfaces, the polymers used for brushes are usually hydrophilic, so water will be attracted into the brush forming a repellent layer in aqueous environment. proteins and microorganisms encountering the brush surface will be repelled by steric hindrance due to the water bound in the brush and the elasticity of the polymer chains [ ] . polyethylene oxide (peo) and polyethylene glycol (peg) are two examples of polymers used to produce hydrophilic brush coatings on biomaterials. in a recent study by. hadjesfandiari et al. [ ] it was proved that peo brushes covalently immobilized reduced % the adhesion of staphylococci and e. coli to a surface. apart from the chemistry, also topography of a surface can be structured to increase anti-adhesive properties. as an attempt to find efficient alternatives over more classic antimicrobials, micro/nanostructured materials present themselves as possible solutions. recently, have been done an attempt to elucidate if alterations on micro or nanotopography of a surface could influence colonization and consequent contamination. an example of structure modification is the application of superficial nanostructures (nanoparticles, nanofibers or nanotubes) reducing the area available for microorganisms to attach [ ] [ ] [ ] . as said previously, mimicking the topography of anti-adhesive surfaces innately present in nature is an innovative way to obtain new self-disinfecting surfaces. many plants have special surface properties namely wettability. for instance, lotus leaves have micropapillae structures covered by nanostructures with fine branch-like shape. these structures along with epicuticular needle-shaped wax tubes that cover them give the lotus leaf a superhydrophobic surface and consequently antifouling properties. water droplets roll down the lotus leaf due to its great hydrophobicity dragging out any possible particles present, and maintaining its surface clean [ ] . other example of nature that inspired anti-adhesive surfaces' coating is sharkskin. sharks have a special scale micropattern, which consist of a rectangular base embedded in the skin with tiny spines on the surface. the ribbed texture of these scales is responsible for the selfcleaning, anti-biofouling, hydrophobic and drag reducing properties of shark skin [ , ] . such properties have made of sharkskin one of the most mimicked surfaces, not only in research environment but in industrial area too. sharklet af (sharklet technologies, alachua, fl, usa) is a company that uses shark skin topography in different surfaces in order to avoid bacterial adhesion and biofilm formation [ , ] . a study comparing sharklet micropattern surfaces with regular surfaces in a clinical simulated scenario proved that shark skin microtopography reduced the number of attached bacteria by about fold [ ] . there are several materials with intrinsic antimicrobial properties, as silver, zinc, cooper, or chitosan. nevertheless, materials can also be modified to acquire bactericidal activity [ ] . chemical or physical changes in the surface can produce antimicrobial effect. chemical substances such as antibiotics or biocides can be incorporated in the materials to give it antimicrobial properties. however, a balance between these properties and biocompatibility must be observed since the materials will be in direct contact with users' organism raising toxicity concerns. photo-activated surfaces are also an example of self-disinfecting materials recently developed. fig. presents some examples of different types of antimicrobial surfaces. some materials have intrinsic antimicrobial properties. they do not need antimicrobial loading to exert its activity since the material by itself has the natural ability to eliminate microorganisms. among the most known natural materials with antimicrobial properties are metals as silver, copper, or zinc and polymers like chitosan [ ] . for many metals with antimicrobial properties, it is the ionic form that presents higher bactericidal activity and not the elemental metal. silver and copper are good examples of that. silver has been used in medical field for many centuries due to its antimicrobial properties. its bactericidal mechanism is based on the binding of silver atoms with thiol (sh) and disulfide (s-s) groups present in the proteins of bacterial cell membranes, leading to its disruption and eventual cell death [ ] . silver has been used for many applications on medical field especially when added in the form of silver sulfadiazine to creams or wound dressings [ ] . several wound dressing containing silver are available on market, however, not always they comply the expectations. cavanagh et al. [ ] analyzed different silver dressings on market and only two of them showed bactericidal activity against s. aureus. apart from that, silver dressing may be related with increased serum silver levels with an associated decrease on white blood cells [ ] . this leads us to conclude that the delivery system of the antimicrobial is very important. a delivery mechanism that acts by contact without leaching silver ions to the surrounding areas or tissues would be good strategy to achieve antimicrobial action without compromise the safety of the product. more recently, silver nanoparticles have been greatly studied and used to produce potential antimicrobial materials such as catheters and surgery sutures [ , ] . however, for biomaterials implanted inside the organism there are some possible risks associated with releasing of silver ions causing local toxicity and possible accumulation in organs [ ] . silver may also have different ranges of efficacy according to the class of bacteria. gram-positive bacteria's cell walls do not have an outer membrane as gram-negative bacteria do, but they have several layers of peptidoglycans, making their cell wall thicker [ ] ; this, along with their negative charge results in the trapping of silver ions (positively charged) preventing their entrance into the bacteria [ ] . agc glass (ags glass uk ltd., rugby, great britain) developed an antibacterial glass by incorporating silver ions inside the glass. the company claims that this glass eliminates % of bacteria and prevents fungi proliferation which they demonstrated in the three bacterial and two fungi strains evaluated [ ] . surfacine development company (tyngsborough, ma, usa) is another company that developed a silver coating (surfacine ® ) that can be applied in several materials, from medical devices to food preparations and packaging industry or water distribution systems. this coating showed good antimicrobial activity against several bacteria, fungi and yeast [ ] . other company, purethread technologies inc. (cary, nc, usa) has developed purethread ® , a system that embeds silver salts into textile fibers, obtaining antimicrobial textiles. according to the company this system does not weaken or disappears during washing and kills . % of microorganisms after four hours of contact with the fabric [ ]. a scientific paper about a study involving hospital curtains incorporating the antimicrobial textile developed by this company proved that those curtains take more time to be contaminated for the first time when compared to regular curtains [ ] . similarly, to silver, copper has been used for many centuries as an antimicrobial agent especially for water treatment and transportation. copper was also very used in the nautical field, to prevent adhesion and growth of organisms in hulls of ships [ ] . the bactericide mechanism of copper is related to the release of copper ions that cause damage in the bacterial envelop and consequent leakage of the cell content and influx of copper ions into the bacteria. this will generate toxic radicals causing oxidative damage to cellular components and dna degradation [ ] . more recently copper came up as an alternative for preventing hospital acquired infections through its application in hospital surfaces and medical equipment [ ] . a study by souli et al. [ ] performed on a hospital intensive care unit, compared two rooms, one with copper coated equipment (beds, side table, intravenous pole stands, etc.) and the other with regular equipment. the copper coated equipment room presented a reduced percentage of colonized surfaces ( . %) compared to the regular compartment ( . %). this study showed both reductions in colonization by gram-negative and gram-positive bacteria and in total bioburden ( . vs . cfu/ cm ). sifri et al. [ ] also developed a study where a section of an acute care unit was equipped with copper impregnated materials, hard surfaces and textiles-patients' gowns, sinks, bed rails, tray tables, sheets and blankets. the results were quite promising with copper equipped section presenting patients' infection reductions of % when caused by c. difficile and % when caused by multi drug resistant organisms, comparing to a non-modified section in the same hospital unit. two companies that collaborate with each other, cupron medical textiles (cupron inc., richmond, va, usa) and cupron enhanced eos surfaces (eos surfaces, norfolk, va, usa) provided the copper impregnated materials used for this study. the product developed is a hard-antimicrobial surface impregnated with copper that continuously kills . % of harmful bacteria in two hours, according to its specifications. according to the company this surface is effective against s. aureus, enterobacter aerogenes, mrsa, e. coli and p. aeruginosa [ ] . there are several substances with antimicrobial activity that can be added to a surface to obtain an antimicrobial surface. antibiotics were one of the first substances to be applied to surfaces such as prosthesis or textiles for wound dressing production, to obtain antimicrobial action. despite the promising results, it is well known that the major disadvantage of antibiotics use is the microorganisms' ability to develop resistance. this drawback has led to an increasing search for alternatives to be used as antimicrobials. quaternary ammonium compounds, chlorhexidine, benzalkonium chloride or nitric oxide are some examples of substances that were already tested as an alternative [ ] [ ] [ ] . quaternary ammonium compounds are disinfectants used in hospitals and healthcare facilities for several clinical purposes as disinfection of surfaces and medical material. however, more recently the quaternary ammonium compounds have been tested as antimicrobial loading agents to different surfaces and materials, namely polymers [ , ] . antimicrobial peptides (amps) are a class of peptides, components of innate immune system, with a broad activity against bacteria, virus, fungus and more, providing a non-specific defense against a broad spectrum of invaders [ ] . recently, numerous studies [ , ] refer its use as antimicrobial loading agents for their excellent characteristics, presenting several advantages over classic antibiotics, namely fast and broad spectrum of action with low susceptibility to induce bacterial resistance [ ] . in contrast with the mechanisms of action of antibiotics, which are based in slow processes of enzymatic inhibition and target specific cellular activities as dna or protein, the amps are effective against different species of microorganisms and also reduce the risk of resistance development [ ] [ ] [ ] . for all these reasons, amps are being applied in the production of antimicrobial surfaces, either by simple adsorption on the surface or covalent immobilization [ ] . nevertheless, studies suggest that covalent immobilization offers many advantages toward physical adsorption, including higher local and long-term stability, and lowering toxicity [ , ] . all those antimicrobial substances are loaded to the surface either by immobilization or by incorporation on the bulk material; recent studies on the application of each type of loading strategy are summarized next. in materials' incorporation process, the antimicrobial substances are added to the ingredients during the phase of production, to obtain a homogeneous mixture of the bulk material with the antimicrobial. this system allows antimicrobial activity throughout the bulk material, even in deeper layers and not only on the surface. this allows the material to retain its antimicrobial activity even when worn out [ , ] . in a very recent study, ferreira et al. [ ] used levofloxacin to load a bone cement, achieving good results against s. aureus not only on its planktonic form but also on biofilm. that was quite positive since this bacterial strain is greatly associated with biofilm formation on bone implants. ciprofloxacin incorporation on textile fibers by electrospinning was tested by li et al. [ ] this strategy aimed to produce a bandage to prevent wound infection and the results in vivo (rats animal model) were quite positive for e. coli and s. aureus. in han et al. [ ] study they incorporated quaternary ammonium methacrylates in dental adhesives to prevent caries by avoiding the accumulation of bacteria and biofilm formation on teeth, testing the presence of three staphyloccocal species (staphylococcus mutans, staphylococcus gordonii, and staphylococcus sanguinis) and the results showed a decreased biofilm formation for the adhesives containing the quaternary ammonium methacrylates. nanoparticles containing quaternary ammonium polyethylenimine were tested in a study about endodontic sealers by barros et al. [ ] . the results were quite positive for enterococcus faecalis, with the endodontic sealers showing good antimicrobial activity. the amp, ll- , was incorporated by cassin et al. [ ] in a membrane of collagen and hyaluronic acid for the production of antiinfective films to cover injured tissues. antimicrobial coatings can be obtained either by adsorption of the antimicrobial substance, by covalently binding it to the material or by immobilizing it using self-assembled monolayers. this strategy can be an advantage for some substances that are too toxic to be used as bulk material but that immobilized in small amounts as surface coatings can exert their antimicrobial activity without the toxicity drawback [ , ] . the coatings can also be divided according to their mechanism of action. they can deliver the antimicrobial substance by leaching, releasing it to the surrounding area or direct contact with the surface where the coating is immobilized. some coatings release substances with antibacterial properties that will interact with microorganisms acting away from the bulk. this leaching mechanism has the advantage of a larger perimeter of action around the surface where it is immobilized. it is important, though, that this release into the surrounding area is controlled to avoid toxic effects by excess of substance [ ] . coatings can also attach certain antimicrobial molecules to the materials' surface. several coatings can be classified as "contact biocides" since they use non-leachable substances that kill by contact with the bacteria and with no need to be released from the surface. this strategy is very interesting for both the self-sterilizing effect and the long-lasting activity, acting just through a direct contact with bacteria without consuming itself releasing from the surface without need [ , ] . alt et al. [ ] realized a study where coating of rifampicin and fosfomycin was applied to prosthesis showing good efficacy against s. aureus, even the methicillin resistant strain, on a rabbit animal model. neut et al. [ ] performed a similar study using a gentamicin-releasing coating that was able to prevent staphylococci growth on a prosthetic hip surface for at least hours, showing promising results. in a work by iyamba et al. [ ] , quaternary ammonium compounds (hexadecyltrimethyl ammonium bromide and hexadecylbetainate chloride) were immobilized on medical catheters reducing s. aureus adhesion to its surface. zanini et al. [ ] also developed a work in which quaternary ammonium silanes were used to coat polyurethane catheters. the results were promising with this coating proving its antimicrobial activity against e. coli bacteria. in a recent study, casciaro et al. [ ] successfully covalently immobilized frog skin derived amp on contact lenses. this strategy not only reduced the adhesion of p. aeruginosa, a species highly associated with contact lenses associated keratitis, but it also proved to be nontoxic to mammalian cells and it did not compromise the lenses physical properties. amps have also been immobilized on nanoparticles as loading strategy. ma et al. [ ] immobilized the amp hhc- on titanium oxide (tio ) nanotubes by physical adsorption with the nanotubes acting as nanocarriers for peptides delivery. they obtained an antimicrobial and anti-fouling surface showing a reduction in s. aureus adhesion and great antimicrobial activity (> % activity). braun et al. [ ] immobilized the human peptide ll- on porous silica nanoparticles, achieving an antimicrobial delivery system. all the studies presented above prove the good potential of modified surfaces to be applied on infection control in different scenarios; still some aspects must be taken into account and more studies are still needed. most studies evaluating the antimicrobial/anti-adhesive activity potential of engineered surfaces do not take into account realistic physiological environments. this can affect the results, since the host's conditioning film may affect the surface in many different ways, for example, covering the surface creating a deterrent layer preventing the antimicrobial substance to leach or to contact directly with the microorganisms, reducing its antimicrobial efficacy [ ] . in addition, the composition of the surface may determine which components of the conditioning film will adhere and that on its turn will influence other cells adhesion, as bacteria. felgueiras et al. [ ] showed that different concentrations of octadecyl acrylate (c ) immobilized on polyurethane surfaces will affect the deposition of albumin and fibrinogen when the surface is in contact with human plasma, that on its turn will affect microorganisms' adhesion to the surface since albumin avoids bacteria adhesion and fibrinogen promotes it. besides, coatings with antimicrobial peptides can be degraded by some components of conditioning films such as proteases [ ] . despite the pointed weaknesses there are already on the market some successful cases of such as biocote limited (coventry, united kingdom) that developed an antimicrobial additive technology, biocote ® , that can be incorporated on textiles, polymers, ceramics and more. these additives are based on different antimicrobial substances such as silver, zinc or phenolic compounds, chosen according to their application and support material [ ] . sanitized ag (burgdorf, switzerland) is another company that developed an antimicrobial additive that can be incorporated in polymers and textiles for different areas of application such as healthcare, public transportation and food industry. this additive technology, sanitized ® , uses different active ingredients as silver, zinc pirithione, silane quat or isothiazolinone that can be added on liquid form, powder or paste according to specific production requirements of the final product [ ] . photo-activated materials have been used in different technological fields namely in antimicrobial surfaces development. the most studied material used in photo-activated surfaces production is tio . tio has high photocatalytic properties and is highly used in cosmetics and skin care products since it is not absorbed by human skin [ ] becoming a very interesting material for development of antimicrobial surfaces. in fact, its antimicrobial efficacy has already been proved against bacteria, fungi, protozoa and virus. the main advantage of this strategy is that tio is not degraded so its activity can be maintained for long periods. tio surfaces are only activated when irradiated by specific photon energy. when this irradiation occurs, reactions of photo-oxidation involving o and h o take place on the surface with consequent formation of oxygen free radicals. these free radicals will attack the cell wall and cytoplasmic membrane of the microorganism by peroxidizing its lipids, leading to leakage of cellular components and lately cell lysis [ , ] . recently, adán et al. [ ] published a study where a water microfiltration system using photocatalytic membranes produced with tio and porous steel showed good inactivation for the retained bacteria. in another study by joost et al. [ ] thin films with tio nanoparticles were illuminated with uv light and after min of exposure no bacteria (e. coli) had survived. the analysis of bacterial cell membrane showed modifications in the chemical structure of unsaturated fatty acids and decomposition of saturated fatty acids faster than normal, confirming the peroxidation of membrane lipids. in the market, there are already some products for microorganism elimination based on tio photocatalytic action. purehealth™ (orion, florence, italy) is a system developed by orion that coats walls and floors and is activated by special lamps of solar spectrum. this system promises to eliminate virus and bacteria [ ] . this review points out the great contribution of surfaces for infection spreading, not only in healthcare facilities but also in other public spaces and food processing facilities. there is an urgent need to pay more attention to surface contamination in different spaces since there is still a lack of guidelines for microbiologic assessment and established safe thresholds for health care facilities. in addition, it is important to establish infection control procedures for public spaces, namely schools since they often present high microbiological charges on surfaces, posing a real risk for children whose immunological system is still developing. both on public spaces and on healthcare facilities, it should be determined how to assess microbiologic presence on surfaces as well as how frequently this assessment should be made, and which methods should be used to obtain a realistic analysis rather than performing visual assessments. the implementation of standard acceptable limits of microorganisms on surfaces is also a key point for a better control of infection spreading. standardized definition of which microorganisms can be considered indicator microorganisms should also be achieved. the existing guidelines for cleaning and disinfection methodologies in healthcare facilities should come to a consensus about the disinfectants to be used in each setting, the frequency of cleaning and the methods to apply. the implementation of self-disinfecting surfaces is a good strategy for infection control, and this review presents some successful research already developed in this area. these surfaces show clear advantages over the regular surfaces with traditional cleaning: the state of continuous disinfection and the antimicrobial activity that permanently eliminates the microorganisms. there are still some issues to improve, like the long-term efficacy of the antimicrobial/anti-adhesive action, or the high cost of implementation of these surfaces in large areas. biomaterials, modified surfaces, or new scientific products in general, have to face some big challenges before they reach the market. problems such as costs of manufacturability, scalability on production, intellectual property issues or regulatory aspects are just some of the main obstacles those products will have to overcome in order to be commercialized [ ] . some products even with great results on laboratory will fail when brought to larger scale due to their high cost of production, making them commercially unattractive. but, looking at the numbers nosocomial bacteremia alone can cost a hospital over million euros per year [ ] , and antibiotic resistance-associated infections may cost more than million euros per year in european union. in fact the societal cost of infections due to the selected antibioticresistant bacteria were estimated at about eur . billion each year, between hospital related costs and losses in productivity due to patients' absence from work [ ] . therefore, maybe it is worthy to invest on new technologies, betting on infection prevention and control not only to avoid higher costs but also to avoid the harm and loss of human lives. self-disinfecting surfaces are a step forward to the future of infection control policies. more studies involving self-disinfecting surfaces should be performed in order to realize its full potential to improve infection control and safety strategies. the reports on the application of these materials in healthcare facilities show promising results and there are already few companies supplying products with self-disinfecting properties showing that investment on prevention may be the best way to reduce the tremendous problem of infection spreading. the authors declare no conflict of interest. microbiology of healthcare-associated infections and the definition accuracy to predict infection by potentially drug resistant pathogens: a systematic review the role of the surface environment in healthcareassociated infections the role played by contaminated surfaces in the transmission of nosocomial pathogens guideline for disinfection and sterilization in health care facilities: what clinicians need to know transfer of multidrug-resistant bacteria to healthcare workers' gloves and gowns after patient contact increases with environmental contamination bacterial contamination on touch surfaces in the public transport system and in public areas of a hospital in london foodborne illness acquired in the united states-major pathogens outbreaks and factors influencing microbiological contamination of fresh produce role of attachment to surfaces on the prevalence and survival of campylobacter through food systems bioluminescence atp monitoring for the routine assessment of food contact surface cleanliness in a university canteen use of rapid microbial kits for m regular monitoring of food-contact surfaces towards hygiene practices commission decision of cleaning and decontamination efficacy of wiping cloths and silver dihydrogen citrate on food contact surfaces biofilm formation in food industries: a food safety concern review of enteric outbreaks in child care centers: effective infection control recommendations presence of pathogenic bacteria and viruses in the daycare environment bacterial contamination of public telephones in the downtown area of sarajevo diversity of bacterial communities of fitness center surfaces in a u.s. metropolitan area elevator buttons as unrecognized sources of bacterial colonization in hospitals how do we assess hospital cleaning? a proposal for microbiological standards for surface hygiene in hospitals a modified atp benchmark for evaluating the cleaning of some hospital environmental surfaces epidemiology and control of nosocomial infections in adult intensive care units improving cleaning of the environment surrounding patients in acute care hospitals improving environmental hygiene in intensive care units to decrease multidrug-resistant bacterial transmission role of hospital surfaces in the transmission of emerging health care-associated pathogens: norovirus, clostridium difficile, and acinetobacter species characteristics of nosocomial infection and its effects on the survival of chemotherapy patients with advanced non-small cell lung cancer contamination of hands with methicillin-resistant staphylococcus aureus after contact with environmental surfaces and after contact with the skin of colonized patients acquisition of spores on gloved hands after contact with the skin of patients with clostridium difficile infection and with environmental surfaces in their rooms hand hygiene in intensive care units: a matter of time? disinfection of the radiologist workstation and radiologist hand hygiene: a single institution practice quality improvement project evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings hospital microbial surface colonization revealed during monitoring of klebsiella spp., pseudomonas aeruginosa, and non-tuberculous mycobacteria recovery of resistant bacteria from mattresses of patients under contact precautions a quantitative approach to defining "high-touch" surfaces in hospitals are hygiene standards useful in assessing infection risk? am rate of contamination of hospital privacy curtains on a burns and plastic surgery ward: a cross-sectional study contamination of portable radiograph equipment with resistant bacteria in the icu microbial contamination of manually reprocessed, ready to use ecg lead wire in intensive care units microbial contamination of hospital reusable cleaning towels interventions to reduce the incidence of hospital-onset clostridium difficile infection: an agent-based modeling approach to evaluate clinical effectiveness in adult acute care hospitals world health organization, practical guidelines for infection control in health care facilities practical guidelines for infection control in health care facilities, world health organization characterization of microbial community composition, antimicrobial resistance and biofilm on intensive care surfaces respiratory viral rna on toys in pediatric office waiting rooms promises and pitfalls of recent advances in chemical means of preventing the spread of nosocomial infections by environmental surfaces use of purified clostridium difficile spores to facilitate evaluation of health care disinfection regimens viscosity is an important factor of resistance to alcohol-based disinfectants by pathogens present in mucus basic infection control and prevention plan for outpatient oncology settings standard infection control precautions literature review: routine cleaning of the environment in the hospital setting visualization of hospital cleanliness in three japanese hospitals with a tendency toward long-term care an evaluation of hospital cleaning regimes and standards norwalk virus: how infectious is it? modern technologies for improving cleaning and disinfection of environmental surfaces in hospitals no touch' technologies for environmental decontamination a review on recent advances in cold plasma technology for the food industry: current applications and future trends medical applications of cold atmospheric plasma: state of the science inhibition of salmonella typhimurium on radish sprouts using nitrogen-cold plasma controlling microbial safety challenges of meat using high voltage atmospheric cold plasma inactivation effect of dielectric barrier discharge plasma against foodborne pathogens on the surfaces of different packaging materials antibacterial surfaces: the quest for a new generation of biomaterials reduced health care-associated infections in an acute care community hospital using a combination of self-disinfecting copperimpregnated composite hard surfaces and linens antibacterial titanium surfaces for medical implants development of a catheter functionalized by a polydopamine peptide coating with antimicrobial and antibiofilm properties cecropin -melittin functionalized polyurethane surfaces prevent staphylococcus epidermidis adhesion without inducing platelet adhesion and activation a novel cationicpeptide coating for the prevention of microbial colonization on contact lenses biomimetic self-cleaning surfaces: synthesis, mechanism and applications antibacterial surfaces based on polymer brushes: investigation on the influence of brush properties on antimicrobial peptide immobilization and antimicrobial activity antibacterial surfaces based on polymer brushes: investigation on the influence of bru polysaccharide-based antibiofilm surfaces natural and bioinspired nanostructured bactericidal surfaces a review of the biomaterials technologies for infection-resistant surfaces integrated antimicrobial and nonfouling zwitterionic polymers zwitteration: coating surfaces with zwitterionic functionality to reduce nonspecific adsorption anti-caries effects of dental adhesives containing quaternary ammonium methacrylates with different chain lengths zwitterionic polyhydroxybutyrate electrospun fibrous membranes with a compromise of bioinert control and tissue-cell growth zwitterionic sulfobetaine polymer-immobilized surface by simple tyrosinasemediated grafting for enhanced antifouling property the biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides polymer brush-based approaches for the development of infection-resistant surfaces carbon nanotube-based antimicrobial biomaterials formed via layer-by-layer assembly with polypeptides new strategies in the development of antimicrobial coatings: the example of increasing usage of silver and silver nanoparticles thermosensitive nanofibers loaded with ciprofloxacin as antibacterial wound dressing materials theoretical explanation of the lotus effect: superhydrophobic property changes by removal of nanostructures from the surface of a lotus leaf bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants the characterization, replication and testing of dermal denticles of scyliorhinus canicula for physical mechanisms of biofouling prevention micropatterned endotracheal tubes reduce secretion-related lumen occlusion surface micropattern resists bacterial contamination transferred by healthcare practitioners silver coordination polymers for prevention of implant infection: thiol interaction, impact on respiratory chain enzymes, and hydroxyl radical induction development of silver sulfadiazine loaded bacterial cellulose/sodium alginate composite films with enhanced antibacterial property evaluating antimicrobial efficacy of new commercially available silver dressings silver absorption in patients with stevens-johnson syndrome and toxic epidermal necrolysis treated with silver-impregnated dressings. a case series biobased silver nanocolloid coating on silk fibers for prevention of post-surgical wound infections antimicrobial activity and cytocompatibility of silver nanoparticles coated catheters via a biomimetic surface functionalization strategy oral toxicity of silver ions, silver nanoparticles and colloidal silver -a review the bacterial cell envelope antibacterial effect of silverzeolite on oral bacteria under anaerobic conditions novel hospital curtains with antimicrobial properties: a randomized, controlled trial antimicrobial applications of copper killing of bacteria by copper, cadmium, and silver surfaces reveals relevant physicochemical parameters role of copper in reducing hospital environment contamination reduction of environmental contamination with multidrug-resistant bacteria by copper-alloy coating of surfaces in a highly endemic setting component release and mechanical properties of endodontic sealers following incorporation of antimicrobial agents adherence of staphylococcus aureus to catheter tubing inhibition by quaternary ammonium compounds inhibition of staphylococcus aureus adhesion to the surface of a reticular heavyweight polypropylene mesh soaked in a combination of chlorhexidine and allicin: an in vitro study bactericidal specificity and resistance profile of poly(quaternary ammonium) polymers and protein-poly(quaternary ammonium) conjugates antibiofilm properties of model composites containing quaternary ammonium methacrylates after surface texture modification will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? esculentin- a derived peptides kill pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface local delivery of antimicrobial peptides using self-organized tio nanotube arrays for peri-implant infections dairy-derived antimicrobial peptides: action mechanisms, pharmaceutical uses and production proposals antimicrobial peptide structure and mechanism of action: a focus on the role of membrane structure antimicrobial properties of membraneactive dodecapeptides derived from msi- covalent immobilization of antimicrobial peptides (amps) onto biomaterial surfaces dhvar antimicrobial peptide (amp) chemoselective covalent immobilization results on higher antiadherence effect than simple physical adsorption antibacterial, physicochemical and mechanical properties of endodontic sealers containing quaternary ammonium polyethylenimine nanoparticles characterization and activities of a bactericidal peg-hydrogel with immobilized antimicrobial peptide levofloxacin-loaded bone cement delivery system: highly effective against intracellular bacteria and staphylococcus aureus biofilms bottom-up fabrication of zwitterionic polymer brushes on intraocular lens for improved biocompatibility the design of antimicrobial ll -modified collagen-hyaluronic acid detachable multilayers octadecyl chains immobilized onto hyaluronic acid coatings by thiol-ene "click chemistry" increase the surface antimicrobial properties and prevent platelet adhesion and activation to polyurethane solid films of blended poly(vinyl alcohol)/poly(vinyl pyrrolidone) for topical s-nitrosoglutathione and nitric oxide release high-density antimicrobial peptide coating with broad activity and low cytotoxicity against human cells rifampicin-fosfomycin coating for cementless endoprostheses: antimicrobial effects against methicillin-sensitive staphylococcus aureus (mssa) and methicillin-resistant staphylococcus aureus (mrsa) a gentamicin-releasing coating for cementless hip prostheses-longitudinal evaluation of efficacy using in vitro bio-optical imaging and its wide-spectrum antibacterial efficacy development of antibacterial quaternary ammonium silane coatings on polyurethane catheters membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides the effects of blood conditioning films on the antimicrobial and retention properties of zirconium-nitride silver surfaces cecropin a-melittin mutant with improved proteolytic stability and enhanced antimicrobial activity against bacteria and fungi associated with gastroenteritis in vitro lack of significant dermal penetration of titanium dioxide from sunscreen formulations containing nano-and submicron-size tio particles photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity understanding the antimicrobial mechanism of tio -based nanocomposite films in a pathogenic bacterium bacterial inactivation and degradation of organic molecules by titanium dioxide supported on porous stainless steel photocatalytic membranes photocatalytic antibacterial activity of nano-tio (anatase)-based thin films: effects on escherichia coli cells and fatty acids translational biomaterials -the journey from the bench to the market -think "product analysis according to microorganism and antimicrobial sensitivity in a university hospital in barcelona the bacterial challenge: time to react contamination of medical charts: an important source of potential infection in hospitals hospital-wide comparison of health care-associated infection among intensive care units: a retrospective analysis for - cleaning and disinfection of biofilms composed of listeria monocytogenes and background microbiota from meat processing surfaces microbial evaluation of foodservice surfaces in texas child-care centers clinical infection in burn patients and the consequences hospital-acquired pneumonia and ventilator-associated pneumonia in adults at siriraj hospital: etiology, clinical outcomes, and impact of antimicrobial resistance relationship between environmental fungal contamination and the incidence of invasive aspergillosis in haematology patients fungal contamination of hospital healthcare workers' overalls high burden of aspergillus fumigatus infection among chronic respiratory diseases rapid fulminant case of aspergillus prosthetic valve endocarditis advances in the diagnosis and treatment of fungal infections of the cns primary cutaneous aspergillosis due to aspergillus tamarii in an immunocompetent host prevalence of pathogens and indicator organisms in home kitchens and correlation with unsafe food handling practices and conditions transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board the microbiological quality of washing-up water and the environment in domestic and commercial kitchens transfer of campylobacter and salmonella from poultry meat onto poultry preparation surfaces etiology of diarrhea among children under the age five in china: results from a five-year surveillance importation, mitigation, and genomic epidemiology of candida auris at a large teaching hospital candida infections of the genitourinary tract supragingival and subgingival microbiota from patients with poor oral hygiene submitted to radiotherapy for head and neck cancer treatment a novel quantitative sampling technique for detection and monitoring of clostridium difficile contamination in the clinical environment identification of clostridium difficile reservoirs in the patient environment and efficacy of aerial hydrogen peroxide decontamination environmental contamination in households of patients with recurrent clostridium difficile infection sanitary status and incidence of methicillin-resistant staphylococcus aureus and clostridium difficile within canadian hotel rooms clostridium difficile infection: a worldwide disease isolation and identification of human coronavirus e from frequently touched environmental surfaces of a university classroom that is cleaned daily detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units severe acute respiratory syndrome coronavirus on hospital surfaces epidemic and emerging coronaviruses (severe acute respiratory syndrome and middle east respiratory syndrome) performance of food safety management systems in poultry meat preparation processing plants in relation to campylobacter spp. contamination the evolving nature of infective endocarditis in spain: a population-based study enterococcal meningitis: a clinical study of cases and review of the literature prospective study on central venous line associated bloodstream infections detection of ctx-m- -producing escherichia coli isolates of lineages st -b and st -a in environmental samples of a tunisian hospital urinary tract infection syndromes. occurrence, recurrence, bacteriology, risk factors, and disease burden van der poll, expression and function of granzymes a and b in escherichia coli peritonitis and sepsis community-acquired escherichia coli enteritis in korean children: the clinical application of a stool polymerase chain reaction assay the occurrence of influenza a virus on household and day care center fomites the environmental deposition of influenza virus from patients infected with influenza a(h n )pdm : implications for infection prevention and control influenza virus contamination of common household surfaces during the influenza a (h n ) pandemic in bangkok, thailand: implications for contact transmission within-host evolution of human influenza virus contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season environmental monitoring for gastroenteric viruses in a pediatric primary immunode ciency unit widespread environmental contamination with norwalk-like viruses (nlv) detected in a prolonged hotel outbreak of gastroenteritis evaluation of a new environmental sampling protocol for detection of human norovirus on inanimate surfaces detection and molecular characterization of enteric viruses in children with acute gastroenteritis in northern italy prevalence, antimicrobial susceptibility, and clonal diversity of pseudomonas aeruginosa in chronic wounds microbiology and antimicrobial susceptibility of otitis externa: a changing pattern of antimicrobial resistance community-associated methicillin-resistant staphylococcus aureus infection in portugal clinical, microbiologic, and epidemiologic characteristics of pseudomonas aeruginosa infections in a university hospital dissemination of human adenoviruses and rotavirus species a on fomites of hospital pediatric units group a rotavirus detection on environmental surfaces in a hospital intensive care unit survival of salmonella in bathrooms and toilets in domestic homes following salmonellosis diversity of bacterial communities on four frequently used surfaces in a large brazilian teaching hospital enteric fever caused by salmonella enterica serovars with reduced susceptibility of fluoroquinolones at a community based teaching hospital of nepal evaluation of food handler practices and microbiological status of ready-to-eat foods in longterm care facilities in the andalusia region of spain the role of primary care prescribers in the diagnosis and management of community-associated methicillin-resistant staphylococcus aureus skin and soft tissue infections community-acquired pneumonia with methicillin-resistant staphylococcus aureus in a patient admitted to the intensive care unit: a therapeutic challenge catheter-related infections in patients with haematological malignancies: novel preventive and therapeutic strategies the authors acknowledge the funding provided by fundação para a ciência e a tecnologia (fct -portugal) through the scholarship sfrh/ bd/ / (micaela machado querido) and the project "b-safecoat -desenvolvimento de novas tintas com propriedades autodesinfetantes" (poci- - -feder- ). key: cord- -idh io v authors: hassan, md. zakiul; sturm-ramirez, katharine; rahman, mohammad ziaur; hossain, kamal; aleem, mohammad abdul; bhuiyan, mejbah uddin; islam, md. muzahidul; rahman, mahmudur; gurley, emily s. title: contamination of hospital surfaces with respiratory pathogens in bangladesh date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: idh io v with limited infection control practices in overcrowded bangladeshi hospitals, surfaces may play an important role in the transmission of respiratory pathogens in hospital wards and pose a serious risk of infection for patients, health care workers, caregivers and visitors. in this study, we aimed to identify if surfaces near hospitalized patients with respiratory infections were contaminated with respiratory pathogens and to identify which surfaces were most commonly contaminated. between september-november , we collected respiratory (nasopharyngeal and oropharyngeal) swabs from patients hospitalized with respiratory illness in adult medicine and paediatric medicine wards at two public tertiary care hospitals in bangladesh. we collected surface swabs from up to five surfaces near each case-patient including: the wall, bed rail, bed sheet, clinical file, and multipurpose towel used for care giving purposes. we tested swabs using real-time multiplex pcr for viral and bacterial pathogens. case-patients with at least one pathogen detected had corresponding surface swabs tested for those same pathogens. of patients tested, had a laboratory-confirmed respiratory pathogen. of the swabs collected from surfaces near these patients, ( %) had evidence of contamination with at least one pathogen. the most commonly contaminated surfaces were the bed sheet and the towel. sixty-two percent of patients with a laboratory-confirmed respiratory pathgen ( / ) had detectable viral or bacterial nucleic acid on at least one surface. klebsiella pneumoniae was the most frequently detected pathogen on both respiratory swabs ( %, / ) and on surfaces near patients positive for this organism ( %, / ). surfaces near patients hospitalized with respiratory infections were frequently contaminated by pathogens, with klebsiella pneumoniae being most common, highlighting the potential for transmission of respiratory pathogens via surfaces. efforts to introduce routine cleaning in wards may be a feasible strategy to improve infection control, given that severe space constraints prohibit cohorting patients with respiratory illness. with limited infection control practices in overcrowded bangladeshi hospitals, surfaces may play an important role in the transmission of respiratory pathogens in hospital wards and pose a serious risk of infection for patients, health care workers, caregivers and visitors. in this study, we aimed to identify if surfaces near hospitalized patients with respiratory infections were contaminated with respiratory pathogens and to identify which surfaces were most commonly contaminated. between september-november , we collected respiratory (nasopharyngeal and oropharyngeal) swabs from patients hospitalized with respiratory illness in adult medicine and paediatric medicine wards at two public tertiary care hospitals in bangladesh. we collected surface swabs from up to five surfaces near each case-patient including: the wall, bed rail, bed sheet, clinical file, and multipurpose towel used for care giving purposes. we tested swabs using real-time multiplex pcr for viral and bacterial pathogens. case-patients with at least one pathogen detected had corresponding surface swabs tested for those same pathogens. of patients tested, had a laboratory-confirmed respiratory pathogen. of the swabs collected from surfaces near these patients, ( %) had evidence of contamination with at least one pathogen. the most commonly contaminated surfaces were the bed sheet and the towel. sixty-two percent of patients with a laboratory-confirmed respiratory pathgen ( / ) had detectable viral or bacterial nucleic acid on at least one surface. klebsiella pneumoniae was the most frequently detected pathogen on both respiratory swabs ( %, / ) and on surfaces near patients positive for this organism ( %, / ). surfaces near patients hospitalized with respiratory infections were frequently contaminated by pathogens, with klebsiella pneumoniae being most common, highlighting the potential for transmission of respiratory pathogens via surfaces. efforts to introduce routine cleaning in wards may be a feasible strategy to improve infection control, given that severe space constraints prohibit cohorting patients with respiratory illness. pathogens present in ill patients' respiratory secretions can contaminate nearby hospital surfaces, such as floors, walls, bedrails and mattresses, through coughing, sneezing and touching [ ] [ ] [ ] [ ] . respiratory viral and bacterial pathogens, including staphylococcus aureus, streptococcus pyogenes, influenza viruses, respiratory syncytial virus, adenovirus, rhinoviruses and novel coronavirus strains, can survive on hospital surfaces for days, weeks or even months. furthermore, touching contaminated surfaces may lead to nosocomial transmission of pathogens between patients, family caregivers, visitors, and healthcare workers [ , , ] . patient care areas in bangladeshi hospitals are open wards with multiple beds in a room and are frequently overcrowded with patients, family caregivers, and visitors [ , ] . a previous study by rimi et al. found a median of four people per sq. feet of floor space in hospital wards in bangladesh and observed a median of five uncovered coughs or sneezes per sq feet per hour [ ] . due to shortage of health care workers, family caregivers (family members who provides hour hands on care to sick patient, including bedside nursing and cleaning) are integral part of inpatient care in bangladeshi public hospitals, contributing crowding of hospital wards [ , ] . the world bank estimated that in only $ of public funds per capita were spent annually on health infrastructure in bangladesh. thus, resources for infection control are severely limited in bangladeshi hospitals [ , ] , making it difficult to implement international infection control guidelines [ ] . the lack of routine infection control practices, including no regular surface cleaning, may increase the transmission of respiratory pathogens via hospital surfaces [ , , , ] . family caregivers, visitors, and hospital staff may acquire respiratory infections either through direct contact with infected patients or via droplets, aerosols or contaminated surfaces. contaminated hospital surfaces can pose a serious risk of infection for patients, health care workers, caregivers and visitors. within this context of scarce resources, describing the magnitude of surface contamination in bangladeshi hospitals, particularly identifying priority areas for decontamination, could influence infection control policy and practice. our objective was to assess the frequency with which patients hospitalized for respiratory illnesses in bangladeshi public hospitals contaminate nearby surfaces, to identify commonly contaminated surfaces, and to determine which pathogens are detected most frequently. we conducted the study in two public tertiary care teaching hospitals in rajshahi and jessore, bangladesh between september and november . rajshahi medical college hospital contains approximately , beds with eight adult medicine wards and four pediatric wards. jessore medical college hospital is a -bed hospital with two adult medicine wards and one pediatric ward. paediatric wards typically admit patients < years of age and older patients are admitted to adult medicine wards. the mean bed occupancy proportion in these hospital wards are consistently > % with patients being treated on the floor and in hallways when beds unavailable [ , ] . study physicians in adult medicine and pediatric wards identified patients aged � years who met the severe acute respiratory illness (sari) case definition of subjective or measured fever (� c˚) within the past seven days with cough or sore throat [ ] . in pediatric wards, physicians identified children < years of age who met the severe pneumonia (sp) case definition: cough or difficulty breathing and at least one danger sign (i.e. chest indrawing, stridor while calm, history of convulsions, inability to drink, lethargy or unconsciousness and/or intractable vomiting) with onset of symptoms within the last seven days [ ] . since case-patients were identified immediately after admission, their illnesses were mostly community acquired. study physicians collected respiratory swabs (nasopharyngeal and oropharyngeal) from identified cases using the world health organization's laboratory safety manual protocol [ ] and pooled them into a single cryovial containing viral transport media (vtm). trained research assistants in each hospital collected one swab sample from five different surfaces near each enrolled case patient: the wall next to the patient's bed, bed rail, bed sheet, clinical record files, and a multipurpose towel. the multipurpose towel is a cloth brought from home by family caregivers and used to clean patient respiratory secretions, wiping the patient's face or head, and to dry caregivers' hands and face [ ] . we selected these surfaces because patients, caregivers, and healthcare workers (hcws) frequently come into contact with them in the hospital wards [ , , ] . the research assistants collected surface swabs between - hours after the case-patients' admission to the hospital. this allowed for adequate time for hospital surfaces to be exposed to possible contamination by respiratory pathogens, while also making sure that surfaces were swabbed before the enrolled patients were discharged or died, as patient turnover in wards was high with a median hospital stay of three days [ ] . the risk for infection from these potentially contaminated surfaces between patients within a room, between patients and healthcare workers, or between patients and family caregivers would vary based on the particular surface and how these different risk groups interacted with the surface. with one sterile rayon swab stick per surface, the research assistant swabbed the area of the wall in contact with the bed cm high from the level of the bed sheet, all surfaces of the bed rail located in the area near the patients' head, half of the bed sheet where the patient's head was including underneath the patient, front and back cover of the patient file and both sides of the multi-purpose towel. not all patients had a wall or bedrail nearby as some patients were cared for on the floor, due to overcrowding. swab samples from each surface area were put into individual cryovials containing vtm and kept in a cool box for up to minutes with a temperature between ˚- ˚c. both the respiratory swabs and surface swab samples were labelled, packaged, stored in a nitrogen dry shipper (- ˚c) and sent to the icddr,b virology laboratory by batch twice a month. the swab samples were thawed and the cryovials containing the sample were vortexed. about μl of the swab supernatant was used for nucleic acid extraction using invimag pathogen kit/kf (stratec molecular, berlin, germany) and the final eluted volume of nucleic acid solution was μl, as per the manufacturer's instruction [ ] . the real-time multiplex pcr assay was performed as per the manufacturer's instructions using an agpath-id ™ one-step rt-pcr kit (ambion) with the fast track diagnostic (ftd) respiratory pathogens kit (fast track diagnostics, luxembourg) for different viruses and bacterial pathogens [ ] . casepatients with at least one pathogen detected in their respiratory swab had corresponding surface swabs tested for those same pathogens. detection of nucleic acid of at least one similar pathogen on respiratory swab and a nearby surface was defined as contamination of that surface. to investigate wider hospital contamination, we also tested the surface swabs collected near casepatients with no pathogens detected in their respiratory swabs for the most commonly detected pathogens identified on surfaces near patients with detected respiratory pathogens. we summarized the data using descriptive statistics. we assessed the difference in proportion of pathogen detection in respiratory swabs and surface swabs between adult and paediatric ward patients using chi-square test considering fisher exact test where appropriate. any association with a p value < . was considered statistically significant. study participants (aged � years) or their legal guardians (if aged < years) provided informed written consent. the institutional review board of icddr,b reviewed and approved the study protocol. the institutional review board at the centers for disease control and prevention (atlanta, ga, usa) deferred to icddr,b's approval. we collected and tested respiratory swabs from patients hospitalized with respiratory illness: sari cases from adult medicine wards and severe pneumonia cases from paediatric medicine wards. the median age of patients in the adult wards was years (iqr - ) and in paediatric wards three months (iqr - ). the male-to-female ratio was . : (table ) . of the patients, ( %) had detectable viral and/or bacterial nucleic acid in their respiratory swabs. paediatric patients more frequently had one or more detectable pathogen in their respiratory swabs than adult patients ( % versus %, p = . ). bacterial pathogens were identified in % of adult respiratory swabs. klebsiella pneumoniae, streptococcus pneumoniae and human cytomegalovirus ( ) ( ) human parainfluenza viruses ( ) ( . ) other viruses b ( ) ( ) ( staphylococcus aureus were most commonly detected during the study period. in contrast, viral pathogens were commonly detected among paediatric patients, including human cytomegalovirus, respiratory syncytial viruses, and human rhinoviruses (table ) . clinical features, mean duration of symptom onset to sample collection ( . days vs days) did not vary between patients with a detectable viral and a detectable bacterial nucleic acid in respiratory swabs. two patients, one with klebsiella pneumoniae and one with streptococcus pneumoniae detected in their respiratory swabs, had been hospitalized at other facilities within two weeks prior to admission to the study hospital, suggesting that these organisms could have been hospital-acquired. both these patients had abnormal chest x-rays and were diagnosed with severe pneumonia in the study hospital. we tested the surrounding hospital surface swabs for each of the patients with evidence of respiratory pathogens for the same viral/bacterial nucleic acid detected in their respiratory swabs. we collected and tested surface swabs near the patients as not all these patients had a wall or bedrail near them. nearly half of the hospital surface swabs ( % [ / ]) had evidence of contamination by at least one pathogen included in the testing panel. the most commonly contaminated surfaces were the bed sheet, the multipurpose towel, and the bed rail. we infrequently detected bacterial or viral nucleic acid on wall surfaces or on patients' clinical record files (fig ) . sixty-two percent of patients ( / ) had detectable viral and/or bacterial nucleic acid on at least one (range: - ) nearby surface, including % of adults ( / ) and % ( / ) of pediatric patients. the most common bacterial pathogen detected on surface swabs was klebsiella pneumoniae and % ( / ) of patients positive for klebsiella pneumoniae had at least one surface with detectable dna. the most frequently detected viral pathogen on surfaces was human cytomegalovirus and % ( / ) of patients positive for human cytomegalovirus had detectable dna on nearby surfaces ( table ) . we tested nearby surfaces for patients ( patients from adult wards and from pediatric wards) without detectable viral/bacterial nucleic acid in their respiratory swabs. we tested these surfaces for six frequently identified pathogens in respiratory swabs of case-patients: klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus aureus, human cytomegalovirus, respiratory syncytial viruses and human rhinoviruses. klebsiella pneumoniae was detected on at least one nearby surface in % ( / ) of these patients, staphylococcus aureus in % ( / ), and streptococcus pneumoniae in % ( / ) patients. viruses, including, human cytomegalovirus ( / ), respiratory syncytial viruses a and b ( / ) and human rhinoviruses ( / ), were rarely detected nearby these patients. nearly two-thirds of the patients hospitalized with laboratory-confirmed acute respiratory infection had at least one nearby contaminated surface. klebsiella pneumoniae was the most commonly detected pathogen in patients' respiratory swabs, and was detected in nearly every environmental swab testing, suggesting widespread hospital contamination from current and previously hospitalized patients. with the ability to spread rapidly in the hospital environment, klebsiella pneumoniae has been linked to several nosocomial outbreaks [ , ] . the most frequently contaminated surfaces were the bed sheet, towel, and bed rail, further highlighting the perils of no routine surface cleaning practices in these hospitals. studies in tertiary care hospitals of bangladesh have shown that one in patients with a hospital stay greater than three days developed a hospital-acquired respiratory infection, and that only % of those infections had a viral aetiology, suggesting a large proportion of these infections might be bacterial [ , ] . since, future work should further investigate the role of klebsiella pneumoniae may be an important nosocomial pathogen in these hospitals. several other studies have identified multidrug resistant klebsiella pneumoniae in hospital environments and its association with severe infections, prolonged hospital stays and increased mortality rates, particularly in debilitated and immunocompromised patients [ , ] . a study in an urban tertiary care hospital in dhaka, bangladesh, reported that % of the clinical specimens (sputum, pus, urine, throat, and vaginal swabs) collected from hospitalized patients were drug resistant [ ] . moreover, our study findings were consistent between the two hospitals despite being located in different geographical areas and may suggest surface contamination with klebsiella pneumoniae as a wider public health problem. the predominant bacterial pathogens we identified, klebsiella pneumoniae, streptococcus pneumoniae, and staphylococcus aureus, can survive on surfaces from a few days to a few months [ , , ] . bacteria, in the presence of low humidity, forms biofilms protecting microorganisms from harsh environmental influences and are difficult to eradicate [ , ] . in bangladesh, hospital surfaces are not adequately cleaned and hospitals report insufficient supplies of cleaning agents [ , , ] . to remove and prevent biofilm formation, hospital decontamination protocols should include strategies such as daily cleaning of surfaces with disinfectant (e.g. . % sodium hypochlorite or % chlorhexidine solutions). among the most commonly detected viral pathogens, rsv was prevalent in respiratory swab of paediatric patients and nearby surfaces. rsv has been a major nosocomial hazard on pediatric wards and has been linked with hospital outbreaks [ , ] . with reported higher case fatality among patient with nosocomial rsv infections (or . , % ci . - ), widespread surface contamination with rsv is concerning for low income hospitals ( , ) . we identified, that towel, was frequently contaminated with respiratory secretions. [ ] . islam et al. reported that family caregivers frequently used a multipurpose towel for patient secretions and for their own use, without cleaning it in between these uses [ ] .this suggests that the towel may act as a potential vehicle for transmission of respiratory viral and bacterial pathogens from patient to caregiver [ ] . infection control could target care giving practices associated with the use of the towel and should test feasible low-cost interventions such as the supply of low cost disinfectant by hospitals that encourage caregivers to clean the towels more frequently and to improve hand washing practices, including the use of hand sanitizer. an important limitation of our study is that we only identified the presence of viral or bacterial nucleic acid on different hospital surfaces, and cannot be sure that the pathogens we detected were viable. a second limitation is that we conducted the study in only two public hospitals, so the findings may not be representative of all public hospitals. however, our findings were consistent between the two typical tertiary care hospitals we studied, located in completely different parts of the country. a third limitation is that we only sampled surfaces once, which limits our ability to comment on duration of contamination, and did not have the ability to observe contamination from seasonal infections. influenza, for example, has a known seasonal pattern in bangladesh, circulating usually between may and september, potentially explaining the low detection rate in our study [ , ] . lastly, we did not investigate drug resistance patterns of the bacteria we detected due to resource constraints. based on evidence from other studies, however, it is likely that many of the pathogens we detected on surfaces were drug resistant and future studies should consider including investigations about drug resistance [ ] . this study identified that hospital surfaces in these bangladeshi hospitals, were frequently contaminated with respiratory pathogens and pose a potential threat for fomite-borne transmission of respiratory infections to patients, healthcare workers and family caregivers. to prevent the spread of klebsiella and other infections between patients, healthcare personnel must follow specific infection control precautions including strict adherence to hand hygiene and the use of gloves. in addition, our data clearly indicate that efforts to regularly disinfect environmental surfaces and ensure clean towels for patient caregiving could reduce risk of exposure to patients, healthcare staff and visitors. the government of bangladesh has taken a number of initiatives to improve infection control in hospitals [ ] . despite this, a nationally representative survey of healthcare facilities showed that healthcare workers performed recommended hand hygiene in only % of opportunities, suggesting low adherence to international standards [ ] . barriers include awareness, training, accountability and appropriate infrastructure (among health facilities, % handwashing locations had no water, % had no soap and - % had no alcohol based sanitizer) to support these behaviours [ , , ] this study highlighted the gaps in practice, as well as the substantial barriers to improvement that will require widespread investments to address. in , the directorate of hospital infection control, directorate general of health services (dghs) at the ministry of health and family welfare in bangladesh has communicated the intention to form infection control committees in each district and tertiary care hospital across the country to improve the safe care [ ] . further investigation to identify the true contribution of fomites in the transmission of respiratory pathogens within hospital settings could be useful to help these committees prioritize efforts to improve hand and surface cleaning. supporting information s file. dataset. (dta) significance of fomites in the spread of respiratory and enteric viral disease environmental contamination makes an important contribution to hospital infection contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? clinical infectious diseases nipah virus contamination of hospital surfaces during outbreaks how long do nosocomial pathogens persist on inanimate surfaces? a systematic review hand-touch contact assessment of high-touch and mutual-touch surfaces among healthcare workers, patients, and visitors infrastructure and contamination of the physical environment in three bangladeshi hospitals: putting infection control into context family caregivers in public tertiary care hospitals in bangladesh: risks and opportunities for infection control the health workforce crisis in bangladesh: shortage, inappropriate skill-mix and inequitable distribution universal health coverage assessment people's republic of bangladesh international nosocomial infection control consortium report, data summary of countries for - : device-associated module incidence of and risk factors for hospital-acquired diarrhea in three tertiary care public hospitals in bangladesh. the american journal of tropical medicine and hygiene who interim global epidemiological surveillance standards for influenza manual for the laboratory diagnosis and virological surveillance of influenza a quantitative approach to defining "high-touch" surfaces in hospitals economic burden of influenza-associated hospitalizations and outpatient visits in bangladesh during . influenza and other respiratory viruses maternal vitamin d supplementation during pregnancy and lactation to prevent acute respiratory infections in infancy in dhaka, bangladesh (mdari trial): protocol for a prospective cohort study nested within a randomized controlled trial ftd respiratory pathogens luxemburg: fast track diagnostics an outbreak of hospital-acquired klebsiella pneumoniae bacteraemia, including strains producing extended-spectrum β-lactamase outbreak of klebsiella pneumoniae producing a new carbapenem-hydrolyzing class a β-lactamase, kpc- , in a new york medical center incidence and viral aetiology of hospital-acquired respiratory infections at three tertiary care hospitals in bangladesh rates of hospital-acquired respiratory illness in bangladeshi tertiary care hospitals: results from a low-cost pilot surveillance strategy detection and treatment options for klebsiella pneumoniae carbapenemases (kpcs): an emerging cause of multidrug-resistant infection predictors of hospital surface contamination with extended-spectrum β-lactamase-producing escherichia coli and klebsiella pneumoniae: patient and organism factors. antimicrobial resistance and infection control prevalence of extended-spectrum βlactamase-producing escherichia coli and klebsiella pneumoniae in an urban hospital in dhaka, bangladesh survival of enterococci and staphylococci on hospital fabrics and plastic survival of enterococci and staphylococci on hospital fabrics and plastic survival strategies of infectious biofilms efficacy of disinfecting solutions in removing biofilms from polyvinyl chloride tracheostomy tubes. the laryngoscope healthcare worker and family caregiver hand hygiene in bangladeshi healthcare facilities: results from the bangladesh national hygiene baseline survey risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influenza and other respiratory viruses the clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato-oncology unit nipah virus contamination of hospital surfaces during outbreaks influenza in outpatient ili case-patients in national hospital-based surveillance estimates of seasonal influenzaassociated mortality in bangladesh bangladesh: dghs, ministry of health & family welfare behaviour change intervention to reduce caregivers' exposure to patients' oral and nasal secretions in bangladesh health worker and family caregiver hand hygiene in bangladesh healthcare facilities: results from a nationally representative survey bangladesh: health economics unit, health sevices division we would like to thank authorities of participating hospitals, all the study participants, the study physicians, field staff and laboratory staff for their contribution. we acknowledge gladys leterme for reviewing and editing this manuscript.icddr,b acknowledges with gratitude the commitment of cdc to its research effort. icddr,b is also grateful to the governments of bangladesh, canada, sweden and the uk for providing core/unrestricted support. key: cord- -bcw f b authors: nan title: abstracts: th ebsa european biophysics congress, august rd– th , budapest, hungary date: - - journal: eur biophys j doi: . /s - - -z sha: doc_id: cord_uid: bcw f b nan o- structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md , usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr , ). together with the light driven clpump halorhodopsin chr is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o- molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station , ecole polytechnique fé dé rale, ch- lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over percent for single junction and percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi- d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale ( - nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier ] - nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier ] meta-stable - nm raft domains that can be turned into stable * - -nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier ]) and facilitated by raft-lipid interactions. [tier ] protein complexes of various sizes ( - nm) and lifetimes. i will also talk about how domains of tiers and are coupled to the membrane partitioning (tier ). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh and daam-fh -fh fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh -fh fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh -fh also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh -fh , remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = , , , , ). d o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about % of the nitroxides in the upper part of the chain (n = , ) are h-bonded by a single water molecule and this fraction reduces to % at the chain terminus (n = , ). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about % to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both -and -sasl in the protein cavity. the temperature dependence of the librations is more marked for -sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol- , -bisphosphate (pip ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf , hela and g tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps . h o) on the malignant cell lines (g , hela, mcf ) irradiated with the jcm - doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps . h o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p- nmr analysis of the respiratory syncytial virus m - protein structure and of its interaction with some of its targets c. sizun the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m - acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m - shares structural similarity with vp , a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m - might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger , , ingo mey and claudia steinem institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs - ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol , bisphosphate (pip ) in the membrane, followed by phosphorylation of the threonine residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b- (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp- - , vega- - . modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. these processes are involved in the pathogenesis of aging and diabetes. non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [ ] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [ ] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, rue becquerel strasbourg, france. cnrs, umr , strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches Ícaro p caruso human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin- -rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration ( lm) constant and stoichiometrically varying the rutin concentration ( - lm) . the emission spectra were obtained in the range of to nm, with the excitation wavelength at nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were . and . m - at and k, respectively. from the modified stern-volmer association constants . (at k) and . m - (at k) were calculated the thermodynamic parameters dh = . kj mol - , dg k = - . kj mol - and dg k = - . kj mol - , and ds = . kj mol - k - . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites . and . , and binding constant . m - and . m - at and k, respectively. the efficient quenching of the trp fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = . nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b lyp functional with - g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about . ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code ao ) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp ) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin - on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's greatest discoveries, ) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph . a : stoichiometry of complexation was detected for both complexes, with association constants of m - and m - for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by mm ttc increased % upon complexation. supported by (fapesp # / - , - ) brazil. p- itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas , dominique burnouf , eric ennifar , sondes guedich , guillaume bec , guillaume hoffmann isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv- reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: ( ) the binding of the tpp ligand and ( ) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least times lower than gfp. this difference is due to an intrinsic mutation of cfp (n i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w (po --bound) and w (non-po --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w . the water h-bond imbalance of - kj mol - is equalized at little enthalpic cost upon formation of a contiguous water network (at - h o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w -dna interactions by additional w -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial -mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh domains of cd ap protein to study interaction with their ligand i.f. herranz-trillo , j.l. ortega-roldan , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin /cms family of adaptor proteins. in this family all three n-terminal sh domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh domains, suggesting an overlapping specificities in binding. cd associated protein (cd ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd ap-sh a/ sh b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over - million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the : and : stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n . microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu + ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu + ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of , , , mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu + complexes in aqueous solution at two ph values, . and . , the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu + complexes. the formation of amb-cu + complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n . membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin- , however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the xhis-tagged protein was immobilised via specific binding to a cu + terminated sensor surface, with a change in frequency indicating approximately % coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix , *, zeger debyser , johan hofkens and yves engelborghs laboratory for biomolecular dynamics, university of leuven, belgium, laboratory for molecular virology and gene therapy, university of leuven, belgium, laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p has well characterized protein and chromatin interacting regions. we studied ledgf/p in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p and the hiv- integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b or c , play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b and c into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [ ] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [ ] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [ , ] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [ ] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t and its distant relative coliphage rb encode co-chaperon proteins (respectively gp and coco) that can replace groes in the chaperonin complex. gp is also required in the folding of the major capsid protein of the phage. prd is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd . we have found evidence that prd protein p can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, svobody sq., kharkiv, , ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin ( mol %) and cholesterol ( mol %). to achieve this purpose, two fluorescent probes with different bilayer location, , -diphenyl- , , -hexatriene (dph) distributing in membrane hydrocarbon core and -lauroyl- -dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range lm [ k d [ nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper , , d. spridon , l.j. van ijzendoorn , m.w.j. prins , eindhoven university of technology, department of applied physics, eindhoven, the netherlands, dutch polymer institute, eindhoven, the netherlands, philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. , p. , ). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a - kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin- (twf ) on the monomeric actin. we determined the affinity of twf to the atp-actin monomer with fluorescence anisotropy measurement (k d = . lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf . in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf . we determined the thermodynamic properties of twf and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe o np was studied in female wistar rats i.v. administered either placebo ( % v/v rat serum in . % nacl), suspension of tio np (positive control, bimodal / nm distribution), or fe o np (bimodal / nm distribution) in doses of . , . or . mg/kg. rats were sacrificed h, -, -and -days after np injection (n= - /each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered mg fe o /kg. plasma mg levels rose in this group and weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe o np. study was supported by fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: . biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in % of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. detailed information on the mechanism and the thermodynamics of the reaction was revealed: first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h po hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin- (gal- ) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal- . our goals are two-fold: ( ) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and ( ) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away -phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (* %) is constituted by amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab( - ) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab( - ) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag , a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of °c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak , b. ró _ zycka-roszak , e. woźniak , r. skrzela , k.a. wilk department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = , , , ) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n . every year over million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ ] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [ ] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [ ] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [ ] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu - -bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse , gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse , matteo castronovo , bojana lucic , alessandro vindigni , giacinto scoles and loredana casalis sincrotrone trieste s.c.p.a., trieste, italy, temple university, philadelphia, usa, protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm . the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt . in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp ) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at °c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween , tween , and pluronic f . znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of . and . mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph . ) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f , to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. , in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano ( ) ( ): , bano et al. nano lett ( ) ( ): - ). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the ( - ) and ( - ) segments of crotamine, a toxin from crotalus durissus terrificus venom [ ] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol : ) and anionic (popg; popc:popg : ) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p * - ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r - , a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r - depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r - to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats and that are involved in the interaction with anionic suvs. results indicate that dys r - constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp- function by occupying a binding site near the active center of the enzyme (bika di et al. ). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp- (cdmmp- ) was expressed in e.coli bl (de ) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp- activity, however, determination of ic % or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp- in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of to da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [ ] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc [ ] in liposomal and planar bilayer systems was possible. abcc -containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc for immobilization via ni + and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met and his residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met bond. this allows the binding of others endogenous ligands forming alternative low spin species , or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h q, h n, and the double one h q/h n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m , to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of . gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin -acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin complex, and study a possible side-binding effect of the leiomodin . the cardiac leiomodin of rattus norvegicus is a kda molecular weight protein, which contains a kda n-terminal, a kda leucin reach repeat (lrr) and a kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin concentration. the nucleator activity of leiomo-din was ionic strength dependent. the data also confirmed that leiomodin is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin . these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc- ( , ', , '-tetrachloro- , ', , '-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh -da ( ', 'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, - , japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri( -n-methylpyridyl)-mono-( -carboxyphenyl)porphyrin and bi-cationic meso- , -bis( -n-methylpyridyl)- , di-( -carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t phage. the wide nucleotide specificity of -phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay , j. vincze , e. balogh hheif, budapest, hungary, nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [ ] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [ , ] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [ , ] . the initially formed cpps show hydrodynamic radii in the range of nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of - nm [ ] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [ ] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna ' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst ) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles ) to detect the expression/activation of critical cell signaling proteins at the single cell level; ) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and ) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b -microlgobulin (b -m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in , cells from localized pca tissue areas (gleason and ) of patients with known metastatic status. the overall median % triple positive for b -m + /pcreb + /ar + cells was . %. the median triple positive for the samples with metastatic potential was % compared with those without metastatic potential (median = %); p = . by a wilcoxon rank sum test. the results were confirmed in pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl- were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o- optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp s and vsfp s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding % dr/r in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr ) slowing with depolarisation and chief (chr /chr hybrid) in contrast, accelerating. new chr point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm ncrna in a sknbe -derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm at its basal level (mock cells) or at . fold higher levels (s cells). the two cell lines shared very similar pools of functional k channels, but s cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s rapid application of gaba elicited a current with a ec = . lm; this current was antagonized by bicuculline ( lm) and potentiated by zaleplon (ec = nm). in mock cells, real time pcr evidenced a high level of gaba a a subunit, while in s cells a significant expression of a and a was detected, whereas a mrna was downregulated by %, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of lm to lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin- (chr ), the panel of excitatory opsins now includes an array of chr variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o- subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav . subunit in pc proximal and distal dendrites, where their density is -fold lower than that found in aiss. a gradual decrease in nav . density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv . voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav . channels, the density of kv . first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca + redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca + response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca + homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca + ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na + concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca + -homeostasis since bz-induced decrease of: in striated muscle ca + release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca + homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca + release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca + release events on images recorded from fibres loaded with fluorescent ca + indicators. to elucidate the effects of ptdinsp-s on ca + release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins p, ptdins p, and ptdins( , )p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca + release events. supported by the hungarian national science fund (otka ), té t. calcium sparks elicited by mmol/l caffeine and by a depolarization to - mv were recorded at high time resolution on both x-y ( frames/s) and line-scan images ( lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, . and . % of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f ; . ± . vs. . ± . ; n = vs. ; mean ± sem, p \ . ) and the full width at half maximum (in lm; parallel with fiber axis: . ± . vs. . ± . ; perpendicular to fiber axis: . ± . vs. . ± . ) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the , dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc . the activation of nfatc dependent genes is consistent with an increase of the il secretion from primary coltures human myotubes from ccd patients and with fibre type predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p- ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew , paula pescador , matthias schade , julian appelfeller , jü rgen liebscher , oliver seitz , daniel huster , andreas herrmann , and anna arbuzova humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, humboldt universitä t zu berlin, institute of chemistry, berlin, germany, universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of nm and - lm and a length of - lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of t -mdr cells. these cell surface transporters have a uic conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least -fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il- is a multifunctional cytokine with pleiotropic effects on t cells. the il- r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il- and - , important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il- and il- r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il- signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il- r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il- r with lipid rafts as well as with the il- r/mhc superclusters was determined by clsm. molecular scale interactions of il- ra with il- r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il- r signaling was assessed by following the effect of cholesterol depletion on il- induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a -mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp and trp ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad , india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij and brij , at °c and °c, and compared them to drms obtained with triton x- (tx ). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx while pe content was decreased in tx drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin- and stomatin) were present in all drms. contrary to tx drms, analysis of electron paramagnetic resonance spectra (with -doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to %, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of - nm diameter and - nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of - ) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg , gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [ nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca + controlled all-or-none like recruitment of synaptotagmin- c ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin- (syt) is the major ca + sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca +-mediated membrane recruitment of the cytosolic c ab domain. the ca +-dependent recruitment of syts c ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c ab and different c ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca +] . this ca + controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t= °c; ph . ). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd , mediating ,,dynamic'' adhesion in cell rolling and icam- , mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd , icam- and the mhci is investigated on the nm-and lmdistance levels on ls t colon carcinoma cells in two different conditions of lymphocyte homing: ( ) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam- and down-regulating that of cd . ( ) crosslinking of cd and icam- representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd and icam- molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann , hetvi gandhi , remigiusz worch , , robert weinmeister , christian bö kel , and petra schwille biophysics research group, technische universitä t dresden, germany, crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only such kinases in humans (jak , , and tyk ), which associate to different cytokine receptor chains. here we study the dynamics of gfp-tagged jak and jak in epithelial cells with fluorescence correlation spectroscopy (fcs). jak and jak behave differently in various aspects: in the absence of receptors, jak still binds the membrane, whereas jak diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak to be ± nm. the association of jak with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak diffuses as a monomer in the rather small cytoplasmic pool, whereas jak diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide (hnp ) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp upon membrane binding/insertion were followed, showing that hnp have quite distinct preferences for mammalian and fungal membrane model systems. hnp showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp membrane interaction process were given by fluorescence quenching measurements using acrylamide, -doxylstearic acid ( ns) or ( ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads ( or nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly % of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the -hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. image gauge v . . the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, stanford street, london se nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb ) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from nm at temperatures below lipids phase transition to nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin- includes a short sequence containing a crac motif (v tkywfyr ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav- (v -l ) nonapeptide encompassing this motif and including the first residue of cav- hydrophobic domain (l ), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., , eur. biophys. j., , - ). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/ / . our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-( -betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of °c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at °c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at °c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica , artur wrona and olga hrydziuszko institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data ( % decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding %) we have shown that the pore radius can measure up to . nm (for a nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to . nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat and stat ) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat and stat in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat and stat . strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [ ] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol ( : : ) with or mol % of ganglioside gm , which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au ( ), although surface modification with -mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [ , ] . in this presentation, we report a detailed mechanism by which ab-( - ) fibrillizes in raft-like lipid bilayers composed of gm /cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e , e , and p ) and non-structural ones (ns , ns , ns a, ns b, ns a and ns b), the latter associated with the membrane originated from the er in the emerging virus. the ns b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns b, a detailed structure determination of this protein is very difficult. the ns b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns b is constituted by two a helices, h (approximately from amino acid to ) and h (approximately from amino acid to ), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu - -bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. , ( )). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c : ceramide) or no (c : ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a : popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability ( - ). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), ( ) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties ( ) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf w. s. aureus lipid extracts from cells grown at ph . and . , studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was * - Å less than ph . . with peptide, bilayer separations of ph . and . extracts were reduced by * Å and * Å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of / of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs Á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr residues and the pi -kinase / akt pathway specific tyr residues in nonconfluent cultures. tyr residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, , rue du loess, strasbourg cedex, f- , france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin- and - receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il- / r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft . t lymphoma cells by increasing the ec. k+ level or by blocking kv . voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il- / r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il- -induced receptor activity, as monitored by measuring stat -phosphorylation, increased upon depolarization, whereas il- induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il- r and il- r with mhc i as well as between il- r and il- r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il- /il- induced phosphorylation of stat transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il- binding and the formation of the receptor complex (il- r alpha -il- r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il- r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin- (il- ) is an important cytokine involved in adaptive immunity. il- binds with high affinity the singlepass transmembrane receptor il- ra. the occupied complex, il- /il- ra then engages either il- rc or il- ra , to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il- ra and il- ra . in contrast, type i complexes containing il- rc could not be observed. however, ectopic expression of gfp-tagged il- rc/jak induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea and rab gtpases. the il- ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il- r heterodimer may require internalization and that early endosomes serve as a platform for il- signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) ( ). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses ( - ). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a -residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the -residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm gangliosides. gm takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup (sup nm) on the mobility of gm on the plasmamembrane of living cells. preformed sup nm aggregates were incubated with cells and gm molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm were then tracked. the mobility of gm was found to decrease dramatically in the presence of sup nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm might imply a consequent loss of function of gm , thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form to . and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega , and , cex sas nanofluid and by esf project no. . understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from to lm x lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe o nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe o . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega , and , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e , e or e , of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including - - proteins. - - proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of - - (eta) with as in lbs. herein we show how - - (eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, - - (eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since - - (eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain ( - ) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m '' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta ( - ) we present an experimental study on the fibril formation of ab( - ) peptide at ph . . the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin - was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin - (amyl - ), reverse amylin - (rev-amyl - ) and scrambled amylin version scr-amyl - . the results show that for amylin - peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [ ] . md show that the x revamyl- - stack has looser selfassembly than the x amyl- - stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [ ] . the results of md show that x amyl- - could have a turn, which consists with ftir data [ ] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [ ] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor (a -lz). first, a -lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a -lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a -lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a -lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble -subunits and -subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega , , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca + pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause % leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m , is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab and ab induce aggregation and toxicity of ab while the behavior of ab is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept and smsept , which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept was able to hydrolyze gtp, whereas smsept was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of °c for smsept and °c for smsept . these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after -st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after -st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients hours after -st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from to bar), and small angle x-ray scattering (saxs) changing the temperature (between - °c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d = . - lm /s corresponding to small oligomeric forms, whereas the slow component with d = . - . lm /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h . the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § - - and - - ) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p -dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm and mcm . furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu , and its mammalian counterpart, gon l. preliminary data showed that udu and gon l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu and gon l can bind to mcm and mcm and they are localized in the nucleus. these data suggest that udu and gon l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, nanyang drive, , singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of -nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position of the histone h . exceptional importance of the h lys acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [ ] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm - ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [ ] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from cm - to cm - . ir spectra show that in ph . - . interval carbonyl (c=o) band at - cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph . significantly decreases band intensity at cm - due to unbounded c =o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in - cm - frequency interval, corresponding to o-h group in-plane bending vibration ( - cm - ). as for acidic conditions, it was observed that under the extreme ph (* . ) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at cm - and cm - corresponding to out-of-plan deformation of nh groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb / also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb , hmgb-domain recombinant proteins and linker histone h . we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb / regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h . based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy -dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh . ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm. plasmid effect on the repair of dna j. vincze , i, francia , g. vincze-tiszay hheif, budapest, hungary, univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm. plasmid on repair of single strand breaks in dna induced by cogamma irradiation in e.coli k ab (wild type) and its different rec mutant cells. the pkm. resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm. , the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm. r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k , k ), derived from the parental hybrid peptide ca( - )m( - ), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [ ] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca( - )m( - ) [ ] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg ( : ) and pope:popg( : ) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: ( ) dioleoylphosphatidylcholine (dopc); ( ) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and , -bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw together with a longrange component of the correlation energy [ ] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some % to % of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter (ctr ), which is the main copper transporter in the human cell, and the kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca + and predicts the blockade of alkali metal ion currents by micromolar ca +. conductance patterns observed in varied mixtures containing na+ and li+, or ba + and ca +, are predicted. ca + is substantially more potent in blocking na+ current than ba +. in apparent contrast to experiments sing buffered ca + solutions, the predicted potency of ca + in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca + for the pore. these experiments depend on the problematic estimation of ca + activity in solutions buffered for ca + and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca + versus na+ is always larger than the dynamical selectivity because ca + ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations . the shift of vibrational fingerprints in the - cm - frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis and compared with the present and previous experimental data . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [ ] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au( ) surfaces by means of the dft [ ] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([ %) differ only at the th position, k (mb-i) and e (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v g (jmr) and d h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng and suet-mien tan biophysics group, a*star institute of high performance computing, and, school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb tm that is absent in the better-studied platelet integrin aiibb [ ] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb within the simulation timescale as compared to its 'lossof-function' b t g mutant or aiibb [ ] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb , suggestive of tighter packing. the likely conservation of this polar interaction across the b integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [ ] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [ ] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [ ] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at to k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm -dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm [ ] [ ] [ ] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm . we have found that the allosteric effect in crm -dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm . light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin- (chr ) and channelrhodopsin- (chr ) , which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels . the n-terminal putative seven-transmembrane region of chr was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr) . as for the majority of membrane proteins, there is no d-structural data for chr available yet. here we present homology models of chr using two high-resolution x-ray template structures of br ( qhj ) respectively asr ( xio ) in order to get structural and functional insights into chr . with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs . . . comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr . here we show that the br-based homology model is a reliable model of chr exhibiting structural features already found experimen-tally . our br-based homology model of chr allows predictions of putative crucial residues within chr . so we proposed several mutations within the chr sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [ ] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [ ] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd- , a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter and its surface nanomodifications . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest , . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio surface . [ ] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin (hsaqp ) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp tetramer (pdb code d s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp tetramer. elucidating the modular structure of the protein g c fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to membrane systems spanning over to us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to * a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than compounds (us nci protocol (dtp) based on obtained directed library over new compounds, among them compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [ , , ] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [ ] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [ ] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [ ] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted ( °respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale ( - ns), however, this process may be affected by unfavorable energy barriers (from to kj mol - ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p y and p y . to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p y is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp , member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp with nd of his , which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a % w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated * such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by - %) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo u and m a . in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [ ] [ ] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [ ] [ ] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [ ] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [ ] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex ( b q) [ ] . ns md trajectories (charmm force field) were analyzed and roles of ca + and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [ ] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [ ] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechan- hiv- protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via all-atom explicit solvent molecular dynamics simulation runs of ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [ ] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [ ] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [ ] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [ ] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [ ] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [ ] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [ ] [ ] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type (hiv- ) gp and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv- entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv- integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p [ ] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [ , ] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv- integrase and block the interaction with ledgf/p . the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p and hiv- integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [ ] currently the most potent inhibitors show an in vivo ic of nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [ ] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level , . this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy - . despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [ ] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [ ] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [ ] , light exposure minimization and object tracking [ ] , or super-resolution strategies. the features and operation of the rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional ( d) volume with a video-rate scanning. we present d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than frames per second lateral scanning with fast refocusing enables the d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz , , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [ ] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated , lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [ ] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [ ] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl- protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u- mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy ( pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di- -anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye ( lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation ( min.; mw/mm ) of dye loaded tissue had no statistically significant effect on apd or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd (by %) and conduction velocity ( %). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m ) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with % saccharose, r and r with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at hours and after days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * - lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution ls) and a pixel array of spads. the spad array has a full frame time resolution of - ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional ( d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution ( . - Å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the . to Å resolution range. a recent example is our Å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex , two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at - Å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all species we investigated ( mammals, fungi, plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra . one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\ ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (* nm) as the load is increased, until it suddenly vanishes as the isometric force is reached ( . ± . pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for * % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle * residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a b ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz , pá l petrovszki , zoltá n kó ta the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at . hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the degrees rotor steps, meaning that the rotation rate of the rotor is around hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [ ] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [ ] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [ ] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [ ] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp of the c subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published in march , and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range . - pn force and - , nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin- family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin- function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin motility is exceptional in the kinesin- family. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of amino acids in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin- kip and of the spindle-organizing protein ase affected cin velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large ( nm) and one small ( nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferÒ and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # - - -a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph . and °c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg + ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* %) and b-sheet (* %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix ). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position , which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop '' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop '' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: ( ) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the human members (hspb - ), this study focused on hspb (hsp involved in some cancers), hspb (lens specific), hspb (lens, muscle, heart, lung) and hspb -r g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [ , ] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [ fe- s] cluster [ , ] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with , and kcal/mol at , and °c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with , and kcal/mol and , and °c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from to mol %), the first transition was shifted to a lower temperature value around °c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type (mfe- ) from different organisms. mfe- is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe- has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe- . mutations in the mfe- -coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe- protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura , - warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov , marko sustarsic , arnold j.m. driessen groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa /alb chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [ ] [ ] . the t ss consists of multiple copies of * different proteins (totalling * mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase (dgat ) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat binding sites (sit and sit ) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit showed to bind more strongly in nonpolar membrane models, while sit was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a nm blue shift in fluorescence emission. the binding of sit and sit peptides to negative liposome gave dissociation constants (k d ) of and . lm, respectively, and a leakage action -fold higher to sit . the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [ ] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp . this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s and s leads to the binding of the - - protein. the - - proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the - - protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the - - :pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by - - binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs at . Å resolution was solved. the stoichiometry of - - f /rgs protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between - - f and rgs protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build d model of - - f /rgs protein complex. our model revealed new details on architecture of complex formed by - - proteins. to date all known structure of - - proteins complexes suggests that the ligand is docked in the central channel of - - protein. our results indicate that the rgs domain of rgs protein is located outside the central channel of - - f protein interacting with less-conserved residues of - - f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca + ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n -( -carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas and pah genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas and pah using biophysical techniques. pah and ceas genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah and ceas were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to °c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at °c. moreover ceas and pah showed to be stable over a wide ph range (ph . - . ). we are currently working on improving ceas crystals which are a promising step towards the elucidation of the ceas structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from to nm, last for min for three scans with a nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g and later as a gap junction-like feeding tube through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha l glyr are clustered, while the alpha k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [ ] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [ ] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution d imaging and d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of nm in the plane and nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol , -bisphosphate (pi( , )p ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi( , )p in synaptic active zones is important for synaptic transmission. however, pi( , )p does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi( , )p is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi( , )p clusters and on synaptic protein-pi( , )p interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about % of the chromosome to be transferred into the forespore. the remaining (* mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * lm /s during nuclear cycles - , both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor (tlr ) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr organisation is required for the signalling process. macrophages natively express tlr at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [ ] , reversible on-off qd photoswitching [ ] , and ph sensing [ ] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [ ] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [ ] [ ] [ ] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [ ] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [ ] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [ ] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [ ] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the or amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than * nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to * nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [ ] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [ ] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a ). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected time-lapse images (time intervals min) of dual-marked a cells under three different light intensities ( . , . and . mw/cm ) and a variety of pulse lengths ( . , . , , . and ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than . mw/cm together with pulse durations longer than ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [ ] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [ ] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [ ] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [ ] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [ ] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb . fluorescence intensity f , f , f and f was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the and hours old seedlings, and bgf intensity decreased of hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße , , germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto was coupled to the halotag ligand (htl) and fast reaction of htl-atto with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for : labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than g proteincoupled receptors (gpcrs); overall, - % of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of * ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk ( ) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph , the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph , repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants ( ) ( ) ( ) ( ) . fss with a polymeric hydrophilic head proved to be mild towards mps ( ) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods ( ) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans ( ). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * k the dynamic response observed in the pico-to nano-second time regime is driven by ch dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [ ] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [ ] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [ ] , preferential exclusion [ ] and water replacement [ ] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of , -dipalmitoyl-phosphatidylcholine (dppc) in d o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & . - . . under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [ , ] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [ ] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [ ] . in recent papers [ , ] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [ , ] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa , , mikio kataoka , japan atomic energy agency, tokai, japan, juelich centre for neutron science, forschungszentrum juelich gmbh, nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, É va juhá sz, jó zsef tímá r, balá zs heged} us nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within hours and the -hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the -hour migration distance ranged from to micron and from to micron in mesothelioma and melanoma cells, respectively. the lowest -hour cell division probability was found to be . in both the melanoma and mesothelioma series while the highest proliferation activity reached . and . in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r= , ; p= , ) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n ba titin isoform as compared to short n b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße , gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p . paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp / protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp / machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal and tip in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea , the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin- motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider , marco tarantola , and andreas janshoff institute of physical chemistry, georg-august-university, gö ttingen, germany, max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor (tgf-b ) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a and mda-mb was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [ ] to fission yeast cells [ ] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea travels at the tips of growing microtubules and is delivered to the cell ends [ ] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain isoform (tmbr- ) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr- to actin was* lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by % in the presence of tmbr- . the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr- seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [ ] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a , um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [ ] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf- , associated to a low metastatic potential showed tether stiffness of pn/um in average. the second cell line, mda-mb , poorly differentiated with a high metastatic potential had a tether stiffness of pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p ) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt and histone deacetylase- , function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. we extensively characterize important liposomal properties, finding diameters between and lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp / complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal nm and br ) on the activity of mdia formin and arp / complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia -, and arp / complexmediated actin assembly. the activity of the arp / complex is inhibited by sktm and tm nm , while tmbr does not have any effect. all three tm isoforms inhibited the activity of mdia . these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with , and lg/ml of collagen type i for h and h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf insect cells) was implemented. initial expression levels were determined by either partial purification using ni ? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain - % a-helices and - % b-sheets in agreement with estimates based on homology with the ca atpase serca . we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v . membrane distribution jimé nez-garduño am , , pardo la , ortega a , stü hmer w unam, mexico-city, mexico, mpi-em, gö ttingen, germany the potassium channel k v . is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v . has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v . in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv . associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv . fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v . current density. we suggest that k v . coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [ ] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , Ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v . , ca v . and ca v . channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v . channels on-gating is shifted to more positive membrane potentials by about mv compare to ca v . and ca v . channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v . and ca v . channels compare to ca v . channels. further, more charge is moved per unit of macroscopic current amplitude in ca v . channels compare to ca v . and ca v . channels. supported by apvv- - and vega / / . the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i e of the adult rat muscle-type sodium channel (rna v . ) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i e was expressed in tsa cells and studied by means of the patch-clamp technique. when applied from the extracellular side lm qx stabilized the slow but not the fast inactivated state in i e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger isoform (nhe ), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe . we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe , these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida , - wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v . ) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v . channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w . w c, w c and w a at the outer tryptophan-rich lip, as well as d c, e c, k c and a c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a -fold inhibition loss was observed, tryptophan is crucial for the reversible blocking effects of pq. as a result, w c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm , tm ) a-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm inner helices are crossed each other near the cytoplasmic side and leu and val in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe in tm outer helices was dragged by lipids, leading to an opening of mscl. thus phe was concluded to be the major tension sensor. during opening, tm inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of * to * kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed to of * - nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length * kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length * kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was * . indicating % colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. %) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn . electrophysiological investigation of the hvdac ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. , d- gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel (hvdac ) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of - %. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ± mv and evaluation of the resulting currents. the hvdac was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y and f were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y a. in this study we provide a structural interpretation for this observation. md simulations on the y a mutant suggest side chain rearrangements of f located one helical turn below y . loss of p-p stacking induces reorientation of f from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y insensitive drugs can still favorably interact with the reoriented f aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s ) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa is very weak (apparent number of gating charges * . versus in k ? channels) and its putative voltage-sensing domain most likely lies outside the s because trpa completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa . in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p- action of double stress on photosystem aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd ? and co ? ) on activity of ps in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd ? ( - m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn -cluster or y z on donor side of ps , is inhibited stronger than at action of only co ? . the steadystate level at cd ? treatment is remain stable, while at co ? action it is increased. simultaneous action of cd ? and photoinhibitory illumination ( lmol photons m - s - ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co ? and excess light. result indicates that damage sites of action cd ? and co ? are donor and acceptor side of ps , accordingly. we assume that binding site of cd ? is y z or camn -cluster, one of the recombination partners with p ? on the donor side of ps . thereby, action of cd ions on donor side of ps leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos , z. gingl and p. maró ti department of medical physics and informatics, university of szeged, hungary, department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up with better time resolution ( ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: ) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, ) electron transfer in the reaction center, cytochrome bc complex and in between obtained from relaxation of the fluorescence and ) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at and nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [ ] and photoprotection [ ] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s state of a carotenoid [ ] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [ ] reveal that each chlorophyll is surrounded by or molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [ ] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [ ] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being : for the rfpcp and : for the hspcp [ ] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( o ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative o sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße - , d- mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q type or qequenching), an additional quenching center responsible for qi type (identical to q center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp and cp we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [ , ] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [ , , ] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence ( ). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae ( ) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated ( ). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s , s). the ratio k k sv • s /s was suggested as a measure of the functional domain size. values in the range of - were found for lhcii macroaggregates and - for native thylakoid membranes, corresponding to domain sizes of - chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r- and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from ls to s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc complex of the bacterium. in the shorter (\ ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r- were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at nm was divided into a ns and a ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. ( ) febs lett. , - ] to spectrally silent conformational changes. the nature of the ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker , szilvia z. tó th , lá szló ková cs , alfred r. holzwarth and gy} oz} o garab institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, * - % of the variable fluorescence intensity relaxes within ms in darkness and can be re-induced within ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, * % of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below - °c), where the q a -/s recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne , edward o'reilly , cecile blanchard , alexandra olaya-castro and james n. sturgis lism, cnrs and aix-marseille university, marseille, france, department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh , lh and lh . here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo , g. bencsik , g. kozak , cs. visy , z. gingl , k. hernadi , k. nagy , gy. varo and l. nagy departments of medical physics and informatics, physical chemistry and materials science, technical informatics and of, applied and environmental chemistry, university of szeged, hungary, institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th , , herbert van amerongen , , gy} oz} o garab , lá szló ková cs institute of plant biology, biological research center, hungarian academy of sciences, hungary, wageningen university, laboratory of biophysics, wageningen, the netherlands, microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp , kocp , kopsbw, kopsbx, dgd ), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan , hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. /c, h- budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl , k.yoshihara university of warmia and mazury, department of physics and biophysics, olsztyn, poland, suntory institute for bioorganic research, wakayamadai, osaka , japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with -fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h o although the reaction can occur in the presence of oxygen with lower rate [ , ] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h o as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch ) -cooh/-s-(ch ) -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h o or d o as solvent, under the variable temperature ( - k) or pressure ( - mpa) conditions, revealing the overall reorganization free energy of . ± . ev, activation volume of - . ± . cm mol - and inverse solvent kinetic isotope effect of . ± . ( °c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode ), or attached to sams through the electrostatic interaction (mode ), or specific ''wiring'' (mode ). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode ). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to -cis, -anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp , proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp to the schiff base, and reprotonation of asp from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [ ] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl to asn (l dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between cm - and cm - to histidine vibrations. these bands show the protonation of a histidine, most likely hisl , during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [ ] [ ] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [ ] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [ ] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [ ] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, -(n-ethyl-n-isopropyl)-amiloride (eipa) [ ] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [ ] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [ ] [ ] . we will present results of the first computational studies of nor (both cnor and qnor types) [ ] [ ] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [ ] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for c-type cytochromes ( ). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions ( ) ( ) ( ) . a putative outer-membrane cytochrome, omcf, is crucial for fe ? and u ? reduction and also for microbial electricity production ( ). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c of photosynthetic algae and cyanobacteria ( ). the structure of oxidized omcf was determined ( ) being the first example of a cytochrome c -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( n and c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( ( )). years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… ( )). in spite of superimposable d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers ( )…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie- and rhodobacter ferroxidans sw both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie- the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for . h, h and h. after . h, aox activity was increased only in specimens incubated in anoxic conditions ( . %). after h, increase in aox activity was significant in both hypoxic and anoxic specimens ( . % and . %, respectively), with even greater increase after h, . % for hypoxic and . % for specimens in anoxic conditions. mycelia treated for h was then oxygenated for minutes. this induced decrease in aox activity of % in anoxic and even . % in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f f -atpase on hydrogenase activity during glycerol fermentation k. trchounian , , g. sawers , a. trchounian department of biophysics, yerevan state university, yerevan, armenia, institute for microbiology, martin-luther university halle-wittenberg, haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd- , hyd- and hyd- have been well characterized. hyd- has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph . [ ] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f f -atpase. indeed, at ph . in an e. coli mutant (dk ) lacking f f overall hyd-activity was reduced to approximately % of the wild type activity; hyd- , but not hyd- , was detected in an in-gel activity assay. f f is therefore suggested to be required for hyd- activity. at ph . in glycerol medium hyd-activity in dk was * % of wild type activity and hyd- and hyd- exhibited only weak activity. this indicated a significant f f contribution towards hyd-activity as ph decreased. furthermore, at ph . hydactivity was negligible and only a very weak activity band corresponding to hyd- could be observed. these results suggest that f f -atpase is essential for hydrogenase activity during glycerol fermentation at ph . . taken together, the results suggest an interdependence between hyd- , hyd- and f f -atpase activity. [ ion channels and transporters control many facets of cancer cell biology and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology , . we have contributed to this field showing that k v . (herg ) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in ). the herg dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia and colorectal cancer , herg overexpression confers a higher malignancy to neoplastic cells. moreover, herg blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg blockers overcame chemoresistance in acute leukemias as well as reduced gi cancer growth, angiogenesis and metastatic spread . the overall message emerging from our data is that the herg protein represents a novel biomarker and drug target in oncology. up to now, herg was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg blockers that exhibit no cardiotoxicity and are more specific for the herg channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl. k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv . gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\ . mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv . gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd and anti-cd stimulation. our results indicate that the biophysical parameters of kv . gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd and anti-cd exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim ) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b -subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using -d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p ). it has been over years since sensory neuron-specific sodium channel na v . was identified. since then na v . has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v . in unmyelinated primary sensory neurons. na v . was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v . in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v . cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd complex and cd . we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv . (tea or anuroctoxin), ikca (tram- ), or the crac channel ( -apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc a ) are associated with several diseases including cancer. however, data about napi b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc a gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc a gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc a in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc a gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in , in and in spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h o and d o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf- breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for , , , and hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca ? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k p channels (twik channels) and k p channels located at the plasma membrane (trek / channels). living at the edge: volume the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca ?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca ? by extracellular ca ? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than %w tested using ll of mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd , ubq, a-, b-casein, and wt, a t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's d orientation. therefore, several techniques have been proposed in order to determine the full d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [ , , ] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in d orientation of single molecules down to a nanosecond timescales. the d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a - % higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from . (physiological) to . (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. , kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic f-labeled amino acid f-phg. using f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. : ]. here, we present novel analogs of gs with different f-prolines in the b-turn region, and with cf -bpg in place of leu. based on these f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h n /victoria/ / influenza virus strain has been submitted to various bpl inactivation conditions (from lm to mm). cell infection ability was progressively reduced and entirely abolished at mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph on gm liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, -hydroxylated fatty acid. minerval ( -hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of -hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, -hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at °c in the presence of triton x- . the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of -hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied -hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c -hpc, in mixtures of popc with and mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional ( d) counterpart of the classical stern-volmer analysis for three-dimensional ( d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of nh and oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = , , , ) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l and a . for cytotoxicity test, the cells were seeded in -well plates ml of Á cells/ml in the culture medium eagle'a or dulbecco with % calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately % of cells was taken as tccd . this work was supported by grant n n . membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre , d. arrais , j. martins , ibb -cbme, faro, portugal, dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a- , glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until . m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above . m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in , usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [ , ] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di( : ), mixed ( : )( : ), and methylated di( : - me). in order to minimize the effect of headgroup electrostatics, measurements where performed in m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from . seconds in the dope lipid to seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n and n n . many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, : - , ( )). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., , , ( )) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after days and reached % conversion in days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz , halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani , , alessandra filabozzi and giovanna fragneto dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d (small momentum-transfer diffractometer) and d (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h- budapest pazmany p. stny. /a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. ( )). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor -prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr and ccr mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain (tmd ) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr and ccr , coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr and ccr that is responsible for cholesterol requirement for cxcr and ccr conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr -using and ccr -using hiv strains. putative crac sequences involve residues v /l -y -k in cxcr and l /v /v -y -k in ccr . in cxcr , crac motif is highly conserved across chordata species, whereas in ccr , crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t and t data showed that there are two reorientation types present for every protein solutions below °c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from to residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) , . in the present work, al ? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a c and a c. the average lifetime of donor in the presence of acceptor increases with the increase of al ? concentration, indicating as adopts a more extended conformation upon al ? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al ? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from ± lm /s as monomer state to ± lm /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly % of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [ ] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [ ] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [ ] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [ , ] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase d structure. measuring hsqcs peaks intensities up to bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the residues of each mutant. so-fast hmqc experiments were also performed to measure folding and unfolding rates from bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i ) and a -residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of - gpa and - °c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p , the tumor suppressor p , and the adenovirus e a oncoprotein. the globular domains of cbp/p are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p . many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p -mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, Á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t - ., budapest, h- , hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around %. this transition is characterized by a scale-free power-law cluster size distribution with an exponent . . we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a well- we focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [ , with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes ( ), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of , and kda which were bound cgmp more intensively. after d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [ ] . helix specific interactions also cause tilt, in one direction, between two dna fragments [ ] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [ ] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [ ] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [ ] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn- and - using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [ ] [ ] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n , the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, ). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science ). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a or a -like subunit [ ] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [ ] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from . % to . %) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb , which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein (nsp ) and non-structural protein (nsp ). this hexadecameric nsp -nsp complex is a hollow, cylinder-like structure assembled from eight copies of nsp and held together by eight nsp molecules [ , ] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp -nsp form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks- with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of * - monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used nm diameter aunp loaded with rhodamine g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. nm and nm diameter aunp loaded with doxorubicin were incubated at different time points with a cell line (human adenocarcinomic alveolar basal epithelial cells). only nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\ nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with - lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of ', '-dichlorofluorescin, the effect being higher for the fenton-modified dnps after -h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [ ] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation ( - lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [ ] . upon illumination, the chromophore retinal isomerizes around the c -c double bond [ ] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [ ] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [ ] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [ ] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan ) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [ , ] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln- , p-c-met and mcl- , which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: ) fixed xenograft tissues from an established ltl castration resistance human prostate cancer or crpc model; and ) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in ) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and ) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) are already quite popular among many scientific and technological disciplines . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les- , , which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les- containing isatine fragment, activated caspase- involved in receptor-mediated apoptosis, while les- possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase- , and les- which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les- and les- were combined into one molecule. in vitro studies showed that such hybrid molecule called les- possessed times higher anticancer potential (ic = lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from ± nm till ± hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe o ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str , , berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection ( nm) vs neurite indentation ( - nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. , d- gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr is also expressed by tumor cells. first experiments show that activation of tlr displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe o -nanoparticles (nps) to stimulate tlr in a cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe o -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [ ] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [ ] . three types of colloids were prepared at various temperatures ( , and °c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of - nm. for agnps synthesized at °c, for example, a bimodal size distribution was observed (about and nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used -mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: ) quality and quantity of structural blocks of nc, and size unimodality; ) branching at specific sites in polymer chain of nc; ) providing nc with reactive chemical groups; ) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: ) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; ) dna for transfection of mammalian, yeast and bacterial cells; ) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants # , # , # . in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\ nm) is a very promising candidate [ ] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [ ] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between and nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as lm. it is generally accepted, that h s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h o . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h o . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a % delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible fold drop of br mean resistance over * cycles of interspersed illuminations at the same gold-br-gold junction when k[ nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at nm were used along with synchronized ultrafast laser pulses at nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, nm, fs, hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within * min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si- ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b f cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b f murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& . s/m), f co of control and gt-cells was found to range from to khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation , inverse emulsion techniques and more recently microfluidic techniques such as double emulsion or jetting method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than %. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range - lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d domain of flagellin subunits, comprising residues - , protrudes at the outer surface of flagellar filaments. the d domain has no significant role in the construction of the filament structure. thus, replacement of d may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g pamam dendrimer was modified with anti-tag nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. dept of chemistry, faculty of science, national university of singapore, singapore, dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll and ficoll is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of to mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, -cell and -cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of -cell and -cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly- hydroxybutyrate (phb) [ ] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at * cm - to that of cell proteins (amide ii band at * cm - ), showing a. brasilense to be able to produce phb up to over % of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d is not essential for filament formation. the concept in this project is to replace the d domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table ) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck , nk , nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated fe ions of differing energies swift heavy ion irradiation of srtio under grazing incidence'' proc. natl. acad. sci. usa forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta -adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin , , viktor foltin , , erich gornik , rumen stainov , , tanya zlateva nü rnberg. icsd e.v. postfach (pob) , d- mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. / a/jan stretch ( - mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins , . acknowledgments this work was supported by project grant ptdc/qui/ / (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: . enlargement of executive committee by a. honorary & presidents (permanent - : moral support & - fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). . implication of interdisciplinary topics to esba/iupab congressprogrammes, . also for biophysical journals. . organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. . co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals .- . could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin , , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon , zsolt má rtonfalvi , pasquale bianco , beá ta vé rtessy , mikló s kellermayer micro-viscosimeter generated and manipulated by light andrá s buzá s , lá szló oroszi , ló rá nd kelemen , pá l ormos temesvá ri krt proc. natl. acad. sci. usa neural signal recordings with a novel multisite silicon probe gergely má rton , anita pongrá cz , lá szló grand , , É va vá zsonyi pé ter pá zmá ny catholic university, faculty of information technology, h- , /a prá ter st multiscale pattern fabrication for life-science applications francesco valle , beatrice chelli , michele bianchi , eva bystrenova , marianna barbalinardo , arian shehu , tobias cramer , mauro murgia , giulia foschi miroslava kuricova , jana tulinska , aurelia liskova , eva neubauerova , maria dusinska , , ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci , jelena ban , elisabetta ruaro , massimo tormen , marco lazzarino , and vincent torre light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: ) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). ) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from . to . and the photochemical rate constant from Á s - to Á s - with an apparent halftime of about - hours after change from anaerobic to aerobic atmosphere. ) the electrogenic signal (absorption change at nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi , m. bokor , p. kamasa , p. tompa , k. tompa research institute for solid state physics and optics, hungarian academy of sciences, pob. , budapest,hungary, institute of enzymology, biological research center, hungarian academy of sciences, pob. , budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at . mhz frequency in the - °c to ? °c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below °c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers and the freely-orbiting magnetic tweezers . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench , . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then , % and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . , thus, notable variations in the rna binding specificity was observed for the divalent ions of mg ? , ca ? and mn ? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m g modification together with the principal stabilizing m a modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the -base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with -base-long indifferent gene segments for use as molecular handles, was cloned into a pet b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of - pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at * , , nm, corresponding to * , , bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert , structural biology brussels, vrije universiteit brussel, pleinlaan , brussel, belgium, department of structural biology, vib, pleinlaan , brussel, belgiumin , scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments ( kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse , osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar and ifnar . detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar and ifnar with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar / in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o- self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse , osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar and ifnar based on mvqd-trisnta were realized on live cell [ ] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u a cells stably expressing ifnar at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat , we observed the dynamic coming-and-going contact between the microcompartments of ifnar and stat .a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the . - hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph . to . . forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted . ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as . these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann , christoph schweiger , feng zhang , wolfgang. j. parak , thomas kissel ,# , pilar rivera_gil ,# biophotonics, institute of physics, philipps university of marburg, pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe o ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe o nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than % of all drugs and are encoded by about % of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) , to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin- will be presented . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is lm wide, lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [ ] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy y, n , ne- c) achieving an extremely accurate guidance [ ] . local surface topography is also known to influence the cell fate [ ] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [ , ] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio nps in concentrations: . ; and lg/cm for h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for h was suppressed significantly (p. , p. ). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p. ). no alteration was found in lymphocyte proliferation treated in vitro with tio nps for h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from to nm. on higher pillar neuronal differentiation reaches * % hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord- - vzwc d authors: nan title: proceedings of scanning /seems charleston, south carolina, usa date: - - journal: scanning doi: . /sca. sha: doc_id: cord_uid: vzwc d nan the widespread application of monte carlo electron trajectory modeling has never been realized in the electron microscopy community because of the computation-intensive nature of the monte carlo algorithm (e.g., many hours of computer time were required to run one simulation which might improve one's ability to interpret data if sufficient statistics were obtained). massively parallel supercomputers with multiple-instruction multiple-data (mimd) architecture are a good platform for monte carlo electron trajectory codes without the complications of vectorization of the computer code required on vector supercomputers (e.g., cray-ymp). the nist monte carlo electron trajectory simulation code has been adapted to run on a massively parallel computer. for the first time, the increased speed of the calculations has made monte carlo calculations a real-time tool for data interpretation. the increased speed achieved by the parallelization of the monte carlo code results in the ability to model small probability events due to the massively parallel monte carlo (mpmc) code's ability to simulate large numbers of electron trajectories quickly. applications include both thin-film and bulk x-ray microanalysis and examples of contrast in backscattered electron images obtained in the sem. this work was supported by the united states department of energy under contract #de-ac - al . in electron probe x-ray microanalysis, "secondary fluorescence" refers to the emission of characteristic x-rays following photoelectric absorption of the primary x-rays generated by the electron beam. while generally a minor contribution to the total x-ray emission, secondary x-ray fluorescence can become an important issue, when the spatial resolution of analysis is of interest, because of the significantly greater range of x-rays than of electrons. thus, for a nickel- % iron alloy excited with a kev electron beam, the range of excitation of the primary radiation will be a hemispheric volume with a diameter of about one micrometer, while the range of secondary fluorescence of iron k-shell radiation by nickel k-shell radiation will be a hemispheric volume with a diameter of approximately micrometers for % of the total secondary radiation. such long-range production of secondary radiation is important when the measurement of trace element distributions near phase boundaries is attempted in situations where an analyte appears at high concentration across the boundary. monte carlo electron trajectory simulation provides a powerful tool for the calculation of the three-dimensional distribution of the primary radiation. to calculate the spatial distribution of the secondary fluorescence due to the absorption of the primary radiation, a second calculation is needed that integrates the x-ray absorption equation in three dimensions. the resulting hybrid calculation can give the total secondary radiation induced for a given structure, such as a planar interface between two materials. the transport phenomenon of incident electrons in solid materials is an important subject in microscopy, microanalysis, and microlithography. it has been treated by either an analytical method, the transport equation, or monte carlo simulation. because of the flexibility of modeling, monte carlo simulation has been developed extensively. various models for monte carlo simulation proposed so far are classified into four groups from the aspect as to how inelastic interactions are treated. the most simple model is the one which uses the inelastic mean free path. this model is applied, for example, to spectroscopic analyses of elastically reflected electrons, auger electrons, and photoelectrons. the most popular model is the single scattering model in which the step length is taking the free path of elastic scattering of electrons, and an energy loss during their traveling is calculated by the continuous slowing-down approximation of bethe, [de/ds] bethe . the most complicated model is the direct simulation model in which all inelastic interactions are taken into consideration as the discrete process. this model requires all differential cross sections for inelastic scattering and, thus, a long computational time. the last one, a compromise model, is the hybrid model which introduces partially the discrete process of inelastic scattering. in the model the continuous energy loss [de/ds] ( ) where [de/ds] dis includes both energy losses due to core electron ionization and valence electron excitation. a variety of the cross sections has been used for inelastic scattering. the fast secondary production model uses the moller equation for the cross section, assuming that all atomic electrons are free (murata et al. ). the authors have published a hybrid model by using the differential inelastic cross sections of vriens for core electron ionization and the moller cross section for free electron excitation (murata et al. ). the mott cross section is used for elastic scattering. typical results for backscattering have shown fairly good agreement with experimental data. the present paper gives a brief survey of monte carlo modeling and discusses the validity of the hybrid model mentioned above. figure shows a typical example of the calculated depth distribution of generated x-rays in an au target in comparison with the experimental result of castaing and descamps ( ) . agreement is good. also shown is the result without the discrete processes of inelastic scattering. an appreciable deviation is seen in the vicinity of the peak. however, the effect of the energy straggling is not so significant. we have also done calculations for other x-rays and have obtained reasonable agreement with experimental data. the effect of the energy straggling will appear more significantly in electron scattering in a thin film. figure shows the energy distribution of transmitted electrons, with a comparison between two monte carlo results and the experimental data of shimizu et al. ( ) . the agreement is not as good; it seems that the disagreement comes from insufficient discrete processes because we still keep the continuous energy loss process. we also propose a new model to improve the discrepancy in figure , caused by energy straggling. the straggling is impor-tant in thin-film analysis, especially at initial energies near the ionization energy for x-ray production. discussions of ways to improve monte carlo simulations have usually concentrated on topics such as the effect of the choice of scattering cross section, or the appropriate model for electron stopping power at low energies. much less attention has been given to considering whether or not we actually have sufficient experimental data to make it possible to demonstrate by a comparison of simulation and experiment that one model, or a portion of that model, actually performs better than another alternative. the earliest experimental data on electron-solid interactions was published years ago (by starke in germany in , and by campbell-swinton in england in ), and numerous workers since then have contributed to the literature on this topic. unfortunately, there never seems to have been any attempt to collect and collate all of this material, and consequently workers seeking data such as the variation of the secondary electron yield with energy for silicon have been forced to conduct a random search of the literature to find what values are available. it is not surprising that such a search typically finishes as soon as a set of data plausibly matching the simulated values is found. in order to try and remedy this deficiency, a systematic search of published data has been carried out to generate material for a rudimentary database, with the hope that this will provide some of the necessary numbers against which monte carlo simulations can be tested. in its current form the database, derived from separate references, is divided into four segments arranged by atomic number (or compound) and comprising secondary electron yields, backscattered electron yields, x-ray ionization cross sections, and electron stopping power. only experimental values are included; thus interpolated, extrapolated, or normalized data sets, or values not specifically indicated by the author to be experimental, have been removed. this restriction unfortunately eliminates most of the voluminous secondary electron data, since such published values have invariably been normalized in order to facilitate fitting to a yield curve equation. no attempt has been made to judge the quality of any of the data since any such assessment would be premature until a sufficient number of independent values are available to permit obviously erroneous, or possibly dubious, results to be safely identified and eliminated. the database is available on request from the author both in printed form, or as a set of cricketgraph ™ files for the apple macintosh. while the quantity and quality of data available for a given element vary widely, they are mostly sparse and scattered. for about half the elements in the periodic table, no experimental values ever appear to have been published, and data are conspicuously absent for even the most common alloys and compounds. even for an element such as silicon ( fig. ) , the scatter in the data is too large to make it possible to be certain to better than a factor of what the se yield is at some energies. thus, unless a major source of quantitative results has been overlooked, it is fair to conclude that the experimental data presently available are not good enough to allow the merits of competing monte carlo models to be judged, or even to permit specific numerical comparisons (e.g., the backscattered yield of carbon, copper, and gold at kev) to be made with any satisfactory level of certainty. on the positive side, however, the database does give some indication of interesting trends (e.g., the variation of secondary yield with atomic number) that have not been easily accessible previously. school of electrical engineering and the national nanofabrication facility, cornell university, ithaca, new york, usa two simulation programs have been developed at cornell, seel [ ] [ ] [ ] (for simulation of electron energy loss) and pyra-mid. seel is a two-dimensional monte carlo program for the simulation of electron trajectories in complex, multi-material nanostructures; the program incorporates energy loss models that include quantum mechanical cross sections for energies down to < ev. the second program, pyramid, , is a very fast electron beam proximity correction program that can perform proximity correction (electron scattering correction) for complex ultra-large-scale integrated circuit patterns with nm minimum feature sizes (mfss). pyramid uses seel to generate the point spread function or radial exposure distribution (red) as the input. seel simulations have been tested against numerous published electron energy loss data and pyramid has been tested on ulsi density ( nm mfs) exposures using . µm pmma. simulation results are compared with experimental data to evaluate pyramid's performance. a more recent application of seel is the evaluation of signal-noise ratio (snr) for time-resolved microscopy of microelectromechanical structures (mems) . for this application, we are interested in estimating the snr for high-speed video recording , of moving high-aspect-ratio mems oscillating > mhz, particularly applications of nanoelectromechanical structures for metrology and time-resolved characterization. iv- scanning vol. , supplement iv ( ) simulation of image formation and detection systems in the sem is a vital link in performing image analysis to obtain precise measurements, to provide the necessary connection between image parameters and structural dimensions, and to reflect important microscope beam and detector parameters. monte carlo methods allow a wide degree of freedom in specifying simulation conditions for sample composition and geometry. published examples often limit the simulation to two-dimensional structures, with a zero-diameter electron beam and all secondary or backscattered electrons (bse) collected. a more useful and realistic approach will take into account the effect of beam diameter, and detector geometry and gain characteristics. these effects for simulated bse images for three-dimensional patterned structures of carbon on a silicon substrate are shown in figure . the monte carlo simulation used here is based on a single-scattering procedure with the rutherford scattering cross section and the bethe energy loss formula modified for low-energy primary electrons. the program is a further development of a single-scattering monte carlo program (in pascal) widely distributed by david joy and modified later by russ et al. the program is written in c language and runs on a sun workstation. the program can scan the beam position in x and y directions over an arbitrary multielement and multilayer sample with topographical features to produce images. using the monte carlo procedure, it is possible to extract information about the position, energy, and direction of the electron at each scattering point. this allows tracking bses as a function of angle. if the dependence of detector gain on electron energy is added, then the details of signal formation can be modeled. the effect of electron beam shape and diameter on the image can be added by convolution after an ideal image is generated. figure shows the matrix representing a gaussian electron beam used in this work. an example of a simulated bse-sem image is shown in figure for a structure with carbon features on a silicon substrate. fig. . contour plot of gaussian shape electron beam with standard deviation of Å was used to convolute the data obtained for a zero-width electron beam. fig. . bse image simulated using a zero-diameter electron beam (left) and after convolution using the gaussian beam width shown in figure (right). the simulation was performed for kev primary electrons with trajectories for each point. the image represents × pixels. monte carlo simulations of electron scattering in a target normally use one of two elastic cross sections, either the screened rutherford cross section or tabulated partial wave expansions of the mott cross section. the screened rutherford cross section gives acceptable results for high energies and low atomic numbers, but mott cross sections are required for low to medium incident energies ( . - kev) and high atomic number targets. however, computations tend to be slow using tabulated data due to the need to interpolate between data points. empirical equations for the total and differential electron/atom elastic scattering cross sections have been found that can be substituted for tabulated mott cross sections in predicting backscattering coefficients. the total elastic mott scattering cross section is fitted by similar form to the screened rutherford cross section but contains three terms in energy in the denominator. the empirical total elastic scattering cross section is valid for atomic numbers up to and for energies from ev to kev: ( ) the fit to the differential mott cross sections is decomposed into two parts, one part being of the same mathematical form as the screened rutherford cross section (σ r ), and the second part being an isotropic distribution (σ i ). the screened rutherford part of the differential scattering cross section is first fitted to the half angle of the mott cross sections. this fit of the differential screened rutherford is in turn reduced to a fit of the screening parameter alone over energy and atomic number. in marked contrast to the screened rutherford cross section, the tabulated mott cross sections show only a small overall downward trend in half angle with increasing atomic number(z). implying an average rutherford screening parameter for all z, with e the electron energy, of: the ratio of the total cross sections (σ r /σ i ) between the screened rutherford part of the differential scattering cross section and the isotropic part of the distribution is fitted to the backscattering coefficients calculated directly from tabulated mott cross sections. the ratio of rutherford to isotropic cross sections is: ( ) figure shows a comparison of the calculated backscattering factors using the present empirical fit (solid lines) with those calculated using mott cross sections. the fit for al, cu, and au is good over the entire energy range. the fit for ag is moderate and the fit for c is high. however, most deviations are similar to differences because of the use of different atomic models in the mott cross sections and are acceptable. there are two major reasons why the simple monotonic eqs. - work well. first, the scattering of the electrons in a solid is a multiple scattering process. thus, many of the complex quantum interference effects are averaged out. second, the elastic backscattering is monotonic with atomic number. these two factors serve to smooth out the effects of the complex multidimensional cross sectional surface that is being fitted over z, e, and θ. reference iv- scanning vol. , supplement iv ( ) σ rutherford σ isotropic = e − z / z + z × e σ t = . × − z . (e + . z . e . + . z / e . ) a scanning interference microscope may be constructed by allowing light which has probed the object and light which has not probed the object (the reference beam) to interfere on a suitable detector. if the detector is large in extent, a conventional scanning interference microscope results, whereas if a point detector is used we have a confocal scanning interference microscope. the image in both cases may be regarded as a superposition of three terms. the first represents a normal conventional or confocal image depending on the kind of interferometer used. the second we will call an interference term image, whereas the third represents a constant background. it is possible to design a system in which the interferometer term image is identical for both conventional and confocal scanning systems in all respects, including optical sectioning. a simple scheme permits the interference term image to be detected separately and then processed in a variety of ways. let us consider two specific geometries. the first is an almost common path confocal system based on a single mode optical fibre. the interferometer in this case is confocal. simple image processing then permits surface profilometry to be performed. the second implementation is a conventional scanning interference system. the interference term image from this conventional image may again be isolated and processed to give a confocal image. in biology, the description and precise representation of microstructural forms is of increasing importance for threedimensional ( -d) computer image understanding, in particular to enhance -d visualization and analysis. the intermediate level of computer vision, located between the bottom layer (signals) and the top layer (model) is best suited as a starting point to improve multidimensional image understanding. we implement the concept of -d topology embedded in the bottom-up structure of data processing to enhance subsequent rendering and specific quantitative analysis. segmented and contoured serial section images require sophisticated algorithms to generate correct surface-rendered views. we have, therefore, developed a method to evaluate connectivities between sections, based on bijective correspondance analysis of the center of gravities of contours. these connections indicate nods and branches of -d structures and can be interactively edited in exploded views of the contour stack. because of the strong data reduction, such procedure can be implemented even in computer graphic systems with entry graphics. the topological skeletons are the backbone to render correctly biological structures of free forms, in particular those featuring frequent branching patterns. examples where such an analysis is successful include microvessel networks, dendritic trees, lung anatomy, or dental root canals (baumann et al. ) . moreover, the automatic labeling of connected structures gives rise to the analysis of topological criteria, which was only available in stereological methods until now. this includes criteria such as connectivity and branching angle, or higher representations of branching schemes. in return, topological connectivities can be used to improve the segmentation of images in the top down-process of data processing, or it can serve as embedded analytical graphics to improve volume renditions. the mammalian central nervous system (cns) contains at least ten times more glial cells than neurons. the three major populations of cns glia are astrocytes, oligodendrocytes, and microglia. astrocytes and oligodendrocytes together are often referred to as macroglial cells and arise from primitive neuroectodermal precursor cells, while microglia are derived from the mesodermal germ layer. it is thought that common precursor cells, known as o- a progenitors, can differentiate into either astrocytes or oligodendrocytes during gliogenesis. in contrast, the origin of microglial cells remains enigmatic, although there is substantial evidence that microglial cells arise from primitive hematopoietic stem cells that gain access to the cns at a very early stage of development. much of our knowledge about glia and glial cell function has been derived from histochemical and electron microscopic studies. astrocytes can be visualized reliably using immunohistochemical methods with antibodies against the glial fibrillary acidic protein (gfap). gfap is an abundant constituent of intermediate filaments which can seen ultrastructurally in most astrocytes. in addition, astrocytes also contain enzymes, such as nadph diaphorase and glucose- -phosphatase, which are readily detected by enzyme histochemical methods. we have focused much of our attention on developing methods for detecting oligodendrocytes and microglial cells in sections of rat brain. lectin histochemistry has been a particularly useful tool in studying these types of cns glial cells. using lectins i and ii from griffonia simplicifolia seeds with carbohydrate specificities against α-d-galacatose and nacetyl-d-galactosamine (glcnac), we were able to demonstrate selective labeling of microglia and oligodendrocytes, respectively. the b -isolectin from griffonia simplicifolia coupled to horseradish peroxidase (gs i-b -hrp) can be used to detect a membrane-bound glycoprotein on the microglial cell surface at all stages of cns development ranging from the early embryonic age to adulthood. in contrast, oligodendrocytes were found to express glcnac-containing glycoproteins in the perinuclear cytoplasm using biotinylated gsl ii. the perinuclear staining was determined ultrastructurally to be associated with golgi complexes. biochemical analyses using tricine/sds-polyacrylamide gel electrophoresis and western blotting with gsl ii showed the glcnac-containing glycoproteins to be insoluble, with molecular masses ranging from to kd. having available specific markers for the three major glial cell groups, we were able to combine lectin histochemistry with immunohistochemistry to perform double-labeling studies demonstrating specificity of each stain for a given glial cell type. following the study of glia in the normal cns, we went on to investigate glial cell reactions that occur as a consequence of nervous system injury or disease. it became immediately ap-parent that microglial cells were the major glial cell type responding to neuron injury. microglia not only proliferate and change their morphology in response to cns damage, but they can also vary their membrane phenotype by expressing new molecules on their surface. we found that antigens of the major histocompatibility complex (mhc), which are largely absent from the normal cns, are expressed de novo on microglia and related perivascular cells responding to neuron injury. our studies, which have included various neuropathologic conditions including stroke and brain tumors, have shown that the expression of mhc antigens, as well as other related immunomolecules, is always restricted to cells of the microglial lineage. these findings strongly suggest a role for microglia as indigenous immunocompetent cells of the cns. jeremiah r. lowney national institute of standards and technology, gaithersburg, maryland, usa a scanning electron microscope (sem) can be used to measure the dimensions of the microlithographic features of integrated circuits. however, without a good model of the electron-beam/specimen interaction, accurate edge location cannot be obtained. a monte carlo code has been developed to model the interaction of an electron beam with lines lithographically produced on a multilayer substrate. the purpose of the code is to enable one to extract the edge position of a line from sem measurements. it is based on prior codes developed at nist but with a new formulation for the atomic scattering cross sections and the inclusion of a method to simulate edge roughness or rounding. the code is currently able to model transmitted and backscattered electrons, and the results from the code have been applied to the analysis of electron transmission through gold lines on a thin silicon substrate, such as used in an x-ray lithographic mask. there is provision for both transmitted and backscattered electron detectors. by comparing the predictions of the code with measured data, it is possible to obtain edge positions to the order of nm, which is needed for the advanced lithography projected for the year . the uncertainty of these measurements is limited by the sample geometry and surface roughness and not by the measurement process. much of the improved code is devoted to the treatment of boundary crossings by the electrons. the present code allows for a substrate of at most three layers and one or two identical lines with a trapezoidal cross section on top. there is also provision for a symmetrical jog (i.e., a discontinuous change in the width of the trapezoid) along the edges to simulate edge roughness and rounding. the three layers that form the substrate, which are typical of an x-ray mask membrane, are silicon, polyamide, and chromium. the lithographically produced lines are gold, and the chromium improves adherence of the lines to the substrate. the code is easily modified to model other media by simply changing the atomic parameters in the input subroutine. the x-ray lithography mask, which has been used as a test sample, is a very good model system for the development of accurate sem standards because it provides a measurement of both transmitted and backscattered electrons for comparison with the predictions of theoretical models. a plateau in the transmitted and backscattered electron signal occurs as the beam traverses the sloping edge of the line trapezoid. this effect can be blurred by edge roughness, and the effects on the plateau of various widths and heights for the jog can be shown as well as the effects of rounding at the bottom of the lines and the calculated noise level for various numbers of trajectories. direct comparisons with measured transmission through an x-ray mask of gold on a silicon membrane can be used to demonstrate the determination of the edge of the gold line. figure shows the transmission (measured downward) along the axis of a gold line indicating the plateaus in the data near the middle of the edges. figure shows a simulation of the data with the plateaus at nearly the same location along the edges as in the data. this work shows how high-resolution metrology of the features produced by advanced lithography can be obtained with an sem. extensions of this code to modeling secondary signals as well as the effects of charging are planned. at philips research, a monte carlo program for electron microscopy is developed, which simulates electron-solid state interactions in the energy range . - kev. it is based on the program published by l. reimer (scanning , , ) . in particular, to ensure proper operation in the low-voltage region, mott cross sections for elastic scattering are calculated by numerically solving the dirac equation. the model for inelastic scattering treats inner shell ionizations as discrete events, described by a scaled gryzinski formula, whereas the effect of valence and/or conduction electrons is incorporated as a continuous bethe loss. this model, which differs from similar models presented in the literature, provides good fits to experimental stopping power data. the program is extended to include multilayer specimens, each layer composed of multiple elements. one of our objectives is the modeling of cd linewidth measurements in a high-resolution low-voltage scanning electron microscope. our monte carlo program can generate top-view backscattered electron (bse) and secondary electron (se) video profiles of lines with variable slope and pitch. we use the se generation model of d.c. joy (j microsc, , , ) , which is extended to account for the regions near the corners of the line profile. furthermore, the recollection of se by the sample is accounted for. as a result, realistic pure se profiles are generated. the primary beam parameters include the probe size and the depth of focus. determination of the top and bottom linewidths via the commonly used heuristic algorithms reveals the systematic errors of these methods. a simple example is the probe size dependence of the peak-to-peak width, which can be related to the strong asymmetry of these peaks. we expect that improved algorithms can be developed, which use a modest database acquired by monte carlo simulation. a scanning electron microscope (sem) fitted with a helium-neon laser interferometer is used to measure the widths of features on photomasks. in this way the magnification of the sem can be known very precisely. algorithms yielding good measurement repeatability, which use the back-scattered electron (bse) signal, have been developed (nunn , nunn and turner ) but in order to be able to relate measurements made on the image to the physical dimensions of the artefact, it has been necessary to model the image formation process. the essential details of the "plural scattering" model used in this work have been described by d.c. joy ( ) . typical geometries and materials of photomasks have been modelled along with a range of accelerating voltages and beam diameters to observe how the image is affected by the different parameters. more important, the modelled image intensity profiles are studied to relate the position of the physical foot of the sloping edges of actual physical lines to the broadened image intensity profile of the edges. although the "single scattering" model, also described by joy ( ) , is more rigorous and more applicable to the type of samples used in this work, it was not used in the first instance because of the much greater computer time required. results from the modelled profiles suggest that the position of the % thresholds of the bse image intensity profiles are located a few tens of nanometres away from the physical position of the edge. the exact offset depends on the angle between the sloping sides and the horizontal. the modelled offset increases from approximately nm for a vertical edge to nm for an edge angled at ˚ to the horizontal. the effect of the bse detector size and geometry on the signal detected has also been modelled. the model shows that for best performance the detectors should collect the largest possible solid angle, but that as long as the detector is placed symmetrically, the only adverse effect of a smaller detector on the image is a loss of signal to noise. measurement comparisons between optical microscopy and the sem corrected by the "plural scattering" modelled offsets have been encouraging (nunn and turner ) ; nevertheless, if time permits, a more rigorous study using more appropriate models should be pursued. audio enthusiasts carefully read the test reports when looking for their new equipment. however, usually no one tries to find out the frequency response, distortion or signal-to-noise ratio of a very expensive, complex system such as a scanning electron microscope (sem). sems were once used mainly as image-gathering devices, and in spite of certain obvious and sometimes serious problems they served as acceptable and effective instruments for many applications. these "image grade" microscopes are now being replaced with much better quality "measurement grade" instruments. the guaranteed resolution is no longer enough to ensure good performance and other factors must be considered as well. even though these newer systems have field emission of lab electron guns, the main advantage is in the built-in, computer controlled, digital image and data-processing capabilities. there are several alternatives to upgrading an existing microscope with an external, usually desktop computer-based measuring system. soon the sem, similar to a scanner or camera, will just be a part of an imaging network, and automatic image enhancement and processing will be commonplace. this presentation deals with a short description of the electrical properties of different components of an sem; the beam scanning circuitry, the video signal chain, sampling, analog to digital (ad) and digital to analog (da) conversion. it will relate their possible influences to the detected and observed signal to improve the imaging. this will also make possible better comparisons of measured data to the computer-modeled data. essentially, the main parts of an sem essentially are the electron source, electromagnetic coils, dc power supplies, (scan) generators, detectors, amplifiers and, finally, the displays. the quality of an sem image depends on the characteristics of the primary electron beam, the correctness of the beam scanning, signal detection, and the fidelity of the video signal chain. the correctness of the beam scanning not only means good linearity and proper amplitude and direction of the deflection of the primary and displaying electron beam, but the displaying or data assigning has to be in correct synchrony also. the deflection coils are fundamentally nonlinear and inherently have hysteresis; hence, correcting circuitry is indispensable. the total distortion caused by improper magnification, nonlinearity, and hysteresis can be greater than % and it is difficult to keep it below % . the fidelity of the video signal chain depends on its transfer function, noise, and distortion figure. the main parts of the video chain of a modern sem are the detector, amplifier, ad converter, computer memory (disk, imaging network), da converter, amplifier, and the display or printer. all of these parts make their contribution and form the overall characteristics of the video chain. to produce good quality images at a certain scan rate, the bandwidth of the video chain must be high enough to show the finest possible details of the image. the transfer function that describes the frequency or bandwidth and phase characteristics of a detector or an amplifier is very useful to characterize the video chain of an sem. for good quality tv frequency imaging, - mhz bandwidth is required, but for slow-speed image collection this bandwidth can be surprisingly low, that is, - khz. noisy signal means loss or lack of information. at a certain noise level, details that otherwise would be in the video signal cannot be seen. the visual effects of noise are closely tied to the resolution, appearance, contrast, brightness, and other aspects of the image, as well as intensity distribution and other properties of the noise itself. a db ( - ), signal-to-noise ratio gives a reasonably clean image, but more noise degrades the ability to differentiate two areas of different brightness by eye. to turn an analog signal to digital data, ad conversion is needed. usually there is a sampling and hold circuitry that keeps the analog signal unchanged for the time of conversion. depending on the design, this results in smaller or larger signal loss because just a fraction of the analog signal is collected due to the relatively short sampling and long conversion time. well-designed circuitry, for example, a gated integrator, can improve the signal-to-noise ratio, but the quantization process in the ad converter itself introduces noise also. modeling data with monte carlo or other techniques has to include the shortcomings of the real, nonideal measuring tool, that is, the sem with all its associated components. by knowing the transfer function, noise, and distortion figure in digital form, it is relatively easy to obtain more accurate comparison of the measured and calculated signal (fig. the calculation of image contrast in the scanning electron microscope (sem) can be done using monte carlo techniques if the electron trajectories can be calculated through the composition profiles in the specimen. this has been done for the case of a discrete one-dimensional composition variation in the incident beam direction , but most of the programs that are available are designed to calculate signal intensities from samples of uniform atomic number and density (e.g., reimer and stelter ) . nevertheless, the calculation of electron trajectories in a three-dimensional heterogeneous microstructure has not been reported. the basic idea behind a monte carlo calculation is that an electron, in penetrating a solid, will undergo a series of predictable elastic collisions (electron-atom) which change the direction and inelastic collision (electron-electron) which change the energy. in figure , which is the example of a calculation in a lead tin eutectic with somewhat unrealistic geometry, the electron enters the sample and is scattered to a new coordinate location. since the directional change and distance scattered are sensitive to the variations of atomic number, atomic weight, and density, the electron might travel to location in the block of the lead-rich position of the pbsn eutectic, scatter into the position , and then go on to position of the tin-rich matrix. thus, depending upon the details of the microstructure, that is, the size and location of the second phase, the image will appear different. the anticipated backscattered intensities from this rather unrealistic microstructure are shown, for example, in figure as the block varies both in size and location below the sample surface. we clearly see that the backscattered signal in this case is much more sensitive to the position in the specimen of the sec- iv- scanning vol. , supplement iv ( ) fig . the geometry of the sample and a schematic of the electron trajectory for the calculation. intensity ond phase rather than the size distribution, but for more realistic microstructural geometries such a calculation would be useful in determining the presence of alternative phases in a complex assemblage. terference microscopy - is based on the observation that a simple single-arm interferometer can be constructed by allowing light from a laser to be back-reflected from a target and to reenter the laser resonant cavity to produce a modulation of the laser light-intensity. [ ] [ ] [ ] the resultant light modulation is dependent on the optical phase (optical path length traveled) and amplitude of the reentry light. if the target is moved over several wavelengths, the laser light intensity displays a cos(z) dependence; for a mirror target, the light modulation index (i-i o /i+i o ) can be as great as . . these observations were denoted as laser-feedback interferometry (lfi); this phenomenon has been characterized and analyzed several times in the past years. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the theory of lfi , , provides an understanding of the harmonic content of lfi signals; they follow a bessel-function dependence, a property that is useful in designing electronic feedback circuits necessary to produce a practical laser-feedback microscope. lfi can employ either diode or gas lasers as the interactive laser source/detector. using a he-ne laser with a high-reflectivity output coupler mirror (≤ %), it is possible to measure surface profiles of highly-reflecting surfaces (e.g., metals or high index-of-refraction materials such as silicon) with axial detail as small as nm and surface vibrations up to - mhz can be measured down to picometer amplitudes. in addition, weakly backscattering materials (e.g., biological cell surfaces and cell components) give sufficient signals to provide a useful method of high-resolution imaging in biology. a versatile laser-feedback microscope (phoebe) has been constructed having the following properties. the he-ne ( . nm) laser incorporated is a milliwatt, linearly-polarized output unit with special mounting to minimize output mirror movement. access to the low-power rear laser beam allows the laser intensity to be measured directly by a silicon photodetector. the main beam is expanded to fill the back aperture of any type of microscope objective (air-, water-, or oil-immersion) suitable for the microscopic examination being undertaken. an x,y scanning stage moves the sample under the laser beam in a raster fashion; pixel × pixel image frames are obtained in < s. axial (z) motion, furnished by a tubular piezoelectric transducer, provides both a modulation signal input for the electronic feedback circuit ( khz) and repositions the sample at each point. the electronic feedback circuit maintains a fixed distance between the output coupler mirror of the laser and a point in the object being imaged. the correction (output) signal of the feedback circuit then gives a measure of the surface topography of the sample. concomitantly, the laser intensity modulation gives a measure of the surface reflectivity of each point on the sample. these two images are digitized and stored in the computer memory. all microscope control functions have been consolidated in a dedicated small computer allowing samples to be run without interruption by the resetting or realignment of the microscope. phoebe scans for biological use range from µm × µm to µm × µm. when an air objective is used, surface topography images display a quantitative measure of height; for fluid-immersion objectives, the measured heights are shortened by a factor dependent on the index-of-refraction of the fluid (water or oil). the confocal property of this scanning microscope is due to the requirement that only backscattered light reentering the cavity-resonator mode (tem oo ) of the laser is effective; the through-focus response of lfm verifies this property in comparison with a confocal pinhole placed at the focus of the laserbeam expanding lens. the lateral resolution of phoebe is nm as determined by imaging silicon-based resolution standards. an important result of measurements with the phoebe lfm is that high-contrast images can be obtained from biological samples placed on a plastic or glass substrate (e.g., a microscope slide or petri dish). in contrast to images of samples with sharply defined index-of-refraction boundaries, what is measured in this case is a point-by-point optical path length difference with the substrate furnishing the majority of the back-reflected, lfi-measured light. other imaging modes include the use of interferometric optical sectioning and the buildup of three-dimensional structures from a series of twodimensional sections. because of the coherence requirement of lfm, the use of fluorescent labels and the detection of fluorescence is precluded; however, other reflective labels may be employed to gain back some of the advantages of that method. - , - ( ) . sarid d, iams d, weissenberger v, bell ls: compact scanningforce microscope using a laser diode. opt lett , lett , - lett , ( university of california, irvine, california, usa nmr microscopy is one of the newly emerging tools for the high resolution three-dimensional ( -d) imaging of live animals and plants for biological as well as medical research. more recent applications and developments include such things as porous materials and microflow, essential for oil research. one of the main interests of nmr microscopy, however, lies in the fact that the method is truly a noninvasive -d high resolution imaging tool with which µm resolution can be achieved. however, nmr imaging, especially nmr microscopy, has a number of formidable difficulties, namely, small signal-tonoise ratio due to the inherently small object size, diffusion and bandwidth limitations, and other inhomogeneity effects such as chemical shifts and susceptibility. among others, diffusion problems due to the random brownian motion of (water) molecules appear to be one of the fundamental physical limitations to nmr imaging, especially in high-resolution microscopy where these molecular diffusion distances are close to the resolution limits. although the diffusion effect in nmr has been studied extensively, the effect on nmr microscopy has not as yet been observed experimentally due to the current limitations of the nmr microscope. theory, however, suggests substantial resolution broadening in nmr microscopy if molecular self-diffusion prevails, especially when we deal with molecules with large diffusion coefficients. resolution limits on nmr microscopy especially include diffusion limits, namely, effects of phase variation due to molecular self-diffusion during data acquisition, effects of signal attenuation and related line broadening, and some diffusion effects of molecules which, when confined in boundaries or walls, exhibit anomalies in resolution observation in nmr microscopy. the first two are based on free-water molecules, while the third is based on the model of water molecules confined by boundaries, which have significant physical consequences. diffusion effects limit resolution in microscopic imaging, as does the phase factor which is related to the finite bandwidth of the imaging instrumentation: that is, the available gradient strength and acquisition time, both of which are intimately related to the diffusion effect. an x-ray microtomographic system is being developed at our laboratories amil & arts. a generalized feldkamp cone-beam reconstruction algorithm was already developed for our system (wang et al. ) . the generalized algorithm is approximate, but quite accurate and computationally efficient. under some feasible conditions, the generalized algorithm produces exact volumetric reconstruction for longitudinal invariant specimens and exact transaxial reconstruction for a point source contained in that transaxial plane. in the generalized feldkamp cone-beam reconstruction, a transaxial slice is reconstructed using projection data collected from a scanning turn of ˚ angular range. in fan-beam reconstruction, there actually are two complete sets of projection data over a full-scan range. exact reconstruction can be achieved using only projection data corresponding to a half-scan. in our cone-beam system configuration, it can be appreciated that there are "approximate redundancies" in data acquired along geometric rays that would be identical after projection onto a transaxial plane. there are various weight functions for half-scan fan-beam reconstruction. with gullberg and zeng's weight function, a half-scan generalized feldkamp cone-beam algorithm is obtained for less longitudinal blurring: ( ) where ( ) ∆ is an additional angle for a smooth transition between essential and duplicated radon regions, and φ(z) is an offset specific to the longitudinal coordinate z. optionally, cone-beam projection values associated with appropriate pairs of opposite rays can be lineraly interpolated to synthesize needed fan-beam projections for transaxial reconstruction. at an additional computational cost, the interpolation-based cone-beam algorithm allows exact reconstruction if the longitudinal specimen variation is linear. numerical simulation results demonstrate the feasibility of our extended algorithms. wang g, lin th, cheng pc, shinozaki dm: a general cone-beam reconstruction algorithm. ieee trans med imag ( ), - ( ) biological specimens can be preserved by rapid freezing, a process which takes just a few milliseconds and is termed "cryofixation." it is an alternative to chemical fixatives which may take many minutes to penetrate a specimen and even longer to fully stabilise cellular components, thereby compromising their ultrastructural and chemical integrity. when cryofixation is performed properly, the water molecules in the specimen do not have time to form ice crystals and the specimen is preserved in a near life-like state. the time-scale of cryofixation means that short-lived phenomena can be captured and preserved. when this aspect is combined with electrical or chemical stimulation in a controlled experiment, then cellular processes can be studied by freezing the experiment at predetermined time intervals, so that a series of transient steps are preserved. besides electrical and chemical stimulation, other methods have been combined with cryofixation; these have involved electrophoresis, electroporation, temperature-jump, and flash photolysis. electrically stimulated muscle was slam-frozen by sjöström et al. ( ) and van harreveld et al. ( ) . their methods held specimens in defined physiologic states: true time-resolved freezing was introduced by heuser et al. ( ) , who showed that ultrastructural differences could be observed in synaptic events at neuromuscular junctions between and ms after stimulation. chemical stimulation is effectively performed by a chemical flow method, involving the rapid mixing of small specimens with a stimulant from different syringes and flowing them for a predetermined time along a reaction tube before spraying the reactants into a coolant, thus quenching the reaction (knoll et al. , rand et al. . electrophoresis involves the movement of particles in an electrical field and was used with freeze fracture to study the diffusion rate of intramembrane particles (sowers and hackenbrock ) . electroporation uses a radio frequency field to induce transient pores in cell membranes (chang and reese ) . optical stimulation has been used to cause a temperature jump to investigate temperature effects, for example, a ˚c jump after ms exposure to a xenon lamp, on the ultrastructure of lipid specimens (chestnut et al. ) . flash photolysis methods have been used to release caged photolabile chemicals in the study of the dynamics of the actomysin cycle (funatsu et al. , ménétret et al. . when the available stimulation methods are considered with the available freezing methods (namely, slam, plunge, jet, and microdroplet-spray freezing), then it becomes clear that there is promising scope for the study of dynamic cellular processes using electron microscopy. the combined methodology integrates the temporal resolution of rapid freezing with the spatial resolution of the electron microscope. chang dc, reese ts: changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. biophys j , - ( - ( ) universität des saarlandes, homburg-saar, germany contrary to standard preparation at ambient temperature, there is some confusion on what is optimal in cryopreparation of biological specimens for subsequent electron microscopy (em) investigation. this results mainly from the diversity of methods as well as instruments described and the rapid development of these techniques in the preceding years. since the big advantages of cryopreparation in various fields are often hidden by the choice of unsuitable or even antiquated techniques, it seems to be justified to report about the recent developments in this field and to classify different methods for different purposes. this tutorial is based mainly on experience in transmission electron microscopy (tem). nevertheless, most of the information may also be useful for scanning electron microscopy, since a proper preparation has the same importance in both fields. without doubt, cf is the most important first step in most cryopreparations with a big variety of different techniques. the simplest procedure (plunging or immersion cryofixation = icf) is best suited for vitrification of thin suspension layers ("bare grid" or "ice embedding") for subsequent cryo-tem in the frozen hydrated state. high pressure freezing (hpf) reduces ice segregation in suitable specimens (e.g., thin plant leaves or rigid tissues such as cartilage). the main problem of hpf in soft tissues results from the indispensable dividing of the specimens into small pieces. impact freezing on a metal mirror (mmf) is well suited for suspensions such as hpf or double jet (djf), but mmf has an advantage for soft tissues, since it is suitable for larger specimens, that is, tissue slices > mm . as far as liquid cryogens are concerned (icf, djf), ln or partially frozen n -slush are not to be recommended; ethane gives the best results. propane is well suited and less expensive for routine applications. as an alternative to cryosectioning, these more time-consuming preparations gain continuously in importance for cytochemistry. even element analysis is possible in certain cases. only for simple morphology or morphometry additives (e.g., oso , uo -acetate, aldehydes) are short drying times in freeze substitution (fs) or freeze drying (fd) possible. proper drying by fs or fd without additives seems only possible for small objects (diameter < . mm) in periods between - days at − ˚ to − ˚c. otherwise, severe artifacts by thermal collapse phenomena and redistribution effects result. a suitable instrumentation is of great importance for this long drying time. the results are excellent and the advantages are striking as long as sufficiently long drying times are employed. in addition, low-temperature embedding (lte) in special acrylics improves the results. care has to be taken in handling these monomeric resins, since some of them are strong allergens. sugar protected specimens are easy to section on the dry knife at − to − (− )˚c for subsequent histochemistry of macromolecular components. overall morphologic preservation is rather poor in comparison with fs/fd/lte. larger areas are mostly not obtainable, but speedy work and results within hours instead of days or weeks are possible. sectioning can be considerably improved by the use of cryo-diamond knives together with an ionizer (useless without antistatic tool). sectioning below − ˚c down to − ˚c is possible with cryo-diamond knives on an ionizer, if the specimen is well frozen (no ice segregation) and the cutting area << . × . mm . trimming with a diamond trimming tool is advantageous. good preconditions are given after hpf or mmf. the advantage of mmf is the mirror-like, well-frozen surface, which can be easily trimmed and orientated to the knife edge. cryotransfer to the tem/stem or proper fd for fedx pose no severe problems (fd for > h at − ˚c). most of the different actual cryomethods (except fresh frozen cryosectioning < − ˚c for cryo-tem/stem) allow routine work, if the most suitable method is carefully selected and modern instrumentation is available. in all cases, the additional effort and investigation are definitely justified by better and more reliable results (literature and reprints on request). paul walther, renÉ hermann, martin mÜller laboratory of em , department of cell biology, eth zurich, zurich, switzerland the reason for using low temperatures for preparation and microscopy is the decreased mobility of atoms and molecules reducing the danger of artefact formation. for scanning electron microscopy (sem) cryotechniques have the following advantages. . cryofixation (rapid freezing) is the fastest way to immobilize a biological sample at a defined physiologic state. thereby, all processes in a cell are arrested within milliseconds. chemical fixation, in contrast, takes seconds or minutes to act, leading to unpredictable osmotic effects and redistribution of cellular compounds. . a frozen biological sample behaves like any other bulk specimen, and redistribution of substances is almost excluded. inner structures can be made amenable to the electron beam by cryofracturing or cryosectioning. . the conductive metal layers have a finer grain size when applied to cold samples, because diffusion of the metal atoms on the surface is reduced. . the frozen sample is analyzed in the sem by use of a cold stage. this prevents volume changes due to drying artifacts. in addition, hydrocarbon contamination due to irradiation by the electron beam is greatly reduced at cold temperatures. for most applications in biology, cryotechniques are only used for some but not for all preparation steps; for example, samples are cryofixed and then dehydrated by either freezedrying or freeze-substitution and afterward stored and imaged at ambient temperature. for high resolution sem, it is advantageous to coat also dry samples at cold temperatures in order to obtain a finer grain size and to observe the samples at cold temperatures in the sem to reduce hydrocarbon contamination. ( ) a major limitation of cryotechniques is distortion of the ultrastructure due to ice crystal formation during freezing. high pressure freezing allows for direct cryofixation of living samples within minimal or no ice crystal artefacts up to a thickness of several µm. ( ) the problems of water vapour contaminants that condense on the sample during preparation and microscopy were well investigated for the tem freeze-etching technique in the s and s. during the last years, this knowledge has been adapted for the construction of cryo preparation systems for the low-temperature sem. ( ) drying artefacts such as shrinkage are omitted by observing fully hydrated frozen samples. on the other hand, the ice covers many structures of interest and therefore often needs to be partially removed, either by freeze-drying or by freezesubstitution. however, removal of water bears the risk of drying artefacts. ( ) hydrated samples are extremely sensitive to the electron beam. the high surface-to-volume ratio inherent in particulate samples, and the fact that we already live in a sea of possibly contaminating particles, necessitates special care and understanding in particle preparation. selection of reagents and handling practices can be critical. for instance, in a hypothetical case, a particle analyst reports that his sample consists of approximately five major inorganic phases. the first has a rounded morphology with crystalline overgrowths, the light element content of the second is depleted, the crystalline structure of the next is damaged, the fourth occurs at a % volume level, and the fifth consists of glass shards. in this example, these results are essentially worthless. phase one was soluble in a liquid used in the preparation process and then reprecipitated upon evaporation. the light elements of the second were leached because of the use of the same solvent. the third was attacked by hf formation during ultrasonification in a fluorinated solvent. the fourth was originally present at a % volume level, but was preferentially attracted to the sides of the storage container prior to sample preparation, biasing the sampling; and the fifth is not part of the original sample, but comes from the ground glass neck of one of the reagent bottles. decisions on reagents and methods are crucial but unfortunately cannot satisfy every concern. for instance, reagents chosen for inorganic preparation should be nonpolar when easily leached elements such as boron or lithium are of interest, or if surface oxidation or ionic dissolution is a concern. but nonpolar solvents can increase particle agglomeration problems due to the lack of charge dissipation. therefore, wise choices in preparation methods are strongly tied to the objectives of the analyst, and sometimes multiple methods performed in parallel with additional analyses may be required to obtain truly representative results. contamination often is a concern, especially when performing high resolution work on a trace particle constituent. suspended urban air particulate is typically - µg/m , mostly in the . - . µm range and usually with less than particles per cubic centimeter above µm. supermicrometer-sized par-ticles can constitute µg/m . these particles may settle out gravitationally, electrostatically, or may simply happen to intercept the surface of the sample mount or sample particles. a large background can accumulate on unprotected substrates. the use of easily charged containers such as polystyrene disposable petri dishes or polyethylene centrifugation cones can further complicate dust collection or an additional problem, sample losses, due to electrostatic forces. the use of light microscopes for particle preparation makes possible a variety of preparation methods including micromanipulation. uses include ensuring even particle distributions on mounts, precise particle positioning, particle size reduction, washing unwanted films or residues off of particles, and many more. a light microscope can also be used to make some helpful observations such as specific gravity, population densities, refractive index, morphology, size, solubility, film thickness, and color. "micro world" effects often have to be provided for when performing preparation on a microscale with a microscope. for example, the polarity of solvents sometimes must be sacrificed for lower evaporation rates. reduction in evaporation rate can be aided by the use of small transparent covers, air movement shields, or by the choice of a combination of substrate and solvent that have poor affinity for one another (the resulting bead-ing-up action decreases the surface-to-volume ratio). the insertion of a "reservoir object" such as a probe tip at °"holding" a liquid droplet against the substrate, or a coverslip from the edge of which a supply of underlying liquid can be obtained, are examples of creative procedures that may be used (fig. ) .recommended polar solvents include heptane and cyclohexane. these avoid hfc environmental concerns, evaporate readily, and avoid halogen interactions. isoamyl acetate, flexible collodion, and formvar in ethylene dichloride are preferred micromanipulation and mounting media. microscale fume "hoods" protect microscope optical coatings and personnel. for containers, glass or static dissipator-coated plastics are preferred to ordinary uncoated plastics. if clean rooms are not available, static dissipators (physical or chemical), laminar flow work benches, single hepa-filter curtained areas, and modified work practices are inexpensive solutions. syringe filters help provide easy to handle point-of-use contamination control for reagents. recommended micromanipulation probes include tungsten and specific animal hairs. intermediate substrate surfaces may employ temporary teflon or paraffin coatings. other procedures include ashing, centrifugation, filtration, ultrasonification, and the exploitation of effects observed during micromanipulation. an approach for the indirect visualization of biological material in transmission electron microscopy (tem) is the freezeetch or freeze-fracture/replica method. the preparations steps ( fig. ) of this purely physical method include: (a) fast fixation and stabilization by quick freezing (in ms vs. min necessary for conventional chemical fixation); (b) creation of clean fracture faces with a fracturing cryotome (high vacuum required); (c) replication with electron beam evaporation (ebe, high vacuum required); (d) three-dimensional imaging of fracture faces and surfaces (with structural resolution of - nm); (e) high stability of the pt-c replicas in the electron beam (selection of relevant details) and possible long-term storage (weeks to months). the important drawbacks are that the replicas have to be cleaned (subsequent changing of several cleaning solutions over hours or days), picked up (disintegration of highly structured replicas happens frequently), and can be viewed only as a result of one single fracturing event. specimens providing iv- scanning vol. , supplement iv ( ) fig highly redundant structures and relatively smooth fractures, such as cell suspensions or o/w emulsions, were investigated using freeze fracture/replication and ambient temperature transmission electron microscopy (at-tem). freeze-drying is comparable to freeze-etching ( fig. ) , but with the important difference that all the water is removed from the specimen by sublimation. in tem, scanning electron microscopy (sem), or scanning tunnelling microscopy (stm), small cell components or particles, such as viruses, can be imaged together with the replication layer on top. in freeze-fracturing for low-temperature sem (lt-sem), most advantages of tem cryopreparation are kept. the preparation includes: (a) quick freezing; (b) fracturing under high vacuum conditions (c) coating with a planar magnetron sputter source (pms, ar as working gas); (d) -d imaging and observation of changed conditions (in situ etching); (e) high signal/noise ratio of pt or w coatings for secondary electron imaging and low yield from cr for backscattered electron detection of immuno-gold labelled specimens. the specimens can be imaged directly eventually after multiple fracturing (i.e., searching of distinct structures) in a different situation (i.e., fully hydrated or partly freeze-dried, coated or uncoated), in a "close to nature state." however, new problems have to be solved in order to get thin reproducible, conductive coatings, without superimposition of specimen and coating film structures and direct imaging, reducing contamination and beam damage, or long-term low temperature storage. moreover, the coating film thickness, the ice in the frozen bulk specimen, and, most important of all, the type of sem are now limiting the structural resolution. the biological relevant details of results obtained with low-temperature sem in a conventional fieldemission sem can be compared with those achieved with routine freeze fracture/replication and tem. according to honig and hook, the physics of water in high vacuum play an ultimate role in the application of these techniques (fig. ) ; therefore, only this knowledge in combination with cold stages and high vacuum technology enable the application of state of the art preparations of biological material for electron microscopy. the cryo-jet freezing technique provides a means for the in situ preservation of diffusible ions in mouse spinal cord explants after trauma. using the technique, the effects of trauma, in particular the intracellular shifts of calcium and other diffusible ions, can be analyzed and recorded by qualitative and quantitative electron probe microanalysis (epma). chemical fixation must be avoided if one intends to attempt the epma localization of diffusible substances in cells and tissues. the goal of the cryo-jet method is to confine axonal components and chemical elements to a biologically natural position. trau- matized mouse embryo cord explants were compared with the intact rat spinal cord trauma model, using epma studies of perturbations of calcium and other ions that are reported to be located in the axonal cytoskeleton. icr mouse embryo spinal cord explants were grown to - -day maturation in maximow chambers. cords that survived stringent elimination examination criteria and exhibited minimal degeneration or spontaneous necrosis were randomly divided into four groups. group i served as experimental controls; group ii was traumatized by dropping a mg weight through a . cm glass pipette positioned directly over the culture and cryojetted h after trauma. representative explants from i and ii were freeze-substituted and embedded in epoxy for comparison. by light microscopic examination of the whole mount, living culture, the site of impact could be determined within - min. the impact zone became markedly dense when compared with the surrounding, uninjured tissue. toluidine blue of the freeze-substituted sections revealed the site of impact on the surface and path of the trauma damage through the explant. control cultures with spontaneous necrosis could be distinguished from the experimental group, because the traumatic necrosis was confluent and often even wedge-shaped. within the traumatized zone, nerve fiber alterations similar to those observed in in vivo spinal cord trauma and calcium toxicity could be found, that is, granular degeneration of axoplasm, pleomorphic spheroids, tubulovesicular profiles, and myelin showing adaxonal, periaxonal, and intramyelinic vacuolization. light microscopy sections from the freeze-substituted explants were noted to be well preserved at the surface and into the culture for - µm (fig. ) ; however, some ice crystal damage was noted deep within the body of the culture. previous studies that compared metal mirror and plunge freezing results with the propane jet methodology reported here confirmed that propane jetting is the method of choice for preserving organotypic spinal cord tissue cultures. x-ray microanalysis of cryosections of the cytoskeletal tubules, cross bridge structures, mitochondria, and the myelin sheath of in vitro spinal cord explants, compared with the in vivo model of spinal cord trauma, has yielded unique data concerning the ionic changes that occur as a result of trauma or experimental manipulation. after trauma, populations of axons were found that could be distinguished by their elemental compositions. one group was similar in elemental composition to controls, although the morphology was dissimilar, while a second group had - % lower k and - -fold elevated ca (fig. ) . the response of axons and the associated myelin was paired, that is, loss of k and gain of na within the axon was always accompanied by comparable changes in the myelin and vice versa. the morphologic appearance of axons was not a predictor of the elemental composition. the cardiomyopathic (cm) hamster manifests cardiac dysfunction from an early age, with the disease ultimately progressing to congestive heart failure. on the basis of significant elevation in the ca + content of the cm hearts, as well as significantly increased ca + in the mitochondrial (mt) fractions and an increased density of voltage-sensitive ca + channels, previous studies suggested a ca + overload in the cm hearts (sen et al. ). evidence has also been presented for an increased sensitivity of cardiac muscle cells of cm hamster hearts to an external ca + "stress," that is, any manipulation which increases ca + influx into the cells (hano and lakatta , sen et al. ). on the other hand, ultrastructural studies have shown localized focal lesions from an early age, believed to be due to local cell injury. the cardiac myocytes within these areas suffer irreversible damage, becoming ca + overloaded as demonstrated by measurements of mt and a-band (ab) ca + content by electron probe microanalysis (epma) in a previous study from our laboratory (bond et al. ). this study also demonstrated that the vast majority of cardiac myocytes throughout the cm hearts not only had a normal ultrastructural appearance, but also showed no elevation of subcellular ca + content. an alternative explanation for impaired contractile function may be a decreased amount or availability of ca + stored in the sarcoplasmic reticulum (sr) for stimulated release and activation of contraction. to investigate this question, we have utilized epma to measure ca + content directly in the junctional sr (jsr), as well as in mt and ab of the cm hamster heart, either under control conditions or, alternatively, after pretreatment with the ca + channel agonist, bay k , in order to increase ca + influx into the cardiac muscle cells. isolated papillary muscles from normal and cm hearts at days of age were stimulated to contract electrically, and parameters of isometric contraction were recorded at l max . muscles were randomly assigned to one of three protocols: ( ) eleven cm and normal papillary muscles were used to construct dose/response curves to the ca + channel agonist, bay k. after stabilization at l max , cumulative doses of bay k ( - m to - m) were added to the muscle bath. all contractile parameters were recorded from the muscle at each dose. ( ) for epma, five cm muscles were frozen, after stabilization at l max , at peak +dt/dt and five muscles during relaxation. ( ) ten cm papillary muscles were incubated with single dose of - m bay k in order to elicit a maximal inotropic effect. once the contractile response to drug addition had stabilized, the muscles were frozen either at peak +dt/dt (n= ) or during relaxation (n= ). ultrathin cryosections were cut from the surface of the frozen muscles and freeze-dried overnight. subcellular ca + content (ab, mt, and jsr) of cm muscles was measured by epma in stem mode in a philips cm scanning transmission electron microscope (fig. ) . measurements of baseline contractile function revealed a significant decrease in develop tension (dt) (from . ± . g in cm muscles to . ± . g in normals), +dt/dt ( . ± . g/s to . ± . g/s) and a decrease in −dt/dt ( . ± . g/s to . ± . g/s) in cm versus normal hamster. there was no significant difference in values of resting tension (rt). the inotropic response to increasing doses of bay k was markedly blunted in the cm muscles compared with controls, suggesting that even when ca + entry into the cardiac muscle cells is increased, force development is still impaired. a comparison of elemental content (na, mg, p, s, cl, k, and ca) of ab and mt between experimental groups revealed no statistically significant differences. in addition, no differences in elemental composition of ab and mt were observed compared with our previous measurement on normal hamsters frozen during contraction and relaxation (moravec and bond ) . the amount of ca + stored in the sr of the cm muscles that were rapidly frozen during relaxation (in absence of bay k) was . ± . mmol/kg dry weight ( fig. ) (left panel, rel); however treatment with the ca + channel agonist bay k significantly increased the size of the store to . ± . mmol/kg dry weight (right panel, rel). thus we conclude that ca + uptake into the sr of cm muscles was enhanced as a result of bay k treatment. the contractile data show that the amount of ca + that can be released from the jsr of cm muscles during a cardiac twitch is very small (equivalent to the difference between the relaxed and contracted values) in "control" (untreated cm hamsters, left panel); however, treatment with bay k increases the sr ca + load in the relaxed muscle, with little demonstrable effect on the amount of ca + remaining in the sr at peak +dt/dt (right panel, cont), resulting in a significant increase in the amount of releasable ca + in the sr store. the total ca + content measured in the relaxed bay k treated muscles was, nevertheless, considerably less than previously measured in normal papillary muscles rapidly frozen during relaxation. in summary, these data suggest that ( ) a ca + deficit, as opposed to a ca + excess or ca + overload, may be an important factor contributing to the cardiac dysfunction in cm hamsters, and ( ) that, specifically, impaired ca + regulation by the sr may result in this ca + deficit. the application of automated scanning electron microscopy (sem) to the analysis of particulate populations is in many ways a unique application of microanalytical instrumentation. when applied to the analysis of large numbers of particles, the sem is used as both a microanalytical and a macroanalytical instrument. as a microanalytical instrument, the sem provides single-particle compositional and morphologic information that is not available from the conventional macroanalytical techniques used in particle analysis such as atomic absorption and instrumental neutron activation analysis. as a macroanalytical instrument, the sem, when used for automated particle analysis, provides information from which population characteristics can be inferred. for example, automated sem information often is used to determine the percent of an aerosol that originates from a given source by extrapolating the single-particle results (e.g., the number of particles containing major fe) to the entire particle population, that is, the air filter. this value is then extrapolated to the sampled air volume. while providing the analyst with a wealth of information, the dual role of the sem in automated particle analysis has several limitations that must be considered when doing an analysis. these limitations involve, among other things, the spatial dispersion of the particle sample; particle size distribution; analytical parameters such as magnification, accelerating voltage and electron dose; and the algorithms for determining particle composition and particle groupings. at nist we have been conducting a series of experiments to study the limitations associated with the quantitative elemental analysis of particles during an automated run. specifically, we have been evaluating the relationship of measured x-ray intensity to the accuracy, precision, and detection limits of automated x-ray analyses. analytical accuracy, precision, and detection will have a pronounced effect on the ability to separate particles with similar but different elemental compositions into groups. for this experiment, we developed an analytical glass series containing six glasses with varying amounts of uranium and lead ( table i) . samples of the different glasses were prepared as bulk-polished specimens and as particles. the results from the analyses of the bulk samples represented the "best case" that could be expected under a given set of analytical conditions since there were no particle effects involved. all analyses were done on an electron probe and a sem at kev and na beam current. dead times for the bulk and particle analyses were between and %. two separate counting times were selected for the experiment , s and s. these times resulted in the x-ray peak intensities for pb and u m xrays that are shown in table ii . glass k- was used as the standard for the quantitative analyses of the different runs. for the particles, both bulk and particle forms of k- were used as standards. the concentration of oxygen was determined by stiochiometry. the results of the experimental runs on the bulk and particle forms of the glasses are shown in figure a and b as plots of the u versus pb concentrations in wt.% (normalized to % total analysis) for the s data. the bulk plot shows a complete separation of the six different glasses, while the particle results show an overlap of adjacent glasses even at the s count time. the s data show a much stronger overlap between adjacent glasses for both bulk and particle morphologies. the analytical data were also processed with a clusteranalysis algorithm to determine the average silhouette width (asw) for both bulk and particle forms of the glasses at the different counting times, (fig. ) (kaufman and rousseeuw ). the asw is a robust measure of the cluster strength with a value approaching representing the maximum association among cluster members. since there are six glasses in the series, the asw for six clusters should be the highest. of all the different runs on both bulk glasses and particles, only the s data on the bulk glasses has the highest asw for six clusters. the results of this study indicate that the uncertainties associated with the shorter counting times in asem analysis may severely limit the ability to distinguish correctly between similar groups of materials. in addition, the uncertainties associated with particle analyses are considerably greater than those from bulk analyses due to absorption and mass effects. these greater uncertainties for particles underscore the need to define the strengths and limitations of asem analysis and to design automated methods that will maximize the information that can be obtained from a sample. botany department, university of georgia, athens, georgia, usa the monoblepharidales (chytridiomycetes) produce asexual motile reproductive structures known as zoospores through the cleavage of cytoplasm in the zoosporangium. although different aspects of zoosporogenesis have been studied in a number of zoosporic fungi, little is known about the events of zoospore formation in the sporangium of the monoblepharidales, or the chytridiomycetes in general. earlier studies of other zoosporic fungi proposed various spore formation events that have been challenged recently (hyde et al. ) , especially with respect to the relationship of vesicles, golgi, and cleavage furrows. hyde et al. ( ) concluded that all eukaryotic cleavage events may need reinvestigation. we are studying the various aspects of zoosporogenesis in monoblepharella using both chemical fixation and cryofixation methods to determine whether the cleavage events in this chytridiomycete are similar to those found in the study by hyde et al. ( ) . questions that remain include how the nuclei accumulate in the sporangium, how the cytoplasmic domains are established, and how each zoospore obtains its usual complement of cellular components. mycelia were induced to form sporangia by transferring the cultures to distilled water. sporangia were chemically fixed at different stages of development using a sequential aldehyde/ osmium fixation protocol. the resulting tissue was embedded in araldite/embed . freeze substitution fixation was also used for comparison of vesicle and cleavage furrow profiles. when the tissue was to be used for cytochemical localizations, osmium was omitted and lr white was used as the embedding medium for both fixation protocols. various dyes, lectins, and antibodies were used to localize organelles within the sporangium. nuclei were close to the plasma membrane as they moved from the subtending hyphal strand to the swelling tip of the developing sporangium. the centrioles were closely associated with the nuclei and oriented toward the periphery of the sporangium. nuclei then moved a short distance from the centriole toward the center of the sporangium. microtubules, originating from the centriolar region ( fig. ), were seen around the nucleus and extended into the cytoplasm, possibly delimiting the boundary of the forming zoospore. membranes forming the cleavage furrows appeared around the forming flagella near the kinetosome (fig. ) and also at specific sites along the periphery of the sporangium. the furrows continue to expand at both sites as extending sheets of membrane. dictyosomes with large numbers of vesicles were closely associated with the nuclei, suggesting a source of the membrane needed for furrow extension. er was first seen in large sheets in the central region of the sporangium. later the strands of er surrounded the nuclei prior to ribosomal aggregation. iv- scanning vol. , supplement iv ( ) fig. nucleus (n) with associated centriole (arrow) and microtubules (arrowheads) at periphery of sporangium. fig. developing cleavage furrow and flagellum (f). note coated region of furrow (arrowhead) and radiating microtubules (arrows). hyde gj, lancelle s, hepler pk, hardham ar: freeze substitution reveals a new model for sporangial cleavage in phytophthora, a result with implications for cytokinesis in other eukaryotes. j cell sci , ( ) center of ultrastructural research, barrow hall, university of georgia, athens, georgia, usa cryptocaryon irritans, a parasitic ciliate of many species of seawater fishes, has a complex life cycle consisting of feeding, resting, dividing, and infective stages. the disease, termed white spot disease, can cause extreme fish loss in marine aquaria and mariculture environments. cryptocaryon irritans attaches to the epidermis of the fish and feeds on epidermal cells. this form of the ciliate termed the "trophont" appears as large white spots on the fish. the trophonts grow in size and eventually leave the host. the free-living trophont settles down to the substrate and secretes a cyst wall. while in this resting stage known as the "tomont," the cell undergoes a series of unequal palintomic divisions to form daughter cells called "tomites." the cyst wall ruptures and free swimming ciliates known as "theronts" are released. the theronts represent the infective stage as they search for new hosts. different procedures were used to prepare tomite, theront, tomont, trophont, and cyst stages. organisms were fixed with glutaraldehyde and osmium tetroxide for transmission electron microscopy (tem). parduc's fixation (oso +saturated hgcl) was applied for the scanning electron microscopic (sem) work. the parasite's cytoskelatal framework was stained by using indirect immunofluorescence technique. for this purpose monoclonal anti-α-tubulin was used as a primary antibody, and rhodamine-labeled goat antimouse igg as a secondary antibody. fluorescently labeled cells were examined by laser scanning confocal microscopy. the trophont has an elongate body shape with a broad anterior end, a tapered posterior end, and is completely covered with somatic cilia. the cytostome is apically located and trophonts often were observed moving with their mouth part leading. kineties were arranged in a parallel fashion along the longitudinal axis of the cell and terminated in a ring around the cytostome. there is no oral membrane at the oral region. there are cirri-like structures around the oral opening as described by cheung et al. ( ) . these cilia are shorter ( - µm) and wider than somatic cilia. the cytopharynx is surrounded by ridges or oral ribs (nonciliated lining). large bundles of microtubules support the oral region. the theronts are oval to teardrop-shaped and completely covered with cilia. they have a ventral mouth with a slit-type structure in the middle. the cytostome covers almost / - / of the body. the cirri-like structures are found around the mouth and are similar to those in trophonts. the oral ribs are present but they are layered. based on sem and tem results, it was found that there were some structural differences in the cytostomes of trophonts and theronts. the mouth probably does not become functional until the theront has entered a host. the short and stiff cilia seem to be used for burrowing and the gathering of food particles. the role of the pellicle and cyto-plasm is discussed in relation to penetration into fish epidermis. we found that there are mucocysts in both trophont and theront, but not in tomont. theront mucocysts are concentrated around the slit type structure of the mouth. the secretion of mucocysts might contain enzymes that help the theront to penetrate into fish epidermis. they also might aid trophonts in feeding on fish tissue and have a function in cyst wall formation at the later stage. penetration into the fish tissue probably is started by mucocyst secretion that either enables the parasite to stick to the tissues or to help it enter the tissue by enzymatic reaction over the irritated areas. the theront may then utilize its relatively stiff oral cilia for burrowing into the these irritated areas. after burrowing into the epidermis of fish, the theront develops its oral apparatus and increases its size. trophonts eventually leave the host when they reach a certain size ( - µm). this study is supported in part by the national aquarium, baltimore, md. over the years that the fbi has practiced sem/edxa, the technology has grown in importance to be considered an essential tool for investigative forensic exams. because of the wide variety of applications, there is a corresponding vast array of preparation methods and analytical techniques. analytic techniques and sample preparation methods are inextricably linked, and although many are routinely applied, often only imaginative approaches serve to fulfill the desired analytic result. the scanning electron microscope (sem) practitioner is expected to be knowledgeable about the applications and proficient at the methods of preparation in order to utilize the sem to its greatest advantage. the main types of analysis practiced at the fbi include ( ) visualization of structure, including surface features and internal structure, ( ) inorganic elemental characterization, ( ) particle analysis, and most recently ( ) the use of a compositional data base for identification and association. sem is a powerful complement to light microscopy (lm) for low magnification morphology characterization and is unsurpassed for applications requiring greater depth of field than are available with lm. toolmark and fracture exams can be enhanced by stereo analysis. preparation can be minimal, and several electronic signals (bei, sei) are available. the preparation of cross sections permits the study and comparison of heterogeneous materials. embedment usually is necessary to support the object during cross sectioning. hard materials generally are polished by an adaption of metallurgical polishing methods, and soft materials generally are microtomed. sectioning often is possible by manual methods, without the use of a microtome. this method can reveal complex structures such as plating layers and document laminates. the most routinely applied application of edxa is the qualitative exam. it is part of the inorganic analysis scheme for material characterization. the qualitative exam frequently is combined with elemental distribution mapping to provide "compositional pictures." particle populations often are indicative of an environment and can be used to associate an item or individual with an activ-ity. too small for individual manipulation, they are most easily sampled by adhesive lift. additional methods involving separation and concentration often are effective. compositional characteristics of materials are stored in a database to permit comparison and identification of a questioned material. standard spectra are collected and a specific peak for each element is integrated above background and ratioed to the sum peaks from all elements. this value representing % x-ray counts is stored in a "periodic table" data base including standard information. in addition, the original spectrum is stored on disk and a hard copy is filed. the standard files include metal alloys, building materials, paints, tape adhesives, fingerprint powders, and cosmetics. the reference list can be queried for comparison to an unknown for alloy matching, identification of an unknown material, or manufacturer identification. the effectiveness of this method depends upon the compositional uniqueness of the material and the variation of composition within the class of materials to which it belongs. this project is in its infancy, with only several hundred entries to date. data entry currently is manual, although software currently is being developed to extract required data from spectra automatically and to export it to the database. the need for a vehicle for information exchange has been expressed within the community of sem users in crime labs. since an electronic medium such as internet was not feasible because most forensic laboratories are not electronically linked, a "newsletter" was produced to link laboratories involved with sem in forensic science and related areas, as well as individuals in industry and academia. timely and informal, it augments the professional publications and attempts to bring practical methods, reprints from obscure journals, translations from foreign publications, and questions/answers directly to the user. hamilton county coroner's laboratory, cincinnati, ohio, usa many examinations in the crime lab involve comparing questioned material from a suspect to known material from a victim. by characterizing the material it may be possible to establish a link between the suspect and victim. our trace evidence section characterizes materials by using a combination of analytical instruments. the infrared microspectrometer is used to determine the organic constituents, and the scanning electron microscope-energy dispersive x-ray spectrometer (sem/edxa) is used to analyze the inorganic composition. this approach is routinely applied to paint particles because paint formulations include both organic and inorganic constituents. analysis by sem/edxa is very valuable when more than one layer is present in the particle. the instrument can then be used in line scan mode to analyze each layer individually without separation. in addition to comparisons, the sem/edxa is used to identify materials. in bombing cases it may be difficult to identify the explosive as well as other components. large amounts of potassium and sulfur in residues from a pipe bomb indicate black powder as an explosive. if chlorine is also present then "pyrodex," a black powder substitute, may have been used. other applications, such as matching small fractures, make use of the superb imaging capabilities of the sem. the capability of maintaining excellent depth of field at high magnifications is particularly important when matching the ends of wires that have been pulled apart. this is exactly what was done in a case of tape players that were jerked out of victims' vehicles. small fractured surfaces also have been encountered in the investigation of hit-and-run accidents when pieces of chrome trim were knocked loose and left at the scene. clearly modern instrumental means of analysis, such as the sem/edxa, are critical to the work of the forensic scientist. the increased sensitivity of the instrumentation, however, may raise questions as to the relevance of the evidence found. if a single, very small paint particle is found on the jeans of a pedestrian struck in a hit-and-run accident, could the particle be from the striking vehicle, or merely from the roadway debris at the scene? such questions indicate that increases in instrument sensitivity require sensitivity on the part of the analyst to questions of contamination and weight of evidence. research division, office of laboratories and scientific services, u.s. customs service, washington, d.c., usa the u.s. customs service laboratory system provides a variety of analytic services to control the commerce and assure enforcement of numerous regulations at the border. any item which is imported into the united states may need to be analyzed to determine the answer to any number of questions. what is the item? is the item correctly described? does the item infringe upon a u.s. patent? the scanning electron microscope (sem) and eds x-ray system can be utilized to answer some of the questions which arise. several examples follow. a u.s. company holds a patent on a feature incorporated into an electronically programmable read-only memory (eprom) cell. they allege to the international trade commission (itc) that another company is incorporating this patented feature in their eproms without the patent holder's permission. they win their case and the itc issues an exclusion order. it is at this point that the u.s. customs service becomes involved. we are charged with enforcement of the exclusion order. the incorporated feature is exceedingly small on a visible scale. how will we know which shipments of eproms should be excluded from entry into the u.s.? now the ability of the sem to produce images easily at very high magnifications comes into play. with the help of the sem, the laboratories are able to determine if the infringing feature is present or not. another u.s. company holds a patent for a denim (textile) finishing process. they bring a complaint before the itc that their patent is being infringed upon. the complaint is upheld by the itc and an exclusion order is issued. the u.s. customs service laboratory system must now develop a method to differentiate among various finishing processes in use. research at the headquarters laboratory found that a combination of imaging with a stereomicroscope and an sem could make the differentiation. the sem samples were au-pd sputtercoated with a hummer vi a sputtering system (anatech ltd.). the sputtercoated denim samples clearly showed distinctive features not easily seen with an optical microscope. a sample purported to be eelskin was submitted to the headquarters laboratory for conformation of its identity. it was thought that the product might be embossed plastic or possibly leather of mammalian origin. the top surface of the sample was au-pd sputtercoated and then examined with the sem. the features displayed by this examination were convincing evidence that the sample was indeed leather. a cross section of the sample was then prepared and au-pd sputtercoated. the sem images showed cell patterns consistent with reptilian or marine origin. another sample arrived courtesy of a foreign customs service. their inspectors had seized a large statue of a roman gladiator and his horse and chariot. the original reason for suspicion was that the declared value for the statue was much higher than one would expect for an object of this type; it appeared to be a cheap plastic statue. it had been dismantled and tested for the presence of drugs. the results were negative. using our princeton gamma-tech eds x-ray system, an elemental analysis of the exterior covering of the portion of the statue we received was performed. it showed the presence of large amounts of silver. this would explain the high declared value of the statue. when the u.s. customs service ran tests on narcotics particle detection systems, it was noted that sampling for heroin was more difficult than sampling for cocaine. a brief study of samples of each narcotic using the sem showed that in general the average particle size for heroin is less than that of cocaine. this may help to account for the sampling difficulty for heroin. allan n. walters u.s. postal inspection service, forensic laboratory, dulles, virginia, usa currently, the two main applications of sem/edax are explosive residue analysis and alloy quantitation. most explosives encountered are from improvised explosive devices (ieds) and commonly are low explosives such as black powder, smokeless powder, pyrodex, and flash powder. pyrodex and smokeless powder are commercially manufactured, while black and flash powder may be either of commercial or improvised (homemade) manufacture. the following table illustrates the composition of the common low explosives: device components are placed in the sem, and edax is performed before disturbing the residue. the resulting elemental profile is then used to guide further analyses with other instrumentation such as xrd, ftir, hplc, tlc, and chemical spot tests. sputter coating of samples is not performed so as to avoid modifying the sample and interfering with subsequent analyses and examinations. quantitative analysis is performed on alloys which are of interest to the u.s. postal service engineering and development center and is required to ensure that the materials meet the re-quired specifications. samples have included lock bodies, lock springs, keys, and lock tumblers. reverse engineering using quantitative analysis of current collectors for mobile electrification systems (mail sorting machines) has been conducted. quantitations are performed using a standardless method. scanned probe microscopy has evolved significantly over the last years. beginning with the first commercial scanning tunneling microscopes (stm) and continuing through the sophisticated "multifunction" microscopes of today, the "probe" has been one of the most critical and, in some instances, the least understood component of these systems. depending on the type and geometry of the sample, the properties of a probe can be optimized to reduce imaging artifacts. examples of this will be given using two well-known techniques, scanning tunneling microscopy (stm) and atomic force microscopy (afm). also, new innovations in probes for these two techniques (and others) will be presented. scanning tunneling microscopy, as first introduced, used a probe constructed of a chemically sharpened tungsten wire. later, it was found that a suitable probe could be formed from a mechanically sharpened wire (usually pt/ir). both of these probes proved that they could produce self-consistent images under specific sample and environmental conditions. however, problems arose with the tungsten probe while imaging in air because of the formation of native oxide. in addition, the mechanically formed probe tended to produce significant imaging artifacts if used on samples with topography > nm. in many cases the distortion produced by these artifacts made the data uninterpretable. a solution to these problems was to use a chemically etched probe (to control the shape) made from an inert material with physical properties suitable for the samples involved. musselman et al. developed the techniques necessary to chemically etch pt/ir wire into tips with a controlled geometry. these probes were capable of imaging surface structures greater than µm peak-to-valley depths with significantly reduced tip-related artifacts. this controlled geometry shape also made it more advantageous for use as a coated tip for electrochemical imaging. still, the aspect ratio of this particular probe was not suitable when imaging high-aspect ratio features such as pits in optical discs, contact vias in ics, fracture surfaces, etc. to obtain images from these types of structures, it was necessary to "machine" (in a controlled way) the probe described above. by using a focused ion beam (fib) as a machining tool, it was possible to create a probe to image the above features ( fig. ) . this fib "nanomachining" technique has opened up many possibilities for specialty probes. the majority of atomic force microscopy still utilizes the basic silicon nitride (si n ) triangular cantilever and pyramidal probe combination. the base of the pyramid is on the order of µm with the sidewalls extending upward at an approximate o angle to the apex. again, for samples with features < nm, such as mica, these probes have provided very good image repeatability. in general, however, for structures much greater than nm, a convolution of the probe and the sample surface will again occur. these probes can be modified, using the fib technique, to increase their aspect ratio significantly. because of their "hollow" design, these modified probes are still limited to topographies of ≤ . µm. most of the advances in probe manufacturing (on a wafer level) have come about by utilizing silicon as the probe material. several silicon probe types are now available, which provide significantly sharper tips with aspect ratios on the order of to . for imaging structures of higher aspect ratios, such as vias or deep trenches, longer and thinner probes are needed. these are made possible by using a combination of fib and electron beam techniques to "grow" a thin probe using an existing si n pyramidal probe as the base (fig. ). these probes are available in lengths up to µm with aspect ratios of ≥ to . utilizing a combination of fib milling of existing structures and electron beam growth, probes with lengths of - µm are feasible. an obvious problem with probes of this type (and with any long, thin structure) is "flexing" during imaging. analysis of one such probes' physical characteristics has shown that the elastic modulus is relatively low (e~ . gpa), while the coefficient of friction on most surfaces is extremely low (µ< . ). thus, probes of this type should be kept as short as possible while exceeding the maximum peak-to-valley distance to be imaged. other scanned probe techniques now in the prototype phase include thermal, magnetic force, near field optical, and probes capable of imaging undercut sidewalls. probes for all of these methods have been shown to be feasible, although manufacturing techniques to provide large quantities, reliably and repeatably, have yet to be developed. when imaging with an atomic force microscope (afm), the image resolution is a complex function of the relative tip and sample geometries. when imaging or measuring high-aspect ratio features, sharp and slender tips offer the possibility of probing down into extremely small topographical features. the most commonly used contact mode afm tips are batchfabricated si n thin-film cantilevers with an integrated pyramidal structure used as the tip. it has been shown that microtips, which are fabricated by electron beam-induced growth of carbonaceous material on the apex of the pyramid, can reduce the artifacts associated with integrated pyramidal afm tips. graph of a whisker of electron beam-grown contamination, or microtip, grown on the apex of an integrated pyramid, is shown in figure . an obvious problem with the use of a long slender microtip is the lateral deflection of the microtip as it is scanned across the sample surface. the proper use and interpretation of artifacts associated with electron beam-grown microtips demands an understanding of the mechanics of microtip deflection. it has been observed that long, slender microtips scanned over flat surfaces (rms roughness of < nm) produce hysteresis in the fast-scan direction. a model has been developed to explain the observed hysteresis loop in terms of a mechanical cantilever beam deflecting because of lateral frictional forces induced by repulsive imaging forces. this model is shown schematically in figure . by applying cantilever beam mechanics to the model of microtip deflection, a method of calculating the elastic modulus of microtip material has been developed. to find the elastic modulus of microtip material, a series of experimentally determined microtip deflection distances and the respective microtip lengths are required. microtip deflection distances have been experimentally determined for different microtip lengths on two different flat surfaces; fusion deposited boro-silicate glass and polished silicon. the elastic modulus of the microtip material has been determined from the deflection data to be approximately . gpa. once the elastic modulus has been determined, the coefficient of friction between microtip material and a sample sur-face can be calculated. the coefficient of friction between a microtip and the sample surface will indicate if the sample material is suitable for afm imaging with electron beam-grown microtips. the elastic modulus of microtip material and the coefficient of friction data lead to a better understanding not only of microtips but of electron beam-induced contamination in general. the low elastic modulus rules out the possibility of the material being diamond-like and suggests a polymeric material. the use of microtips can greatly improve image resolution; however, it is important to note that since microtip deflection increases with increasing microtip length, the microtip used to image a sample surface should be as short as possible while remaining long enough to image the largest peak-valley structure on the sample surface. the magnitude of observed microtip deflection should be reduced substantially by the use of ac mode microscopes where surface friction is less of a concern. because of their ability to achieve high resolution simultaneously in all three dimensions in a wide range of ambient conditions, scanning probe microscopes are promising candidates for performing measurements of surface topography. crosssectional and perspective views can be generated, nondestructively, at any location once an image has been acquired. surface topography measurements fall into two basic classes: position (or pitch) measurement and size (or critical dimension) measurement. the ability of a microscope to perform position measurement depends more on the quality of its design and construction than on the fundamental interaction of probing beam or stylus with the sample. size measurement, on the other hand, depends strongly on the probe-sample interaction. modern manufacturing, especially semiconductor lithography, often produces high-aspect ratio, submicron structures whose size and shape must be known with tiny uncertainties. surprisingly, stylus profilometers in the guise of scanning probe microscopes can perform some of these measurements at a level unmatched by any other type of microscope. to obtain size and shape measurements in semiconductor manufacture, we have developed a scanning probe microscope with several refinements, depicted in the figure. we use capacitance-based sensors for probe force sensing iv- scanning vol. , supplement iv ( ) cantilever and microtip at rest and for probe position measurement. , many of the samples that we scan are at least partially electrically insulating. for this reason, our microscope is used primarily as a scanning force microscope, although it is capable of operating as a tunneling microscope also. our force sensor employs a small silicon beam that pivots in one dimension about a pair of magnetically constrained ball bearings. the beam forms a pair of capacitors that both sense the position of the beam and maintain its balance with a suitable servo loop. this force-balance technique allows high-force sensitivity without sacrificing the stiffness required to resist surface forces. capacitors are also used to measure the position of the probe tip. the piezoceramic tube used as a scan actuator exhibits strong hysteresis and creep, so the drive voltage is an unreliable measure of probe position. the capacitors monitor the probe position in all three dimensions during the scan, and these data are collected along with the topograph. the most important factor determining the quality of the measurements is the shape of the probe tip. geometry alone makes the probe-sample interaction strongly nonlinear. in surface roughness measurements, a blunt probe can severely limit the range of spatial frequencies that can be detected. in scans of high-aspect ratio features, the probe shape determines what parts of the features can be measured. when scanning deep trenches and holes on a patterned surface, we use either a conical probe or a cylindrical probe, depending of what part of the feature is most important. the conical probes are made using focused ion beam sputtering of iridium. a chemical etch is used to form the cylindrical probes. if the probe shape is well known, then it is possible to determine what parts of a scan were distorted by the probe tip and, in some cases, this distortion can be removed. , the probe microscope itself can be used to determine the probe tip shape if a suitable structure is available for probe characterization. since the pioneering work of binnig and rohrer in the early s on scanning tunneling microscopy (stm), the stm has evolved into a powerful tool for spectroscopy, metrology, electrochemistry, and nanolithography. many other instruments have also evolved from the stm technology under the family of scanning probe microscopes (spms) with applications in atomic force, electric potentiometry, and magnetic force imaging. in the magnetic force microscopy (mfm) mode, the technique has been applied to the imaging of magnetic bit patterns in recording media (grütter et al. , mamin et al. ) and the mapping of static and dynamic magnetic fields of recording heads (martin and wickramasinghe ) . mfm is typically performed in the noncontact atomic force microscopy (afm) mode using a silicon cantilever which is coated with a thin film magnetic material, usually co, ni-fe alloy, or co-pt-cr alloy. the force exerted on the magnetic tip by stray fields from the sample causes the deflection of the cantilever which is subsequently measured. using a novel variation of the stm technique with a flexible iron tip, rice and moreland ( ) have also performed mfm imaging on magnetic bit patterns on a hard disk in a tunneling-stabilized mfm (tsmfm) mode. standard mfm images, however, reflect both topographic and magnetic information, with the relative strengths of each signal depending on the tip-to-sample spacing. using a differential interferometric technique, schönenberger et al. ( ) have shown that the topographic and magnetic information can be reasonably separated. in this abstract, results of some applications of the spm for topographical and magnetic force imaging of magnetic materials are presented. as the critical dimensions of magnetic devices are getting smaller, the surface topography and magnetic morphology of the recording head and media are becoming increasingly important with respect to optimization for best performance. the ability of the spm to obtain submicron topographical and magnetic information makes this an invaluable metrology and failure analysis tool for the magnetic recording industry. compared with other techniques for highresolution magnetic imaging, such as lorentz microscopy, electron holography, scanning electron microscopy with polarization analysis, mfm has the advantages of ease of sample preparation and the ability to operate in air. typical resolutions of - nm are obtainable in the mfm mode, although resolutions of - nm have also been achieved with considerable effort. in our work, topographical imaging was performed in the contact mode using a v-shape silicon nitride cantilever of length l = µm, width w = µm, thickness t = µm and force constant k = . n/m. the sensing tip at the end of the cantilever is pyramidal in shape, with a × µm square base and : aspect ratio. the tip radius is typically smaller than Å. for magnetic force imaging, the instrument was operated in the noncontact amplitude resonance mode by oscillating the cantilever and measuring changes in its resonant frequency using either phase or amplitude detection. this method of detection is responsive to the force gradient. for each point of the recorded image, the cantilever was first brought to a large tipto-sample distance of nm or greater to acquire the magnetic force image and subsequently moved close to the sample surface to acquire the topography image. this allows the simultaneous acquisition of topography and magnetic force images. a - v specimen bias was also applied to provide a linearizing and stabilizing force to the servo feedback loop. the cantilever used for the mfm imaging is a diving boardshape probe made of ( ) silicon. the geometry of the cantilever are l = µm, w = µm, t = µm and k = n/m. the sensing tip is conical in shape with a height of about µm, an aspect ratio of : , and a tip radius of < Å. the cantilever, which was sputter-coated with a Å thin-film cobalt material and magnetized inside a - gauss solenoid field, has a typical resonant frequency of khz. figure shows a three-dimensional topographic profile of a defective thin-film recording head, in which the pole tips protrude slightly from the surface, using the contact afm mode. the dimensions of the two rectangular pole tips are about µm × µm and µm × µm. the gap width was measured to be about . µm. dirt particles can also be seen and these are due to the cleaning process during sample preparation whereby the head was lightly swabbed with a cotton tip soaked in methanol. figure shows the magnetic force image of the surface of a recorded computer hard disk. the bright and dark lines represent regions of different magnetization or bits on the hard disk surface. the separation between each pair of bright-dark lines was measured to be about . µm and the track width is about µm. the striations in the topographic image (not shown), representing the texture lines, are approximately perpendicular to the recorded bit patterns. the line profile of the bit patterns in figure (not shown) is very similar to the calculated force gradient contours by mamin et al. ( ) in which both the horizontal and vertical magnetization components of the tip are are being sensed. iv- scanning vol. , supplement iv ( ) fig. three-dimensional topographic profile of a defective thin-film recording head using contact afm, in which the pole tips protrude slightly from the surface. in a good recording head, the pole-tips are supposed to be slightly recessed from the surrounding region. image resolution in traditional far-field microscopy is limited by diffraction caused by the primary aperture in the optical system of the microscope being used. in practice, diffraction limits lateral resolution to approximately λ/ , or about . micron for optical microscopes. to image smaller cellular structures and biological materials which are smaller than . micron, biologists have used microscopies which "illuminate" the specimen with radiation of smaller wavelength, such as an electron beam (i.e., using an electron microscope). the nearfield scanning optical microscope (nsom) breaks the diffraction limit to lateral resolution by illuminating (or collecting light from) the specimen through a sub-wavelength aperture which is held a small fraction of a wavelength above the surface of the specimen. using this nearfield technique, resolution far better than λ/ has been demonstrated on biological specimens and resolution better than λ/ has been demonstrated on standards (betzig and trautman ) . with nsom we have recently obtained optical images of tobacco mosaic viruses ( nm diameter) and of photoreceptor rod outer segments at a sub-wavelength resolution. betzig e, trautman jk: near-field optics; microscopy spectroscopy and surface modification beyond the diffraction limit. science , in june , the initial incident involving a report of a syringe found in a canned pepsi ® product received nationwide publicity. during the following months, law enforcement authorities investigated hundreds of additional claims of tampering from nearly every state in the united states. more than cases involving foreign objects allegedly found in soft drink containers were processed by the national forensic chemistry center (nfcc). to date, the forensic and law enforcement efforts of the fda have resulted in numerous arrests and convictions for charges related to product tampering and felonious reporting of product tampering. scanning electron microscopy (sem) has been valuable, and in several cases crucial, in providing conclusive evidence of fraudulent reports of product tampering. the items reportedly found inside of suspect cans were primarily hypodermic syringes (with and without needles), but other reported objects included nails, screws, bullets, pins, sewing needles, tacks, glass and plastic shards, and rodent carcasses. since most of the cases involved syringes, primarily insulin-type syringes, the first analyses were designed to determine if the syringes were contaminated and if they contained human blood, tissue, drug, or insulin residue. syringe/needle rinses were examined by a number of techniques including sem, which was used to find any intact blood cells and/or tissue. energy dispersive x-ray analysis (edxa) was performed on residue preparations. edxa was able to detect a small k αl peak for zinc in several syringe rinses which was consistent with edxa analysis of dried residues containing some types of insulin. type identification of insulin from syringe rinsing was performed by lc/mass spectrometry in a method developed at nfcc. in another case, a broken pin was reportedly found in a soft drink can. a cross-sectional analysis of the suspect pin by edxa showed the pin was a nickel-plated, iron-core straight pin. the absence of observed iron corrosion provided evidence that the object had not been in contact with the soft drink from the time the canned product was produced until the time the pin was allegedly discovered. the most common question requested by investigators was "how long had the syringe been in the soft drink can?" to answer part of that question, a number of experiments were conducted at nfcc. one sem/edxa method investigated the corrosion of the aluminum crimp (found on some syringe needles) submerged in diet cola. new aluminum crimp needles were sealed in diet cola and removed at -h intervals. stereoscopic light microscopy was initially used to examine each needle crimp. it was discovered that the submersion produced a brown to black "corrosion flower" in less than h of soaking. of specific interest, the submersion repeatedly produced a single point of corrosion on the crimp. the single point of corrosion suggested possible electrolysis of the aluminum crimp in the soft drink. this corrosion point continued to enlarge and penetrate more deeply into the aluminum crimp over time. backscattered electron (bse) imaging and x-ray mapping were used to highlight the region of corrosion in the sem. figure shows the secondary and backscattered electron image (sei and bei) of an aluminum crimp on a syringe needle after h submersion in diet cola. the sei shows the corrosion of the aluminum crimp as pitting in the crimp surface. the bei shows the corrosion area as a darker region caused by surface oxides blocking bse generation from the aluminum below. the study also demonstrated that the corrosion of the aluminum crimp was dependent upon the amount of time the crimp was submerged in the pressurized soft drink container. figure shows a point of corrosion on an aluminum crimp after weeks of submersion. at this time interval, several points of corrosion often developed. the pitting of the aluminum mater-ial immediately beneath the corrosion was extensive. sem/edxa analyses have shown that the aluminum crimp on syringes submerged in diet cola produced a characteristic single point of corrosion after only h. the corrosion resulted in damage to the crimp surface and the production of a metal oxide. the location of the oxide was identified and plotted by x-ray mapping for oxygen (via edxa). the analysis of samples related to the pepsi "tampering" cases of are continuing to date. in many cases, the circumstances and sample condition are unique and generated specific questions which require further forensic research using additional time-related studies, multielement x-ray mapping, and/or image analysis. confocal microscopy can be used to investigate the properties of rough surfaces. based on the kirchhoff approximation (sheppard et al. a,b) , the three-dimensional ( -d) confocal image can be modelled using the -d coherent transfer function (ctf). the form of the -d ctf for confocal reflection and transmission have been derived using a high-angle scalar theory (sheppard et al. ) . in both cases the ctfs can be expressed analytically. using these allows the profile of the rough surface to be reconstructed. in many cases we need to know only the statistics of the rough surface, rather than the actual profile. ways in which the statistical properties can be extracted directly have therefore also been considered (sheppard iv- scanning vol. , supplement iv ( ) fig . a. castenholz in previous in vivo studies based on vital microscopic and fluorescence microscopic techniques it was possible to observe lymph flow in initial lymphatics and regional lymph nodes. [ ] [ ] [ ] [ ] as an expression of immunological mechanisms, the studies carried out in the rat tongue under various issues showed some remarkable phenomena such as cell traffic along lymphatic pathways and phagocytotic activity of the lymphatic endothelium. since these phenomena could not be defined with traditional fluorescence microscopy on the cytological level, confocal laser scanning microscopy (clsm) was applied to the living tissue and fixed specimens). moreover, in morphologic studies on the lymphatic and blood vascular system, clsm has proved a very suitable tool also for the representation of resin-injected tissue, which is commonly processed as corrosion casts for scanning electron microscopy. all these approaches, also including the application of special fluorescent markers, should be outlined here. in vivo studies with the clsm have been designed for the functional morphologic analysis under normal conditions and in a state of inflammation and experimental edema. after staining the lymphatic endothelium with dimethyl-carboxfluorescein, clsm revealed a distinct pattern of the so-labelled initial lymphatics consisting of bright (cytoplasm) and dark zones (nuclear portions). for labelling tissue macrophages passing the lymphatic pathways and other phagocytotic cells, fluorescent microbeads (latex standard particles and liposomes) were applied by interstitial injection. thus, clsm enabled a certain identification of moving particle-laden cells and also gave evidence of phagocytotic activity of the endothelium of initial lymphatics. in long-term experiments, phago-cytosis of cells lining the sinuses of lymph nodes could be clearly recognized. tissue (tongue, skin) and lymph nodes from different sites of the rat were examined in the clsm as unfixed or fixed (glutardialdehyde) thick sections. cells labelled with fluorescent microbeads or stained with fluorescent dyes thus could be easily detected and identified. thereby, it was possible to determine the location of single beads at the endothelial surface or within the endothelial cytoplasm (fig. ). if the tissue was conventionally stained with hematoxilin eosin or acridine orange, optimum information could be obtained from unlabelled structures in these specimens as well. by means of the two-detection system, clsm also was able to distinguish two or more different fractions of microbeads incorporated by macro-phages ( fig. ) or lying in tissue spaces as free elements. in hemal lymph nodes of rats, erythrophagocytosis related to sinus macrophages was successfully represented by the clsm after vital staining of erythrocytes with the fluorescent celllinker pkh . in this approach, mercox (acrylate) stained with rhodamin and fluorescent yellow was used to create high fluorescence in the lumen of blood or lymphatic vessels. if the resin was injected into the tissue, the interstitial spaces were filled up by resin. clsm of sections from such resin-injected specimens enabled exact distinction between casts related to the blood or lymphatic system and those of the interstitial spaces, when two differently stained resins were simultaneously injected from the arterial system and into the interstice. clsm of resin-injected specimens was also applied as a suitable method for the control of corrosion casts of sem. proceedings of scanning /seems iv- today, clsm has become an established tool in many fields of biological sciences. because of its abilities to produce images with optimum resolution and clarity, the application in experimental lymphology is striking as well. some experiments in techniques related to in vivo experiments, supra vital and fixed tissue, and resin-injected specimens have been reported here. these approaches, also comprising the application of new fluorescent markers for living cells, may demonstrate how wide the spectrum of application is spanned for clsm in experimental lymphology and immune research. this study was supported by a grant of dfg (deutsche forschungsgemeinschaft, bonn) daniel chin, ph.d agouron institute, la jolla, california, usa the regulated metabolism and distribution of nucleic acids is required for endogenous antisense or rna regulatory systems. recent interest has focused upon using exogenous agents as antisense therapeutics to treat viral infections or metastatic diseases. both endogenous and therapeutic antisense regulation of gene expression requires the formation of a hybrid between the antisense molecule and the message or gene sequence. it is not surprising that detection of such hybrids in living cells has not been reported to date. we have used fluorescence resonance energy transfer (fret) to study hybrid formation and dissociation after microinjection of oligonucleotides (odns) into living cells. two systems were examined: one system characterized the kinetics of hybrid dissociation of two synthetic odns while a second system examined the distribution of hybrid complexes formed by hybridization of injected odns with endogenous mrna. in the first system, a -mer phosphodiester odn (+pd) was synthesized and labeled with a ′ rhodamine (+pd-r). the complementary, antisense ′-fluorescein labeled phosphorothioate odn (-pt-f) was specifically quenched by addition of the +pd-r, as detected by both absorbance and fret in solution. rapid and specific hybridization between the odns occurred at µm within min and the preformed hybrid slowly dissociated (t / ≈ h) in the presence of a -fold excess of the unlabeled odn. upon microinjection into the cytoplasm of cells, preformed fluorescent hybrids dissociated with a halftime of min, which is attributed to the degradation of the phosphodiester. formation of the hybrid from sequentially injected odns was detected by fret transiently in the cytoplasm and later in the cell nucleus, where nearly all injected odns accumulate. diffuse, specific nucleoplasmic fret could be distinguished from nonspecific fret in punctate nuclear structures which may result from concentration effects similar to fret seen in late endosomes or lysosomes between endocytosed, inert fitc-dextran and +pt-r odn. a second set of experiments with two adjacent -mer pt odns complementary to mouse b-actin mrna was performed in mouse t fibroblasts or n neuroblastomas. the upstream odn was ′ labeled with fitc and the downstream odn was ′ labeled with rhodamine. when hybridized to synthetic actin mrna or a complementary -mer pd odn, quenching of fitc and fret was observed in solution. injection studies in t cells showed transient odn accumulation and fret in filopodia and extended processes. in n neuroblastomas, fitc quenching was dependent upon cytoplasmic compartmentalization. after min, both odns accumulated into the nucleus. in summary, these experiments suggest that antisense odns can hybridize to intracellular targets in both the cytoplasm and the nucleus. the goal of this work was to increase the sensitivity of the electron backscattering pattern (ebsp) technique by introducing an electron energy filter to increase contrast and improve pattern visibility. energy filtering previously has been shown to increase contrast in selected area channelling patterns (joy ) . energy filtering differs from arithmetic background subtraction in signal processing, because filtering before detection has a physical basis and eliminates the unwanted background without increasing the noise component in the signal. the electrostatic retarding filter used in this experiment was constructed as a cone with five coaxial electrodes. the primary purpose of the electrodes was to create symmetric fields that would not distort the pattern, yet allow only backscattered electrons with the least energy loss to contribute to the pattern. analysis (courtesy e. munro, micro electron beam software, ltd.) of the electron trajectories indicated that for kev electrons, filtering voltages of up to kv could be used before chromatic aberrations became unacceptable. the angular field-of-view of the filter was approximately o . another feature of the detector is the use of a microchannel plate as the initial sensing component. microchannel plates have high gain and are very sensitive to the low-energy electrons that pass through the retarding field filter. in a configuration similar to that of venables ( ) , the output of the microchannel plates was proximity-focussed to a phosphor screen that displayed the microdiffraction patterns. the gain of the assembly exceeded a simple phosphor by more than times. the light output of the assembly was focused on a kodak megaplus . ccd camera using either a macrolens alone or a hybrid fiber optic-lens combination. no vignetting was observed across the field. the digital output of the camera was displayed and analyzed on a macintosh iix using nih image. results were obtained on a variety of materials including single crystal silicon, al-sic metal matrix material, alumina, aluminum foil, and aluminum used in aluminum cans. the high voltage design limited the maximum retarding potential to − kv so that meaningful data were obtained with incident electron beam energies between and kev. filter potentials above % of the primary beam accelerating voltage increased the kikuchi band contrast relative to the background. figure contains a very low contrast pattern for alumina obtained with essentially no filtering. the effect of filtering to within kv of the beam accelerating voltage is shown in figure for the same specimen. the best patterns were obtained when the filter voltages were between and % of the accelerating potential. filtering above % degraded pattern resolution while less filtering usually produced less contrast. up to four-fold improvements in the contrast ratio were achieved on some specimens. the detector also had the ability to obtain energy-filtered backscattered electron images in sem raster mode. one advantage of this configuration is that the sem characterization of a given feature and the ebsp data obtained from it can be obtained from the same perspective. another is that filtered im-ages appeared to have improved surface detail and greater crystallographic contrast. venables ( ) showed that a detector of this kind could be used for "dark field" sem imaging. this work demonstrated the feasibility of employing an energy-filtering detector with high gain to increase the sensitivity of microdiffraction measurements in the sem. further work is planned to optimize the filter and improve pattern quality and the range of filtering voltages. the authors gratefully acknowledge the contributions of munro electron beam software, ltd. and of mark vaudin at nist, as well as the support by the national science foundation under award number iii- , and the department of commerce under contract number -dkna- - . submicron-sized elements are of great interest in physics and technology. there exists a variety of methods to produce them by using conventional microelectronics technology as well as new methods for deposition of such elements directly from the desired material onto a substrate. the current onestage maskless techniques, for example, laser-induced cvd or laser-induced etching , do not ensure resolution below nm because of their physical limitations imposed by the laser wavelength. focused ion beams, which generally allow the formation of submicron-sized elements, often are unacceptable as they can cause radiation damage in the material. moreover, the cost of the necessary equipment is high. these circumstances stimulate the development of techniques for direct formation of submicron-sized patterns with the necessary geometry from an arbitrarily chosen material directly at substrates based on electron-beam-induced cvd . the cvd experiments were performed in a temscan jem- cx electron microscope at an accelerating voltage of kev and a beam diameter ~ nm. the electron microscope was equipped with a specially designed pressure cell and an oil-free system for pumping and reagent vapor inlet. this allows to maintain a well-defined atmosphere of chemical compounds (metal carbonyls and halides, freons) around the specimen. two electronically controlled needle valves are capable of supplying two different reagents simultaneously into the specimen holder. the sample itself is placed inside a heater which provides temperatures up to ˚c required for the local electron-beam-induced etching. contacts are made to the sample, which allow in situ measurement of its electrical characteristics. the holder is supplied with two differential apertures (through which the electron beam passes) placed above and beneath the sample. these apertures keep the gas pressure around the sample up to pa without breaking the vacuum in the microscope column. w(co) and rez(co) were used as reagents. structure and composition analysis both were performed in a jem- fx electron microscope equipped with a link an- / s system for edx analysis. self-supporting rods of - nm in width, containing tungsten or rhenium, respectively, were grown up to nm long at a speed of the electron-beam movement - nm/s and at a vapor pressure near the sample of . pa. provided a constant speed of the beam is maintained, the thickness of the produced rods was found to be inversely proportional to the beam speed but decreasing towards the rod's end. this result is similar to that obtained by electron-beam-induced fabrication of self-supporting carbon-containing rods electron microscopic observation of the inner structure of these self-supporting metal-containing rods revealed many individual fibers mutually aligned in parallel. thus, the mor- iv- scanning vol. , supplement iv ( ) fig. tem image (a) and sad pattern (b) from a self-supporting tungsten containing rod section after annealing in vacuum at ˚c for min. the accelerating voltage is kv, the diffraction length on the (b) is cm. phology of these rods is similar to that of the well-known carbon rods . this observation suggests similar formation mechanisms independent from the rod material. selected area diffraction (sad) patterns show the prepared rods as being amorphous. in situ annealing of the tungsten-containing rods at ˚c for min in the heating holder inside the microscope column transforms the amorphous structure into a nanocrystalline one (fig. ) . sad patterns reveal the presence of a set of different phases: in addition to pure crystalline tungsten there are various tungsten-containing compounds. among them, in particular, two new cubic phases with lattice constants ± pm and ± pm could be identified. after annealing chemical and phase compositions of the rods grown from w(co) and rez(co) were found to differ depending on the conditions of their formation (reagent vapor pressure, electron current, beam speed). the rods of nano-sized width, grown from rez(co) are considered to be promising as tips for scanning tunneling microscopes because of their chemical stability. an important requirement for developing high-speed highpower devices is fabrication of thermally stable ohmic contacts with low resistance and smooth interfaces. metal semiconductor interface inhomogeneities, such as lateral interface phases and spiking protrusions, lead to nonuniform current flow and consumption of the gaas substrate. this type of interface morphology is not acceptable for device applications where a large electric field or shallow contact is required. in particular, devices such as a heterojunction bipolar transistor (hbt) cannot tolerate contacts with lateral and vertical interface inhomogeneities. recently, we have introduced a novel metallization scheme, pt/ti/ge/pd, which yields thermally stable and low resistance ohmic contacts to both n and p + -gaas (han et. al. ) .to insure processing reproducibility and contact reliability, one must identify and understand the mechanisms responsible for this contact's electrical per-formance. this study employed cross-sectional transmission electron microscopy (tem), auger electron spectroscopy (aes), and electrical measurements (transmission line mode) to investigate the structural, chemical, and electrical properties of this contact. the interface morphology, phase composition, and elemental diffusion were examined and correlated with the measured contact resistances at annealing temperatures which yielded the best slight degradation and the worst electrical performance. annealing at ˚c yielded the lowest specific contact resistance, ~ . × - Ω-cm . the metal-semiconductor interface was planar and structurally abrupt. the pd and ge reacted to form a pdge phase. directly beneath the pdge was a thin, discontinuous, ga-rich pd-ga-as ternary phase. the presence of this ga-rich ternary compound has important implications for contact formation on the n-gaas substrate. formation of this interface phase creates excess ga vacancies in the gaas substrate. upon heating, ge diffuses into the gaas, occupying ga vacancies at dopant levels, to form an n + surface layer, thus allowing considerable tunneling at the metal-semiconductor interface. the existence of the ga vacancies is crucial, because otherwise ge would not readily diffuse into the n-gaas to form this n + -gaas layer. for the p + -gaas, this ge indiffusion occurs also, but the ge concentration level is not such that it has a detrimental effect, that is, it does not approach that of carbon (the p-type dopant) ~ × cm - . the ti/pt layers remained stable, hence no surface degradation was present. annealing at ˚c resulted in a slightly higher specific contact resistance, ~ . × - Ω-cm . there was significant elemental diffusion within the contact metal and minor elemental diffusion into the substrate. the interface exhibited a roughness on the order of nm and possessed large areas where spiking single-phased pdgega protrusions spatially dominated the metal-semiconductor interface region. the nonplanar nature of the interface lends itself to several explanations: ( ) nonuniformity in the heating associated with the annealing process, ( ) material defects or pitting in the gaas substrate such that the contact metallization covered and filled the pitted area as it would a flat gaas surface, and/or ( ) new phases being formed as a result of the ti layer beginning to break down as a diffusion barrier. apparently, this interface nonuniformity (spiking) does not cause significant deterioration in the specific contact resistance, since the contact resistance maintains reasonable electrical integrity. it is speculated that the compositional uniformity of the protrusion phase, which is close to that of the pdge phase formed during the ˚c anneal, is responsible for this behavior. annealing at ˚c proved to have a detrimental effect on the specific contact resistance, ~ - Ω-cm . this degradation was accompanied by strong chemical intermixing between the contact and the substrate, resulting in laterally continuous and vertically deep (> nm) multiphased protrusions spiking into the gaas substrate. the surface of this contact possesses surface anomalies (~ . µm in size) with a density of ~ %. the anomalies are somewhat enriched in pt and as and depleted in ge. this nonuniform surface morphology demonstrates that pt no longer represents a smooth surface for bonding. our results demonstrate that annealing temperatures between - ˚c are of practical interest for hbt device processing. the thin base region ( Å) and narrow distance between the contact and emitter ( . - . µm) make the ˚c anneal impractical for the hbt. specifically, the composition, extent, and magnitude of the interface spiking would be totally detrimental to this device design, that is, spiking areas would penetrate through the base region, into the collector, and short the device. the present paper is a review of diagnostic availabilities as well as of physical data taken by color cathodoluminescence scanning electron microscopy (ccl-sem) used for investigation of structural, polytypic, and impurity inhomogeneities of sic crystals, epitaxial layers, and devices (saparin and obyden ) . as examined objects, the "alpha" and "beta" sic crystals, different polytypes ( h, h, c, r) sic epitaxial layers and devices have been studied. the epitaxial layers were grown by the sublimation "sandwich" method (vodakov et al. ) in the vacuum or ar-media under temperatures between and ˚c. from results of experimental studies it is possible to enumerate the following that are impossible to prove by other techniques: ( ) space distribution of impurities; ( ) static and dynamic characterization of polytype transformation. multitransformation of polytypes during the growth process was observed by way r → c → h. it was concluded that the growth conditions of epitaxial layers and surface perfection of the substrate markedly influence polytype variations. ( ) space distribution of radiation defects on dependence of annealing temperature; ( ) action of mechanical defects on the spatial distribution of luminescence centers; ( ) availability to observe the transient layers in sic devices; ( ) -d analysis of epitaxial layers with polytype transformation. saparin gv, obyden sk: colour display of video information in scanning electron microscopy: principles and applications to physics, geology, soil science, biology, and medicine. scanning , - the examination of chemical vapour deposition (cvd) diamond films by the scanning electron microscope (sem) (secondary electron mode with spatial resolution - nm) shows that the morphologies of films are strongly affected by the synthesis conditions, especially the substrate temperature, the methane concentration, total pressure in the deposition chamber, etc. studies demonstrate that the changes of ch for concentrations in the range of . - % vol. create the shape variation of crystals determined by the ratio of the apparent growth rates of the ( ) and ( ) faces r( )/r( ) in cubo-octaedrons interval from . - . . the exact cubooctahedral shape observed very frequently is determined by the value of ratio . and preferable orientation of the ( )-or ( ) faces. thus, the secondary electron mode of the sem is a very useful diagnostic technique for diamond films. less frequently researchers use the second diagnostic availability of the sem (saparin and obyden ) : cathodoluminescence (cl) mode (spatial resolution . - . mkm) with monochromatic and panchromatic (real color) images and local cl spectra. diagnostic possibilities of this mode identify the quality of diamond films in comparison with natural diamond. the cl spectra of undoped epitaxial films show the dependence on the face upon which the epitaxy was done. cl discriminates between different types of diamond. there are distinct spectral differences between natural, synthetic diamonds and cvd films. the cl emission of the a-band was associated with donor-acceptor pair recombination (blue and green regions); vacancy-rich material emits in the red spectral region ( - nm). we would like to note that the cl spectra of natural diamonds (types ia, ib, iia, and iib) lie in the range of - nm. variations in these cl spectra were attributed to differences in the defect structures formed during the growth of material. cl images and local spectra allow one to recognize, for doped and undoped films, the small amounts of impurities, such as al( ppm) and b( . ppm) and nitrogen remaining in the gas, which could be a possible source of donors. thus, the sem investigations of morphology, crystallinity, and cl emission of films led to the correlative estima-tion of diamond film quality in comparison with the natural diamond as standard. the etching process of a crystal surface during its dissolution is well-known. the process of etch pits formation, as a result of crystals dissolution, usually is explained by scientists by the presence of dislocations inside the crystal structure. however, the same process occurring near the crystal edges is studied less, although such investigations can give additional scientific knowledge for a better understanding of the dissolution mechanisms of solids. this brief communication continues the series of investigations (dorozhkin , dorozhkin et al. the results show that here can be various surface structures taking place under geometric interaction among the growing etch pits and dissolving fap crystal surfaces. as the fap has a hexagonal crystal structure, the growing etch pits mainly have a hexagonal shape also (dorozhkin , dorozhkin et al. ). on the other hand, the investigated fap crystals were obtained from natural fap-containing rock after its disintegration and concentration stages. therefore, the crystals had a very irregular shape and a lot of dislocations inside. figure shows an example of a typical hexagonal etch pit having only three faces instead of the necessary six; three lacking faces (left side) have already been dissolved by the acid. a very similar situation is presented in figure . this is another example of a former hexagonal etch pit having only three faces (center). it is easy to restore events which occurred with the pits some minutes before. the pits began to form and grow from the moment when the outputs of dislocations on the fap crystal surface began to interact with the acid solution. as the pits are always faced only by fast dissolving faces, their dissolution rate is greater than the one for the crystal surface in common. on the other hand, the growing of etch pits and the fap crystal surface dissolution are always occurring simultaneously. as a result, a layer of fap substance between the growing pit, which is situated close to any vicinal dissolving crystal face, and the vicinal crystal face itself were getting increasingly thinner. finally, this layer disappeared completely, resulting in the pits' formation having only three faces (figs. , ) . if one had dissolved the fap crystal for a few more seconds, its surface would have reached the bottom of the pits and they would have disappeared entirely. figure shows an example of interaction between etch pits with an irregular shape and a surface of the fap crystal. for this purpose a very thin and sharp spallation fragment of the fap crystal was chosen which was then etched by phosphoric acid solution. inasmuch as the spallation fragment was equal to a random and unknown crystallographic face, the irregular etch pits were obtained. it is easy to see four different states of such pits (fig. ) : they point to a pit which ( ) ( ) is only running through the fap crystal but is situated relatively far from the dissolving crystal face. finally it should be noted that all the above-mentioned cases of interaction among pits and crystal surface may only take place when directions of dislocations inside the dissolving crystals are close to lines parallel to any nearest vicinal crystal face. k. habib and p. g. caceres* materials applications department; *central analytical laboratory kisr, safat, kuwait a fundamental study of co-based metallic glasses has been conducted. the study focused on understanding the changes of the properties and structures of an fe-b-si glass as a function of co, co-ni, co-mn, and co-ni-mo additions. the separate addition of co, co-ni,co-mn, and co-ni-mo elements was successful in a way four new metallic glasses were produced. the compositions of the new glasses are fe co b si, co fe ni b si , co fe mn b si , and co fe ni mo b si . consequently, an evaluation of the physical and magnetic properties was determined. furthermore, the internal and surface structures of the glasses have been characterized by a transmission electron microscope and a scanning tunneling microscope, respectively. a comparison between the internal and surface structures of the glasses was carried out on both amorphous and crystallined forms. as a result, a correlation between the properties and structures of the glasses is established. for instance, figures a and a show surface structures of the co fe ni mo b si metallic glass in the amorphous and annealed crystalline forms, respectively. on the other hand, figures b and b show the corresponding x-ray diffraction patterns of the amorphous (fig. a) and the annealed structures (fig. a) , respectively. figures a and a are basically three dimensional line plots of the surface profile. it is clear that the amorphous structure (fig. a) represents a complete rough surface along nm × nm scanning area. in contrary, the annealed structure (fig. a) exhibits a surface profile with less surface roughness than the annealed structure. this observation is in agreement with work done by the author on other metallic glasses cited elsewhere (habib et al. ). the current status of external funding for most academic and research facilities throughout this country is meager at best. many institutions are being forced to seek financial support from sources other than the conventional governmental agencies, private funding, or contractual agreements. this facility has, for the past years, derived the bulk of its operational budget through third-party payments for services rendered as hospital charges for diagnostic pathology services. while we have been fairly successful in maintaining a relatively consistent level of service, the overall cost of this operation continues to rise. as cost containment became the buzz word and hospital admissions declined, the number of requests for diagnostic procedures also began to decline. this pattern was observed not only in the electron microscopy (em) lab but in many other specialized laboratories throughout the hospital and our affiliated clinics. as the health care debate accelerated and new concepts such as hmos, emergency care clinics, managed competition, and regional alliances all geared to assure "more health care for the dollar came into being," it became apparent that an entire new concept was needed to provide high-quality diagnostic services for these newer group practices and smaller clinics that were being created to meet these new demands. the diagnostic referral service * has been established in the department of pathology, medical college of georgia, to offer a range of state-of-the-art diagnostic services to external referral sources, including private practitioners, group practices, pathology laboratories, and hospitals. these specialized services are designed to supplement other routine analyses and assist in the diagnosis, prognosis, and clinical management of patients with a wide range of diseases. the laboratories involved are fully cap-accredited, and all diagnoses are evaluated by a pathologist certified by the american board of pathology. the following is a listing of the laboratories contributing to this endeavor and a brief summary of some of the diagnostic offerings. this laboratory provides cytogenetic analyses to identify specific chromosomal abnormalities. these are useful not only in establishing a diagnosis of malignancy, but also in classifying certain malignant disorders, monitoring remission and progression, deciding on treatment regimen, and estimating prognosis. chromosomal analysis can be conducted on bone marrow aspirates, peripheral blood, and lymph node biopsies. this laboratory offers standardized transmission electron microscopic analysis of biopsies from any organ or tumor, as well as specialized procedures for examination of cell suspensions such as bone marrow and lung aspirates and blood samples. em analysis is particularly useful in conjunction with light and/or immunohistochemical microscopy for the diagnosis of tumors which cannot be classified by conventional light microscopy. working in close collaboration with the histology laboratory, the immunohistochemical laboratory, and the renal biopsy service, the em laboratory provides standardized, reproducible ultrastructural analysis which allows a consistent comprehensive approach to the interpretation of biopsy pathology. immunophenotyping by flow cytometry details the presence or absence of surface antigen markers of cellular maturation which define cellular subsets present in specific forms of leukemia and lymphomas. this technique provides precise information to aid in the diagnosis, prognosis, and clinical management of patients thought to have varying forms of lymphoproliferative disorders. in addition, quantitative dna ploidy and cell cycle analysis in combination with standard histopathologic and cytochemical methods provides the most comprehensive assessment of clinicopathologic status, tumor aggressiveness, and the likelihood of disease progression for patients with breast, colon, and ovarian cancer. this facility performs state-of-the-art diagnostic immunohistochemical and in situ hybridization techniques for the morphologic analysis of cellular and molecular events. these techniques are adjuncts to histopathologic examination and can be applied to the study of neoplastic, infectious, and other diseases. the laboratory offers more than immunohistochemical markers as special decision-making tests to resolve differential diagnoses. special histochemical stains and molecular probes for colorimetric in situ hybridization are also provided by this laboratory. this laboratory offers a full service for examination and consultation on kidney biopsies involving a wide range of disease processes. in addition to routine histopathology, all renal biopsies are examined by immunofluorescence for the identification of immunoglobulins, albumin, and c deposits, and by transmission electron microscopy for the detection of early or submicroscopic abnormalities. this laboratory presently offers complete dau screening and gc/ms confirmation for pre-employment/employee drug testing. upon completion of certification as a forensic urine drug lab, this will be the only such certified lab in this area. department of pathology, duke university and va medical centers, durham, north carolina, usa microprobe analysis in biomedicine is usually done on an electron microscope (em) equipped with an energy-dispersive x-ray detector (edx). this type of analysis is commonly referred to as electron probe microanalysis (epma). there are also other fundamentally different new techniques for microprobe analysis which involve the use of laser or ion beams. however, these are not yet commonly employed for diagnostic studies (ingram et al. ) . whereas biomedical epma primarily used to be a research tool, that is no longer the case. epma findings now often have diagnostic, therapeutic, and/or legal significance for the patient (baker et al. , shelburne ). in addition, since much of the current work involves conditions such as the pneumoconioses, the findings frequently have public health and/or industrial medicine implications as well as implications for single patients (shelburne et al. ) . currently the most commonly studied clinical conditions include the pneumoconioses, especially asbestosis and related conditions, "hard metal" pulmonary fibrosis, and other min-eral-induced pneumoconioses (roggli and shelburne ). a second major application is the use of this technology for the analysis of stones, particularly renal stones. microprobe analysis can be more sensitive than x-ray diffraction or chemical techniques, particularly for the identification of small components of complex stones. another major application is the use of microprobe analysis to identify unexplained pigments or deposits and to study unexplained granulomas (kupke et al. , pickett et al. . as is evident from the foregoing discussion, most applications involve the study of insoluble particulates. accordingly, conventional histologic processing with chemical fixation and paraffin or plastic embedding is acceptable. an obvious limitation of this approach is that electrolytes cannot be studied. currently we are exploring the feasibility of utilizing flash freezing techniques to permit studies on electrolytes utilizing cryoultramicrotomy. one way to gauge the usefulness of this technology is to study the use of microprobe analysis in a single hospital system, that of the veterans administration medical centers. currently there are va medical centers in the united states. within these hospitals there are diagnostic electron microscopy laboratories. of these, only five currently utilize epma as a diagnostic technique. at each of these laboratories, conventional transmission electron microscopic studies are the predominant type of analysis. epma studies constitute less than % of our electron microscopy laboratory workload. nevertheless, as the chemical information available from epma is better understood by clinicians, and as cryotechniques are shown to be useful, we anticipate increased usage. in conducting these studies, it is important to be aware of several artifacts that are common problems. the major types are those caused by poor specimen fixation. not only does traditional chemical fixation remove electrolytes from the tissue, it is common in electron microscopy laboratories to add heavy metals such as osmium, uranium, and lead. these elements may produce peaks in the final spectrum that can obscure important elements of physiologic significance. for example, the m-alpha line for lead obscures the k-alpha line for sulfur. in conducting an epma study, it is important to identify all peaks obtained so that the investigator is not mislead by a contaminant. in addition, it is important to utilize several controls. the investigator should not only probe the feature of interest, but also the cytoplasm adjacent to that feature and the blank stub. only in this manner can artifacts contributed by, for example, metal in the microscope column be understood and eliminated from consideration. all living things are infected/affected by viruses. whether the subject is a tissue culture, an animal being used in research, or a human, it behaves differently when infected with a virus. in research subjects virus identification is important to prevent erroneous data due to the presence of a foreign organism. in the case of human viral illness, it is increasingly important to identify pathogens so that appropriate viral therapy can be initiated. several antiviral agents are already on the market, and many more are presently in clinical trials. advantages of using electron microscopy (em) in viral diagnosis are that it is rapid; specific standards and reagents are unnecessary; and infectious particles are not required. disadvantages include the facts that a fairly high concentration of virions must be present in liquid samples to visualize them, and that solid tissues may have focal infections that must be included in the sampling. identification of viruses by direct em is performed by two techniques: negative staining of liquid samples and thin sectioning of tissues, cells, and tissue cultures. negative stains most used in virology are phosphotungstic acid (pta) and uranyl acetate, although many others have been described. for thin sectioning, any fixation method used successfully for em of tissue will preserve viruses; this includes some form of glutaraldehyde and osmium fixation, usually followed by uranyl acetate. detailed preparatory techniques have been described. in negative stains, morphology questions used in identification are: is the virus naked (always icosahedral) or enveloped (pleomorphic); if naked, what is its size, and does it have distinguishing capsid (outer) markings; if enveloped, does it have visible spikes or fuzz around the outside; what is its size, and is the nucleocapsid (the core) visible in particles that have been penetrated by stain; what is the shape of the nucleocapsid, if visible? naked human viruses are all icosahedral; these pathogens fall into three size ranges: - nm, - nm, and - nm. the small viruses may or may not have surface substructure; those that do not are not morphologically distinguishable and have been referred to as small round viruses (srv) (fig. a) if smooth, or small round structured viruses (srsv) if rough. others may have characteristic surface markings that permit precise morphologic identification. the medium-sized (fig. b) and large (fig. c, d) viruses are identifiable. enveloped viruses (those that have a pliable covering) are harder to identify, especially if mixed together with cellular debris. if they have surface fuzz or spikes (fig. e) , they are more readily distinguished. the genetic material inside is sometimes packaged into a distinct form such as an icosahedron, similar to the naked viruses (fig. a-d) or helical filament (fig. f ). if the negative stain penetrates the membrane, this nucleocapsid may be recognizable. however, some viruses do not have a morphologically characteristic nucleocapsid. in thin sections of infected cells, dna viruses are usually seen in the nucleus ( fig. a, b) where they are constructed, and rna viruses are usually found in the cytoplasm where they are formed (fig. c, d) , but there are exceptions. enveloped viruses can be seen associated with or budding through cell membranes; the membrane type is a further clue to identity. finally, the shape of the nucleocapsid within enveloped viruses is a key. possibilities are icosahedral (round in sections, fig. a, b) , helical or filamentous (like worms in sections, fig. c ), complex (pox viruses), or morphologically nondescript. recognition of viral particles and differentiation from cellular components and debris is paramount. once the presence of a virus has been determined, one may consult an atlas to confirm identification. [ ] [ ] [ ] [ ] [ ] for specific concentration or identification of viruses, some antiviral antibodies are available. these reagents can be used to aggregate, to coat, or to gold-label viruses. use of antibodies requires an a priori hint of the identity of the potential pathogen for selection of the proper reagent. charles d. humphrey, cynthia s. goldsmith, luanne h. division of viral and rickettsial diseases, cdc, atlanta, georgia, usa an unexplained acute pulmonary illness resulting in the death of previously healthy individuals was recognized in may . the cause of the illness was quickly identified serologically, pathologically, and genetically as a close relative of prospect hill virus (a rodent-transmitted hantavirus). recently, we isolated the virus from trapped rodents near the homes of patients, cultivated it in vero e cells, and determined that it was identical genetically and morphologically to the causative infectious agent. preparations for electron microscopy (em) were made by extracting, clarifying, and concentrating the virus from unfixed and . % glutaraldehyde-fixed, supernatant fluids of infected vero e cells . uninfected supernatants were prepared similarly as controls. concentrated virus suspensions were applied to glow-discharge treated formvar-carbon grids, blotted, and stained with . % uranyl acetate (ua) or with % phosphotungstic acid (pta), ph . . infected and noninfected cells were prepared for thin section by washing with . m phosphate buffer (po ) ph . , fixing in situ with . % glutaraldehyde in po , scraping, and pelleting. cell pellets were postfixed en bloc in po buffered % osmium tetroxide, stained in . % ua, dehydrated, infiltrated, and embedded in epon-substitute araldite. thin sections were stained with ua and in lead citrate. the extracted negatively stained virus resembled a hantavirus. the nm - + nm viruses were spherical, generally nonelongated particles with typical hantavirus grid-like surface features to which surface projections were attached (fig. ) . iv- scanning vol. , supplement iv ( ) fig our previous studies have demonstrated that application of scanning electron microscopy (sem) imaging for examination of transplanted hearts gave new possibilities for evaluation and early detection of acute rejection (jakobczak et al. ) . continuation of our studies confirmed the benefits of sem for diagnostics and basic investigations in cardiac transplantations. the aim of this study was to investigate coronary vessels during acute rejection in experimentally transplanted hearts. allogeneic heterotopic heart transplants were performed in ether anaesthetized rats, using microsurgical techniques according to the ono-lindsey method. male rats of the inbred long-evans strain were used as recipients, and male inbred sprague-dawley rats served as donors. the anastomoses-the donor aorta to the recipient abdominal aorta and the donor pulmonary artery to the inferior vena cava of the recipient (end to the side)-were performed under the operating microscope (wild m ) with - microsutures (davis-geck). cardiac allograft survival was estimated daily by electrocardiogram and palpation of ventricular contractions. rejection was considered complete at the time of cessation of a palpable cardiac beat. rejection was confirmed with laparotomy and histologic examination. the animals did not receive immunosuppression. allograft survival ranged from to days. transplanted hearts were perfused with . % nacl and . % glutaraldehyde solution in . m sodium cacodylate buffer ( mosm; ph . ). hearts fixed in glutaraldehyde were cut into thin sections, mm thick slides orientated perpendicularly to interventricular and interatrial septums. slides were treated with % osmium tetroxide for h at room temperature. then the specimens were dehydrated in an ascending series of ethanol solutions, ending in rinses in % ethanol and in pure acetone. thereafter, slides were criticalpoint dried using co , mounted on aluminum stubs with conductive silver paint and coated with a thin layer of gold. for examination a jeol jsm c sem was used. microcorrosion casts of the coronary arteries and veins and myocardial microvasculature of transplanted hearts were prepared by infusion of methacrylate casting medium (mercox). following infusion of approximately ml of casting material for one heart, the preparations were left for min to allow polymerization to occur. then, the tissue was macerated, leav- empty nm particles were often seen. virus particles were seen by thin section em as shells coated with barely distinct surface projections and enclosing hair-like strands of nucleocapsid material (fig. ) . particle size ( - nm) was smaller than that seen by negative stain em. spherical particles with elongated tubules (fig. ) were often observed by both negative stain and thin-section em. we conclude that deer mice trapped near the homes of humans with unexplained acute pulmonary illness harbor hantaviruses that likely are the causative agent for the human illness. ing a microcorrosion cast. dried preparations were mounted on stubs, coated with gold, and examined with sem (murakami , potter et al. . investigations of the coronary vessels of the right and left ventricular walls, septum, and the apex region were performed. tissue fragments were taken from various heart regions each day during the first postoperative week for studies with sem, and remnant tissues were used for histologic procedure. grading of the severity of cardiac graft rejection was based upon the stanford classification. in addition, the coronary vessels of transplanted hearts (of the other experimental group) were reproduced with a casting medium and examined with sem. the observations were compared with views of nontransplanted and syngeneically transplanted control hearts. the applied vascular casting method enables one to study the three-dimensional architecture of the vascular network of the rejecting myocardium. besides the large areas with minimal damage and readily distinguishable impressions made in the cast material by endothelial nuclei, there were the regions where various signals of vascular pathology were present: changes of diameter of coronary arteries and veins; unusual size and shape of the vessels; indentations on the cast surface caused by adhesion of blood cells to the vessel wall; characteristic occlusion of the vessels; irregularity of capillary network with changes in diameter and cast surface; capillary destruction with accompanying extrusion of casting material into the interstitial tissue. the observed vasculopathies varied in mild, moderate, and severe rejection and in several areas of the hearts. studies of coronary vessels of transplanted hearts using sem imaging have made significant advances in the understanding of rejection vasculopathy possible. sem observations of sectioned transplanted hearts correlate with results obtained by application of the microcorrosion casting technique. data showed that the highest rate of acute rejection occurs in the interventricular septum, and the development of rejection in various heart regions differs significantly. observed differences in localization and dynamics of development of vasculopathies in the septum and left and right ventricular walls of transplanted hearts confirm the unequal character of rejection. sem imaging is a valuable method for investigation of vascular pathology during acute rejection in transplanted hearts. the authors are very grateful to prof. a. miodonski, director of the sem laboratory of ent dept. of the n. copernicus academy of medicine, cracow, for the enabling of sem observations. preservation of the ureteral blood supply in kidney transplant surgery and ureteral reconstruction is of paramount importance in preventing urological complications of the ureter. the incidence of complications in ureteral surgery has declined in recent years, in part due to greater understanding of the ureteral blood supply and improved surgical technique. however, the ureteral vasculature has been described primarily at the gross level and only recently received attention at the microvascular level. because of its critical role in the success of renal transplants and its potential vulnerability to surgical trauma, the ureteral vasculature merits further investigation. we studied the microvascular anatomy of the ureteral vasculature (uv) in male new zealand rabbits using transmission electron microscopy (tem) and scanning electron microscopy (sem) of vascular corrosion casts and alkalitreated tissue samples. the uv was perfuse-fixed with buffered glutaraldehyde via the abdominal aorta, washed in buffer, and treated with alkali according to the method of takahashi-iwanaga for sem. for tem, fixed tissue was embedded in epon, sectioned, and stained with uranium and lead. for vascular corrosion casts, the uv was washed free of blood with buffered saline and filled with plastic resin (mercox•/methylmethacrylate, / ) at physiologic temperature and pressure. resin-filled tissues were digested with % koh, washed in water, critical-point dried, mounted on stubs, and sputtercoated with gold palladium for sem examination. the blood supply to the male rabbit ureter originates primarily from ureteric branches of the renal artery, the testicular artery, and the vesicular artery. these intrinsic vessels run within the wall of the adventitia the full length of the ureter. perforating arterioles and venules pass through the muscle wall and divide further in the lamina propria to supply a dense plexus of continuous capillaries in the mucosa. although numerous arterioles and venules populate the lamina propria, the ureteral musculature does not possess a rich capillary bed. the capillary plexus is positioned between the transitional epithelium and the lamina propria and uniformly extends the entire circumference of the ureter. capillary orientation occasionally follows the longitudinal axis of the ureter, but is primarily plexiform and exhibits multiple "y" and "t" shaped anastomoses (fig. ) . at the points of junction with the underlying arterioles and venules of the lamina propria, the capillaries commonly exhibit "kinks" and "bends." alkali treatment revealed that the capillary bed is supported by a dense, fibrous network of collagen (fig. ) . pericytes were occasionally associated with the capillary network. intercapillary distance ranged from - µm and capillary diameters typically were - µm. venous valves were not observed in the ureter. the combination of vascular corrosion casts, alkali treat-ment, and tem provides a clear and comprehensive perspective of the microvasculature of the rabbit ureter. the limited number of intrinsic vessels supplying the ureter wall and capillary bed emphasize the importance of understanding the blood flow in this organ in related surgical procedures. rabbits were perfused with glutaraldehyde and mercox ® via the abdominal aorta and subsequently processed for preparation of vascular replicas according to a previously described method. the angioarchitecture of the tibia was studied with use of the scanning electron microscope. at low magnification the pattern of vascularisation to the tibia could be established. they are richly supplied with arteries which pass into the bone substance proper from the periosteum. the main supply is from the nutrient arteries, which divide longitudinally in a dichotomous manner after passing through a foramina in the compact bone, major branches running both proximally and distally. this is the general case in all long bones. these branches reach the epiphysis and diaphysis of the tibia. in addition, separate arterial entities supply specific regions of the tibia: i. periostal vessels, ii. epiphyseal vessels, and iii. diaphyseal vessels. arteries and veins can be readily distinguished due to their characteristic nuclear impression present on the surface of the replicas. arterial nuclear impressions are oriented longitudinally to the long axis of the vessel, whereas in veins they are situated circumferentially (miodonski et al. ) . for further details about methods, see syed ali et al. . at higher magnification it was possible to demonstrate the microangioarchitecture of the epiphyseal region, which consisted of a very fine meshwork of capillaries arising from a main epiphyseal artery. a connection between arteries of the epiphysis was observed and consisted of medium-sized arteries. the capillaries of the epiphyseal meshwork show a rich anastomosing network building open, sinus-like dilatations. capillaries are also richly present in the areas of the growth plate and stressed areas of the epiphysis of the tibia. the so-called sinuses are situated under the cartilaginous plate. the venous drainage takes place through the vessels running parallel to the corresponding arteries. the animals were premedicated with chloroform and were then anesthetized with ketanest ( . ml/kg body weight) via intraperitoneal injection. after examining the abdominal reflex the rabbits were fixed on an operating table. all preparations were carried out at room temperature. the abdomen was shaved and opened cranially at the xyphoid process down to the pubic symphysis. after gently displacing the intestines and mesenterial convolutes, the abdominal aorta and the inferior vena cava were exposed very carefully caudal to the kidney vessels. a knob cannula was inserted through a small incision in the abdominal aorta and held in place by a ligation around the cannula distal to the point of incision. these procedures should be performed very quickly, otherwise the blood pressure will fall. the hindlimb vessels were washed through the cannula with an isotonic nacl solution containing heparin ( i.e.) and sodium nitroprusside ( mg/l) in ml. the inferior vena cava was opened with an incision as exit for fixing fluid and clamped when required. soon thereafter, . % glutaraldehyde in phosphate buffer ( . m, ph . ) was passed through the same cannula. after completing the perfusion, about ml mercox (methacrylate) was applied through the cannula and observed until the mercox came out of the inferior vena cava. it was then clamped to avoid unnecessary backflow. the animals were left overnight at room temperature and then placed in a water bath for h for rapid polymerization. the bones were dissected free from other tissues and left in a % koh solution, which was changed every day. the specimens were washed with distilled water very carefully; it is very important to avoid every kind of mechanical disturbance. the solution and water can be changed with the help of a small water pump. the best results were achieved with an alternative change of % koh and % trichlor tetra acetic acid solutions. after complete maceration, the preparation was checked under a stereo microscope to eliminate all rest tissue. it was then frozen at − °c and freeze-dried. the preparation was contrasted with % os o vapour in a desiccator for about h to increase the contrast in the microscope. the specimens were fixed on scanning stubs with conducting silver and sputtered with gold ( min, - ma). they were then viewed in a psem (philips) microscope and photographed with kodak film. iv- scanning vol. , supplement iv ( ) fig. low magnification view of a rabbit tibia from the epiphysial zone with its specific end capillaries arrangement, the proliferation zone, and the connecting capillaries between the epiphysis and metaphysis ( apart from the ability to capture three-dimensional ( -d) images of microscopic structures, a confocal laser scanning microscope (clsm) equipped with multiple detectors allows one to add an extra dimension to the data acquisition process. a typical example are the ongoing studies in our laboratories. in this paper, we discuss the channel spill-over problem associated with acquiring two-color clsm images and present image processing techniques that analyze multichannel image data. for double labeling of dna replication sites, mouse t cells, exponentially grown on cover slips or synchronized at specific times in -phase, were pulsed for brief times ( - min) with cldu (chlorodeoxyuridune). the pulsed cells were then fixed and processed for fluorescence microscopy using monoclonal antibodies, appropriate extraction conditions, and fluorochrome-conjugated secondary antibodies which enabled differential recognition of sites of cldu versus idu incorporation into newly replicated dna. under the conditions used, there is no measurable cross reaction between the antibodies for cldu-versus idu-labeled replication sites. optical sections of labeled cells were collected with an olympus lsm gb- clsm with a m w ar ion laser. the microscope is operated in high resolution mode ( × pixels) with three fluorescence channels and a transmission channel. the microscope is controlled by a - mhz computer with mb ram and . gb hard drive. the digital confocal optical sections were transferred via ethernet to a dedicated sun sparc / with mb memory for further analysis. during image capture, a combination of band-pass and high-pass filters are used to mask unwanted emission at each detector. a major problem with the dyes used is their overlapping emission spectra. this seemed to contribute a significant amount of spill-over from the green channel (fitc) to the red channel (texas red or rhodamine) and only an insignificant amount in the other direction. because of the unidirectional nature of the spill-over between red and green channels, we were able to apply a correction factor to the red channel based on the green-channel intensity. to obtain the correction coefficients, a set of calibration images were obtained by using a sample which is identically labeled in both color fluorescent dyes and captured at different gain and offset parameters. for each of these images, the mean spill-over value in to the red channel (r i ) is calculated for each pixel value (i) in the green channel as follows: ( ) where i r (x,y,x) and i g (x,y,z) denotes the image intensities of the red and green channels, respectively, and n i indicates the number of pixels in the green channel having value i. a third order polynomial is fitted to the data set (<i, r i >,i= , , ... ) and the corresponding coefficients (a o a ,a ,a ) are estimated using the least square error criteria. the correction factor for each pixel of the red channel is then computed as follows and subtracted from the original intensity of the red channel to obtain the corrected image. ∆i r (x,y,z)= a o + a i g (x,y,z) + a i g (x,y,z) + a i g (x,y,z) ( ) figure shows the channel spill-over for different filter sets. the corrected images were analyzed using a set of image processing routines developed at our laboratory to obtain the boundary of replication sites in individual channels. these boundary data are then used to detect the overlapping particles between channels, and the overlap area is calculated for each overlapping particle. the results are presented in several forms including a two-dimensional histogram of particle volume and overlap percentage. the image processing routines are able to generate boundary data of a × × image with particles in approximately mins and the overlap estimation using boundary data takes approximately s. currently we are working on visualization techniques of raw and processed multichannel -d images. these fringes, these dark, granular, linear structures shed dark particles which then fused with opposite lines. a stream of particles trafficking between cells in contract, particularly at overlaps, was noticed. this pointed to a rather intense exchange of very small particles between cells of the same origin on contact. we conclude that interference reflection mode, video-rate, laser scanning confocal microscopy is a useful tool for intravital analysis of the intracellular structural dynamics in relationship to cell type, function and pathophysiological state. department of plant pathology and department of botany, university of georgia, athens, georgia, usa powdery mildew diseases of plants are caused by a group of obligately parasitic fungi belonging to the order erysiphales, class ascomycetes. the somatic hyphae of most of these fungi grow exclusively on the surfaces of infected plant organs, most typically leaves. these hyphae form specialized structures known as appressoria that attach to the host surface. each appressorium gives rise to a tiny penetration peg that penetrates the wall of the underlying epidermal cell and invaginates the host cell plasma membrane. the penetration peg then develops into a specialized structure known as a haustorium that absorbs nutrients from the host cell. in this study transmission electron microscopy was used to examine the haustoria of the powdery mildew fungus erysiphe lagerstromemiae and the relationship of these structures to epidermal cells of infected leaves of crape-myrtle (lagerstromemia indica). the haustorium of e. lagerstromemiae (fig. ) possesses a slender neck region and an expanded body that contains a single prominent nucleus. much of the neck is surrounded by a collar of host cell wall material. a single septum with a tiny central pore with which woronin bodies are associated is present in the distal portion of the neck near the haustorial body. the haustorial body is divided into numerous small, coiled branches. the entire haustorial apparatus is separated from the host cell cytoplasm by an extrahaustorial membrane (figs. , ) . though continuous with the host cell plasma membrane, the extrahaustorial membrane is much thicker than the plasma membrane and is highly convoluted in certain regions. the haustorial branches are separated from the extrahaustorial membrane by a region known as the extrahaustorial matrix. at this point it is unclear whether the finely fibrillar material comprising this matrix originates from the fungus, the host cell or both. although the haustorial apparatus of e. lagerstromemiae occupies a considerable portion of the overall volume of an invaded cell, the host cell organelles appear normal. however, numerous golgi bodies and many structures resembling microbodies are concentrated in the host cytoplasm very near the extrahaustorial membrane (fig. ) . pavel vesely and alan boyde* institute of molecular genetics, academy of sciences of the czech republic, prague, czech republic; *department of anatomy and developmental biology, university college, london, u.k. video-rate laser scanning confocal microscopy in the reflection interference mode (irm) enables the visualisation of fine intracellular structures in living cells in vitro and the observation of rapid changes of shapes, the trafficking of very small particles, and exchange of material. the dynamics of this intracellular activity can be established by optical sectioning with objectives of high magnification and numerical aperture, from within the bottom level of the cell contact to the substrate through to the top cell surface: these levels may be up to µm apart for large and/or tall cells. in the present studies, three video rate confocal microscope systems (lasertec lm , noran odyssey and biorad dvc ) were used to compare the ultramicroanatomy and dynamics of motion of intracellular structures in established cell lines (k , k , t , and a on). k cells are clonal descendents of spontaneously in vitro transformed lew/cub rat fibroblasts which give rise to low metastatic sarcomas in vivo: k cells are rous sarcoma virus transformants of the k line and both t and a on cells developed from k by neoplastic progression to a higher metastatic capacity. previously observed differences in the incidence of cells with rapidly oscillating and trafficking particles between k and the other cell lines were analysed at improved resolution. using the noran odyssey system, with zoom factor and a / . nikon lens, this phenomenon was observed in almost all the cells of all the cell lines compared. this finding provided an explanation of the previously described difference between k and the other cells which was obtained using a nikon / . wi lens (but not with an olympus / . wi using the lasertec system, zoom factor ) from the level of the cell periphery adjacent to the culture surface only. the present observations indicate that there may be a difference in the extent of this type of intracellular activity towards the periphery of the cell. more accurate mechanical targetting of the optical probe will be needed before it will be possible to measure this effect. at the free, top surface of some k cells, small pinocytic opening and closing was seen. similar images of opening and closing near the bottom sides of some cells in all cell lines were evidently produced by vertical oscillatory movements of particles in small clusters. when a . mm pinhole diameter (odyssey) was used to improve confocality, the interference fringes were observed to move, during focussing, along the steep slope of the high bodies of the k cells, optically transforming granular, linear, or randomly oriented structures inside the cell into concentric lines. above and below florida institute of technology, department of biological sciences, melbourne, florida, usa acid phosphatase (ap) is well known as one of the representative lysosomal enzymes. ap activity is recognized at the light microscopic level as intensely stained granules. although the localization and distribution of ap activity in the mammalian inner ear were researched by several investigators, we found only one report about the occurrence of ap in the avian inner ear (marmo ) . the objective of the present study is to demonstrate the occurrence of ap activity in the membranous labyrinth of the chick's inner ear, using the simultaneous coupling azo dye method (barka and anderson ) , and to discuss the significance of these findings. after newly hatched chicks were sacrificed, their ears were fixed for h in . % glutaraldehyde (ga) or . % paraformaldehyde (pfa). the membranous labyrinths were dissected out, dehydrated in either a graded series of acetone (used only for . % ga fixation specimens) or n,n-dimethylformamide (dmf), and embedded in jb- (polysciences, warrington, penna.), a methacrylate plastic. these sections were incubated for or h at °c in an incubation medium (ph . ) containing naphthol as-bi phosphate as substrate and hexazonium pararosaniline as a coupler that used the azo-coupling method. in some instances, intact, fixed whole specimens were incubated in an aliquot of the same incubation medium as that of sectioned specimens and were then dehydrated with dmf, embedded in jb- or lr white, and sectioned. for control specimens, either a substrate-deficient medium or an incubation medium containing cupric sulfate ( . m) was used. methyl green was used as a nuclear stain. intense ap activity, as represented by dense dye deposits, was detectable in the supranuclear area of almost all hair cells in the basilar papilla and vestibular sensory hair cells (fig. ). this result was in agreement with the finding of marmo ( ) . there were no differences in the distribution pattern of ap activity of the hair cells from the distal to the proximal region in the basilar papilla. in particular, hair cells of lagenar macula were most often characterized by intense ap activity in the subcuticular area (fig. ) . the functional significance of high levels of ap in the hair cell is still a matter of conjecture. for example, ishii and balogh ( ) demonstrated no morphologic evidence for phagocytic or secretory activities in hair cells, although their electron micrographs of hair cells reveal lipofuscin-like substances within lysosomes. it has long been contended that nonsensory hair cells such as stria vascularis, external spiral sulcus cells, and those around the macula and cristae ampullares help to regulate the ionic composition of endolymph (kimura et al. , kikuchi and hilding ) . in the present study, the columnar cells and the cells of the tegmentum vasculosum showed moderate to strong ap activity, and the transitional epithelia of the cristae ampullares also showed strong ap activity . the dark cells help to regulate the ionic composition of endolymph and perilymph (kimura ). in the present study, intense ap activity of dark cells was concentrated in the supranuclear area or diffusely in the cytoplasm. these results show that ap activity is highest in cells with highest levels of transport activities on the production of endolymph (marmo ) , although the metabolic linkage between ap and the transport enzymes is uncertain. the wall cells and supporting cells of the vestibular labyrinth showed no enzyme reaction. the statoacoustic and vestibular ganglion cells showed various degrees of ap activity. the sections of statoacoustic and vestibular ganglion cells of specimens that had been embedded in lr white displayed more intense ap activity than those sections from specimens embedded in jb- . ap activity was also stronger in specimens fixed in . % pfa than in . % ga. the syncytiotrophoblast layer covers the fetal villous tree which is in direct contact with maternal blood. it contains many microvilli, resembling a carpet surface, which are responsible for the absorption, excretion, and synthesis of many key hormones which are important for fetal development, secretion, and exchange of gases; of course, many earlier scanning electron microscopy (sem) studies of normal and pathologic placental villi have been described and many investigations on the histology, ultrastructure, and three-dimensional features of human placental villi have been carried out. [ ] [ ] [ ] [ ] technical handicaps causing structural deformations on the placental villi have been indicated. all of these studies depended on the normal development of placental villi but were not found to be comparable with tubal pregnancies. the purpose of this study is to describe the development of the human placental villous trees emerging from chorionic plate during the early periods of uterinal and tubal pregnancies, and to compare their three-dimensional structures using sem technique. iv- scanning vol. , supplement iv ( ) fig samples of human placentas have been studied. six early specimens obtained by curettage or by hysterectomy have been staged as described in our previous study. four additional specimens dated according to anamnestic data in comparison with the measurement of fetal weight and length at , , , and weeks p.c. were obtained from clinically normal pregnancies interrupted by legal curettage or by hysterectomy, and two samples aged and weeks p.c. from tubal ectopic pregnancies. all specimens were fixed and prepared for sem. the specimens were dehydrated in ascending concentrations of ethanol, critical-point (bio-red e ) and air dried, and coated with a layer of gold particles using a bio-red sc super coater. observations were made with a jeol-jem sem. ectopic tubal placental villi trees are not as well developed as normal uterinal villi trees. villi formation and ramification were very rare depending on the physiologic causes. some villi shoots were thinned gradually from base to its apex, and at the top these developing villi showed very interesting properties; they were curved, thinned, and crossed each other (fig. ) . the predominant villi types were immature, intermediate, and mesenchymal villi. [ ] [ ] [ ] the surface of these villi trees was completely covered by small and deformative curved microvilli and by fibrinoid particles compared with the normal uterinal villi trees. the trophoblastic shell of some villi was peeled off and degenerated. developmental retardation of placental villus trees is clearly seen. some villi were curved and crossing, and gradually thinned to the apex; on the villi surface, many wrinkles and furrows resembling very old skin were observed (fig. ) . it is well known that the placental development shows a parallel regulation due to the desidual properties. cellular and extracellular interactions take place between uterine and trophoblast during initial stages of placentation. , - khong and robertson suggested that the deficient decidua is the main reason why tubal pregnancies do not reach to term. the findings they describe may be explained as being due to absent decidua rather than being its cause. moreover, another reason for the usual failure of ectopic pregnancies to reach term is placenta accreta which becomes placenta percreta with bleeding. according to our sem observations, in tubal pregnancies the formation and development of placental villi trees were found in some patterns observed in uterinal placental samples. villi formation patterns-buds, tendrils, and shoots-are very rare on the surface of villi trees (fig. ) . placental villi shoots do not show uniform composition compared with to the uterinal villi samples. these are very thinned to the apex of villi, and are compressed in a common configuration, resembling a racket which does not appear as an alive condition. these observations are very interesting and original. the university of iowa central electron microscopy research facility (cemrf) was established in to support all faculty, staff, and students needing this technology. initially, the cemrf operated with one transmission electron microscope (tem), one scanning electron microscope (sem), and three staff members, and it supported about projects annually. during the past years, all instrumentation predating has been replaced and now includes two tems, two sems, two eds systems, a scanning probe microscope, a laser scanning confocal microscope, and all necessary supporting equipment. the facility presently consists of staff members and supports over projects yearly from departments in colleges and industrial laboratories. one of the unique strengths of the cemrf is that both biomedical and physical scientists use the facility. the development of the university of iowa cemrf was made possible due, in large part, to the central administration's support of equipment acquisition over the past decade. of the $ , , invested in equipment, more than % was provided by the graduate college and the vice president for research, with the other % obtained through equipment grants and user fees. of the operating expenses for the facility, % are recovered from a large and well-funded group of investigators who are charged on an at-need, fee-for-service, first-come, first-served basis. faculty recognize that they have a facility available to them that provides immediate access to state-of-the-art equipment and techniques, as well as one that provides training and supervision. in addition to supporting research, the cemrf offers four formal courses, as well as assisting with sections of eight other classes. the facility annually organizes at least two workshops and provides about tours for visiting scientists, faculty and student recruitment, high school students, business groups, and politicians. the facility also serves as the business office for the iowa microscopy society. concurrent with the development and success of the cemrf, several departments voluntarily divested themselves of their own electron microscopy (em) equipment ( ems on campus in compared with ems in ). this represents a savings of more than $ , annually ( dollars), as well as the release of or more rooms for other use. in addition, the interaction between the remaining six em laboratories is more positive as a result of this downsizing and centralization. the availability of user-friendly quality equipment in the cemrf assures that em is now accessible to all university faculty, staff, and students. in addition, with many investigators sharing instruments, it is easier to justify acquisition of new equipment for the cemrf. accurate dimensional metrology of the submicrometer gold absorber structures of x-ray masks can be accomplished in the scanning electron microscope (sem) with the use of electron beam modeling (postek ) . accurate metrology is possible because the x-ray masks present a unique measurement object from most other semiconductor structures viewed in the sem. this occurs because the silicon support membrane is x-ray transparent by design. this characteristic can be used as a distinct advantage in electron beam-based mask metrology since, depending upon the incident electron beam energies, substrate composition, and substrate thickness, the membrane can also be essentially electron-transparent. the areas of the mask where the absorber structures are located are essentially x-rayopaque, as well as electron-opaque. viewing the sample from a perspective below the mask by placing an electron detector beneath the mask provides excellent electron signal contrast between the absorber structure and the base membrane. the present technique utilizes a broad acceptance angle detector which is different in concept from other transmission electron (te) detectors used in the sem. in this case, the broad angle is used to detect as many of the transmitted electrons as possible (i.e., whether scattered or not) that have an energy above some predetermined threshold which is usually several kiloelectron volts. then, the electrons are physically filtered both by the signal threshold characteristics of the detector and an by an electron energy filter in front of the detector. energy filtering of the transmitted electrons excludes the highly scattered and thus lower-energy electrons from entrance into the detector. this greatly improves the contrast level over the conventional transmitted-electron detection mode for this type of application, and greatly simplifies the required electron beam/sample interaction modeling necessary for edge determination. it is, in fact, this change in electron detection philosophy that makes the present te approach so attractive for dimensional metrology and inspection of x-ray masks. monte carlo modeling of the transmitted electron signal was used extensively to support this work in order to determine the optimum electron detector position and characteristics, as well as to determine the position of the edge in the image profile. the monte carlo modeling is more accurate in this work, in contrast to the secondary electron detection mode, because in the transmitted electron detection mode the modeling of the electron beam/specimen interaction becomes far less difficult than in the modeling of typical secondary electron images of other opaque objects. the generated low-energy secondary electrons (which are complex to model) are excluded from the detector and, therefore, do not need to be included in the calculation. combining all of these factors provides a modeled signal that is extremely sensitive to wall slope. wall slope variation can result in large differences in the modeled profiles. the comparison between the data from the theoretically modeled electron beam interaction and actual fitted experimental data is shown in figure for a wall angle of ˚. the theoretical profiles were shown to agree extremely well with experiment, particularly with regard to the wall slope characteristics of the structure obtained from the sem images and video profiles. a plateau in the transmission is seen in the modeled profile as the beam traverses the edge, which can be used to identify the loca-tion of the edge of the absorber line and thus allows accurate measurements to be made. this work provides an approach to improved x-ray mask linewidth metrology and a more precise edge location algorithm for measurement of feature sizes on x-ray masks in commercial instrumentation. the transmitted electron detection mode is also useful in both mask inspection and mask repair, because the high contrast of the image allows for rapid determination of mask defects and high-density contamination particle detection because the transmitted electrons simulate transmitted x-rays. this work represents the first time that electron beam modeling has been used to determine the accurate edge location in an sem image. this also represents an initial step toward the first sem-based accurate linewidth measurement standard from nist, as well as providing a viable metrology for linewidth measurement instruments of x-ray masks for the x-ray lithography community. postek using monte carlo models that take into account the gaussian beam width of the incident electron beam, a noticeable broadening of the linewidth measurements is simulated for aluminium linewidths under glass. this simulated effect correlates closely with measurements of linewidth using energy dispersive x-ray analysis of an aluminium ka line under glassivation. detailed simulation of a full linewidth measurement has been performed using a point source and gaussian incident electron beam for the multiple scattering and single scattering models. the modeling effort was undertaken to better understand the effect that incident electron beam width would have on the accuracy of scanning electron microscope measurements. the multiple scattering (ms) model was adapted from pas-cal code for a single material to simulate layered materials, specifically, sio over aluminium. further, application-specific code was added to simulate an electron beam scanning across a linewidth on a fabricated microelectronic circuit. the linewidth selected for these simulations was one micrometer and one micrometer thick with a selectable sio thickness for the overcoat. in this case, the sio thickness was . micrometers. the single scattering (ss) model was adapted also from available pascal code for layered materials with the same application-specific code used in the multiple scattering model. the differences in the results from the two models used for the simulation are shown in figures and . the results for the ms and ss models at k trajectories are shown in table i . the gaussian beam width was selected to be . micrometers. the gaussian beam noticeably broadens linewidth measurement over point source, based on the results in table i . the linewidth measurement was taken at fwhm by extrapolation between points. the large differences between the ss and ms models is under further study. the calculation of the image contrast from samples with surface topography can be done using monte carlo techniques as long as the electron trajectories can be calculated through a surface profile. the image simulations that are described here were done using the same methodology that has been applied to the calculation of the electron backscattered signal from samples where the composition variation is taken into account (ly and howitt and these proceedings) . the design of the program is such that the scattering cross sections are reassessed as the electron passes from one region of the specimen into another, which in this case includes free space. the program takes longer to run than that for a homogeneous specimen with a flat surface because the parameters, such as the atomic number and atomic weight and density, need to be constantly updated at each point in the calculation. depending upon the image resolution that is required, we divide the three-dimensional specimen into blocks of either specimen or vacuum. the modifications to the conventional monte carlo approach to such calculations include not only the specimen geometry but also the determination of the energy of the electron that is backscattered and the direction in which it travels. in this way the signals at the backscattered and secondary detectors can be distinguished because almost all the low-energy electrons find their way to the secondary electron detector, whereas only those in the line of sight to the backscattered detector contribute to this signal. in most practical situations, where the geometry of the specimen is difficult to predict, it is useful to have specimens of standard shape, such as a sphere cone or box, to compare directly with the images. the calculation of the image at the secondary electron detector for spheres of the same size but of different atomic weight is shown as an example in figure . the spheres are assumed to be on a graphite substrate and the electron-beam energy is kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. we have also found that pseudo color images, where the various gray levels are replaced with different colors, are very useful when it comes to comparing calculated and experimental images. calculated secondary electron images from nm spheres of silicon, titanium, nickel, and molybdenum on a carbon substrate at an electron beam energy of kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. pierre hovington, dominique drouin, raynald gauvin, david c. joy * department of mechanical engineering, university de sherbrooke, sherbrooke, québec, canada; * em facility, university of tennessee, knoxville, tennessee, usa quantitative analysis of resolution < nm can be achieved at low accelerating voltages. however, to exploit all the emitted signals (x-rays, secondary, and backscattered images) fully, specialized programs have to be used. in this paper we present some results that can be achieved with such programs. it is shown that our single scattering monte carlo program can effectively predict the effect of surface oxidation on aluminum (al), model the backscattered coefficient even at very low energy (< kv) and accurately predict the backscattered profile around a spherical inclusion. at low voltages, mott elastic cross section has to be used. the tabulated mott scattering cross section of czyzewski et al. ( ) , combined with the energy loss of joy and luo ( ) , is used as the physical basis of the program. hence, the program can be used accurately for simulation even at very low energy (e < kv) (fig. ) . numerical experiments can be made with one or several regions of different composition and shapes. the regions can be defined as horizontal (i.e., layered samples) or vertical (i.e., grain boundary). in addition, based on the results of gauvin et al. ( ) , spherical inclusions can also be modeled. the program is written in c language and makes use of a fully graphic environment both for input and output of data. in figure , we present a typical output of the program of a simulation on a multilayer electronic component [a nm ga ( % at ) al ( % at) as ( % at) on a gaas substrat] covered with nm of contaminated carbon. the effect of the backscattered electrons on the resolution can be clearly determined since their paths are marked with a different color. in addition, at the end of the simulation, the interaction volume ranges at , , , and % are plotted. to increase the range of applicability of our program, backscattered, secondary, and x-ray images (k, l, and m lines) can be generated. generated and experimental images can then be used for metrology and microanalysis. we present in figure a backscattered linescan of a nm diameter beam (eo = kv) with a hypothetical nm diameter hemispherical mns inclusion in a fe matrix. it is important to note in figure that the difference between the "screen dimension" and the "real dimension" is over % . in figure , we present an experimental backscattered profile taken across an mns inclusion in steel at kv. we note the similarity of the theoretical and the simulated backscattered profile (slight decrease between the center and the end of the inclusion and the presence of peaks at the interface between the matrix and the inclusion). iv- scanning vol. , supplement iv ( ) fig . when low voltages are used, the geometry and composition of the surfaces greatly influence the resulting signals. in figure , we present an experimental spectrum from a pure aluminum (al) sample taken at kv with a windowless eds detector and a theoretical generated spectrum (hovington et al. ) . the difference between the two spectra is mainly due to the presence of oxide (al o ) at the surface of the sample. in figure , we compute the j(rz) curves for an oxide thickness of and nm and for a nonoxidized sample. the decrease of the al k intensity for the and nm compared with the nonoxidized sample is and %, respectively. the experimental to theoretical ratio found on the spectra (fig. ) is approximately %, indicating that the oxide layer is < nm thick. it is important to note that because the standard and the unknown may not have the same thickness of al o , the use of theoretical stan-dards may become critical in quantitative x-ray microanalysis at low voltages. material investigation methods based on the interaction of the electron beam with a solid requires detailed information about the electron distribution. among the various approaches which allow one to calculate the electron distribution, the monte carlo simulation seems to be most suitable. this is due to the fact that the monte carlo method generally can be applied to any target. the main problem of this approach is the accuracy of the electron distribution approximation. one important criterion of the quality of the approximation is the coincidence of the calculated and experimentally observed x-rays in depth distribution. note that the correct calculation of this function plays the essential role in epma data interpretation. as have been shown in the monte carlo process developed by murata et al., which is based on the mott cross section for elastic and on a knock-on model for inelastic interactions, it provides a rather good agreement between the calculated and experimental φ(ρz) function for cdlα line in al and au targets at the electron beam energy e = kev. but a more careful comparison of φ(ρz), obtained according to this model with the experimental data for sikα in al, ni, ag, and au at e = , , kev, respectively, and cukα at e = , kev, reveals some discrepancy, especially in the tail part. this discrepancy increases when the atomic number grows and the electron beam energy decreases. the origin of this discrepancy can be connected with the errors of the approximation of the differential cross sections for elastic and inelastic interactions, as well as with the ionization cross section. as the mott formula for elastic cross section uses the model of the atomic potential, then the approximation of this potential can influence the results of the scattering. to avoid the errors connected with the choice of the atomic potential approximation, we have used the elastic differential cross section obtained by riley et al. with the help of the static approximation theory (sat). the main features of the developed monte carlo program are as follows: . the elastic interaction is calculated according to the sat cross-section . the energy dissipation process is described by the knockon model. the bethe formula for de/ds is used in the high-energy region (e > . j i ). in the low-energy region (e < . j i ), the equation used in [ ] is exploited instead of the bethe law . the fast secondary electrons produced by electron-electron interactions in an inelastic collision are taken into account . the mean ionization potential is chosen according to the berger and seltzer formula . to calculate the ionization cross section, the formula of gryzinski is used. we have calculated the energy and angular spectra of the electrons transmitted through the films of the various compositions and thicknesses. comparison of these results with the data obtained with the help of the mott cross section shows that the use of the sat approximation for the elastic interaction does not influence essentially the electron distribution and cannot improve the accuracy of its determination. the comparison of φ(ρz) functions which are found in accordance with these two models confirms this conclusion. therefore, one can state that the details of the mott cross section together with the errors in atomic potential approximation cannot influence the electron distribution in targets. the factor which can affect the φ(ρz) in targets independently of electron distribution is the ionization cross section. to take into account this factor, we have used the experimental results obtained by long et al. during differentiation of the heart there appears to be a sequential pattern to the formation of individual muscle fibers. phenotypic changes result in the expression, synthesis, and organization of complex proteins to form the terminally differentiated myocytes. the formation of the basic pattern of myofibers ultimately determines the physiologic performance of the final form of the heart. pattern formation involves both intracellular events associated with myofibrillogenesis and extracellular events associated with cell:cell and cell:matrix interactions. this basic pattern is repeated to form complex layers which results in the final form of the heart. it is essential to understand the sequence of expression associated with these patterns of fibers in order to understand errors that may result in congenital defects in heart formation. in this study time pregnant ( . - . days of gestation, ed) sprague dawley rats were obtained from harlan sprague dawley laboratories (indianapolis, ind.). animals were anesthetized with % chloral hydrate in normal saline solution and the individual embryos, including uterine tube, were removed and placed in . m phosphate buffered saline solution containing . % azide (pbs-a) at °c. the embryos were removed from the uterine tube under a dissecting microscope and fixed for - h in % paraformaldehyde in . m hepes (n- -hydroxyethylpiperazine-n'- -ethanesulfonic acid) at ph . . the embryos were scored and gestational ages were assigned. the embryos ( . - . days of gestation) were stained using a modification of the procedure described by tokuyasu and maher ( ) . after fixation, the embryos were placed in pbs-a for h and immersed in . % triton x- in pbs-a for h. after washing for an additional hour in pbs-a, the embryos were incubated in units/ml bovine testicular hyaluronidase for min, washed in . % triton x- with . m glycine in pbs-a for h and immersed in % bovine serum albumin (bsa) in pbs-a for h. the individual embryos were incubated overnight with rhodamine-labelled phalloidin from molecular probe, eugene, ore. ( µl of stock solution/embryo), or control buffer. following incubation, the embryos were washed in pbs-a at °c for h and immersed in % bsa for h. hearts from . - . day gestation embryos were removed and fixed as above. after fixation, the embryos were encased in % polyacrylamide gel using a modification of the procedure described by hansen and dryer ( ) . after fixa-tion, the hearts were washed in pbs-a for h and immersed in . % triton x- in pbs-a for / h. the hearts were embedded individually in blocks containing % polyacrylamide with . % bis-diamine which was polymerized with % ammonium persulfate. the polyacrylamide blocks containing the hearts were sectioned at µ with an oxford vibratome model g before being stained as described above. the tissue was examined using a bio-rad mrc- confocal laser scanning microscope (biorad, cambridge, mass.) using × (na= . ) and × (na= . ) objectives. at ed . to . , the cells of the myocardium are round and tightly packed with no discernable pattern. the first indication of cell orientation into fiber patterns occurred in the outflow tract and ventricle. the outflow tract myofibers developed circumferentially and maintained this pattern throughout the study time. the ventricular myofibers also developed circumferentially; however, they gradually changed into a net-like pattern ( fig. ) followed by a thickening of the ventricular wall and protrusion of trabeculae. with continued maturation, the ventricular trabeculae appeared to coalesce especially in the re-gion of the muscular interventricular septum (fig. ) . the myofiber pattern developed later in the atria. the atria expressed the same early pattern seen in the ventricles; however, they retained the net-like appearance throughout development. the results indicate that there are region-specific differential changes in the orientation of myocytes that result in the unique myofiber patterns of the heart. these regional pattern changes may be correlated with mechanical forces and hemodynamic alterations during development. the preparation of thick, optically clear sections of fragile tissue structures greatly aids the power of confocal laser scanning microscopy in imaging three-dimensional structures. hale and matsumoto ( ) have presented confocal images of agarose-embedded, vibratome-sectioned tissues; earlier workers have embedded soft tissues in polyacrylamide gels for frozen sectioning and lectin or immunohistochemical staining of structures in - micron sections (bronson et al. , hausen and dreyer , johnson and blanks . we report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. in general, infiltration of - mm specimens for - h in a mixture of - % acrylamide monomer ( part bisacrylamide cross linker to parts monomer) yields, upon polymerization (with . volume of % ammonium persulfate), blocks that cut easily by vibratome between - microns. these conditions work well for tissues previously stained (with fluorescent probes or dii tattoos) or for staining gel sections water-soluble fluorochromes with low molecular weight (e.g., propidium iodide, phalloidin). for immunostaining after sectioning, the acrylamide concentration must be reduced to - % acrylamide to facilitate access of immunoglobulins to antigenic sites, and the gel must be supplemented with % agarose to aid sectioning and handling. illustrated below are confocal images from acrylamide sections of a stage chick embryo, fluorescence-immunostained in whole mount for cadherins. this specimen was fixed in % dmso/ % methanol (dent and klymkowsky ) and incubated overnight with a : dilution of commercial antiserum against a synthetic peptide common to all known cadherins (sigma, pan cadherin, #c ). rinsing preceded overnight reaction with tritc-conjugated secondary antibody, brief formaldehyde fixation, and embedment in % acrylamide. a survey ventral view was taken before transverse vibratome sectioning across the entire embryo at micron intervals, yielding eight sections that were spanning the thoracic region and were mounted in % glycerol for closer inspection. figure illustrates a stereo pair of projections from a series of confocal images across the atrioventricular junction. this gel-embedding and vibratome-sectioning method yields abundant, optically clear, and easily handled sections for confocal examination of fluorescent structures in water-miscible media. greater detail concerning procedures and technical problems with this technique are provided elsewhere (germroth et al. in press) . department of anatomy and cell biology, suny health science center, syracuse, new york, usa endothelial cells arise in the early embryo from precurser cells called angioblasts. in the quail embryo, the emergence of these cells can be observed as an epithelial to mesenchymal conversion of cells from the mesoderm which may be observed by scanning electron microscopy (sem) after removal of the endoderm or by immunolabelling the whole embryo or sections with a monoclonal antibody (qh- ) which labels angioblasts. the process of angioblast migration and assembly into the solid cords, which are the rudiments of the earliest blood vessels, is called vasculogenesis. simple embryological manipulations have been used to distinguish the role of cell migration in the early vessel rudiments. the dorsal aortae arise ventrolateral to the forming somites, and inserting blockages revealed that little cell migration is involved in their formation. the endocardial cells which form the endothelial cell lining of the heart, in contrast, undergo an extensive migration from more lateral regions of the embryo which has been studied by blockages and the construction of quail/chick chimeras. iv- scanning vol. , supplement iv ( ) the first rudiments of veins in the embryo are the cardinal veins. these form beneath the ectoderm of the embryo in an area with few angioblasts. the early stages of cardinal vein formation have been studied by using immunogold-labelled secondary antibodies after qh- labelling and imaging in the sem backscatter mode after silver enhancement. these studies revealed that small groups of angioblasts appear over the lateral mesoderm, possibly by migration from the angioblastrich mesoderm adjacent to the endoderm, and then migrate medially to assemble into a solid cord of cells in association with the developing wolffian duct. the ability of scanning probe technology to image atomic topography of a surface, to manipulate individual atoms, and even to probe the internal structure of a molecule confirms the significance of these super-resolution microscopies used at the nanometer-scale analysis of molecular systems. so far, a number of interactions between the sample and the probe tip gives access to a variety of local properties (wickramasinghe ) . the latest applications of the local probe microscopies on organic molecules include photoemission, tunneling potentiometry, electrostatic, elastic and tribo logic properties, or near-field thermal measurements and as many other phenomena on which various contrast formation mechanisms are based. topics include imaging of individual biomolecules, highly ordered molecular assemblies, and polymeric materials under various environments. the development of additional imaging techniques,based on near-field electromagnetic interaction between the probe tip and the surface (betzig and trautman ) , or even by means of magnetic resonance (rugar et al. ) , indicates that new technologies with subnanometer spatial resolution could be achieved in principle. recent advances in near-field optical microscopy (nsom) confirmed spatial resolution in the range of - nm but still limited by the diameter and optical penetration depth of the aperture. here, we demonstrated a new concept for an aperture near-field scanning optical microscope which combines force microscopy and optical scattering for imaging the sample at subwavelength resolution without the use of an aperture. the end of a silicon tip is illuminated in transmission mode by a laser beam through a transparent substrate. both the tip and the sample undergo perpendicular motion, each at a different frequency, with amplitudes chosen comparable to the desired measurement resolution. the scattered light from the tip and the surface is detected at the difference frequency for imaging and sample at sub-wavelength resolution without the use of an aperture. we describe the novel experimental scheme and present the most recent results obtained from our system. the resolution demonstrated to date is nm using helium-neon wavelength and optical line scans are shown in figure . finally, a consideration of the basic theory demonstrates that much higher resolution can be easily anticipated. these preliminary results firmly establish the great potential of this new near-field optical microscopy for biological research. high-resolution scanning electron microscopy (sem) studies of enamel crystals from remineralized enamel have provided clues as to the changes in crystal diameters within specific zones of artificially created carious-like lesions. , these data have supported the evidence obtained through polarized light microscopy of zones of demineralized and remineralized enamel. the dark zone, viewed in polarized light, has been identified as the zone of remineralization. these initial studies were conducted using sputtered coatings of gold/palladium (au/pd). high-resolution images of the crystals within the zone of remineralization revealed large crystallites in excess of µm, often with what appeared to be remineralized "nodules." however, it was difficult to determine whether these were one crystal, or two crystals which had fused during the remineralization phase. a study was conducted using an ultrathin coating of sputtered chromium (cr) film and se-i imaging to provide topographic contrasts of crystal surfaces. since the majority of the se-i information should be produced close to the surface, the resultant image is specimen-surface specific. , artificial caries-like lesions were prepared in noncarious human teeth (second and third molars) and sectioned into µm sections which were then viewed in polarized light to identify the dark zone of remineralization. the sections were then fractured through the dark zone producing a spicule of enamel with an edge of fractured enamel free from sectioning artifacts. each specimen was then mounted onto supports with silver paste with its fractured edge facing up. measurements were then made to determine the location of the dark zone so that it would be easily located within the sem. specimens were degassed and then sputtercoated with nm au/pd and nm cr. , specimens were imaged at high magnification in the se-i mode by placement on the condenser/objective (c/o) lens stage of an isi ds- /lab sem at kv and a hitachi s- cold cathode field emission (fe) sem operated at kv. high magnification se-i images (s- fe sem) of au/pd coated enamel crystal surfaces within the dark zone, revealed particulate features decorated by metal giving it a continuous granular appearance (fig. ) . analysis of a specimen coated with a . nm cr film revealed crystal surfaces with well defined particulate features that were clearly delineated and separate from one another. this study documents the effect au/pd coating has upon the examination and evaluation of enamel crystals at high magnifications. results obtained document that an ultrathin coating of sputtered chromium film of - nm does not produce the coating artifacts found with conventional sputtered au/pd. an ultrathin coating of cr, together with the use of se-i imaging, allows surface features to be more accurately imaged and measured. iv- scanning vol. , supplement iv ( ) fig . previous results have shown that the ability of intercalative dyes to modulate the antiviral activity of poly r(a-u) was related to the groove through which the dyes intercalated into the poly r(a-u). when poly r(a-u) was combined with the minor groove intercalating dyes such as ethidium bromide (eb) or the minor/major groove intercalating dyes, optimum enhancement of antiviral activity was observed at a dye/ribonucleotide ratio of / . no enhancement of antiviral activity was observed when poly r(a-u) was combined with the major groove intercalating dyes. when eb was combined with poly r(a-u) and then added to human foreskin fibroblast (hsf), the % effective dose of the poly r(a-u) was -fold lower. the results of additional studies demonstrated that the enhanced antiviral activity was not due to superinduction of interferon, direct viral inactivation, or host cell cytotoxicity. phase contrast, fluorescence, and confocal micrographs of hsf cells following a -h exposure to µm eb alone or a µm eb/ µm poly r(a-u) combination stained the nucleolus, but not the chromatin. negatively stained transmission electron microscopy (tem) preparations (fig. a) and replicas (fig. b) (fig. a,b) which may be associated with the enhanced antiviral activity of this combination. under the conditions employed in this study, poly r(a-u) exhibits an elongated conformation ( - nm in length) that possesses a number of hairpin loops as well as single-stranded and double-stranded domains (fig. a,b) . the double-stranded domains are found predominantly at the base of nm hairpin loops. in contrast to the poly r(a-u) alone, micrographs of the eb/poly r(a-u) combination illustrate the presence of condensed structures with diameters ranging from to nm. results from scanning force microscopy corroborate the results of both tem preparations. tem of unstained and uranyl-stained eb/poly r(a-u)-treated hsf cells illustrate the endocytosis of electrondense material into acidic compartments of the hsf cells. subsequently, the electron-dense material escaped from the acidic compartment and formed electron-dense bodies with dimensions that closely approximate the dimensions of the eb/poly r(a-u) combination visualized in the negative staining preparations. these electron-dense bodies are detected near the nuclear membrane and in the nucleolus. the nucleolus of an unstained, eb/poly r(a-u)-treated hsf cell demonstrates the segregation of the nucleolar components so that the fine fibrillar component, which comprises the nucleolar organizer region, is being peripheral to a dense granular material occupying the major central area of the nucleolus. the results of the current study and our previous work suggest that the elevated antiviral activity of the eb/poly r(a-u) combination may be related to the ability of the eb to complex with poly r(a-u) and condense it into a conformation with dimensions that can be accommodated by endocytotic vesicles. exit from the acidic compartment is promoted by unbound eb that induces endosomal or lysosomal swelling. subsequently, changes in the topology and surface charge of the poly r(a-u) induced by the eb allow increased access to the nucleolus which results in the modulation of additional cellular processes, especially rrna synthesis and processing. aromatic constituents in plant cell walls are associated with other wall components and affect strength and other characteristics of plant cell walls. ultraviolet (uv) absorption microspectrophotometry has been useful in characterizing aromatics within specific cell types in plant organs. , , this technique was applied to walls of bran and endosperm cells in a series of hard and soft wheats. kernels from hard and soft cultivars were fixed in glutaraldehyde ( % in . m cacodylate buffer at ph . ), and sections were cut with a microtome at µm thickness. the sections were mounted on quartz slides in glycerin and covered with a quartz cover slip. cell types in the sections were analyzed for uv absorption using a computer-controlled zeiss umsp microspectrophotometry system. transmitted illumination was provided by a xenon lamp (xbo w) with a connecting grating monochromator using a bandwidth of nm. a -× quartz objective lens with a final aperture of . µm, which was delimited within about onethird of the area of a field-limiting diaphragm to reduce stray light, was positioned over walls of selected cell types. absorbance of transmitted uv illumination was recorded over a range from - nm at -nm increments. the system was standardized at µm. spectra were collected, displayed, and evaluated using the zeiss lambda scan software. uv absorption maxima or distinct shoulders occurred near , , and nm. while the absorption near nm is not defined, absorption near nm is believed to be due to lignin (i.e., polymerized phenolic constituents) and that near likely is due to ester-linked ferulic acid. synthetic lignin, from polymerization of coniferyl alcohol with horseradish peroxidase, has a strong absorption at nm and no absorption at higher nm. further, ferulic acid linked to arabinoxylans, which has been isolated from bermudagrass cell walls, gave a shoulder near nm and a strong max at nm. other work, using the addition of cinnamic acids to milled lignins or removal of phenolic esters by alkali treatment, supports the above spectral interpretations. the kernel of wheat consists of several distinct cell types (fig. ) . spectra were obtained of walls of these various cell types (fig. ) . epidermal and parenchymal walls gave the highest absorption and both had spectra indicative of lignin (near nm) and also of ester-linked ferulic acid (near nm). nucellar epidermis walls have spectral patterns similar to the cell types above, but absorption was much less. aleurone walls gave spectral patterns indicative of mostly ester-linked ferulic acid and less lignin than previous cell types; the side walls gave a considerably higher absorbance than upper or lower walls. endosperm cell walls, which enclose the starch/protein matrix, were totally lacking in uv absorption maxima, indicating no aromatic constituents in these walls. variations occurred in uv absorption and spectral patterns for walls of various cell types in wheat kernels. however, no consistent differences occurred among the various kernels that might relate to hard-or soft-rated wheats. iv- scanning vol. , supplement iv ( ) fig . food microscopists are continually searching for noninvasive methods of examining the internal structure of foods, especially those with delicate labile structures. x-radiation is of considerable potential because of the great penetrative power and the minimal need for sample preparation. examination can be carried out at atmospheric pressure, and there is no intrinsic reason why temperature-controlled or temperature-ramped studies are not possible. the term x-ray microscopy covers several techniques and can be defined as the use of x-rays to produce a magnified image, or to give information about specific microscopically identifiable parts of a specimen. our current equipment consist of an electron gun, two electromagnetic lenses (condenser and objective), and x-ray target. x-rays are generated from a µm spot on a tungsten target. the target usually is positioned so that the microfocused electron beam glances the side of the target. x-rays from the microfocal spot form a cone which diverges through a thin window. focusing is achieved by placing a grid with bar widths of and µm into the beam and adjusting the current in the condenser and objective lenses. the image is captured on a tv monitor. finally, test radiographs are recorded on x-ray sensitive film. the x-ray radiograph is a shadowgraph and, where the sample is thick, the overlaying shadows will be complicated to interpret. two methods can be used to help interpret the image. one method is to take stereo pairs of the sample and view these images with a suitable stereo viewer which will show a threedimensional image. the other method is tomography. in the projection x-ray microscope, the entire specimen is in focus at once with the same resolution, determined by the spot size, although different depths within the specimen are magnified by different amounts. three-dimensional images can be made by moving the specimen either by tilting or translating it between exposures in the divergent x-ray beam. these images are then viewed with a suitable stereo viewer which will show a three-dimensional image of the structure removing some of the confusing overlay of detail to be found. we have also obtained images from pieces of tissue subject to tensile elongation. in the design of our x-ray microscope there is considerable distance separating the x-ray film from the specimen, and this space allows the apparatus for tomography to be set up. tomography is a form of imaging whereby a section or "tome" can be taken through a specimen by using x-rays, and details above and below the sectional area of interest will be blurred out, giving a fairly clear image of this area. this technique is nondestructive to the sample. the method is similar to that de- scribed by lindegaard-andersen and thuesen, originally proposed by watson. in their method, the x-ray source is stationary and the subject and recording film are rotated synchronously. the x-ray beam strikes the film at near glancing incidence producing transaxial summation images. however, the associated point spread function has a /r radial dependency, which means that smearing of detail will have occurred. the results are sufficiently encouraging, nevertheless, to stimulate the search for improved contrast and resolution, with provision for three-dimensional mapping of internal structures. the images shown here are of food products containing large air cells, giving maximal contrast in the x-ray beam. we have obtained comparable success with water-filled cellular structures such as vegetable and fruit tissue. in the future, we anticipate further enhancement of contrast and resolution by the use of improved x-ray flux, image capture, and image processing. centre for food and animal research, agriculture and agri-food canada, ottawa, ontario, canada over the past years, electron microscopy (em) has proven to be a useful tool in assessing the effects of various physical and chemical treatments on the microstructure of egg components (related to the production of egg products, baking, behaviour in various food systems, nutrient bioavailability, etc.) much of this information has been acquired using transmission electron microscopy (tem) to view yolk samples which have been biochemically extracted after removal from the egg. the author studied microstructural changes in yolk during its in vivo digestion by the epidermal cells of the chick embryo yolk sac. yolk is digested within these cells, as signified by its presence in varying degrees of microstructural alteration, compartmentalized within cell organelles. a temporal sequence can be inferred with the degree of microstructural change observed. it was possible to follow the fate of yolk granules and low density lipoprotein (ldl) particles during digestion, because yolk granules retain their integrity during preparation for em. ldl particles were made visible by crosslinking action of the fixatives and by the use of a specific enhancement technique. initial work focused on the microstructure of native, undiluted yolk which had been fixed using conventional and novel fixatives. thin sections were examined by tem. these experiments allowed us to optimize fixation conditions and confirmed earlier results by other workers that yolk is composed of granules which have a subgranular structure and lack a boundary membrane. the granules sit in a suspension of closely packed ldl particles. yolk granules from incubated eggs were identical to those from unincubated eggs and showed no microstructural changes to indicate that digestion had taken place outside of the cell. after clarifying that yolk is digested intracellularly, the next phase of the study was the microstructural description of intracellular yolk and yolk sac epithelial cells during incubation, prepared using half-strength karnovsky's fixative and the imidazole-buffered osmium tetroxide protocol (ibo) of angermuller and fahimi ( ) which enhanced lipid staining. epithelial cells were examined systematically using em, from apex to base, to study the three processes associated with digestion: uptake, breakdown, and transport. using tem, granules and ldl particles were observed to enter the cells in uptake structures, the microplicae and coated pits, respectively. when samples were viewed using sem (fig. ) , these uptake structures appeared as fossae of different sizes, which have not previously been described in the literature. yolk granules and ldl particles were observed by tem, within the cells, in phagosomes, endosomes, and acid phosphatase-containing vacuoles (secondary lysosomes), components of an active intracellular digestion system as it is presently known. the secondary lysosomes contained yolk granules exhibiting various degrees of microstructural alteration (fig. ) . the microstructure of these intermediates in yolk digestion appears to be very similar to that appearing in the literature describing known biochemical manipulations of yolk ex ovo, and indicates that a re- iv- scanning vol. , supplement iv ( ) fig. sem of apices of yolk sac epithelial cells from an embryo incubated for days. yolk granules have entered the cell, and a fossa (f) is observed. mp = microplicae. lationship may exist among structural, functional, and biochemical information. the results of these individual experiments were used to propose a scheme of yolk digestion based on progressive microstructural changes of intracellular yolk. in addition, a transport system for yolk lipid and its digestion products to the embryo was microstructurally demonstrated using ibo. this transport system shared ultrastructural characteristics with that reported for lipid transport in intestinal cells, especially with respect to the formation and transport of chylomicronlike particles. laser scanning confocal microscopy and digital image processing were used to visualize the pattern of de novo blood vessel formation in the quail embryo. stage quail embryos were fixed on the yolk, excised, then immunolabeled with qh , an antibody marker for angioblasts and vascular endothelial cells . after incubation in fluorescein-conjugated secondary antibodies, the embryos were mounted in pbs/glycerol ( : ratio) under a # glass coverslip. specimens were examined on a biorad mrc confocal laser scanning device equipped with zeiss optics. a × objective lens was used to image the entire caudal half of each embryo during the laser scanning step. ten µm optical sections were acquired in a plane parallel to the embryonic axis (en face). the ten optical sections were then collapsed into one composite image using biorad's proprietary software (fig. ) . the image was imported into photoshop ™ software on a macintosh quadra . using this software, the single composite fluorescence image was processed with the "emboss" tool (fig. ) . other image processing routines such as edge tracing, sharpening, and contrast enhancement can also be applied with ease (not shown). in contrast to conventional microscopy, this procedure yields a map of the entire endothelial network of the quail embryo in sharp focus, and with a highly favorable signal-to-noise ratio (fig. ) . while some geometric distortion occurs when ten sections are collapsed into one imaginary plane, the flat- ( ) and ldl particles ( ) are found in separate organelles, and are also found together within the same organelle ( ) . myelin bodies (m) and lipid drops (l), products of intracellular digestion of yolk, are also observed. fig . this immunofluorescence image depicts a map of all the vasculature elements in the caudal half of a developing quail embryo (stage ). the wide vessels on each side of the embryonic axis are the dorsal aortae; lateral to the aortae are two fields of rapidly forming vascular networks. tened nature of the early avian embryo minimizes this problem. also, since the qh epitope (a carbohydrate) is present on multiple cell surface molecules, the image gives a reasonable approximation of vessel morphology and the protrusions of the vascular endothelial cells. the rendering of the data into a pseudo three-dimensional relief provides the observer with a more familiar visual format (fig. ). the type of image shown in figure facilitates comparison of endothelial sprouts and anastomotic foci during formation of the first vasculature in the quail embryo. based on images from embryos at progressively older developmental stages, we suggest that morphogenesis of the lateral anastomotic network occurs by mechanisms that involve angioblastic tractional structuring of the extracellular matrix (madri et al. , montesano et al. , stopack and harris . this hypothetical mechanism more recently has been elaborated upon by vernon and colleagues ( ) . according to the latter workers, cellular responses to morphogenic cues within the highly planar extracellular matrix underlie early vascular patterning. in addition, the digital imaging approach shown here offers an improved method of comparing normal vasculogenesis with experimentally produced malformations (drake et al. ) . ( . - . ). chemical composition and ph did not change during extended heating. the shmp sample was the firmest and the sc sample was initially the softest. the firmness of the sc and tsp samples increased with extended heating whereas the firmness of the dsp sample did not change. meltability decreased with extended heating in all samples, except the shmp sample which had the lowest meltability of all samples throughout the experiment. there were marked differences in the microstructure of the process cheese protein matrices after heating. osmiophilic areas gradually developed during heating and their incidence was related to the melting salt used. after h of heating, they were numerous but considerably smaller in the dsp sample ( fig. ) than in the tsp sample (fig. ) . the highest and the lowest incidence was in the shmp and the sc samples, respectively, which were the samples with the lowest and highest meltability. new york, usa mozzarella cheese contains parallel protein fibres created by stretching the curd in hot water during manufacture. the protein fibres are oriented parallel to the direction of extrusion and are separated by milkfat or whey (kaláb , taneya et al. . by varying manufacturing parameters and storage time, cheeses with different sensory/functional properties, as well as with different microstructural characteristics, can be produced (kiely et al. ). in the present study, the impact of varying the stretching temperature ( °c, °c, and °c) and storage time ( days, weeks, and weeks) at °c on the microstructure of mozzarella cheese was investigated using light microscopy (lm) and scanning electron microscopy (sem). for both lm and sem, samples were fixed in % glutaraldehyde in mm pipes containing mm cacl , before cryosectioning ( µm sections). sections were cut both parallel to the protein fibres (longitudinal) and across the fibres. for lm, sections were stained with ice-cold oil red o and methyl- ene blue. because the fat is not well fixed by aldehyde fixation, slides were held on a bed of ice during staining to prevent fat migration. for sem, the sections were stained with uranyl acetate in methanol, washed in methanol, and stained with lead citrate, dried, mounted on aluminum stubs, and gold-coated. this procedure removed most of the free fat and water/whey, allowing the association of the protein fibres to be clearly visualized. increased stretching temperature increased the size of the fat globules, which was particularly noticeable in cross sections. with °c stretching, the fat globules were relatively small and uniformly distributed across the section, while with °c and °c stretching there were almost two populations of globules: some small and some very large. in longitudinal sections, the protein strands formed during stretching at °c and °c were slender and smooth, while those formed during stretching at °c were thicker and less regular. little difference was seen with storage time in either longitudinal or cross sections for the three different stretching temperatures using lm. with sem, however, after removal of the fat, the ability of the fibres to hold together decreased with storage time. this is best seen in longitudinal sections, where the protein fibres are closely associated in cheese stored for days (fig. ) , and are able to withstand the stress of drying, while in cheese stored for weeks (fig. ) , the fibres have been weakened (presumably by proteolytic activity from coagulant and culture enzymes) and pulled apart as the section dried. indian dairy association, sector-iv, r.k. puram, new delhi, india milk is a canvas of seemingly silent molecular structure. there is a large variety of "resident molecules" with molecular harmony in milk which are of mammary gland origin. in general, the "structural matrix" of milk comprises three classes of biomolecules: molecules in suspension (casein micelles), molecules in solution (lactose, proteins, vitamins, and salts) , and molecules in emulsion (fat globules). because of its excellent nutritive composition, milk is ubiquitous and the most popular "ready-to-consume" food from time immemorial. today, in iv- scanning vol. , supplement iv ( ) fig . different parts of the world, milk from various species of animals is used for food. in the u.s., however, the cow furnishes virtually all of the available market milk, whereas in my country more than % of the total milk produced is of buffalo origin. hence, structural studies particularly on milk and milkfood products made from various animal species are more fascinating and challenging from a global angle. it is the local consumer habits that determine the final structural orientation of the product in the country of origin, especially in a country such as india where to % of milk produced is converted into a variety ( plus) of traditional milk products, using processes such as coagulation (heat and/or acid), desiccation, and formulation. hence, a presentation on the "structural style" of milkfoods which are developed on the basis of "environment-friendly green technology" in the tropical countries might generate a new dimensional need for future studies on milkfood products. a comprehensive literature scan of published papers in the areas of "food" and "food structure" during the last years reveals the following publication status: during the period to : . global: of the total number of , food articles published, , are related to "food structure." the number on "food microstructure" is . when i think of their contributions, i claim no merit for my presentation in this conference today. these data provide a "food-for-thought" why food structural studies do not receive the desired attention. we may ponder this issue. while preparing this presentation, the excellent review article by kalab on food structure and milk products was extensively consulted. keeping in view the existing and reported studies on the subject, the situation in the tropical countries, particularly regarding indian milk such as buffalo milk, deserves special attention. according to kalab the structure of milk and milkfoods determines their properties such as firmness, spreadability, elasticity, viscosity, and susceptibility to syneresis, which are globally recognized as texture. the understanding of the processes both conventional and traditional and their relationship between "structural style" and "textural mood" is important in product development. in the tropical countries, the preprocessing exposure of milk to the environment, that is, temperature, microbes, and humidity has to receive extra attention. the dynamic status and kinetics of molecular interaction in different species of milk should be a vital area of structural studies in asian countries. the structural studies on milk and milkfood are generally conducted by use of microscopy. the methods used are optical microscopy such as fluorescence microscopy and confocal scanning laser microscopy, and electron microscopy consisting of two major types: scanning electron microscopy (sem) and transmission electron microscopy (tem). these techniques meet the particular requirements of the study. the application and findings derived using sem and tem in various milk food systems have been extensively covered recently by kalab . repeating and reproducing the published data may not be necessary for the participants in this conference. however, a brief microcosm might be refreshing, and is hence documented below: . milk casein micelles ( - nm), being small, cannot be seen using a light microscope, but tem shows them as globules that are apparently composed of submicelles. . the casein micelles, if deprived of their stabilizing factor, aggregate and form a gel of a regular structural "crowd." . in the case of acid-gel, the micelles start disintegration as a result of the solubilisation of their structural partners such as calcium phosphate. . there is an interstructural and intrastructural relationship between the suspended casein micelles and soluble blactoglobulins. heat disturbs their stability equilibrium and their molecular relationship. . among the milkfoods, cultural milk products like yoghurt are highly influenced by heat in relation to their "set-style" and "stirred-style." the indian counterpart, "dahi," offers its advantage when made from buffalo milk because of its high curd tension due to high calcium level. this area deserves a close look using sem technique. . as far as cheese is concerned, because of its inherent multinatural processing steps using chymosin on one hand and starter culture on the other, it is an evergreen exploratory platform using either sem or tem. . dried milkfoods as spray-dried milk particles are globular of larger diameter fat-globules, however, they undergo some changes as revealed by sem studies. lactose plays a commanding role in a so-called instantisation process. while making a presentation before a "highly-structured" qualified scientist using sem and tem, it is tempting for me to present our own work of limited nature. in our laboratory, during the last decade, we carried out a sem study on buffalo milk and its casein. the micelles are of larger size and have higher-bound calcium unlike cow casein micelles. regarding the casein fractions, an elaborate study on the b-casein in relation to structure and function as affected by heat and enzymic cleavage were carried out. work relating to microstructure development on another indian milk product, namely "paneer," from buffalo and cow milk has been reported. more recently, while working on casein micelles in csiro dairy research laboratory in australia, the author made an original contribution when ascertaining the micellar integrity of casein micelles in milk. he used a very simple technology not dependent on sem or tem. it was on the "colour communication" property of molecules as an integrated system or in isolation. using this new technique, it is possible to declare the miscellar integrity and to identify milk from different animal species, based on the different miscellar structure-dependent light-reflecting phenomena. the time has come to develop a "global strategy" on milk structural studies with a view first to understand the local product and then to ensure marketing of "rightly-structured milkfoods" of the right type to meet the countries' marketing needs. starch is a major component of many foods and is commonly used to modify the texture of a system. starch gels are composites of swollen gelatinized granules embedded in a continuous amylose network. the rheological characteristics of the starch pastes or gels depend on the shape and swelling power of the granules, amount of amylose and amylopectin leached outside the granule, network entanglement, and interaction between the paste components. many food systems also include in their formulations a lipid phase, thus, starch-lipid interaction becomes a major concern in starch paste rheology. the interactions of triglycerides might be different from those of monoglycerides due to their amphiphilic character, dispersion states, and steric hindrances. triglycerides owe their characteristics to their fatty acid composition (chain length and insaturation) and distribution (davis et al. ) . the objectives of this work were to analyze the effect of different lipid phases on the swelling power of the starch granules, as well as to analyze the rheological behavior of the starch pastes (apparent viscosity and viscoelasticity). commercial corn starch (cs) (refinerías de maíz, argentina) was used as thickener. the lipid phases used were: ( ) shortening (sh) (molinos río de la plata s.a., argentina) containing . % of mono-and . % of polyunsaturated and . % of saturated fatty acids; ( ) sunflower oil (so) (molinos río de la plata s.a., argentina) containing . % of monoand % of polyunsaturated and % of saturated fatty acids, and ( ) commercial glycerol monostearate (gms) mivaplex (eastman kodak co., u.s.a.), which contained % of monoglycerides and % of diglycerides. seven percent starch suspensions with and without lipid phase ( % of sh or so or % of gms) were prepared using a modification of the method of eliasson ( ) . swelling power was calculated as the weight of sedimented gel divided by the original dry weight of starch. samples were placed on slides and micrographed in a leitz ortholux ii microscope with a photographic camera leitz vario orthomat (leitz, germany). the suspensions were gelatinized in a thermostatic bath at ± . °c under standardized conditions. a rotational viscometer haake rotovisko rv (germany) with a sensor mvip of concentric cylinders was used. the measurements were performed at °c and the transient shear stress curves (τ vs. time) were obtained at different constant shear rates (d) from to s − . apparent viscosities were calculated as τ/d ratio at d= s − . iv- scanning vol. , supplement iv ( ) table i shows the swelling power obtained from the starch suspensions with and without lipid phase. when either of both triglycerides, sh or so, was added, the swelling power values were higher than the value of the control (cs). the so, with the highest insaturation content, presented the higher swelling power. the addition of the monoglyceride (gms) led to the lowest value. the relative sizes of the swollen granules shown on the micrographs of figure confirm these observations. gms forms a helical inclusion complex with amylose, which might delay the transport of water into the granule and consequently decreases the swelling power (eliasson ) . the formation of inclusion complexes between the triglycerides and the amylose seems to be difficult because of the steric characteristics of these lipids. apparent viscosities correlated well with the swelling power (table i) ; the larger the swelling power the higher the viscosity obtained from the rheological curves at long shear times. bird-leider model (dickie and kokini ) was used to analyze the viscoelastic behavior and the shear time dependence of the different pastes: where n and m are the power law parameters and b and c are adjustable parameters related to the viscoelasticity and to the structure breakdown of the samples. a satisfactory goodness of fit was obtained (r min= . ) (fig. ) . when the swelling power increases, b parameter decreases (table i) . since viscoelasticity (b) depends mainly on the network entanglement (leached amylose) and the volume of the swollen granules, both factors should be considered to explain this behavior. because of the health risk and other environmental factors that airborne grain dust presents to the working population, our laboratory has initiated studies on the isolation, identification, and characterization of bacteria that possess multiple resistance to a series of antibiotics and other compounds (e.g., insecticides and pesticides) at the molecular level. samples of grain dust were collected from various grain elevators in the duluth-superior regions of the u.s. during the diverse growing seasons. each sample collected consisted of a heterogenous population of constituents that vary with encountered geographic, climatic, and handling differences. in addition, the geographical growth regions and the mechanism of storage of grains appear to be directly associated with the microbial flora and occurrence of toxic substances. scanning electron micrographs of the concentrated grain dusts were morphologically consistent with the observation of previous investigators. the dust from various plants consisted of a distinct assortment of particles; small husk fragments or pericarp (seed coat in case of flax) and "trichrome-like particles" were also present. numerous bacteria spores were seen at high magnification, particularly durum wheat and barley. light photomicrographs showed a heterogenous population of both gram negative and gram positive bacteria. the bacteria consisted of different shapes such as short rods, long rods, and cocci. transmission electron photomicrographs revealed the isolated strains to consist of one or more flagella attached to the membrane surfaces. at least three of the bacterial strains isolated were encapsulated. we exposed, selectively, twelve of the isolated bacterial strains to a variety of antibodies, pesticides, and insecticides. as a result, of the bacterial strains tested showed resistance to both ampicillin and bacitracin ( µl/mg). of the strains, showed resistance to insecticides (sevin) and the pesticides (enforcer) as high as µl/mg. three of the five strains that were resistant to both ampicillin and bacitracin were also resistant to the insecticide sevin at high concentrations ( µl/mg). these data suggest that bacteria found in grain dust may be directly or indirectly related to occupational health disorders. chlordiazepoxide (librium) is a commercial antianxiety agent, but its use has not been associated with any striking health improvements. nevertheless, millions of chlordiazepoxide (cdz) prescriptions have been written and the drug is widely employed clinically as a muscle relaxant, anticonvulsant, anxiolytic, and hypnotic. adverse effects vary from skin rash, nausea, headache, and impaired sexual functions to vertigo and lightheadedness, as well as complications from the drug's administration during pregnancy. the ciliated protozoan, tetrahymena pyriformis, has been of significant importance to research since its successful axenic culture years ago. the drug's tranquilizing effects on humans may possibly be elucidated by investigating cdz-induced impairment of the protozoan's growth and motility through modifying microtubular-directed ciliary function (bell et al. ) . here, uptake, recovery, and attempted localization of cdz, as well as its alterations of cellular ultrastructure are reported. thin layer chromatography of cdz (hoffman laroche, nutley, n.j.) in three different mobile phases revealed optimal cdz stability when dissolved and stored in isoamyl alcohol; benzene. after days of liquid culture, t. pyriformis removed % of the radioactivity from [ c] -cdz in the growth medium. liquid scintillation counting of cell washes during the -day time course suggested surface, nonspecifically-bound radioactivity. following days of culture, [ c] -cdz together with its metabolites and/or breakdown products were recovered from homogenates of tetrahymena. to detect the intracellular sites of cdz localization and possible action, both transmission electron microscopy and immunoelectron microscopy were performed. the former revealed that cdz disrupted cytoplas-buffer or cacodylate-buffered dopa revealed dopa/ppo reaction product but failed to reveal a definitive, substrate localization of the enzyme. instead, cytoplasmic morphologic distortions of buffered dopa-treated hyphae were apparent, mandating modification of the employed higher-plant, cytochemical procedure for localizing fungal ppo. thus, attempts to establish the route of ppo secretion to the growth medium utilizing liquid cultured hyphae may be of limited value as the sheath appears to be sloughed during shake culture. in this connection, c. versicolor grows upon a solid substratum when decaying timber in situ. support: doe-bctr program. the evaluation of surface topography is conventionally done with the scanning electron microscope (sem) by visually reconstructing stereo images in a stereo viewer. the monocular clues in an sem image, shown for example in the image in figure , can also be used to map the topography of a sample surface. the first attempt to identify the surface gradient from a secondary electron detector in this way was reported by suganuma ( ) who found that ( ) and described the inclination of the surface to the specimen plane, where a and b are the signal outputs from the two detectors and a n and b n are the signal outputs from the same material when it is perfectly horizontal. although it is possible to add additional detectors and to modify their signal outputs in a conventional electron microscope, the technique can also be implemented in a straightforward manner by using the differences in signal intensity from the two halves of a split backscattered detector. thus if a and b are the signal outputs from the two different halves of the detector, the backscattered signal, which is usually collected as topographic information (a − b) or composition information (a + b), can be multiplied together (a − b ) to produce the basis of the empirical relationship described by suganuma iv- scanning vol. , supplement iv ( ) fig . ( ) to fit the contrast variations. indeed, since the denominator in this equation is also a constant and there is invariably a flat spot somewhere in the image, it is easier to simply use the expression to try and describe the surface gradient. this function can then be scaled to an absolute magnitude of about to take maximum advantage of the contrast variations that exist. the simplest way to evaluate the surface topography without actually modifying the microscope is to process a digital image in a computer. such an image can be collected directly or a polaroid micrograph can simply be scanned into the machine. in either event the processing is fairly straightforward to accomplish the use of software such as the nih image program. the surface profile is obtained by integrating the surface gradient over the scan distance, and in figure we show, by way of an example, a faceted surface displayed as a conventional secondary image, as a topographic profile, and as a grey scale image indicating the faceted surfaces with the same orientation to the electron beam direction. suganuma t: measurement of surface topography using sem with two secondary electron detectors. j electron microsc ( ), - ( ) corn kernels of an unknown italian cultivar were surfacesterilized in full strength bleach ( . % sodium hypochlorite), rinsed in sterile distilled water, and germinated on difco potato dextrose agar (pda) under fluorescent light at room temperature. isolation of bacteria was made either directly from the rhizosphere of seedlings or from colonies produced on agar. bacteria were identified by their fatty acid profiles (microbial identification system, newark, del.) and diagnostic biochemical test (micro-id, durham, n.c.) . fusarium moniliforme used to test for bacterial antagonism were isolated from corn. experiments designed to infect seedling roots with the isolated bacterium were performed on corn kernels that were subjected to a double sterilization process in which the kernels were surface-sterilized with bleach and then subjected to a mild heat treatment to remove internal bacteria and fungi. this process produced sterile seedlings which remained so at least up to the to leaf stages of growth. the corn cultivars used were trucker's favorite, silver queen, reid's yellow dent, and an inbred cultivar, pr. samples were fixed in % glutaraldehyde in sodium cacodylate buffer and postfixed in % osmium tetroxide in buffer. the tissues were dehydrated in an ethanol series, critical-point dried and coated with gold-palladium or embedded in spurr's medium and sectioned. samples were examined using a philips scanning or jeol cx transmission electron microscope. the bacteria isolated from roots of the italian corn cultivar were gram-negative rods and were further characterized as being positive for voges-proskauer reaction, nitrate reductase, and ornithine decarboxylase and b-galactosidase. the isolate fermented arabinose and malonate. it was negative for phenylalanine deaminase, lysine decarboxylase, urease, h s, and indole production. it lacked the ability to utilize adonitol, inositol, and sorbitol, and was negative for esculin hydrolysis. these biochemical characteristics served to place this bacterium in the family enterobacteriaceae, the tribe klebsielleae, and it was identified as e. cloacae according to ewing ( ) . microscopic studies established that the bacterium was endophytically associated with the roots of the corn cultivar. scanning and transmission electron microscopy demonstrated that the bacterium was distributed uniformly over the corn root epidermis but was randomly distributed intercellularly in the root cortex and outer margin of the pericycle, usually adjacent to phloem cells. although there was a proliferation of bacterial cells in the intercellular spaces of roots (figs. , ) , there was no evidence of damage to host cells, decline in seed germination, nor seedling growth from e. cloacae infection during the week observation period. the presence of the bacterium on kernels of several corn cultivars enhanced the growth of corn seedlings and inhibited growth of f. moniliforme when the bacteria-infected kernels were germinated in fungal-amended soil. this indicated that e. cloacae is biologically associated with corn plants. the bacterium exhibited strong antagonism to several f. moniliforme isolates when grown on nutrient agar and pda plates. the fungus growth was inhibited and restricted to aerial growth on agar plates. the endophytic nature of e. cloacae in corn roots and its antagonism against isolates of f. moniliforme indicate that this bacterium has potential for the biocontrol of f. moniliforme, a corn pathogen. tracheal organ cultures have been widely used for the demonstration of the cell-and organ-destroying capacities of bacteria and viruses colonizing the respiratory tract. however, the assessment of the cell-damaging abilities of fish parasites, the gill organ culture appeared to be a suitable tool (stadtländer and kirchhoff ) . piscine gill epithelium represents the relevant target tissue for cytotoxicity studies of fish parasites, but this tissue is diffi- iv- scanning vol. , supplement iv ( ) fig cult to cultivate in vitro. excessive mucus production complicates the investigation with the light and electron microscope. this is even more difficult when infected specimens are investigated. here, cell debris due to released cells and cell fragments require special attention of the investigator to obtain interpretable electron micrographs. a fine balance is required between preservation of the status quo of the in vitro situation and the necessary procedures (multi-step preparation) for scanning electron microscopy (sem). in this abstract, we describe the detailed and improved preparation of piscine gill epithelium for sem. this will allow other investigators using piscine tissue for research to obtain scanning electron micrographs with high resolution at high magnification. gill filaments were obtained from rainbow trouts (salmo gairdneri, richardson) and were infected in vitro with mycoplasma mobile k, a wall-less prokaryote causing severe damage in gill organ culture (stadtländer and kirchhoff ) . each gill filament (noninfected and infected) was rinsed in several changes of . m cacodylate-trihydrate [cacodylic acid buffer (cb)] at ph . to free the tissue surface from mucilage, medium, and unattached mycoplasmas. the washing step turned out to be critical for obtaining satisfactory results. excessive washing led to complete removal of cell debris and did not reveal the same status quo of infection as seen with the light microscope during the in vitro cultivation of gill filaments. on the other hand, insufficient washing complicated the interpretation of electron micrographs, especially the identification of m. mobile on gill epithelium, due to excessive mucus and cell debris released from infected tissue. the pre-fixation was performed with . % glutaraldehyde (v/v) (in cb) for h at ˚c. the aldehyde was removed by careful rinsing in cb (three times for min). samples were not postfixed, but dehydrated in acetone covering a graded series of solutions (acetone/water) from through % (v/v). all specimens were critical-point dried (cpd) using carbon dioxide as the transitional fluid. cpd was performed in a critical point drying apparatus e (polaron equipment ltd., england) by filling the chamber with / of liquid carbon dioxide. the heating process was conducted in ˚c per min steps until the critical point was reached ( o c and bar). samples were immediately placed in an exsiccator containing anhydrous calcium chloride to avoid rehydration of samples by air contact. after being attached to aluminum mounting stubs using double-sided sticky tape, each specimen was coated with a layer of to nm of gold in a hummer v sputter coater (technics inc., alexandria, va., usa). samples were examined in an etec auto scan b (etec, calif., usa) operating at an accelerating voltage of . - kv. results were documented on a polaroid × land film (type , positive-negative). the method described above allows the detailed investigation of noninfected and infected piscine gill epithelium at high magnification with an sem (fig. , ) . good structural preservation of noninfected, apparently intact secondary lamellae (fig. ) and destroyed lamellae after infection with m. mobile (fig. ) document the usefulness of the described method for the study of the infectious process with this mycoplasma on fish tissue and will certainly also give convincing results in investigations with other fish pathogens. the arrows in figure indicate the presence of the flask-shaped mycoplasma on damaged piscine gill epithelium. bars represent figure : µm, figure : µm. for this study, the sem was superior to light microscopy (lm) and transmission electron microscopy (tem). lm does not demonstrate details in the damaged tissue and tem is much more laborious and time consuming. the mammalian bladder functions in urine storage and expulsion and is thus subject to alternating distension and contraction. blood flow in the bladder wall is compromised by distension (dunn , levin et al. , and acute and chronic overdistension result in ischemia and necrosis, respectively. the latter often leads to loss of mucosal integrity. yet, the vasculature of the bladder wall has rarely been studied (inoue and gabella ) except at the gross level. in this study we describe several unique features of the bladder microvascular anatomy using light microscopy (lm), transmission and scanning electron microscopy (tem and sem), vascular corrosion casting, and alkali digestion (takahashi-iwanaga ). twenty four male new zealand white rabbits were anticoagulated, anesthetized, and cannulated via the abdominal aorta. for routine lm, tem, and sem, the bladder vasculature was flushed with buffered saline at physiologic temperature and pressure, then perfuse-fixed with buffered glutaraldehyde; for corrosion casting, the vasculature was filled with resin (mercox/methylmethacrylate monomer, / ); and for alkali digestion, tissue was treated with n naoh at ˚c for min. thin sections were stained with lead and uranium, and casts and digested tissues were mounted on sem stubs with silver paste or colloidal carbon and viewed at - kv. the bladder is supplied by left and right vesicular arteries, branches of the internal ( %) or external ( %) iliac arteries. within the adventitia, the vesicular arteries send coiled branches dorsally and ventrally over the bladder surface. secondary arteries penetrate the muscularis to supply a rich capillary plexus (fig. ) closely apposed to and located in grooves within the base of the transitional epithelium (fig. ) . capillaries measure - µm in diameter and are often fenestrated and invested with pericytes. veins exhibit abundant valves primarily in the basal half of the bladder. the lamina propria of the bladder consists of very loose, flexible, collagenous connective tissue. unusual capillary "glomeruli," associated with accessory vessels paralleling the primary bladder vessels, are present in the adventitia on either side of the bladder wall. these glomeruli consist of one to four contiguous capillary spheres. these various methods provide a clear, three-dimensional view of the microvasculature of the rabbit bladder, reveal the very close association between the urothelium and the underlying capillary plexus, and describe the fine structure of the mucosal capillaries. several unique features of the bladder vasculature including capillary glomeruli require further char- iv- scanning vol. , supplement iv ( ) acterization. the latter may be associated with sensory ganglia related to pressure sensation, but their function has not been determined. these results form the basis for comparison of normal bladder vasculature with that of experimentally compromised vasculature. the epithelial cell layer lining the nasal turbinates of humans is functionally and histologically consistent with that of the lower airways. this anatomic site is easily sampled using an inexpensive, disposable curette, and the epithelium obtained can be evaluated for both clinical and experimental objectives. the original rationale for ultrastructural evaluation of clinical specimens of nasal epithelium in this facility was to document index ciliary lesions consistent with the diagnosis of primary ciliary dyskinesia (pcd) (figs. , ) among individuals considered at risk. patients referred for study encompassed both adult and pediatric populations and had lifelong histories of chronic sinusitis, bronchiectasis, and/or otitis media, but with normal immunoglobulin levels. several patients presenting with situs inversus, polysplenia, or infertility problems-clinical findings considered risk factors for pcd-also were evaluated. approximately nasal biopsies are submitted annually for evaluation, of which approximately % demonstrate impaired ciliary motion and ultrastructural abnormalities that could be the basis for altered ciliary motion and a diagnosis of pcd. this finding is consistent with the suspected prevalence of pcd in the general population although the rate appears higher among individuals of scandinavian descent, pacific islanders, and inbred populations. the diagnosis of pcd is confirmed by the electron microscopic documentation of any of three ultrastructural level lesions of airway cilia. these lesions are: ( ) dysmorphology of dynein arms, ( ) absent radial spokes, and ( ) microtubular transpositions. in our experience, dysmorphology of the dynein arms represents the major form of ciliary abnormality associated with pcd. missing radial spokes and microtubular transpositions have not been documented among any of our patients. these clinical studies also have provided a unique opportunity for defining a spectrum of ciliary abnormalities which distinguish the heritable primary ciliary abnormalities associated with pcd from acquired cil- iary abnormalities associated with chronic disease or acute injury due to infectious processes or exposure to irritant gases in the ambient air. in addition, other histopathologic features such as mucus cell hyperplasia have been encountered occasionally among children presenting with chronic respiratory disease and referred for evaluation. in summary, our experience shows that ultrastructural evaluation of nasal epithelium can provide a clinically significant perspective on respiratory health. this work was supported by scor grants hl , hl , and hl from the national heart, lung, and blood institute and by u. s. environmental protection agency cooperative agreement cr to philip a. bromberg. this is an abstract of a proposed presentation and does not necessarily reflect epa policy. using conventional light microscopy, silicone has been described as a translucent, clear, mucoid, refractile material which is often difficult to visualize. it is not birefringent, as is silica, by polarized light microscopy. silicone gel tends to form homogenous, rounded "globules," "vacuoles," or "droplets" unlike the angulated, sharp spicules observed with silica. in paraffin-embedded tissue sections, silicone gel often appears to be slightly out of the plane of focus. in addition, silicone gel "globules" occasionally drop out of standard µm histologic sections during tissue processing, leaving partially or totally vacant holes or "cysts." because silicone extravasation and deposition into surrounding fibrous breast capsules is difficult to visualize by standard light microscopy techniques, the electron microscopist must often "blindly" examine multiple fields/grids in a labor-intensive fashion. to decrease the time generally required for the positive identification of silicon by electron probe microanalysis (epma), alternative light microscopy techniques for preliminary screening purposes were investigated. six periprosthetic capsulectomy specimens, synovium from a previously reported silicone breast augmentation patient with arthritic pain and a silicone granuloma from another patient with a ruptured prosthesis were utilized in this study. sections were cut at , , , and µm and mounted on glass slides. in addition to standard permount mounting media, aqua-mount was stained with ink preparations: black stamp pad ink, india ink and aniline artist dyes-black, blue, brown in a : mixture for coverslip application. each series was stained with a battery of common histochemical stains. the sections were then viewed with a zeiss axioplan microscope utilizing conventional incidental light, polarized, non-koehler, phase contrast, and darkfield microscopy. a commercial silicone gel and silicone gel extracted from a previously implanted silicone breast prosthesis were smeared and examined unstained or stained with papanicolaou and "diff quik." silicone was noted to be refractile, nonpolarizable, and nonstainable. the results confirm the refractile, nonpolarizable, and nonstainable properties of silicone in histologic and cytologic preparations (fig. , scale bar = µm). decolorizing techniques (toluidine blue o, etc.) that were not completely differentiated, especially with thicker sections, occasionally demonstrated nonspecific dye "trapping" on the larger silicone globules. the relative ease of silicone localization was greatly increased in histologic specimens with non-koehler, phase contrast, and darkfield microscopy when compared with conventional light microscopy. although standard h&e staining was adequate for silicone localization in tissue sections, uniform dark staining with toluidine blue o increased the contrast between the stained tissue and the unstained refractile silicone. the contrast was also enhanced by aqua mount black stamp pad ink mounting media, with the silicone appearing milky white. in thicker sections, negative staining was slightly accentuated because of the increased concentration of stain as well as thicker, more refractile silicone globules, especially with darkfield microscopy. by utilizing these sensitive screening techniques, we were then able to sample paraffin-embedded tissue selectively, which dramatically decreased the time and effort for correlating confirmatory identification of silicon by epma (fig. ) iv- scanning vol. , supplement iv ( ) the majority of biological samples are high in water content. preparing tissues for scanning electron microscopy (sem) requires removal of this water and often produces severe structural distortions due to surface tension forces during phase transitions. this can result in bulk shrinkage artifact, surface cracking, curled cell borders, clumping or flattening of cilia, and collapse of surface vesicles. failure to postfix biological material with oso can lead to extraction of surface membrane lipids during solvent dehydration. it is likely that many shrinkage artifacts are related to incomplete or improper fixation and drying. critical-point drying (cpd) has become the standard procedure for most biological materials. although it produces a relatively intact end product, wollweber et al. have reported shrinkage of as much as % after cpd of glutaraldehyde and osmium-fixed macrophages and lymphocytes. the use of mordant techniques to complement or enhance oso and uranyl binding may aid in preserving fine structural details regardless of the drying method. numerous substitution/transition fluids have been introduced for both critical-point and direct-evaporative drying, some with more success than others. an advantage in using solvent drying techniques as alternatives to cpd is the ability to process large numbers of samples simultaneously. early attempts at direct drying of nonosmicated samples from ethanol and other solvents produced a generalized cell shrinkage and collapse. freon evaporative drying was shown to be a useful rapid drying technique by liepins and de harven. gamliel further refined the freon direct-drying technique to include the use of guanidine hcl as a bifunctional mordant, prior to osmification, to minimize shrinkage of leukocytes. direct drying from hexamethyldisilazane(hmds) was introduced by nation to dry insect tissue and has subsequently been used for drying various other tissues (adams et al. ) . peldri ii has since been shown to be an effective solvent drying medium. it is a solid at room temperature. a comparative study by bray et al. of peldri ii, hmds, and cpd, using both plant and animal tissues, has produced identical results with animal tissues but not plant tissues. the purpose of this study is to introduce acetonitrile as a potential solvent for direct evaporative drying. acetonitrile has been used as a less toxic propylene oxide replacement transition fluid for transmission electron microscopy (tem) dehydration and infiltration (edwards et al. ) . to date, no studies have been published indicating the of use of acetonitrile as both a dehydration and intermediate transition fluid for direct solvent-drying of tissue-cultured cells for sem. this study compares solvent drying of kb (hela) cells from freon , peldri ii, hmds, acetonitrile, and ethanol to critical pointdried cells grown on thermanox coverslips. the mordant (gtgo) technique of gamliel is included to illustrate its usefulness in shrinkage control. solvent-plasticware compatibility should always be tested before preparing cultured cells for sem; whenever possible, the use of glass is best. cells grown on thermanox coverslips were fixed in . % glutaraldehyde, . m cacodylate, . m sucrose, . mm cacl , ph . . the gtgo fixation protocol of gamliel was found to be the most useful one in preserving cell architecture (figs. , ) . in figure , cells were evaporative-dried under a mild aspirator vacuum after gtgo fixation and dehydration in acetonitrile ( %, %, %, %- ×, min each). as a comparison, cells in figure were identically fixed and criticalpoint dried after ethanol dehydration. the overall appearance of the cells from both treatments is similar at low magnification (figs. a, a) , with some shrinkage evident in either case. under these conditions, it appears that the cpd cells retain more fine structural details than those evaporative dried from an (figs. b, b) . the results of this study are encouraging and may lead to future studies to develop a simple fixation protocol which enables evaporative drying from solvents directly miscible with water such as acetonitrile. snow, which may occasionally cover up to % of the earth's land, supplies about one-third of the water that is used for irrigation and the growth of crops (gray ) . for this reason, estimating the amount of water in winter snow pack is an extremely important forecast activity that attempts to predict the amount of water that may be available for the following growing season. unfortunately these estimates can be easily iv- scanning vol. , supplement iv ( ) fig . confounded by the sizes and shapes of the snow crystals that comprise the snowpack. a snow crystal is a single frozen ice grain that generally results from a process known as nucleation in which atmospheric water vapor condenses on a solid particle or nucleus at temperatures below °c. when nucleation occurs, the water molecules form a hexagonal crystal lattice resulting from the specific orientation and binding that occurs between the oxygen and hydrogen atoms. depending on the temperature and moisture that prevails during formation and descent of snow crystals, nucleation may result in plates, stellar crystals, columns, needles, or dendrites-all of which are based on the hexagonal lattice structure. an individual snow crystal may range in size from µm to mm (gray ); aggregations of two or more of these crystals form a snowflake. the shapes of snow crystals have been extensively studied and photographed with the light microscope (bentley and humphreys , nakaya ) . although these studies have resulted in a classification system that currently recognizes distinct classes and over subclasses of snow crystals (hobbs ), detailed examinations have been hampered by the difficulty of working with a frozen specimen, which is susceptible to sublimation and melting, and by the limiting resolution of the light microscope. for these reasons, an attempt was made to determine whether snow crystals could be collected/stored and prepared for observation and recording in the low-temperature sem. attempts to allow snowflakes merely to settle on a precooled specimen holder were unsuccessful; the snowflakes tended to "bounce" off the holder and those that did alight did not remain attached during subsequent handling. a successful procedure consisted of placing a thin layer of methyl cellulose solution on a holder and precooling it to the prevailing outside temperature during a snow fall. next, snowflakes were allowed to settle on the surface of the methyl cellulose solution. after a few minutes, the holder was plunged into liquid nitrogen and transferred to the laboratory where it was retrieved from liquid nitrogen, mounted on the transfer rod of an oxford ct- hr cryosystem, moved into the prechamber for sputter coating with au/pd, and then inserted into a hitachi s- field emission scanning electron microscope (sem) equipped with a cold stage that was maintained at − °c. these procedures allowed us to observe several forms of the individual snow crystals as well as their nucleation centers. at low magnification, the specimens, which did not appear to be altered by the sputter coating, resembled those that had been previously photographed with the light microscope. the snow crystals were stable in the beam, did not sublime, and could be observed at magnifications of , × or more to reveal microcrystalline water deposits or rime on the surface of some the snow crystals. this procedure, which was used to collect specimens during several snow falls in beltsville, maryland during the - winter season, was also capable of preserving sleet, graupel, and hail. furthermore, storage holders were devised that allowed capture of the snowflakes and their storage in liquid nitrogen until the specimens could be processed for examination in the sem. finally, the specimen stage of the sem allowed specimen tilt so that stereo images of the snow crystals could be recorded (fig. ) . in conclusion, low-temperature sem is a viable technique for examining snow crystals at magnifications that far exceed the resolution of the light microscope. furthermore, the ability to collect and store samples enables investigators to accumulate samples from numerous locations or different time intervals so that detailed observations and comparisons can be done in a convenient and orderly manner. these were identified and returned into the corresponding reconstruction sections in an automatic tracing procedure, programmed and executed on vidas . (zeiss/kontron germany). in addition it is possible to obtain a plastic, -d-like impression by applying the rcm with oblique illumination. this is achieved by decentralization of stach's slide (central diaphragm). . two main problems in -d reconstruction of histologic specimens are the horizontal distortion during the preparation of serial thin-tissue slides and the following vertical readjustment (alignment). we have recently shown that optical sections can be obtained by rcm within thick tissue-slides. its confocal-like principle provides an alternative to mechanical slices. the preserved integrity of the examined object allows the precise movement within the optical axis in one (vertical) dimension, thus avoiding manual alignment. applying the rcm on histochemical and immunohistochemical stains, reflections can be observed within the tissue. the depth of penetration of the light beam amounts to approx. µm, equivalent to about optical tomolevels (fig. ) . using the example of neurons of the supraoptic nucleus of the rat, we demonstrate this new technique on chrome-alum haematoxylin stained neurosecretion. the reflections of the dye particles associated with neurosecretory granules allow the precise localization of these subcellular structures. the visualization of the neurosecretion and its distribution is more distinct and of sharper contrast than in bright-field microscopy. reflected light is like a binary signal and therefore generates a suitable prerequisite for automatic discrimination in greyscale image analysis. thus identified black and white negatives were reconstructed with the module rec d on vidas . . this paper introduces two extended applications of the rcm for -d reconstruction. the generated optical slices and isohypses allow qualitative and quantitative investigations of intracellular structures and surfaces. quantitative chemotaxis is of great interest in a broad field of cell research (e.g., receptor-ligand interaction, ionic channels, cytoskeletal and metabolic processes, embryogenesis) as well as in clinical studies (e.g., immune reaction, wound healing, infection, tumor invasion). the most accepted assay to measure chemotactic behaviour of cells is the boyden filter assay, in which cells move against a gradient of chemical or biological substances. the chemotactic response is analyzed by measuring either the distance travelled by the leading front of cells or by quantifying the number of cells on the lower surface of the filter. this method is laborious and limited by the observer's subjective errors. we have developed a computer-based image analysis system to estimate the three-dimensional distribution of the migrated cells inside the filter. using one-micron optical sectioning, we can determine the position, size, and shape of many individual cells. within min, the position of thousands of cells can be recorded and the migration profile in the filter can be determined. it is possible to distinguish between different cell populations by selecting particular cell parameters. the system consists of a nikon labophot- a microscope with video microscopy and programmable focus control, used in connection with a hasotec-image processor with fast data acquisition and user-friendly software (mswindows). the high throughput, the consistent accuracy, and the simple operation of the system optimize all aspects of routine chemotaxis analysis in basic research and clinical studies. the atomic force microscope (afm) is being used increasingly in the life-sciences field. with this increase in usage, a concomitant increase in the need for both better developed specimen preparation techniques and better defined operational parameters for the afm instrument has occurred. lifesciences afm methodology can be divided into three main areas: ( ) choosing the appropriate support substrate for the specimen, ( ) choosing the most appropriate immobilization techniques for attaching the specimen to the support substrate, and ( ) selection of the optimum instrument scan parameters to ensure reliable transfer of data from the specimen to the image. of central importance to life-science afm is the nature of the substrate to be used. the most reliable substrates are those with a well-documented surface, whose features are at least an order of magnitude smaller than the specimens of interest. furthermore, the material should be transparent so that other forms of microscopy can be used (i.e., light, fluorescence, near-field scanning optical, etc.) and the surface chemistry should be both well documented and susceptible to chemical derivitization. the two most common substrates to fit the above descriptions are glass and freshly cleaved mica. both of these surfaces can easily be manipulated to produce a wealth of reactive primary amines, either by coating the substrate with poly-l-lysine or by treatment with -aminopropyltriethoxy silane (aptes). an analysis of the modified and unmodified substrates show that treatment with either aptes or poly-l-lysine resulted in an increase in surface roughness (ra). based on the roughness information, we recommend modified mica as being suitable for biological material ranging from whole cells to dna, whereas modified glass is unsuitable for samples with heights < . nm. the red blood cell (rbc) cytoskeleton was examined by atomic force microscopy (afm). samples were placed on either glass or mica and imaged in air. no fixative or stain was used; this allowed modification of the samples between images so that molecular components could be identified. the meshlike structure, which is observed when the rbcs are lysed, is identified as a complex of the cytoskeletal integral proteinsspectrin, actin, and band . . the identification was accomplished by imaging the intact cytoskeleton, then treating the sample to selectively remove these proteins and re-imaging the sample. to support the identification of the cytoskeletal proteins further, images were obtained of samples which had been treated with detergent to remove the lipid membrane and leave the cytoskeleton behind. this is the first study of the intact rbc cytoskeleton which identifies specific proteins. more generally, it shows that afm is a useful tool for examining biological systems in their native state since sample preparation is simple and, once attached to the substrate, the sample can be treated in a variety of ways. we have reported some features about the structural and morphologic changes in nylon transformed from β-alanine single crystal and nylon polymerized from ε-amino-ncaproic acid one, which are members of the ω-amino acid family, through the solid-state polycondensation procedure. it was also found that p-aminohippuric acid [n-( -aminobenzoyl)glycine] single crystal could be converted into an aromatic polyamide crystal by heat treatment below its melting point. in this report, crystalline and morphologic structures of polyglycine produced from glycine (the simplest amino acid) single crystal were examined mainly by means of scanning electron microscopy (sem) and x-ray diffraction technique. the monomer solution was prepared by dissolving g of commercial glycine powder into ml of distilled water at °c. the monomer single crystal was precipitated at °c from the solution, which was transparent and prism-shaped. definitive cleavage planes were observed in the monomer crystal, which is found to be parallel to the a-c plane in the αform crystal of glycine. it has been reported that such cleavage is caused by the characteristic structure of α-form glycine crystal. the monomer single crystal was used as the original specimen for the polycondensation reaction, where the original specimen was annealed in decaline at and °c up to h. morphology of the polymerized material was observed by using a hitachi s- sem with accelerating voltage of kv after being coated with gold. x-ray photographs were taken by a flat camera mounted on an x-ray generator with ni-filtered cu-kα radiation, where the crystal took three orientational positions so that a-c (cleavage plane), a-b, and b-c planes made a right angle with the incident beam, respectively. figure shows two types of x-ray diffraction patterns from a specimen polymerized at °c for h with the a-c plane perpendicular (fig. la) and parallel (fig. lb) to the incident beam, where patterns reflected from the polymerized material were observed as well to have spots from the original monomer single crystal. in figure la , two strong arcs are observed, which are indexed as (inner reflection) and (outer reflection) from type-i modification of polyglycine crystal. a more complicated diffraction pattern is shown in figure lb , which seems to be a fiber diagram. almost all molecular chains in polyglycine-i crystal are considered to be normal to the a-c plane and parallel to the hydrogen bond in the original monomer single crystal. figure a shows a sem photograph for the surface of the original monomer crystal. lamination layers of lamellae are observed on the cleavage plane, edges of which are parallel to the b-axis of the monomer crystal. voids were observed in the grain boundary or crack region. a sem picture of the specimen polymerized at °c for h is shown in figure b , where the laminal materials are seen to overlap each other crosswise. such intersecting laminal structure seems to be responsible for the biaxial crystalline orientation observed in the x-ray diffraction pattern shown in figure la. in the case of the specimen polymerized at higher temperatures, the fibrillar structure was observed over the surface. figure c shows the results for the specimen annealed at °c for h. hiroshi toyoda, takashi itoh, hiroshi sakabe, takashi konishi department of polymer science, kyoto institute of technology, kyoto, japan it is well known that polytetrafluoroethylene (ptfe) is produced by emulsification polymerization in the form of powder or aqueous dispersion to be processed industrially. the dispersion-type material is mainly composed of fibrillar and/or lamellar crystallites, where the spherulite structure generally is unseen because of stiffness of the molecular chain. such morphologic feature is considered to be responsible for the remarkable repellent property of the polymer to liquid. the authors prepared thin films of ptfe from the aqueous dispersion to observe change in the fibrillar structure through heat treatment, considering that from such a viewpoint the fibrillar morphology in ptfe is the aspect that is most different from other polymeric materials. the polymerized ptfe used in this study was produced by dupont ltd. a glass plate was dipped into the solution and then withdrawn vertically. the thin film was prepared by drying the solution on the plate at room temperature. the heat treatment was performed at , , , , and °c for h in an air-drying oven. the samples were immediately quenched in ice water (cooling rate: , °c/min) or cooled in air at controlled rates ( , , l, . , and . °c/min). any serious oxidation and/or degradation effects were not observed even during annealing at °c. a scanning electron microscope (sem)(jeol, jsm- lv) was mainly used to observe the au-coated surface structure of the sample, when an accelerating voltage was set at kv to make the resolution of the sem high. composite materials were produced by coating ptfe on textile of glass-fiber and that of carbon fiber. after the coating and subsequent annealing at °c for min, the material was cooled to room temperature. the surface was found to be covered with balls of a diameter of about nm, composed of several hundred nodules of a diameter of about nm as shown in figure . in this sample, the degree of crystallinity of ptfe was about % and no fibrillar structure was observed. by increasing the annealing temperature to > °c, the fiber bun- iv- scanning vol. , supplement iv ( ) dles grew up. such phenomena may be explained by stiffness of the molecular chain including f instead of h, that is, the chain does not fold even if the molecular motion is stimulated at elevated temperatures. any sheaf-like or microspherulitic structure (a precursor of the spherulite), which is often observed in thermoplastic polymers such as polyethylene, did not appear in ptfe. when decreasing the cooling rate, the fibrils grow laterally and interfibrillar space tends to become larger. it is unknown how such space affects the physical properties of the material. thinner fibrils appeared, which connected the original thick ones, in the case of . °c/min cooling as shown in figure . such morphologic structure characteristic of ptfe seems to be responsible for the processing efficiency. schott glaswerke, department of instrumental analysis and mineralogy, mainz, germany fused-cast refractories of the zac-type (zirconia-alumina cast) are of great importance in the manufacturing of glass. especially glass-tanks for the melting of specialty glasses are built with these materials. zac refractories are produced by melting the raw materials at very high temperatures and subsequently casting them into suitable molds. on cooling, part of the material crystallizes, forming al o (corundum) and zro (zirconia, baddeleyite), where the zirconia crystals are also growing within or into the corundum crystals. besides the crystalline phases there also exists a glassy phase, composed mainly of sio , al o , and zro , and small amounts of alkali (na o, k o), tio , and fe o . this microstructure of the material must have an influence on the corrosion behavior when subjected to the aggressive melt in a glass tank. the aim of this work was to establish a method to characterize quantitatively the microstructure of these refractories. the inhomogeneous nature of the material can already be discerned in the light microscope; however, the resolution is not sufficient to measure the sometimes very small (< µm) crystal sizes of zro , not to mention the determination of a form factor! therefore, the electron microprobe (epma) was used to perform this work. the instrumentation used was a jeol jsm- scanning electron microscope, equipped with an optical microscope for reflected and transmitted light, motorized stage (x, y, z), and coupled with a combined eds/image analyzing system voy-ager from noran, which also controls the stage movement. the following parameters of the microstructure, for example, the geometric and chemical properties of the crystalline and glassy phases, were to be determined: maximum and minimum diameters, area of the individual phases and fractional area within the material, form factors and orientation to a reference plane, and the chemical composition of the glass phase. furthermore, it was necessary to distinguish between the zirconia within the corundum and the more isolated crystals. since the material is inhomogeneous, a rather large number of image fields had to be analyzed (at magnifications of × and higher the individual image field is very small!); as a consequence, the whole procedure had to be automated as much as possible. sample preparation: a flat and polished surface of a section of the material had to be produced. although the grinding and polishing was done with diamond wheels and paste, a certain amount of relief between the hard corundum and the "soft" glass is unavoidable. the first step in analyzing the microstructure is to recognize/discriminate the three phases in the material. the signal used for image formation and subsequent phase discrimination is the backscattered electron (bse) image; however, the abovementioned relief due to differences in hardness made the distinction between glass and corundum impossible, since (a) brightness differences between glass and corundum are not very strong in the first place, and (b) edge effects in the corundum crystals showed brightness values of the glass, and vice versa (discrimination, i.e., tranformation of a particular phase into a binary image, is based on such brightness differences). contrast enhancement or image filters were not sufficient for clear separation of these two phases. therefore, for the recognition of the glass phase, an element distribution for si (x-ray map) was used; combined with the image of zro , which is easily discriminated from other phases because of its high average atomic number/high backscatter signal, it yields the inverse of the corundum image. this sounds quite simple; however, since the resolution of the x-ray signal is considerably less than the bse signal, quite some effort had to be put into the treatment of the si-distribution to give correct areas and outlines of the glass phase. another rather complicated step was the recognition of the zirconia crystals grown within or into the corundum. crystals fully enclosed by the corundum presented no problem, but those growing from the edge into the al o were difficult to discriminate as to belonging to this particular structure. the problem here is to tell the machine what the eye and judgement of the operator consider to be part of this feature. an additional treatment of the glass phase (binary image filters) was necessary to solve this task. the binary image of the glass phase is used as a template for the determination of the chemical data of the glass; it provides the control for stepping the electron beam only over the desired areas while acquiring data with the energy dispersive spectrometer (eds). all image analysis procedures, including the storage of images and eds spectra together with the evaluation set-ups (selection of properties to be determined), were combined within a schedule. the stage control and automation software then connects the stage movement to predetermined points (or a number of points along a line) with the execution of the schedule at each of these points on the sample. after completion of the analysis run, which needs about hours for image fields and is therefore done unattended overnight, the large amount of stored data are statistically evaluated (for the zirconia particles alone, there are easily more than , data sets, consisting of seven measured or derived properties each!). this task is performed using the lotus - - calculation program. the chemical data are extracted from the stored spectra with the zaf-correction procedure to yield wt-% oxide data, and also statistically evaluated with the lotus program. although the aim of this work was only to set up an analysis procedure, the test runs on various samples have already shown differences in the microstructure as determined by the geometric and chemical properties of the individual phases. the application of this procedure to "real" samples will then establish correlations between the microstructure and the properties of the material with respect to their use in glass melting. approximately , tons of electric furnace flue dust accumulated in an industrial area in tifton, georgia. vehicles transporting the flue dust, classified as k hazardous waste, initially dumped the material in a warehouse. once the warehouse was full, the flue dust was dumped in an uncovered pile. run-off from the pile and wind-driven particles contaminated nearby industries, residential buildings, and soils over a period of many years. scanning electron microscopy-energy dispersive x-ray spectrometry (sem-eds) was used to compare the morphology and chemical composition of fly ash dust from the suspect pile ( fig. ) with samples collected from the surrounding buildings and soil. post-it notes (millette et al. ) , modified with a strip of conductive carbon tape, were used to collect dust that had accumulated in buildings surrounding the fly ash dump site. suspect dust particles were analyzed by sem-eds to compare with known dust particles from the fly ash pile. soil samples were sieved, with "fines" from the dry soil analyzed by sem-eds and compared with samples from the fly ash pile. particles similar in chemical composition and morphology were identified in most of the buildings sampled that surround the fly ash dump site. soil samples from areas surrounding the dump site were also found to contain fly ash iv- scanning vol. , supplement iv ( ) fig. backscattered electron image of fly ash spheres from the flue dust pile. scale bar = µm. particles similar in morphology and chemical composition to fly ash from the suspect pile. in conclusion, soil and dust samples taken from homes and outdoor areas surrounding the fly ash pile were found to contain particles similar in morphology and chemical composition to particles from the fly ash pile. scanning electron microscopes (sems) are widely used in detection and quantification of material microstructure and imperfections. results obtain with sems need a high expertise to be fully exploited. specialized programs such as a single-scattering monte carlo simulation can effectively predict the electron beam interaction with solids and thus help the quantification. one of the most powerful advantages of monte carlo simulation to help microscopists is to generate images. with high-speed computers we can now simulate images in a reasonable amount of time. in this paper we present images of spherical inclusion of mns in a fe matrix. it is shown that there exists a difference between the image dimension and the real dimension. also, it is shown that geometric effects can alter the resulting image. the monte carlo program used for low-energy simulation is described elsewhere . figures - show simulated image of mns inclusions (the dark center of the figures) in a fe matrix (lighter part). to build this image we need to simulate , primary electron trajectories and then calculate the associate backscattering coefficient for each pixel of the screen. the image will then need , simulations for a total time of approximately , cpu min on a risc workstation. because of the symmetry of the image we can use an better method. starting from the center of the inclusion we simulate points moving on a radius to the border. then we rotate this line on °and add random noise for the monte carlo simulation to obtain the full image. this image is coded in windows bitmap format and can then be converted to tiff or another format. for our simulation, we used a diameter of nm for the electron beam, at which value the incident beam current would be × − amps in our jeol sem. figure shows a spherical inclusion with a radius and at a depth of nm simulated with a beam energy of kev. figure shows the same inclusion but simulation at kev. figures and show the inclusion with a ratio depth/radius of . , simulated at and kev, respectively. in figure we can observe the discrepancy between the image dimension and the real dimension as a function of the ratio depth/radius. as was expected, the error increased with the depth/radius ratio. it is also interesting to note that increasing the energy of the incident electrons increases the image error. the geometry of the solids affect the backscattering coefficient. we can obseve a white border in figures - a similar problem occurs in the middle of the inclusion which appears lighter. it is interesting to see the same phenomena in figure for an image of a real inclusion. the photograph of the embedded particulates in a matrix taken by sem in backscattering mode should be analysed carefully because this method usually overestimates the dimension of such particulates. the measurement of strain, using lattice parameter changes, can be determined from channeling patterns in the scanning electron microscope (sem) using holz lines. the method used by kuzubowski, for example, in the [ ] orientation of silicon, utilizes the change of the height of the triangle from the intersections of the ( ) and two { } holz lines. more recently we have been investigating the channeling patterns for silicon to calibrate accurately the voltage in an sem and we have utilized a pin wheel pattern formed from the { } holz lines in [ ] pattern at . kev. at this voltage, the ( - - - ) and ( ) are very close to each other, within . degrees, and these two lines split as the voltage increases or decreases. the width of the splitting is quite sensitive to any voltage changes as are the { } intersections with these lines. an experimental electron channeling pattern (ecp) of these lines, along with a simulation are shown in figure . the voltage calibrated from the simulation is . kev as shown in figure c . these same holz lines are, of course, also appropriate for strain measurement since the variation in the voltage is equivalent to an isotropic strain field that would uniformly change the lattice spacings. the shifting of the holz lines in the electron channeling patterns due to strain are from both the change in the magnitude of bragg angle and the change in the orientation of the diffracting planes. the former corresponds to a change in the distance of reciprocal lattice point from the origin(i.e., d spacing) and the latter is due to the rotation of reciprocal lattice point about the origin. thus the total angular change can be written as where θ b is the bragg angle, ∆θ p is the rotation of the plane, g is the diffraction vector, λ is the electron wavelength, and | | represents the length of a vector. e is a matrix equal to ε + i where ε is the strain tensor and i a unit matrix. the approximation ~ is due to the small angle approximation for sin(∆θ b )~∆θ b and − ∆θ b ~ . the holz lines toward the center of the channeling patterns are more sensitive to lattice parameter variations because they have a larger value of g and the change of the bragg angle is in proportion to this magnitude. the angular iv- scanning vol. , supplement iv ( ) ∆θ = ∆θ b + ∆θ p~e g − g widths of these channeling lines are also smaller and so they are well defined. when the strain is not isotropic, the sensitivity is also determined by the orientation relationship between the strain ellipsoid and the g vector. thus the appropriate choice of the g vector can be used to maximize the second term in the equation. in figure a , for example, we show the simulation of a channeling pattern for a strain along only one direction (e.g., [ ] ) which breaks the four-fold symmetry of the [ ] pattern. it can be seen that the strain effects on the { } are all the same since all the planes in this family are symmetrical to the strain direction. however, the sensitivities to the strain in the { } are not all equal, ( ) being more sensitive than ( ). the comparison of the sensitivity can be seen from the simulation and from figure b , in which the angles of splitting are plotted as a function of the strain. for years lab has been the industry standard for thermionic emission cathode material. in , fei introduced ceb as an improved alternative to lab . ceb directly replaces lab and has certain distinct benefits. ceb has a lower volatility than lab , increasing the lifetime of the cathode. this reduces the frequency of cathode purchases and replacements. in addition, greater beam stability and faster startup are achievable from ceb 's greater resistance to contamination. however, to gain the benefits of ceb , it must be operated correctly. studies have shown that the operating characteristics of ceb , such as total emission current, are different from lab . proper operation of ceb comes from understanding the operating characteristics of ceb and how they interact with the system in which it is operating. to compare the operating characteristics of ceb and lab , we performed parametric studies in a jeol scanning electron microscope. the jeol is a self-biased system where the bias voltage (vb) is dependent upon the total emission current as vb = ie*rb, where rb is the bias resistor and ie is the total emission current. the results show that ceb operates at a lower total emission current than that of lab (fig. ) . therefore, for the same bias resistor settings, the bias voltage on ceb is less than that on lab (fig. ) . please note that the lower emission current of ceb does not imply a lower probe current. ceb has high transmission and has been shown to provide probe currents similar to lab . to optimize the operation of ceb in a self-biased system, the operating conditions must be optimized for a low emission current source. these operating conditions include the bias resistor value and wehnelt-to-tip spacing. we have found that increasing the bias resistor improves the performance and lifetime of ceb . other experiments show that adjustments to the wehnelt-to-crystal tip distance further improve the performance of ceb . the effects of adjusting the wehnelt-to-tip distance vary for different electron microscope systems. for independently biased systems, we recommend setting the filament temperature to k (the corresponding filament current is provided with the cathode) and subsequently adjusting the bias until the desired emission image is obtained. this is possible because the emission image of ceb is the same as the lab emission image. the wehnelt-to-tip distance should be set to the distance recommended for lab . in transmission electron microscopes (tems), the operating point is set by viewing the source image. because the emission image of ceb is similar to lab , the procedures for obtaining the lab operating point in tems still apply to ceb . however, the total emission current at the operating point will be lower than with lab . by following these guidelines, the operation of ceb can be optimized and with this optimized operation, the benefits from additional lifetime and increased stability over lab can be achieved. takeshi hatsuzawa, yoshihisa tanimura, kouji toyoda, makoto nara*, syuuji toyonaga*, shin-ya hara*, hirotaka iwasaki*, kazuhiko kondou* national research laboratory of metrology, miti tsukuba; *nikon corporation, tokyo, japan a compact laser interferometer with a piezo-driven scanner has been developed for metrological micro-linewidth measurement in regular scanning electron microscopes (sems). so far, special sems combined with various scanners and interferometers (postek , hatsuzawa et al. ) are necessary to perform absolute and precise measurements; however, this device solved the problem by miniaturizing a one-dimensional mechanical scanner and a multi-optical path interferometer. the arrangements of optical components are illustrated in figure . a mechanical scanner is constructed in the center of a mm diameter base disk. the square part, slit by using a electric discharging machine, is suspended by thin elastic suspensions at each corner. the table is driven by a piezo-electric actuator of a traveling length of micrometers. a he-ne laser beam is introduced on the disk by a single mode fiber through a collimator lens, and it is split and deflected by beam splitters and lenses so that two beams are facing each other and form a differential interferometer. at both ends of the table, right-angle prisms are facing each other so that the laser beam goes back and forth five times. by using the differential arrangement and optical-path multiplication technique, the resolution of the interferometer is improved ten times from the original michelson's arrangement. the reflected beams are surperimposed by a half-mirror to generate an interferometric signal, and it is detected by four photo diodes (pd -pd ) after changing its phase by ˚ through a half-and a quarter-wave plate arrangement. this detection method and an electrically operational processing enhance the resolution iv- scanning vol. , supplement iv ( ) times from its original. eventually, by using physical and electrical methods, the resolution of the compact interferometer is . nm (âλ/ ). to evaluate the performance of the compact interferometer, it was installed in the vacuum chamber of a regular sem (joel jsm- a) as shown in figure . the fiber and electric wires are introduced through two flanges next to the chamber. a software servosystem is constructed by using the interferometer and a piezo driver. according to the positional information read through the interferometer counter, a d/a converter commands the piezo driver to change table position precisely, allowing simultaneous sampling of the secondary electron intensity distribution. in the system, the resolution is improved to . nm (âλ/ ) by using the counter function. thus, a precise measurement system is realized for the absolute measurement of micro-linewidth in a regular sem. a maximum drift of the interferometer counter of nm/h was observed by fluctuations in room temperature, however, the influence of the drift can be neglected in actual measurements since a line-scan is finished within a dozen seconds. a comparison of measurements between the metrological sem (hatsuzawa et al. ) and the compact system was made by using silicon micro-line artifacts, ranging from . to . micrometer. the measured linewidths agree within a couple of nm in both measurements, although the measurement conditions are different in acceleration voltage, etc. the results show that the measurement system using the compact interferometer has the same performance as the metrological sem. this means that absolute and accurate measurements can be obtained everywhere by using the compact laser interferometer and a regular sem. this device can be applied to various types of scanning probing microscopes as well as to optical microscopes by improving the table mechanism and the arrangement of optics. institute of ecology and department of botany, university of georgia, athens, georgia recent studies of lake lanier, georgia, revealed a fungal epidemic on the planktonic alga, synedra acus. clonal isolates of the fungus were identified as zygorhizidium planktonicum (chytridiomycetes), an obligate parasite of freshwater diatoms. although frequently present in lakes and reservoirs of western europe, the occurrence of z. planktonicum in north america has not been previously confirmed. earlier studies have described the morphology of z. planktonicum on asterionella formosa; however, little is known of the fine structure and infection process on s. acus. as a basis for further investigations, morphology and mechanism of infection were characterized by scanning and transmission electron microscopy. these observations provided exceptional accounts of germinating spores and developing thalli. moreover, conjugation was characterized as the fusion of heterogametangia by means of an extended smooth-walled tube, emanated from the smaller "male" gametangium. upon attachment, the club-shaped conjugation tube adhered to a small region of the adjoining thallus. this point of contact became continuous with the maturing "female" gametangium which appeared smooth-walled and often highly vacuolate. ultrastructural examinations also illustrated a shared cytoplasm between conjugating gametangia and apparent migration of organelles; however, fusion of nuclei was not observed. the mechanism of infection on s. acus appeared identical to earlier descriptions of z. planktonicum on a. formosa (beakes et al. ) . following encystment, spores typically produced a single germ tube which grew over the frustule valve in an unwavering, linear fashion. penetration occurred between an overlapping region of the outer and inner frustule. at the point of intrusion, the rhizoid appeared slightly swollen, often further displacing the outer diatom wall. a program for monte carlo simulation of electron energy loss in nanostructures a monte carlo calculation of the backscattering coefficient for a multilayer sample monte carlo program for minicomputers using mott cross sections an improved method of measuring biological submicron motion and displacement using laser amplified motion detection and analysis friedlander sk: smoke, dust and haze, fundamentals of aerosol behavior subcommittee on airborne particles: airborne particles micromanipulators and micromanipulation moor h: recent progress in the freeze-etching technique optimization and application of jet-freezing platinum-iridium/carbon: a high-resolution shadowing material for tem, stm, and sem of biological macromolecular structures freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit scu cryo-preparation and planar magnetron sputtering for low temperature scanning electron microscopy imaging of intramembranous particles in frozen hydrated cells (saccharomyces cerevisiae) vapor pressure data for some common gases. r.c.a. review an improved cryo-jet freezing method in vitro spinal cord trauma early post trauma changes in rat spinal cord: electron probe microanalysis surface studies by stm fabrication technique for tips with controlled geometry for scanning tunnelling microscopy scanning probe metrology low temperature thermal oxidation sharpening of microcast tips microfabrication of afm tips using focused ion and electron beam techniques dimensional metrology with scanning probe microscopes a rocking beam electrostatic balance for the measurement of small forces a scanning tunneling microscope with a capacitance-based position monitor scanning probe tips formed by focused ion beams envelope reconstruction of probe microscope images surface recovery in scanning probe microscopy probe characterization for scanning probe metrology comparison of diffraction techniques for the sem. scan electr microsc electron channelling in the sem a review of excimer laser projection lithography m: direct electron-beam patterning for nanolithography direct stem fabrication and characterization of selfsupporting carbon structures for nanoelectronics die entstehung einer vielzahl von kontaminationsfäden unter der electronen-mikrosonde transition from chemical etching to chemical polishing studied by the sem chemical preparation of dielectrics for studying their microtopography by the sem microprobe analysis in human pathology shelburne jd: preparation of biological tissue sections for correlative ion, electron and light microscopy negative staining: applications and methods detection and identification of viruses by electron microscopy diagnosis of viral infection by electron microscopy electron microscopy in diagnostic virology electron microscopy in viral diagnosis genetic identification of a hantavirus associated with an outbreak of acute respiratory illness isolation of muerto canyon virus, causative agent of hantavirus pulmonary syndrome distinction between bunyaviridae genera by surface structure and comparison with hantaan virus using negative stain electron microscopy anatomic complications of abdominal surgery with special reference to the ureter urological complications of renal transplantation can be prevented or controlled the microvasculature of the guinea pig ureter. a scanning electron microscopic investigation application of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver sem blood vessel cast-analysis microangioarchitecture of the islets of langerhans in the snakes, naja naja, vipera russelli and echis carinatus histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler acid phosphatase activity in the inner ear the development of the stria vascularis in the mouse kimura rs: distribution, structure and function of dark cells in the vestibular labyrinth secretory epithelial linings in the ampullae of the guinea pig labyrinth la fosfatasi acida del labirinto membranoso dell'embrione di pollo durante lo sviluppo the development of human placental villous tree scanning electron microscopic observations on the surfaces of chorionic villi of young and mature placentas ultrastructure of the epithelium of the chorionic villi of the human placenta some new findings about hofbauer cells in the chorionic villi of the human placenta the fine structure of human placental villus as revealed by scanning electron microscopy monte carlo simulation with ebic a monte carlo calculation backscattering coefficients calculations of mott scattering cross section simulation of sem screen image by a monte carlo method quantitative x-ray microanalysis of spherical inclusions embedded in a matrix using a sem and monte carlo simulations a standard procedure for the modeling of the decrease in detection efficiency with time for low-energy eds spectra an empirical stopping power relationship for low-energy electrons measuring the backscattering coefficient and secondary electron yield inside a scanning electron microscope applications of a knock-on process monte carlo simulation based on the mott cross section to quantitative electron microprobe analysis x-ray production as a function of depth for low electron energies theoretical electron-atom elastic scattering cross section cross section for k-shell ionization by electron impact the use of polyacrylamide as an embedding medium for immunohistochemical studies or embryonic tissue immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos i: presence of immunofluorescent titin spots in premyofibril stages novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods whole-mount analyses of cytoskeletal reorganization and function during oogenesis and early embryogenesis in xenopus confocal microscopy of thick sections from acrylamide gel embedded embryos resolution of subcellular detail in thick tissue sections: immunohistochemical preparation and fluorescence confocal microscopy the use of polyacrylamide as an embedding medium for immunohistochemical studies of embryonic tissues application of acrylamide as an embedding medium in studies of lectin and antibody binding in the vertebrate retina developmental angiogenesis: quail embryonic vasculature embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern endothelial cell origin and migration in embryonic heart and cranial blood vessel development morphogenetic mechanisms in avian vascular development near-field optics: microscopy, spectroscopy and surface modification beyond the diffraction limit mechanical detection of magnetic resonance related scanning techniques morphology and diameters of crystallites in remineralized enamel the shape of enamel crystal within human enamel densitometric study of polarized light images from carious lesions conditions required for detection of specimen specific se- secondary electrons in an analytical sem a high resolution se- sem study of enamel crystal morphology high resolution topographic imaging of enamel crystal surfaces analysis of metal films suitable for high resolution se- microscopy antiviral activity of rnadye combinations microspectrophotometry and digestibility of alkali-treated walls in bermudagrass cell types simplified highly efficient apparatus for photographic transaxial x-ray tomography embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos antibodies to b -integrins cause alterations of aortic vasculogenesis, in vivo capillary endothelial cell cultures: phenotypic modulation by matrix components in vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices connective tissue morphogenesis by fibroblast traction. i. tissue culture observations reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro consumption of various process cheese products has been steadily increasing in the u.s.a. and worldwide. these products are manufactured in various styles depending on composition and physical properties sodium citrate (sc), trisodium phosphate (tsp), disodium phosphate (dsp), and sodium hexametaphosphate (shmp) were used at . % levels as melting salts. after cooking, the samples were held at °c for up to h. they were removed after , , . , and h, cooled at °c, and stored for days, after which they were analyzed for moisture, fat, protein, ph, firmness, and meltability. for transmission electron microscopy, small samples ( mm ) were fixed in . % glutaraldehyde and % osmium tetroxide, embedded in spurr's low-viscosity medium, sectioned ( nm thick sections), stained with uranyl acetate and lead citrate, and examined at kv accelerating voltage the data in figure were converted to a pseudo three-dimensional relief map of the embryonic vessels using digital image processing software. it can be argued that this digital rendering, which contains apparent overhead illumination (and shadows below), provides a more comfortable format for assessing visual information. references caric m, kaláb m: processed cheese products textural properties and microstructure of process cheese food rework microstructure of processed cheese products milk gel structure. vi. cheese texture and microstructure effect of draw ph on the development of curd structure during the manufacture of mozzarella cheese structure and rheology of string cheese mozzarella cheese: impact of coagulant type on functional properties encyclopedia of food science & technology food texture and microstructure electron microscopic observations on the casein micelles of buffalo milk: a preliminary study development of microstructure in raw, fried, and fried cooked paneer made from buffalo, cow and mixed milks the role of casein micelles in changes in the colour of milk microstructural evaluation of model starch systems containing different types of oils use of the bird-leider equation in food rheology starch gelatinization in the presence of emulsifiers. a morphological study of wheat starch fixation analysis of tetrahymena pyriformis ultrastructure distribution of polyphenol oxidase in organelles of hyphae of the wood-deteriorating fungus, coriolus versicolor. biodeterioration research a study of the bladder blood flow during distention in rabbits a vascular network closely linked to the epithelium of the urinary bladder of the rat the effects of acute overdistention of the rabbit bladder applications of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver light microscopy techniques for the demonstration of silicone synovial metaplasia of a periprosthetic breast capsule demonstration of silicon in sites of connective tissue disease in patients with silicone-gel breast implants biological specimen preparation for sem by a method other than critical point drying comparison of hexamethyldisilazane (hmds), peldri ii, and critical point drying methods for scanning electron microscopy of biological specimens acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine optimum conditions may allow air drying of soft biological specimens with minimum cell shrinkage and maximum preservation of surface features a rapid method for cell drying for scanning electron microscopy nation jl: a new method using hexamethyldisilazane for preparation of soft insect tissue for scanning electron microscopy the use of a simple method to avoid cell shrinkage during sem preparation handbook of snow: principles, processes, management and use hobbs pv: ice physics snow crystals: natural and artificial interference reflection microscopy in cell biology: methodology and applications rekonstruktion des oberflächenreliefs von erythrozyten mit hilfe der leitz-reflexionskontrast-einrichtung. leitz-mitt wiss tech vii/ reflection contrast microscopy within chrome-alum haematoxylin stained thick tissueslides reflexionskontrastmikroskopie in der immunhistochemie lichtmikroskopische untersuchungen zum einfluss von atrialem natriuretischem peptid (anp) am nucleus supraopticus scanning electron microscopy of post-it ™ notes used for environmental sampling development of a monte carlo program for low energy work measurement of small elastic strains in silicon using electron channeling patterns electron channeling patterns in the scanning electron microscope a metrological electron microscope system for microfeatures of very large scale integrated circuits scanning electron microscope-based metrological electron microscope system and new prototype of scanning electron microscope magnification standards comparative ultrastructural ontogeny of zoosporangia of zygorhizidium affluens and z. planktonicum, chytrid parasites of the diatom asterionella formosa iv- scanning heterogametangia attached by a fully developed conjugation tube (arrowhead) infection sites of diatom host showing intruding germ tubes (arrowheads) and developing thalli. scale bar = µm the author is grateful to dr. david howell for critically reviewing this manuscript and to ms. lara muffley for technical assistance.this research is supported in part by nih grant nddk r dk - . the authors gratefully acknowledge dr. steven armstrong for his assistance in animal preparation. mic integrity and induced mitochondrial hypertrophy without altering microtubular ultrastructure (fig. a, b) . to localize exogenously administered cdz, a polyclonal antibody to the drug conjugated to keyhole limpet hemocyanin (pierce, rockford, ill.) was prepared by immunizing new zealand rabbits and subsequent bleeds. the antibody titer was both detected and quantified by an indirect elisa assay (pierce elisa starter kit). the detected antibody was separated from other serum proteins by immunoaffinity chromatography utilizing pharmacia's mab trap g and then tagged with nm immunogold particles. transmission immunoelectron microscopy employing tagged antibody and glutaraldehyde/ paraformaldehyde-fixed, dmf dehydrated, and lowicryl-embedded cdz-treated and nontreated tetrahymena revealed no immunogold association with either microtubules or any other cytoplasmic organelle. this suggested that intracellular cdz was cytosolic and leached out during em processing. thus, cdz appears to impair growth and motility through an effect on general metabolism, for example, protein synthesis and/or respiration, rather than a direct action upon microtubules. however, isolation, purification, and characterization of microtubular proteins from tetrahymena cultured with and without cdz are required to substantiate this tentative conclusion. support: nsf-rimi grant no. rii- . coriolus versicolor, a white-rot, wood-decay basidiomycetous fungus, elaborates extracellular ligno-cellulolytic enzymes which possess marked industrial and agricultural applications. thus, we have been attempting to overproduce and enhance/regulate the secretion of these enzymes employing polyphenol oxidase (ppo) as a model enzyme. it catalyzes the conversion of o-diphenols (tree-generated resistance factors) to o-diquinones and oligomerizes syringic acid, alignin derivative. previously, we (moore et al. ) employed biochemistry and immunoelectron microscopy to map the route of ppo secretion through intracellular endomembrane and possible wall-associated components for hyphae cultured in defined liquid (biochemistry) and solid (microscopy) media. here, the ultrastructures of c. versicolor hyphae cultured in kirk and kelman's defined liquid or solid media are compared. in addition, ultrastructural cytochemistry to define further the intracellular route of ppo secretion to the growth medium in liquid cultured hyphae is described. hyphae of various culture ages ( - days) were prefixed min in . - . % glutaraldehyde buffered with . m cacodylate/cacodylic acid, ph . and after washing with buffer postfixed for h in buffered % s . for cytochemistry, prefixed hyphae were washed and treated with either cacodylate buffer or buffered mg ml - tlc pure, dihydroxyphenylalanine (dopa) on ice, followed by h at °c and then s postfixed. the hyphae were dehydrated through a graded acetone series and embedded in spurr's low epoxy resin. the ultrastructures of hyphae cultured in defined medium containing or lacking agar were similar (fig. a , b) except that hyphae grown upon agar possessed a sheath (hs) external to the cell wall. comparisons of numerous micrographs of aldehyde-fixed hyphae treated with cacodylate christian h. rickert and timm j. filler institute of anatomy, westfälische wilhelms-universität, münster, germany until today, quantification in cytochemistry has mainly been performed on supracellular level and by subjective estimation which makes it difficult to compare results of different investigations, even for standardized cytochemical procedures. reasons for this are the lack either or of calibrations or of relative reference points. greyscale image analysis principally allows the quantification of dye-density, but in practice stain intensities depend on many technical circumstances, that is, slide thickness, density of materials, light conditions, or costaining effects, and do not allow automatic identification because of weak grey-contrast. thus, greyscale image analysis for cytochemical quantification on subcellular level necessitates four prerequisites: ( ) applicability within tissue-slides combined with high resolving power, ( ) thin optical tomolevels to avoid superimposition of stained structures, ( ) clear distinction of structures (specificity), and ( ) high contrast. the reflection contrast microscope (rcm; leica germany) meets the above mentioned requirements. it combines effective suppression of aspecific reflected light with epi-illumination. because of its confocal-like principle, the rcm can be applied on thick tissue slides to obtain distinct optical sections. , at a magnification of ×, the depth of these sections amounts to approximately - µm, circumventing an accumulative effect as seen in transillumination. some cytochemical stains show specific reflections in the rcm, increasing the detection sensitivity to a level of objects nm in size. , reflections are like a binary signal (all-or-nothing principle) and deliver an intense contrast against a dark background, thus facilitating image processing by substituting the common density measurements with field measurement corresponding to areas of reflections.we designed an analysis program on vidas . (zeiss/kontron germany) for quantification of rcm images. one of the main parts of this application handles the greyvalue manipulation. apart from standard routines for image optimization and automatic region selection, the mean greyvalue of the whole image was determined. this was used as a reference parameter for the identification threshold in order to minimize the deviation of the identified area from the real area of reflection caused by inconsistencies of the light intensity.applying the rcm with consecutive image analysis on gomori-stained neurons of the supraoptic nucleus (son), we verified the validity of our measuring routine by employing it on a recognized system. a linear correlation between the process of neurosecretion and nuclear volume of the son is well known. switching from rcm to bright field, it is possible to obtain topographically identical images of the chosen tissue region. thus, we photographed reflecting neurosecretory granules in the former while karyometry was performed on the latter. the nuclei were classified by area and matched with the corresponding nucleus area of the same cell. a linear regression of these parameters was computed within a % confidence interval. our results in general confirm former findings about the measurable relationship between nuclear size and specific cell activity, surpassing earlier methods by increasing the sensitivity and decreasing the duration.this paper introduces the rcm combined with digital processing as a useful tool for quantification in the investigative gap between light and electron microscopy. the reflection contrast microscope (rcm; leica germany) is a light microscopic instrument, making reflections along interfaces visible by means of centrally polarized epi-illumination. these reflections cause interference patterns that are suitable for the analysis of superficies or detection of contact zones . we present two properties of this widely unknown technique allowing three-dimensional ( -d) reconstructions in the following manners: . applying the rcm on the surface of air-dried unstained erythrocytes, we made use of the phenomenon that neighbouring zones of equal altitude are joined by closed interference fringes; these can be interpreted as isohypses. to quantify the angle of declivity within a period of the resulting dark-light pattern, it is necessary to know the mean wavelength. the difference of layer thickness between two interference lines of equal tone is about nm for monochromatic light of λ= nm. the advantage of this technique is the simultaneous presentation of the profile of all the isohypses in just one picture (fig. ) . key: cord- - z rcy authors: cerofolini, linda; fragai, marco; luchinat, claudio; ravera, enrico title: orientation of immobilized antigens on common surfaces by a simple computational model: exposition of sars-cov- spike protein rbd epitopes date: - - journal: biophys chem doi: . /j.bpc. . sha: doc_id: cord_uid: z rcy the possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. for both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. however, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. to predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. in this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the sars-cov- spike protein on surfaces commonly used in lateral-flow devices. these calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays. the activity and reactivity of an immobilized protein strongly depend on its orientation with respect to the surface of the support in -or on -which it is immobilized. this holds true for enzymes, as well as for antibodies and antigens. therefore, the possibility to control and manipulate the exposition of the relevant residues and protein surfaces plays an important role in the rational design of devices based on immobilized proteins. among these devices, immunosensors represent an expanding space for research and market opportunities. while the path to reach a technologically relevant product must rely upon a strong experimental characterization, [ , ] possibly relying upon atomic-level methodologies, [ ] [ ] [ ] [ ] [ ] [ ] [ ] the preparation/isolation of the protein of interest, its immobilization, and the characterization of the resulting composite are complex and time-consuming, therefore it is also true that guidelines for achieving optimal orientations could improve the efficiency of the r&d connected to protein immobilization. [ , ] however, simplified simulation models that would allow for a rapid prediction of the most plausible orientations are not particularly common. in this manuscript we apply a very simple method based on a unitedresidue modelling of protein-surface interactions, to specifically address the problem of determining the orientation of the sars-cov- spike protein receptor binding domain (rbd) on a few prototypical surfaces for biomedical use. united residue modelling of protein-surface interactions is a rather effective model to screen the poses of protein molecules with respect to surfaces. [ , ] the method we apply is based on the works by jiang, zhou and del monte-martinez, [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and encompasses van der waals and electrostatic interactions, as well as covalent immobilization. the choice of the target protein is motivated by the recent emergence of a new infectious disease (covid- ) caused by a coronavirus (sars-cov- ). [ ] this infectious disease has spread significantly throughout the world, counting . . infected people and a death toll of . as of july . [ ] models suggests that it will remain circulating and active for several months, [ , ] and there is a marked possibility that reinfection is possible, [ , ] thus increasing the time of the circulation of the virus. this pandemic outbreak has had a major impact on world economics, with a very long outlook. [ ] a capillary control of the diffusion of the infection has proven crucial, [ ] and serological tests are expected to have a key role in mass screening. [ , ] methods j o u r n a l p r e -p r o o f the structures of the proteins were downloaded from the protein databank (pdb), [ ] the pka values of reactive groups were calculated using propka, [ , ] and the interfaces were calculated using the pdbe pisa server. [ ] the non-bonded interaction of a residue of type i is represented with a lennard-jones (lj) potential: [ , ] where r is the nearest distance between the residue and the surface, is the energy at the minimum position, is the equivalent van der waals radius of each residue and is a size parameter taken from the literature (see tables s -s , parameters are taken from [ , , [ ] [ ] [ ] [ ] , ] , as indicated in the table captions). the electrostatic interaction is represented through the gouy-chapman potential [ , , ] ( ) where r is the nearest distance between the i-th residue with charge and the surface, is the surface charge density, is the inverse debye length calculated from the ionic strength i as √ , and the relative permittivity of the medium is assumed to be distance-dependent ( ). [ , ] a : buffer salt concentration of . mol dm - is assumed. for silica, the surface charge density is estimated to be - . c m - . [ ] for self-assembled charged monolayers (sam), the charge density is set to + . c m - for the amino-capped monolayer (sam-nh ) and to - . c m - for the carboxyl-capped monolayer (sam-co h). [ , ] the formation of a covalent bond is treated with the following potential: where is the bond energy and is set to kj mol - for imino bonds [ ] and kj mol - for gold-thiol bonds, [ ] [ ] [ ] regardless of the starting oxidation state of the thiol. [ ] the desolvation energy is already accounted for in the vdw term. lj parameters for epoxide-glyoxyl functionalization is assumed to be equal to sam-co h, whereas for gold the parameters have been adapted from reference [ ] . the sampling of the relative protein-surface orientations is performed by rotating a plane around the center of mass of the protein. the plane is initially parallel to the z= plane. only two rotations are necessary, as all the rotations around the normal to the plane will j o u r n a l p r e -p r o o f yield the same energy. the first rotation by an angle [ ] is applied around the y-axis, followed by another rotation of an angle [ ] around the z-axis, and then a translation is applied to optimize the position, similarly to what is done in the popular pales software. [ ] [ ] [ ] the sampling of the pairs is made uniform by using repulsion angular sampling. [ ] [ ] [ ] the distance of the plane to the protein is then set by minimizing the energy terms described above. the most important feature of a composite thought for immunochemical applications is that the orientation of the antigen with respect to the surface must ensure the accessibility of the epitope to the antibodies, to guarantee the recognition. therefore, we have selected the crystallographic structure of rbd in complex with a fragment (fab) of the human antibody cr (pdb id: w ), [ ] and identified the interface residues relevant for the interaction ( figure and table s ). in the analysis of the orientations, we assume that the full length antibody will have the same accessibility as the fab because of the high flexibility of the linkers of the heavy chains (see figure s ). [ ] [ ] [ ] [ ] it is also important to note that, while the spike protein is highly glycosylated at n-and o-positions, [ , ] the structured part of the rbd which is recognized by the antibody only carries one glycation at position [ ] (pink in figure ), and the glycation site faces away from the antibody binding site. on these grounds, we have not considered glycation (experimentally, this would be done expressing recombinant rbd in prokaryotic cells, whereas glycation would be obtained in human cells [ ] ). the fragment of the human antibody cr is shown in blue. hydrophobic adsorption occurs selectively on hydrophobic carriers at low ionic strength. [ ] it is a rather common immobilization protocol, because of its simplicity. the also this immobilization strategy is rather common because of its simplicity. it is slightly less general, because the outcome strongly depends on the nature of the protein and of the surface. the electrostatic interaction of the i-th residue with the uniformly charged surface with a given charge density is estimated by the gouy-chapman potential, [ , ] which is added to a lj term. the parameters defining each system are listed in tables s -s . we have considered the following surfaces: ) silica -a common chromatographic support with high negative surface charge; ) positively charged self-assembled monolayer (sam), with amino capping of the chains; [ , ] ) negatively charged sam, with carboxylic capping of the chains. [ , ] supports # and # imply the possibility of colorimetric detection through gold, [ ] vide infra. the most probable orientations are shown in figure . degrees from the one with highest relative population, except one that is more tilted, yielding a larger accessibility, with a relative population around % ( figure s ). it is apparent that only negatively charged surfaces allow for the exposition of the epitope, and this is anyway relatively marginal. these results suggest that it would be nontrivial to achieve a good orientation relying upon adsorption, either based on hydrophobic or on charge interactions. therefore, we have considered directed approaches based on stronger interactions. in particular, we have considered epoxide-glyoxyl (directed at primary amine moieties) [ ] and gold (thiols and disulfide bridges). [ ] the glyoxyl-based approach is quite popular for multipoint orientation-selective immobilization of proteins on surfaces. it involves a two-step mechanism, in the first step, the primary amine groups of the protein are allowed to react with the aldehyde groups to form schiff base bonds, in the second step the bonds are reduced with sodium borohydride. this kind of immobilization has been simulated in a similar way as described by del monte-martìnez et al., [ ] assuming a working ph = . , to maximize the reactivity of the n-terminus and at the same time limiting the reactivity of lysine residues (see table s ). the choice of gold is also extremely popular, because of two reasons: the strong plasmonic response of gold, which causes a purple coloring of the bioconjugate, and because of the relatively easy manipulation required. current sars-cov serological tests are indeed based on gold conjugates. [ ] the conjugation to the surface is simulated in the same way as the amine-glyoxyl reaction, assuming that all cysteines are equally reactive towards gold (disulfide bridges can interact with gold to a comparable extent as thiols). [ ] the resulting orientation has % relative population. colloidal gold has a net negative surface charge, [ ] but including the electrostatic term has no impact on the recovered orientation. in the epoxide-glyoxyl strategy, the conjugation appears to be mostly directed at the nterminus, which is facing away from the recognition interface but is not topologically very remote. therefore, the epitope will only be partially exposed, whereas for the gold conjugation, ample access to the epitope is possible in the most probable orientation. finally, a completely different strategy could be applied for conjugation to (e.g.) gold nanoparticles: the use of a avidin-biotin affinity system. [ ] biotinylation can be achieved through amine-specific reagents, [ ] and improvement in the selectivity can be achieved with minimal engineering of the sequence. [ ] given that there is a rather substantial difference in the calculated pkas for the different amine sites (see table s ), it can be expected that, for ph values lower than , all lysine residues will be protonated and thus less reactive with probability higher than %. the n-terminus is not facing the interaction site (see figure ). therefore, selective biotinylation at the n-terminus is expected to be possible. in this case, the accessibility of the epitope is warranted if the interaction between the antigen and streptavidin, if at all possible, is sufficiently weak. to explore this possibility we have performed an initial-stage docking using zdock, [ ] and inspected the first two elements that had a significantly higher zdock score ( figure s ). the possible interaction between the rbd and streptavidin was investigated also using haddock . . [ ] the protein-protein interface residues were predicted with cport, [ ] and then used as "active" and "passive" residues in the haddock calculation. about lowly populated clusters with weak energy were obtained; the most significant three with the lowest haddock-scores are reported in figure s and their energies in table s . both dockings indicate that, should the interaction occur, it would occur in a position that does not interfere with the antigen-antibody recognition. in this work, we describe the use of united-residue modelling for the prediction of the orientation of the receptor binding domain of the spike protein of the novel coronavirus sars-cov- , a protein of high immunological relevance at the most commonly used surfaces for the preparation of lateral-flow immunochemical devices. with this simple, yet very flexible approach, we find that immobilization on silica, or through glyoxyl reaction of amine residues, or on gold yield orientations compatible with antibody recognition, with gold granting the highest exposition. in this way, we can explain why random conjugation of the rbd to a gold surface yields responsive immunosensors, which are now routinely used. a more detailed experimental verification of the predictions of protein orientation at surfaces represents a significant challenge for the current biophysical methodologies. [ ] one can expect that cryo-electron transmission microscopy will be limited by the fact that, in most cases, the surface has higher electron density than that of the protein. confocal laser scanning microscopy can be used to assess the positioning of the protein with respect to the support, and super-resolution microscopic techniques, such as total internal reflection fluorescence microscopy also allow for the detection of discrete molecular events (e.g., desorption, unfolding, lateral diffusion, …), [ ] but the orientation is still a high-hanging fruit by these methodologies. conversely, the interaction between the protein and the interface can be probed at the atomic level through the application of solid-state nmr, [ , [ ] [ ] [ ] [ ] [ ] [ ] effort which is being started in our lab. our results suggest that very simple modelling approaches can provide significant hints towards rationally orienting j o u r n a l p r e -p r o o f antigens in a way to maximize the exposition of epitopes, and therefore help in the initial moments of the design of conjugates for immunologic applications, when a rapid response to emergency is vital. this is also testified by the emergence of theoretical modelling of several molecular aspects of viral infection and inhibition mechanisms. [ ] [ ] [ ] [ ] [ ] [ ] overall, the expected short-time impact of our work is to provide guidelines to avoid the experimental exploration of immobilization pathways that are less promising. advanced characterization of immobilized enzymes as heterogeneous biocatalysts on the relationship between structure and catalytic effectiveness in solid surface-immobilized enzymes: advances in methodology and the quest for a singlemolecule perspective trapping rnase a on mcm pores: effects on structure stability, product inhibition and overall enzymatic activity proton-detected solid-state nmr spectroscopy of bone with ultrafast magic angle spinning high-resolution structural characterization of a heterogeneous biocatalyst using solid-state nmr conformations and intermolecular interactions in cellulose/silk fibroin blend films: a solid-state nmr perspective ubiquitin immobilized on mesoporous mcm silica surfaces -analysis by solid-state nmr with biophysical and surface characterization engineering l-asparaginase for spontaneous formation of calcium phosphate bioinspired microreactors structural characterization of a protein adsorbed on aluminum hydroxide adjuvant in vaccine formulation, npj vaccines computer-aided design of bromelain and papain covalent immobilization rational design strategy as a novel immobilization methodology applied to lipases and phospholipases orientation of adsorbed antibodies on charged surfaces by computer simulation based on a united-residue model parallel tempering monte carlo simulations of lysozyme orientation on charged surfaces molecular simulation studies of the orientation and conformation of cytochrome c adsorbed on self-assembled monolayers multiscale simulations of protein g b adsorbed on charged self-assembled monolayers computer simulations of fibronectin adsorption on hydroxyapatite surfaces lipase adsorption on different nanomaterials: a multi-scale simulation study ribonuclease a adsorption onto charged self-assembled monolayers: a multiscale simulation study who statement regarding cluster of pneumonia cases in an interactive web-based dashboard to track covid- in real time analysis and forecast of covid- spreading in china covid- : the unreasonable effectiveness of simple models the dynamics of humoral immune responses following sars-cov- infection and the potential for reinfection coronavirus protective immunity is short-lasting ea measures to mitigate the economic, financial and social effects of coronavirus suppression of covid- outbreak in the municipality of the important role of serology for covid- control poolkeh finds the optimal pooling strategy for a populationwide covid- testing (israel, uk, and us as test cases the protein data bank very fast empirical prediction and rationalization of protein pka values improved treatment of ligands and coupling effects in empirical calculation and rationalization of pka values inference of macromolecular assemblies from crystalline state residue-residue potentials with a favorable contact pair term and an unfavorable high packing density term, for simulation and threading coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding generic coarse-grained model for protein folding and aggregation low-resolution models for the interaction dynamics of coated gold nanoparticles with β -microglobulin understanding the curvature effect of silica nanoparticles on lysozyme adsorption orientation and conformation: a mesoscopic coarse-grained simulation study electrostatic interactions of a string-like particle with a charged plate atkins' physical chemistry use of electroactive thiols to study the formation and exchange of alkanethiol monolayers on gold the gold-sulfur interface at the nanoscale quantifying thiol-gold interactions towards the efficient strength control thiols and disulfides on the au( ) surface: the headgroup−gold interaction prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by nmr prediction of charge-induced molecular alignment of biomolecules dissolved in dilute liquid-crystalline phases nmr: prediction of molecular alignment from structure using the pales software repulsion, a novel approach to efficient powder averaging in solid-state nmr simpson: a general simulation program for solid-state nmr spectroscopy computerintensive simulation of solid-state nmr experiments using simpson a highly conserved cryptic epitope in the receptor-binding domains of sars-cov- and sars-cov refined structure of an intact igg a monoclonal antibody crystallographic structure of an intact igg monoclonal antibody edited by crystal structure of a neutralizing human igg against hiv- : a template for vaccine design structure of full-length human anti-pd therapeutic igg antibody pembrolizumab deducing the n-and o-glycosylation profile of the spike protein of novel coronavirus sars-cov- , glycobiology structure, function, and antigenicity of the sars-cov- spike glycoprotein hydrolysis of edible oils by lipases immobilized on hydrophobic supports: effects of internal support structure conjugation of nanoparticles to proteins stabilization of enzymes by multipoint covalent immobilization on supports activated with glyoxyl groups development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis determination of the surface charge density of colloidal gold nanoparticles using second harmonic generation colloidal gold/streptavidin methods preferential labeling of α-amino nterminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays selective n-terminal acylation of peptides and proteins with a gly-his tag sequence zdock server: interactive docking prediction of protein-protein complexes and symmetric multimers the haddock . web server: user-friendly integrative modeling of biomolecular complexes cport: a consensus interface predictor and its performance in prediction-driven docking with haddock atomic structural details of a protein grafted onto gold nanoparticles h-detected solid-state nmr of proteins entrapped in bioinspired silica: a new tool for biomaterials characterization probing the transmembrane structure and dynamics of microsomal nadphcytochrome p oxidoreductase by solid-state nmr reckoning a fungal metabolite, pyranonigrin a as a potential main protease (mpro) inhibitor of novel sars-cov- virus identified using docking and molecular dynamics simulation identification of a novel dual-target scaffold for clpro and rdrp proteins of sars-cov- using d-similarity search, molecular docking, molecular dynamics and admet evaluation inhibition of the main protease cl-pro of the coronavirus disease via structure-based ligand design and molecular modeling identification of potential binders of the main protease clpro of the covid- via structure-based ligand design and molecular modeling interaction of hydroxychloroquine with sars-cov functional proteins using all-atoms non-equilibrium alchemical simulations delving deep into the structural aspects of a furin cleavage site inserted into the spike protein of sars-cov- : a structural biophysical perspective key: cord- -nvyddekm authors: li, dingsheng; sangion, alessandro; li, li title: evaluating consumer exposure to disinfecting chemicals against coronavirus disease (covid- ) and associated health risks date: - - journal: environ int doi: . /j.envint. . sha: doc_id: cord_uid: nvyddekm disinfection of surfaces has been recommended as one of the most effective ways to combat the spread of novel coronavirus (sars-cov- ) that causes coronavirus disease (covid- ). however, overexposure to disinfecting chemicals may lead to unintended human health risks. here, using an indoor fate and chemical exposure model, we estimate human exposure to disinfecting chemicals on the lists recommended by various governmental agencies against covid- , resulting from contact with disinfected surfaces and handwashing. three near-field exposure routes, i.e., mouthing-mediated oral ingestion, inhalation, and dermal absorption, are considered to calculate the whole-body uptake doses and blood concentrations caused by single use per day for three age groups ( , , and -year-old). we also assess the health risks by comparing the predicted whole-body uptake doses with in vivo toxicological data and the predicted blood concentrations with in vitro bioactivity data. our results indicate that both the total exposure and relative contribution from each exposure route vary considerably among the disinfecting chemicals due to their diverse physicochemical properties. -year-old children have consistent higher exposure than other age groups, especially in the scenario of contact with disinfected surfaces, due to their more frequent hand contact and mouthing activities. due to the short duration of handwashing, we do not expect any health risk from the use of disinfecting chemicals in handwashing. in contrast, exposure from contact with disinfected surfaces may result in health risks for certain age groups especially children, even the surfaces are disinfected once a day. interestingly, risk assessments based on whole-body uptake doses and in vivo toxicological data tend to give higher risk estimates than do those based on blood concentrations and in vitro bioactivity data. our results reveal the most important exposure routes for disinfecting chemicals used in the indoor environment; they also highlight the need for more accurate data for both chemical properties and toxicity to better understand the risks associated with the increased use of disinfecting chemicals in the pandemic. disinfection is recommended as a best practice measure to prevent the transmission of the novel coronavirus (sars-cov- ) that causes coronavirus disease through close contact with fomites (u.s. centers for disease control and prevention b). this is because sars-cov- can remain viable on hard surfaces such as plastic, stainless steel, and cardboard for hours and even days (van doremalen et al. ) . to this end, health and environmental authorities in the united states, china, canada, singapore, and many other countries have published a series of guidance documents or official lists to recommend disinfecting products with possible or proven viricidal efficacy. typical viricidal ingredients in these products include alcohols, quaternary ammonium salts, phenolic compounds, diols, and biguanides. while all offer viricidal efficacy, these disinfecting chemicals differ substantially in structure, properties, and environmental behavior: for instance, quaternary ammonium salts are permanently charged and thus involatile, whereas phenolic compounds are generally volatile and more hydrophilic. these disinfecting chemicals can be added into rinse-off liquid hand soaps or rinse-free hand sanitizers for hand sanitization; they can also be used in pre-saturated wipes or sprays for disinfecting the impervious surfaces of furniture or high-touch objects in homes or offices, such as tables, countertops, desks, sinks, toys, and keyboards (chen ) . while accidental exposure to disinfecting chemicals because of misuse or improper use is of increasing concern since the outbreak of covid- (chang et al. ) , it should be noted that humans can also be exposed to disinfecting chemicals during and post proper disinfectant use. during handwashing, disinfecting chemicals penetrate the hand epidermis and enter the circulatory system. the exposure is limited if hand soaps are soon washed off, because of short contact time, or more considerable if the rinse-free hand sanitizers are left over. more often, without being wiped off, disinfectants remain on the disinfected hard surfaces or objects all day long, either as surface residues or bound to settled dust. touching or rubbing the treated hard surfaces dislodges disinfecting chemicals to hands (surface-to-hand transfer); subsequent mouthing, licking, or biting of hands further transfers these compounds to the gastrointestinal tract (hand-to-mouth transfer). direct mouthing of disinfected objects such as toys also results in the oral ingestion of disinfecting chemicals (object-to-mouth transfer). such mouthing-mediated exposure can be of greater concern for infants and toddlers as they mouth almost everything when exploring the environment. exposure to disinfecting chemicals may be associated with adverse health outcomes. for example, a class of quaternary ammonium salts named benzalkonium chlorides have been found to be irritant to animals (choi et al. ) ; the veterinary poisons information service in london has received reports on benzalkonium chlorides per year since , concerning the buccal irritation of cats caused by their grooming after they "accidentally walk across treated surfaces" (campbell and chapman ) . earlier epidemiological evidence has also suggested possible links between human disease with the use of disinfecting products. for instance, weinmann et al. ( ) showed that compared with no use, high use of disinfectants was associated with a more than twofold increased odds of incident asthma, and low/medium use of disinfectants was associated with remittent asthma. in addition, the health risk of disinfecting chemicals can be disputable, because a chemical recommended by one authority may be viewed as poisonous in another country. for example, while antiseptic hand soaps containing triclocarban and triclosan have been removed from the list of ingredients "generally recognized as safe (gras)" by the u.s. food and drug administration (u.s. food and drug administration ), they are authorized by health canada's list of hand sanitizers against sars-cov- because they "meet health canada's requirements for safety, effectiveness and quality" (health canada b). for these reasons, it is imperative to thoroughly understand the magnitude of human exposure to disinfecting chemicals during and post the application and potential adverse health outcomes associated with the exposure, if we want to take preventive measures to avoid the secondary health risks arising from the reduction of pandemic risk. this understanding is exceptionally important since covid- is not likely to be eradicated in a short time and regular household disinfection is believed to be a new normal in the post covid- world. in this work, we evaluate human exposure and health risks associated with the proper use of active ingredients in recommended disinfecting products against sars-cov- . specifically, we estimate the use rates of disinfecting chemicals in two application scenarios, one describing the disinfection of indoor surfaces and objects ("surface application") and the other describing handwashing ("hand hygiene"). for the "surface application" scenario, we simulate the fate and transport of disinfecting chemicals between indoor compartments, human post-application uptake doses, as well as resulting blood concentrations, using a published process-based model named production-to-exposure (protex) (li et al. a; li et al. b) . for the "hand hygiene" scenario, we simulate dermal uptake during handwashing and resulting blood concentrations with protex. we then evaluate the exposure-related health risks by comparing the modeled uptake doses with in vivo toxicological data, and the modeled blood concentrations with in vitro bioactivity data. the protex model (li et al. a; li et al. b ) contains (i) an indoor chemical mass balance module, describing the fate and distribution of applied disinfecting chemicals between five indoor compartments (indoor air, carpet, flooring, hard surfaces, and walls and ceilings) and resultant chemical loadings on indoor surface compartments, and (ii) a human exposure and toxicokinetic module, describing the entry of disinfecting chemicals into the human body through three routes, i.e., mouthing-mediated route (hand-to-mouth and object-to-mouth contact), inhalation of indoor air, and dermal absorption, and resultant body concentrations. in the protex model, "hard surfaces" encompass the impervious surfaces of both furniture (e.g., tables, countertops, desks, and sinks) and high-touch objects (e.g., such as toys, and keyboards). in this work, we consider two scenarios of disinfectant application. first, a "surface application" scenario means the application of disinfecting products, e.g., diluted solutions or disposable pre-saturated wipes, to the "hard surfaces" compartment through broadcasting, spraying, or wiping. earlier work estimated that disinfecting m of hard surfaces consumes on average . g (following a normal distribution) of disinfecting products (supplementary material, text s ) (weerdesteijn et al. ) . the disinfected hard surfaces are often air-dried, with the disinfecting products not being wiped off. active ingredients in disinfecting products remain on the disinfected surfaces for a long time, which thus causes the potential for continuous exposure post the application. we assume the disinfecting chemicals first enter the "hard surfaces" compartment of the indoor chemical mass balance module and then undergo multicompartmental transport and distribution. protex assumes that the modeled individual touches a different fresh location of indoor surfaces during each surface-to-hand contact, and thus contact with an indoor surface does not alter the loadings of chemicals thereon. in this scenario, human exposure is an aggregation of chemical uptake through a mouthing-mediated route (surface-to-hand-to-mouth and object-to-mouth transfer of both dust-bound chemicals and surface residue), inhalation of indoor air, and dermal absorption. we do not consider the dermal absorption of disinfecting chemicals during disinfection, because wearing gloves is a recommended best practice for disinfection (u.s. centers for disease control and prevention a) and, therefore, adherence of disinfecting chemicals to the hand skin is minimal (popendorf and selim ) . we do not consider inhalation exposure during the spraying of disinfecting products because the investigated disinfecting chemicals are mostly minimally volatile and the inhalation exposure is small compared with the post-application exposure (supplementary material, text s ). second, a "hand hygiene" scenario describes the cleaning of hands with a rinse-off hand washer. surveyed data shows that on average . to . g (following a uniform distribution) of hand washers are used in hand hygiene (sanderson et al. ) (supplementary material, text s ). we assume the disinfecting chemicals are directly applied to the "skin" compartment of the human exposure and toxicokinetic module. in this scenario, we consider human exposure through dermal absorption, as inhalation is minimal given that evaporation of disinfecting chemicals is suppressed in when chemicals are dissolved in aqueous solutions (i.e., hand washer). the u.s. centers for disease control and prevention recommends scrubbing hands for seconds before rinsing hands under running water. we assume that the residue of disinfecting chemicals on hand skin is negligible after rinsing, i.e., dermal absorption discontinues at the end of the hand scrubbing. in this work, the protex model is parameterized to represent the exposure of a typical female white american in an "average" home in the u.s. specifically, the modeled home holds a family of . persons (the average u.s. family size) and has a floor area of m (the average u.s. home size) (u.s. environmental protection agency ), with % of the floor area covered by carpet and the rest % by flooring (defaults in protex). hard surfaces, including furniture and objects, account for % of the total floor area (~ m ), in which on average m (~ %) needs to be disinfected (mccready et al. ). the hard surfaces and walls and ceiling are coated with a -nm layer of organic film (weschler and nazaroff ) . the home is ventilated with a u.s. average air exchange rate of . h - (u.s. environmental protection agency ). we evaluate the exposure of the modeled individual at the ages of (young childhood), (adolescence), and (adulthood) because our earlier work shows that chemical exposure varies marginally between ages throughout adulthood (li et al. a ). u.s.specific anthropometric and behavioral parameters of the modeled individual are age-dependent. for instance, as shown in supplementary material figure s , at ages , and , the modeled individual weighs . , , and kg, possesses a skin surface of . , . , and . m , mouths hands (fingers and palms) . , . , and time per hour, and mouths various objects . , . , and time per hour. since the disinfected area is ~ % of the total area of the "hard surfaces" compartment, we assume that only one-tenth of surface-to-hand and object-to-mouth contacts contribute to mouthing-mediated exposure. for other unspecified parameters, we use default values built in the original protex model for calculation. underlying this practice is an assumption that human exposure factors (e.g., the human activity pattern) remain the same as normal during the covid pandemic, as there is presently no information available on the change in human activity pattern during the pandemic time. for instance, the original protex model adopts a u.s. average scenario that the carpet and flooring are cleaned up twice a month and hard surfaces are cleaned ten times a month (li et al. b) , in which cleanup means to remove settled dust, dirt, and impurities from the room but not to use any chemicals to kill pathogens, according to the u.s. cdc's definition (u.s. centers for disease control and prevention a). we assume that during the covid pandemic, the frequencies of cleanup remain the same, but additional disinfection is introduced, which aims to kill pathogens but not necessarily to clean dirty surfaces (u.s. centers for disease control and prevention a). the original protex model also adopts a normal case that individuals spend % of their time indoors; it is currently unclear the extent to which americans' indoor stay would be extended due to the "stay-at-home" order in each state. since the protex model is mechanistic and process-based, it is possible to remediate the potential issues arising from these assumptions if more realistic data become available in the future. we select chemicals used as active ingredients in disinfectants (for both surface and hand sanitization) recommended by the health and environmental authorities in the u.s. (u.s. environmental protection agency b), china (china's national health commission ), canada (health canada a; health canada b), and singapore (national environment agency ). their molecular structures, names, and abbreviations are shown in figure . these chemicals include quaternary ammonium salts, four phenolic compounds (chloroxylenol, thymol, o-phenylphenol, triclosan), a urea (triclocarban), two diols (triethylene glycol, and bronopol), and a bisbiguanide (chlorhexidine gluconate). note that not all chemicals are recommended to be used in all countries. for instance, chlorhexidine gluconate is judged to be "not effective" against sars-cov- and thus not recommended by china's guidance (china's national health commission ), whereas triclocarban and triclosan are not permitted being used in disinfecting products sold in the u.s. text s in the supplementary material also derives the weight fractions of disinfecting chemicals in disinfectant products (isaacs et al. ) , which follows a triangular distribution and ranges from the th percentile of . % to the th percentile of . %, with a median of . %. in each scenario, we calculate the use rates of the investigated disinfecting chemicals (medians and th percentiles) as the product of the total amount of disinfecting products used in each disinfection (see section . ) and corresponding weight fractions. we do not consider the dilution of disinfectants during use. the protex model requires inputs of a chemical's molar mass, partition coefficients between water, octanol, and air (k ow , k oa , and k aw ), the reaction rate constant with [oh] in the indoor air, reaction half-lives in indoor surfaces (carpet, flooring, hard surfaces, and the organic film), and biotransformation half-life in the human body. supplementary material table s shows the values of these parameters. specifically, when measured values (as cited in table s ) are unavailable, the octanol-water partition coefficients (k ow ) and pka are computed using acd/labs software (advanced chemistry development inc., canada), as hodges et al. ( ) indicated that acd/labs predictions for cationic chemicals are in slightly better agreement with measured values than other quantitative structure-property relationship (qspr) models are. the air-water partition coefficients (k aw ) are estimated using the ntp interagency center for the evaluation of alternative toxicological methods (niceatm) models (zang et al. ) and open structure-activity/property relationship app (opera) (mansouri et al. ) when measured values are unavailable. for ionogenic organic chemicals such as quaternary ammonium salts, the model calculation builds on "distribution ratios" (d ow , d oa , and d aw ), i.e., combined partition coefficients of the neutral and ionized species of an ionogenic organic chemical, rather than partition coefficients. we derive the octanol-water (d ow ) and air-water (d aw ) distribution ratios based on pka of ionogenic organic chemicals using the henderson-hasselbalch equation (schwarzenbach et al. ) , whereas the octanol-air (d oa ) distribution ratios are calculated as the ratios between d ow and d aw based on the air-wateroctanol triangle (mackay et al. ) . the reaction rate constants with [oh] are consensus values of predictions of aopwin (built-in in episuite tm ) and opera. presently, there is no experimental evidence indicating the reaction of the investigated disinfecting chemicals on indoor surfaces. as such, we adopt an assumption frequently used in earlier modeling studies (bennett and furtaw ; zhang et al. ) that the reaction of chemicals on indoor surfaces is negligible. while this assumption seems plausible because a recent study shows that atmospheric reaction of chemicals on surfaces can be "entirely suppressed" by a layer of organic film thicker than nm (zhou et al. ) , we conduct a sensitivity analysis later this work to illustrate the response of modeling results to this assumption. the model uses default values of internal energies and activation energies to adjust the partition coefficients and reaction rate constant to a certain temperature. in addition, human biotransformation-halves are computed using the human biotransformation qsar models by papa et al. ( ) implemented in the qsarins-chem module (gramatica et al. ) of the software qsar-insubria (qsarins) (gramatica et al. ) . since the training set in papa et al. models contains few ionogenic organic chemicals, we calculate both the best estimates and th percentiles to characterize the uncertainty associated with the predicted biotransformation halves. we characterize human exposure using protex-predicted (i) whole-body uptake doses (in μg chem /kg bodyweight /d), i.e., the amount of chemical crossing the inner absorption barrier (e.g., the epithelial layers in the gastrointestinal tract and pulmonary alveoli) and entering the circulatory system (zartarian et al. ) , and (ii) blood concentrations (in μm), of disinfecting chemicals resulting from a single disinfection event each day. uptake doses are presented in the form of (i) best estimates calculated using the medians of the use rates, and (ii) th percentiles calculated using the th percentiles of the use rates. blood concentrations are presented in the form of (i) best estimates calculated using the respective medians of the use rates and human biotransformation half-lives, and (ii) th percentiles calculated using the respective th percentiles of the use rates and human biotransformation half-lives. for cases that hard surfaces or hands are disinfected more than once a day, our "unit" predictions can be scaled directly to the realistic frequency of disinfection due to the linearity of the protex model. note that blood concentration can be time-variant in the case of intermittent exposure; the blood concentrations calculated here are averages over the entire day. we evaluate the health risks of the investigated disinfecting chemicals by (i) comparing the protex-predicted uptake doses with maximum acceptable doses derived from in vivo animal-based toxicological data, and (ii) comparing the protex-predicted blood concentrations with in vitro bioactivity thresholds. if the predicted exposure is lower than the corresponding effect threshold then we consider there is no risk associated with the use of a disinfecting chemical. for the maximum acceptable doses, we use the reference doses (rfd) curated by the u.s. epa's comptox chemistry dashboard (u.s. environmental protection agency a) and the derived no-effect levels (dnel) curated by european chemical agency (european chemical agency ). both the rfd and dnel are derived from available no-effect threshold exposure level observed in animal studies and account for the uncertainty rooted in possible intraspecies differences, interspecies variation, duration of the study, and original data quality (ecetoc ; u.s. environmental protection agency ). due to the high similarity in the designs and definitions of rfd and dnel, we consider them equivalent in this study. in addition, the european chemicals agency reports dnels for both workers and the general population when the dnel for workers are consistently higher than that for the general population. we consider both types of dnels, as several investigated disinfecting chemicals only have worker dnels. multiple reported rfd values in the u.s. epa's comptox chemistry dashboard are also compiled. in this work, the maximum acceptable dose of each disinfecting chemical is depicted as a range to allow for the variability in multiple estimates, with the lowest rfd or dnel being the lower estimate and the highest rfd or dnel being the upper estimate. for in vitro bioactivity thresholds, assay-specific toxcast data (version ) for the disinfectants are downloaded from the u.s. epa's comptox chemistry dashboard (u.s. environmental protection agency a) and subsequently processed in a way similar to a previous study (turley et al. ) . in brief, toxcast records cell line responses to chemicals assessed by bioassays and report the ac values (µm, representing the concentration at which half of the maximal active achieved) for each assay. the active assays indicate the adverse effects posed by a tested chemical on the cells, such as cytotoxicity, disruption to transporter function, interference with dna binding (supplementary material table s ). we first remove all background and control assays from the retrieved data. then a cytotoxicity center is calculated by taking the median of the ac values of all active cytotoxicity assays (cytotoxicity as the intended target sub-family) as the non-specific and least sensitive internal toxicity endpoint. the cytotoxicity limit reported in toxcast is used as the non-specific but more sensitive internal toxicity endpoint. for more specific and sensitive toxicity responses, active assays with ac values lower than the cytotoxicity limit are collected and the th percentile values are used subsequently to represent the most conservative endpoint. such a filter of active assays by the cytotoxicity limit is necessary since cytotoxicity can confound the results: when cells are stressed to trigger cytotoxicity, the observed response may not be specific for the investigated effect ). the th percentile ac is the lower estimate and the cytotoxicity center is the upper estimate for no-effect threshold. before presenting and interpreting the modeling results, we first evaluate the performance of the protex model. since monitoring or biomonitoring data are not available for most disinfecting chemicals investigated here, we compare our predictions with those from a commonly used consumer exposure model called consexpo (web version . . , last update: march , ) (delmaar et al. ) to evaluate the model's performance. consexpo supports simulating human exposure to chemicals used in consumer products through inhalation, dermal permeation, and direct oral ingestion (e.g., chemicals migrating from packaging materials) under a range of consumer use scenarios. for the "hand hygiene" scenario, we compare the protex and consexpo predictions of the uptake doses after a single handwashing event (supplementary material, figure s ). in addition to the physicochemical properties shared with protex, consexpo additionally requires user-defined skin permeability (in m/h) of each disinfecting chemical, which is predicted using the approach outlined by weschler and nazaroff ( ) . figure s shows that the protex and consexpo predictions are in general agreement with each other for the investigated chemicals, with a difference within an order of magnitude for chemicals and two orders of magnitude for chemicals. the two model predictions are closest to each other for triclosan (a factor of . ), dodac (a factor of . ), and c bac (a factor of . ). however, the discrepancy between two model predictions is most prominent for diols (triethylene glycol and bronopol), where consexpo gives -and times higher estimates, respectively, than protex. for the "surface application" scenario, we compare the protex and consexpo predictions of chemical loadings adhering to the hand skin after hand rubbing (supplementary material, figure s ). here, the chemical loadings instead of uptake dose are used for comparison because consexpo does not support the prediction of oral ingestion associated with hand-to-mouth contact. chemicals other than quaternary ammonium salts are excluded for comparison, as they are somewhat volatile and can evaporate from the hard surfaces, whereas consexpo does not consider the evaporation loss of chemicals post the application. as shown in figure s , protex predicts chemical-specific dermal loadings, ranging from . to . mg/cm . this range overlaps with the estimate by consexpo, i.e., a mean of . mg/cm with a % confidence interval between . and . mg/cm , which is a "generic" value for all chemicals rubbed from a chemical-treated surface. in sum, protex gives estimates comparable to those from consexpo. in addition, our protex modeling predicts that the use of a disinfecting hand washer for s once a day leads to dermal absorption of . × - and . × - mg/kg/d (medians) of triclosan by a -and -year-old females, respectively. these values are in the same order of magnitude as the daily exposure of the general americans to triclosan (medians of . × - mg/kg/d for ages - and . × - mg/kg/d for ages - ), which were back-calculated from biomonitoring data in the national health and nutrition examination survey (nhanes) . while this agreement suggests the fidelity of protex to realistic human exposure, it should be noted that sources other than handwashing may also contribute to human triclosan exposure. given that monitoring and biomonitoring data are lacking for a more thorough evaluation of the model, measuring the occurrence of disinfecting chemicals in various indoor compartments and human tissues are warranted in the future. as the first step toward an understanding of human exposure to disinfecting chemicals, we simulate the indoor fate and distribution of these chemicals. since the volatility of chemicals is the dominant factor governing chemicals' indoor fate and distribution, for illustration we select two chemicals: the permanently charged and hence involatile dodac (i.e., with an infinite d oa ), and the moderately volatile o-phenylphenol (i.e., with a d oa of . at ph= ). figure a depicts the transport of dodac between different indoor compartments. over % of dodac remains on the hard surface compartment where it is first applied, which can be either present as surface residues or absorbed onto the settled dust on the hard surfaces. while this fraction can be efficiently removed through regular surface cleanup ( times a month as assumed in protex), it is available for mouthing-mediated human exposure between the cleanup events. approximately . % of dodac, most of which is bound to resuspended dust, enters the indoor air compartment and is subsequently deposited on other indoor surfaces such as carpet, flooring, and the organic film on walls and ceiling. therefore, while the application of dodac targets the hard surface compartment, it is not impossible to observe its occurrence in other nontargeted indoor compartments. yet, dodac's low volatility leads its concentration in the nontargeted indoor compartments to be orders of magnitude lower than that on hard surfaces. for instance, our protex modeling shows that dodac concentration on hard surfaces is ~ and times higher than that in the organic film and flooring, respectively. such inter-compartmental migration has also been found for other indoors-used chemicals. a similar instance is that the broadcast application of chlorpyrifos (k oa = . according to the opera model) resulted in a detectable level of this compound on nontargeted furniture surfaces, which was times lower than that on the targeted carpet surfaces (lu and fenske ) . note that the above calculation assumes that dodac is permanently charged given its application in the form of a water solution. however, the dissociation of dodac might be suppressed as the disinfected hard surface dries. it remains unknown the minimum level of humidity that maintains the dissociation of dodac. to explore whether our above calculation is still valid if the indoor environment is completely dry, we perform an additional simulation by assuming an extreme situation where neutral dodac does not dissociate at all. supplementary material, figure s shows that despite being more volatile, neutral dodac behaves similarly as the dodac cation in the indoor environment, with the dominant share ( %) of the applied amount remaining on the disinfected hard surfaces and a small fraction ( %) migrating between indoor compartments. the fraction remaining on the hard surfaces is still a relevant source for mouthing-mediated exposure. likewise, surface cleanup is the dominant mechanism whereby neutral dodac is eliminated from the room. as such, while the assumption of the reduced dissociation is uncertain, the main conclusions present in this work do not change. for comparison, figure b depicts the transport of o-phenylphenol between different indoor compartments. unlike dodac, more than % of the applied o-phenylphenol readily evaporates from the disinfected hard surfaces and enters the indoor air, with . % remaining on the hard surfaces and available for mouthing-mediated exposure. compared with dodac, o-phenylphenol is more abundant in the indoor air. over % of the evaporated o-phenylphenol will be ventilated out from the room, whereas the rest % will be degraded through radical reactions. in this case, we can see relatively rapid dissipation, or reduced persistence, of o-phenylphenol in the indoor environment. furthermore, the relatively higher volatility of o-phenylphenol results in a more equal distribution of this compound among indoor compartments. for example, our protex modeling indicates that o-phenylphenol concentration on the treated hard surface is merely . and times higher than that in the untreated organic film and flooring, respectively. protex predicted the relative contribution of three routes of entry (mouthing-mediated ingestion, dermal absorption, and inhalation of indoor air) to the aggregate exposure of a -year-old child to various disinfecting chemicals in the "surface application" scenario ( figure ). for comparison, results for a -year-old adult are presented in the supplementary material, figure s . the relative contribution is chemical-specific and thus a reflection of their partition properties. figure shows that mouthing-mediated ingestion dominates the aggregate exposure of the modeled child to quaternary ammonium salts, triclocarban, and triclosan, which is also the case for the modeled adult ( figure s ). these chemicals are either involatile (the permanently charged quaternary ammonium salts) or minimally volatile (with a d oa of . and . for triclocarban and triclosan, respectively, at ph= ). as discussed in section . , these high d oa chemicals tend to adhere to the hard surfaces after surface application. furthermore, their low volatility favors their transfer from the treated hard surface to hand skin during surface-hand contact. we can, therefore, anticipate that mouthing is a remarkable contributor to human exposure to these chemicals. this anticipation echoes an earlier general conclusion that "indoor exposure of chemicals with high k oa is predominantly the result of nondietary ingestion of dust and surface contact" (zhang et al. ) . by contrast, the rest of the investigated disinfecting chemicals are moderately volatile (with k oa ranging from to ) and thus have a limited potential for migration from treated hard surface to hands. figure and figure s in supplementary material show that dermal absorption dominates human exposure to phenolic chemicals and bronopol, which partition almost equally between water and organic phases (with a d ow ranging from to at ph= , based on the neutral values present in table s ), whereas inhalation contributes most to exposure to chlorhexidine gluconate and triethylene glycol, which are extremely hydrophilic (with a d ow lower than - at ph= ). in general, the anatomical structure of hand skin poses permeation resistance to both highly hydrophobic and hydrophilic chemicals: the lipid matrix of stratum corneum retards the permeation of highly hydrophobic chemicals, whereas the aqueous intracellular fluid in the viable epidermis and extracellular fluid retard the permeation of highly hydrophilic chemicals (brown et al. ). as such, chemicals with a moderate k ow or d ow , neither too large nor too small, have the highest capability of permeating through hand skin. for instance, li et al. ( ) found that when chemicals are released exclusively into the indoor air, dermal absorption from indoor air contributes to up to % of aggregate human exposure to chemicals with a k oa from and and a k ow from we then calculate the uptake doses of the investigated disinfecting chemicals after a single use per day in the "surface application" (figure a ) and "hand hygiene" (figure b ) scenario. in both scenarios, the predicted uptake doses vary by around one order of magnitude from the medians to th percentiles (figure ). in the "surface application" scenario (figure a) , the modeled individuals are exposed to quaternary ammonium salts (notably c to c bac) the highest, regardless of age group, because these chemicals are most strongly retained on the treated hard surfaces. by contrast, in the "hand hygiene" scenario (figure a) , the modeled individuals are exposed to phenolic compounds the highest, regardless of age group, as these chemicals are neither too hydrophobic nor too hydrophilic and thus demonstrate a strong dermal absorption capability. in both scenarios, exposure to chlorhexidine gluconate is the lowest among all disinfecting chemicals. a comparison between figures a and b indicates that for the single use of all the investigated disinfecting chemicals, exposure after surface application outweighs that through handwashing. the reason behind this contrast is intuitive: washing hands under running water rinses the disinfecting chemicals off, hence limiting the duration of dermal exposure ( s), whereas disinfecting chemicals can reside on the treated hard surfaces all day long, having time" for humans to touch, before they are removed by the regular cleanup. the difference in exposure between two scenarios is most prominent for chlorhexidine gluconate ( orders of magnitude), followed by quaternary ammonium salts such as benzethonium chloride and c adbas ( orders of magnitude), whereas it is moderate for phenolic chemicals such as chloroxylenol and o-phenylphenol (~ times). we can, therefore, conclude that the post-application exposure is more remarkable than instant exposure during handwashing when the investigated disinfecting chemicals are employed to cope with the sars-cov- virus. modeled uptake of disinfecting chemicals by a -year-old child, a -year-old teenager, and a year-old adult in the "surface application" (panel a) and "hand hygiene" (panel b) scenarios, and the comparison with in vivo toxicological data. in both scenarios, the modeled -year-old child has generally higher uptake doses than the -year-old teenager and the -year-old adult (figure ). in the "surface application" scenario (figure a) , the age-specific difference in uptake is most remarkable for quaternary ammonium salts, by a factor ranging from for c bac to for benzethonium chloride between ages and , but smaller for other compounds, especially phenolic chemicals. as discussed above, human exposure to quaternary ammonium salts mostly come from mouthing-mediated ingestion. compared with teenagers and adults, children have much more frequent surface-to-hand and hand-tomouth contacts, which results in an elevated contribution of mouthing-mediated ingestion. as such, we can expect an order of magnitude higher uptake of quaternary ammonium salts by children than teenagers and adults. by contrast, in the "hand hygiene" scenario (figure b) , human uptake is more consistent among age groups, with a difference generally within a factor of . this difference reflects mainly age-dependent differences in bodyweight and the area of hand surface (different masses of handwasher used). in sum, our modeling results demonstrate that children tend to be exposed to higher doses of disinfecting chemicals than teenagers and adults; the higher contribution of mouthing-mediated ingestion to the aggregate exposure, the larger such an age-dependent difference can be. we also predict the blood concentration of the investigated disinfecting chemicals after a single use per day ( figure ) . the blood concentration reflects the combined effects of uptake of disinfecting chemicals and elimination (e.g., biotransformation, renal clearance, and fecal egestion). in both scenarios, the predicted blood concentrations vary by around one order of magnitude from the medians to th percentiles ( figure ). in the "surface application" scenario (figure a ), triclocarban and triclosan have the highest concentrations (respective best estimates of . and . μm) despite their modest uptake (ranking and , respectively; figure a ), as they are an order of magnitude more resistant to biotransformation than the other disinfecting chemicals (table s in supplementary material). in comparison, while the modeled individuals have the highest uptake of c to c bacs, the blood concentrations of these compounds are not in top rank (ranking to ) because they are predicted to be relatively rapidly eliminated from the human body (table s ). here, we should confess that qsar prediction of biotransformation half-lives of quaternary ammonium salts is challenging and uncertain because they are seldom included in the training sets for developing the currently used qsar tools. while the qsar used in this work (papa et al. ) recognizes that c bac and c bac fall within its applicability domain and warns that c bac and c bac exceed it, readers should be with caution when interpreting the predicted blood concentrations of quaternary ammonium salts. nevertheless, our predicted relative order of elimination rates of the investigated disinfecting chemicals seems to agree with experimental studies and thus be somewhat reasonable. for instance, the half-life of total elimination of bacs was found to be . - . hours in rats (xue et al. ) , which is an order of magnitude faster than that of triclosan of - hours in rats, as reviewed in . in the "hand hygiene" scenario (figure b) , the relative order of predicted blood concentrations of the investigated disinfecting chemicals more closely mimics that of the predicted uptakes. note that the above conclusion builds on an assumption that the disinfecting chemicals staying on indoor surfaces are not degradable. earlier experimental studies have demonstrated that the organic film on indoor surfaces can protect chemicals therein from atmospheric oxidation (zhou et al. ; zhou et al. ) . since [oh] radicals and other oxidants must first oxidize the organic layer before reacting with the chemicals "dissolved" in it, the rate of oxidation becomes the limiting step of the reaction of chemicals (alwarda et al. ) . the latest estimate (alwarda et al. ) indicates that oxidizing a -nm-thick organic layer in indoor conditions would take to days, which means the disinfecting chemicals in the typical -nm thick organic film assumed in protex would be preserved for (lower estimate) to (higher estimate) days. to illustrate the influence of the potential degradation of the disinfecting chemicals on modeled exposure, we conduct a sensitivity analysis, using dodac (involatile) and o-phenylphenol (volatile) as the example, as shown in the supplementary material, table s . should the lower estimate be used, the estimated exposures to dodac and o-phenylphenol would drop by % and %, respectively, relative to the calculation based on default assumption, whereas should the higher estimate be used, the estimated exposures are almost unchanged. that is, in the worst case, our assumption of no degradation on indoor surface compartments leads to an overestimation of exposure to highly involatile chemicals by a factor of five. figure . modeled blood concentrations of disinfecting chemicals in a -year-old child, a -year-old teenager, and a -year-old adult in the "surface application" (panel a) and "hand hygiene" (panel b) scenarios, and the comparison with in vitro bioactivity data. not all disinfecting chemicals investigated have effect thresholds publicly available from the sources used in this study (tables s and s in supplementary material) . out of the disinfecting chemicals, have rfd and/or dnel data ( figure ); in addition, the european chemicals agency database indicates that there is "no hazard identified", instead of simply without any information, for c bac, c bac, c bac, and triethylene glycol. irritation is the most common hazard identified for these chemicals while skeletal toxicity, hepatoxicity, and disruption to homeostasis are possible for some chemicals (table s in supplementary material). in comparison, in vitro bioactivity thresholds from toxcast provides wider coverage with ac values available for all the chemicals mentioned above and another four chemicals ( figure ). it should be noted that among these chemicals, chloroxylenol and triethylene glycol have no active assays with ac lower than their cytotoxicity limits. triethylene glycol also has a very low cytotoxicity center ( . µm) and its cytotoxicity limit is µm, according to toxcast. a total of targets on the macromolecular and cellular level are found among active assays with ac below cytotoxicity limit for these chemicals (table s in supplementary material). comparing the estimated exposure with these effect thresholds enables risk assessment for these chemicals in the studied scenarios. regardless of the age group and effect thresholds, the "hand hygiene" scenario is found to pose no risks to human health. as shown in figure b and figure b , the predicted exposure is three to twelve orders of magnitude lower than even the lowest level that can potentially cause adverse effects. comparing the uptake doses with maximum acceptable doses (figure a ), we can find that, in the "surface application" scenario, all three age groups are at risk for cetrimonium bromide after a single application, while they are not at risk for chloroxylenol, thymol, triethylene glycol, and chlorhexidine gluconate despite variations in exposures and maximum acceptable doses. if the lower estimate for maximum acceptable doses and the th percentile of exposure are used for a more conservative risk assessment, dodac, dddac, bronopol, triclocarban, and triclosan pose risk for all age groups. for o-phenylphenol, the -year-old adult would have little risk, while the -year-old teenager at the th percentile of exposure could experience some risk if the lower estimate for maximum acceptable doses is used, and the -old-year young child could be more likely to be at risk due to the higher exposure. on the other hand, if the upper estimate for maximum acceptable doses is used, in addition to cetrimonium bromide, only bronopol and triclocarban will pose risk to teenagers and adults except for those with lower exposure. however, risk assessment results for young children stay generally the same (except for o-phenlphenol for which young children will not experience risk) due to their much higher exposure levels. by contrast, comparing the predicted exposures with in vitro bioactivity thresholds gives a substantially different profile of risks (figure a ). our results indicate there is little risk for all chemicals for teenagers and adults, except for triclocarban when the lower estimate for bioactivity threshold is used and the modeled individuals are in the higher range of exposure estimates. the -year-old child is at risk for triclocarban no matter the variation in exposure or bioactivity threshold. by using the most sensitive bioactivity threshold ( th percentile of ac ), many children could be at risk for c bac, c bac, benzethonium chloride. only children with th percentile of exposure may be at risk for c bac, dodac, cetrimonium bromide, and triclosan. we should again emphasize that the above results are based on a single disinfection event a day, and that the actual human exposure can be proportional to the frequency of disinfection. for instance, if we evaluate the risk based on th percentile of exposure and the lower estimate for bioactivity threshold, the exposed individual may be at risk because of exposure to triclosan if the hard surface is disinfected five times a day, and because of exposures to c bac and cetrimonium bromide if the hard surface is disinfected six times a day. high-throughput in vitro toxicity testing like toxcast creates a new pathway for bridging the gap between the availably of human-relevant toxicity data and the ever-growing number of chemicals circulating in commerce to better inform regulatory decisions national research council ; punt et al. ) . since its creation, toxcast has been mainly used as a screening level tool to identify "hot spots" where chemical exposure and toxicity may overlap , rank chemicals of concerns based on potential hazards (tilley et al. ) , and prioritize the further analysis of chemicals exhibiting in vitro bioactivity for certain health outcomes (auerbach et al. ; karmaus et al. ) . to our knowledge, there is still a void for side-by-side quantitative examination of in vitro bioactivity data-based risk assessment and in vivo toxicological data-based, or traditional, risk assessment. by such an examination, our study offers insights into the application of highthroughput in vitro bioactivity data in risk assessment. ideally, a comparison between blood concentration with in vitro bioactivity data should yield similar results from the comparison between the uptake doses with in vivo toxicological data if both the exposure and toxicity are accurately reflecting the underlying biomechanisms. however, the risk assessment results from in vivo and in vitro in this study are not in good agreement. results from comparing daily uptake and in vivo toxicological data showed higher risks in contrast with the comparison of blood concentration and in vitro bioactivity data in the "surface application" scenario. even for the "hand washing" scenario, the gap between exposure and effect thresholds indicated by in vitro bioactivity data is several orders of magnitude lower than that indicated by in vivo toxicological data. this disagreement underscores two aspects of high uncertainty in the field of risk assessment. first, there is a need for a better understanding of the biodistribution of disinfecting chemicals within the body. despite recent developments in estimating key biodistribution determinants, a lack of accurate data for such determinants remains as one of the most challenging issues in the in vitro-in vivo extrapolation (ivive) for exposure (wambaugh et al. ; wambaugh et al. ) . indeed, the biotransformation data used in this study is derived from qsar models and carries corresponding uncertainty, especially for the quaternary ammonium salts that fall outside the applicability domain. an overestimate of biotransformation could then result in an underestimate of blood concentration, which would lead to a lower assessment of risk, everything else being equal. second, there is currently a lack of conciliation between traditional toxicity endpoints (i.e., rfd and dnel) and in vitro bioactivity endpoints to achieve more consistent results in the context of risk assessment. for traditional toxicity endpoints that have been extrapolated from animal studies, the point of departure is usually divided by an arbitrary factor of ten to account for interspecies extrapolation as a conservative approach (ecetoc ; u.s. environmental protection agency ). while investigations on a few selected chemicals showed inconsistency between oral equivalent dose extrapolated from bioactivity data in human cell lines and the point of departure observed from animal studies (silva et al. ; turley et al. ), a recent examination on approximately chemicals indicated that there may not be systematic differences between these two metrics (wang ) . if one is to believe human cell line results are more relevant to human health, this could suggest that the approach for deriving traditional toxicity endpoints may be overly cautious and overestimating the risks from the same exposure. nevertheless, in vitro bioactivity data is not without its own shortcomings. cross examinations between toxcast test results with in vivo toxicity data show that chemicals with assay results in toxcast reporting inactive for specific outcomes may indeed induce the corresponding outcomes in vivo (pham et al. ; silva et al. ) . in this work, we present the first thorough overview of human exposure and health risk for disinfecting chemicals recommended to prevent the spread of sars-cov- in scenarios of surface application and hand hygiene. our study shows that the wide range of physicochemical properties results in vastly different exposure patterns for different disinfecting chemicals and different age groups. while no health risks are identified with the hand hygiene scenario for any investigated disinfecting chemicals, some may pose risks with the surface application, especially for young children. as such, it is of potential health concern to leave disinfecting chemicals on high touch hard surfaces without wiping off, although the residuals of some disinfectants are believed to provide continued efficacy of suppressing the replication or survival of viruses, even after dryness (brown et al. ) . this health concern can be viewed as a secondary risk created by the response to the risk of the covid- pandemic. although our assessment indicates health risks arising from the use of disinfectants, this should not be taken as a recommendation of not using disinfectants to contain the threat of covid- pandemic. it should also be emphasized that our assessment reflects the maximum health risks associated with the proper use of disinfecting chemicals, as we assume that disinfectants are used at full strength without dilution, the modeled individual is exposed to disinfecting chemicals through direct hand contact with disinfected surfaces and objects, and that disinfectants are not wiped off after surface application. while it for the first time informs us of the potential health outcomes of the consumer use of disinfecting chemicals, conclusions present in this work can be preliminary because of insufficient information and methodological limitation. therefore, we encourage future work to further elucidate the fate, exposure, and toxicity of disinfecting chemicals. first, since most disinfecting chemicals are ionogenic organic chemicals (iocs), and that the indoor fate and the human biodistribution processes of iocs are less understood at the current moment, future work is encouraged to investigate more in depth the behavior of iocs in the environment and human body. for instance, it remains unclear whether, in what condition, and to what extent, iocs can ionize on indoor surfaces and the human skin, and whether the proportionate combination of neutral and charged forms of iocs can really represent the overall behavior of the partially ionized iocs. it also remains unknown whether the application of iocs, e.g., the disinfectants investigated in this work, can disturb the integrity of the stratum corneum (e.g., hyperkeratosis) and hence influences the mechanisms of absorption and toxicity. in addition, we have little information on whether the transporter-facilitated "active" absorption contributes to the uptake of mouthed iocs in the gastrointestinal tract. there is currently a lack of qsar tools for iocs. with emerging information being available, modification to protex and other existing models to accommodate the possible unique behavior of iocs is warranted. previous reviews have provided a pioneering guide on possible ways of expanding models towards this direction (armitage et al. ; bonnell et al. ) . second, since toxicological data are either missing or associated with uncertainties, advancement in highthroughput toxicity testing is still in great need to close the gap between the number of chemicals with available data and the number of chemicals that humans are potentially exposed. in addition, it is important to develop new knowledge and best practice of how to use this high-throughput toxicity data in risk assessment, given the inconsistent results from traditional risk assessment methods using animal-based toxicity data. despite pioneering works utilizing high-throughput in vitro bioactivity data in risk prioritization (shin et al. ; wegner et al. ; wetmore et al. ) , the value from the rapidly increasing amount of high-throughput toxicity data is unlikely to be fully realized without accompanying methodologies of its application in human health risk assessment that is recognized by a wide range of stakeholders. third, there is a need for a more thorough assessment of possible environmental and health impacts of disinfecting chemicals to avoid possible risk-risk trade-off, i.e., reducing the risk of the pandemic by creating new risks of consumer and ecological exposure. for instance, while our work recognizes that handwashing causes low human exposures, the large fraction of disinfectants washed off can be a relevant source to its presence in municipal wastewater, and by extension, the aquatic environment. monitoring studies have reported surprisingly high levels of total bac concentrations (c to c ) in sewage sludge in china (on average . μg/g) (li et al. ) and surficial sediments in urban estuarine in the united states (on average . μg/g in new jessy and new york) (li and brownawell ) . it remains unknown whether these levels can lead to significant adverse ecological impacts, and to what extent the levels surge after the covid pandemic given that new jessy and new york are the epicenters of the united states. future monitoring work is also encouraged to determine the occurrence of disinfecting chemicals in various indoor compartments (e.g., settled dust, hard surfaces, objects) and human body tissues. joint efforts between (bio)monitoring, modeling, and policy analysis may bring about best practice of disinfectant use to balance their benefits and impacts on human health and prepare us to better respond to public health emergencies in the future. lastly, it is worth investigating further on the viricidal efficacy of the disinfectants (rabenau et al. ) and considering this information in determining the safety of disinfectants. in this study, we assumed the same usage for all disinfectants, which may not be true. it is possible that while some disinfectants are shown to pose lower risks to human health in our assessment, their health risks could be high in reality because their lower viricidal efficacy requires more frequent uses or uses in a larger quantity to achieve the same viricidal performance as others. therefore, a more comprehensive evaluation should be done based on the desired function and the varying amount of disinfectants associated with this function. there is also a need for a more thorough examination of the risk-benefit trade-off, by considering a broader range of medical and health evidence such as the probability of contracting covid- from hard surfaces across different population without the use of disinfectants, the reduction of this probability with different use patterns of various disinfectants, and individual health outcome and impact from covid- . heterogeneous oxidation of indoor surfaces by gas-phase hydroxyl radicals assessing the bioaccumulation potential of ionizable organic compounds: current knowledge and research priorities a weight-of-evidence approach for the bioaccumulation assessment of triclosan in aquatic species prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using toxcast™ high-throughput data fugacity-based indoor residential pesticide fate model fate and exposure modeling in regulatory chemical evaluation: new directions from retrospection influence of drying time on prewetted disinfectant towelettes to disinfect glass surfaces dermal permeation data and models for the prioritization and screening-level exposure assessment of organic chemicals handbook of poisoning in dogs and cats cleaning and disinfectant chemical exposures and temporal associations with covid- -national poison data system reducing covid- transmission through cleaning and disinfecting household surfaces. vancouver, bc: national collaborating centre for environmental health beijing: china's national health commission risk assessment of benzalkonium chloride in cosmetic products consumer exposure and uptake models guidance on assessment factors to derive a dnel. brussels: european centre for ecotoxicology and toxicology of chemicals qsarins-chem: insubria datasets and new qsar/qspr models for environmental pollutants in qsarins qsarins: a new software for the development, analysis, and validation of qsar mlr models hard-surface disinfectants and hand sanitizers (covid- ): list of disinfectants for use against covid- hard-surface disinfectants and hand sanitizers (covid- ): list of hand sanitizers authorized by health canada a comparison of log k-ow (noctanol-water partition coefficient) values for non-ionic, anionic, cationic and amphoteric surfactants determined using predictions and experimental methods consumer product chemical weight fractions from ingredient lists in vitro and modelling approaches to risk assessment from the u.s. environmental protection agency toxcast programme editor's highlight: analysis of the effects of evaluation of food-relevant chemicals in the toxcast high-throughput screening program revisiting the contributions of far-and near-field routes to aggregate human exposure to polychlorinated biphenyls (pcbs) towards a systematic understanding of the dynamic fate of polychlorinated biphenyls in indoor, urban and rural environments how are humans exposed to organic chemicals released to indoor air? quaternary ammonium compounds in urban estuarine sediment environments -a class of contaminants in need of increased attention? occurrence of quaternary ammonium compounds (qacs) and their application as a tracer for sewage derived pollution in urban estuarine sediments air and surface chlorpyrifos residues following residential broadcast and aerosol pesticide applications the evolution and future of environmental partition coefficients opera models for predicting physicochemical properties and environmental fate endpoints potential sequential exposures to -butoxyethanol after use of a hard-surface cleaner interim list of household products and active ingredients for disinfection of the covid- virus toxicity testing in the st century: a vision and a strategy development of human biotransformation qsars and application for pbt assessment refinement using toxcast to explore chemical activities and hazard traits: a case study with ortho-phthalates exposures while applying commercial disinfectants potential of toxcast data in the safety assessment of food chemicals efficacy of various disinfectants against sars coronavirus exposure and prioritization -human screening data and methods for high production volume chemicals in consumer products: amine oxides a case study risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays a comparison of toxcast test results with in vivo and other in vitro endpoints for neuro, endocrine, and developmental toxicities: a case study using endosulfan and methidathion incorporating toxcast and tox datasets to rank biological activity of chemicals at superfund sites in north carolina incorporating new approach methodologies in toxicity testing and exposure assessment for tiered risk assessment using the risk approach: case studies on food contact chemicals centers for disease control and prevention. interim recommendations for u.s. households with suspected or confirmed coronavirus disease reference dose (rfd): description and use in health risk assessments exposure factors handbook comptox chemistry dashboard list n: disinfectants for use against sars-cov- (covid- safety and effectiveness of consumer antiseptics; topical antimicrobial drug products for over-the-counter human use. cfr part aerosol and surface stability of sars-cov- as compared with sars-cov- evaluating in vitro-in vivo extrapolation of toxicokinetics high throughput heuristics for prioritizing human exposure to environmental chemicals toxicokinetic triage for environmental chemicals infer the in vivo point of departure with toxcast in vitro assay data using a robust learning approach hygienic cleaning products used in the kitchen: exposure and risks (rivm report high-throughput screening tools facilitate calculation of a combined exposure-bioactivity index for chemicals with endocrine activity association of household cleaning agents and disinfectants with asthma in young german adults svoc exposure indoors: fresh look at dermal pathways. indoor air growth of organic films on indoor surfaces. indoor air incorporating high-throughput exposure predictions with dosimetry-adjusted in vitro bioactivity to inform chemical toxicity testing distribution and disposition of benzalkonium chloride following various routes of administration in rats in silico prediction of physicochemical properties of environmental chemicals using molecular fingerprints and machine learning adoption of an official isea glossary model for screening-level assessment of near-field human exposure to neutral organic chemicals released indoors multiphase reactivity of polycyclic aromatic hydrocarbons is driven by phase separation and diffusion limitations burial effects of organic coatings on the heterogeneous reactivity of particle-borne benzo[a]pyrene (bap) toward ozone the authors thank yuchao lu for data collection and jon. a. arnot for assistance with pka calculation. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -vm nh lc authors: perez espitia, paula judith; de fátima ferreira soares, nilda; dos reis coimbra, jane sélia; de andrade, nélio josé; souza cruz, renato; alves medeiros, eber antonio title: bioactive peptides: synthesis, properties, and applications in the packaging and preservation of food date: - - journal: compr rev food sci food saf doi: . /j. - . . .x sha: doc_id: cord_uid: vm nh lc abstract: bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living beings. peptides have shown several useful properties for human health, including antimicrobial, antifungal, antiviral, and antitumor activities. these compounds are produced by almost all species of life. however, they are produced in limited quantities in nature. as a result, researchers have tried to synthesize bioactive peptides to study their properties and applications in various areas. among their applications in food preservation, peptides have been incorporated into packaging materials. this review begins with a brief description of the methods used for the synthesis, purification, and characterization of peptides. also, the main bioproperties and mechanisms of action of peptides are discussed. finally, some applications of peptides are presented, especially their use in active packaging, their effects on the polymeric matrix, and peptide migration. food safety is a growing concern of great importance worldwide. recently, the estimated costs of diseases caused by foodborne pathogens was about $ billion in the united states (scharff ) , and it is estimated that in the united states alone about . million illness cases, hospitalizations and deaths will be caused by foodborne pathogens in . the consumption of processed foods with chemical preservatives has led to increased consumer concern and the demand for more natural and minimally processed foods. as a result, researchers have shown a growing interest in natural antimicrobial agents such as certain peptides. bioactive peptides are defined as specific protein fragments that have a positive impact on the functioning or conditions of living beings, thereby improving their health (korhonen and pihlanto ) . the beneficial effects are attributed to different properties found in peptides such as antimicrobial (reddy and others ; rajanbabu and chen ) , antioxidant (sarmadi and ismail ) , antithrombotic (wang and ng ) , anti-hypertensive (erdmann and others ) , and immunomodulatory activities (st georgiev ; gauthier and others ) , among others. ms submitted / / , accepted / / . authors espitia, soares, coimbra, de andrade, and medeiros are with food technology dept., federal univ. of viçosa, av. p. h. rolfs, s/n, campus univ., - . viçosa, minas gerais, brazil. author cruz is with food technology dept., state univ. of feira de santana, av. transnordestina, s/n, campus univ., - . feira de santana, bahía, brazil. direct inquiries to author soares (e-mail: nfsoares @gmail.com) . peptides with antimicrobial properties are used as the first chemical barrier against microbial attack, being synthesized in response to bacterial infections. they are produced by almost all species of life, from microorganisms, plants and animals, to humans (st georgiev ; hancock and diamond ) . in animals, antimicrobial peptides are produced mainly in those tissues exposed to adverse conditions such as skin, eyes, and lungs, which are more likely to be in contact with microorganisms (zasloff ; papo and shai ) . more than antimicrobial peptides have been reported, showing significant variations with respect to their sequence, length, and structure (papo and shai ) . antimicrobial peptides have found many applications, including those in biomedical devices, food processing equipment, and food preservation. in food preservation, peptides can be incorporated into materials to create antimicrobial packaging (appendini and hotchkiss ) . in this way, antimicrobial packaging plays an important role in maintaining the safety and quality of food, since the aim is to prolong food shelf life and to reduce bacterial growth on the product surface (soares and others a) . this type of active packaging interacts with the product and/or the headspace inside to reduce, inhibit, or retard the growth of microorganisms that may be present (soares and others b) . this review highlights the main methods of peptide synthesis and noteworthy peptide bioproperties. also, specific peptide applications in food preservation are reviewed, focusing on their incorporation in polymeric matrices. finally, the effects of peptide incorporation on packaging characteristics as well as their migration into food are discussed. borgia and fields ( ) . copyright ( ) , elsevier. peptides are biomolecules that contain between to several dozen of amino acid residues joined by peptide bonds. the discovery of the different peptide activities has generated enormous interest in this class of compounds and in the methods of isolation, analysis, purification, identification, and quantification. these methods have been systematically studied and improved. however, most sources of natural peptides are poor in these compounds, thus preventing their isolation in sufficient quantities for research. as a result, there was a growing need to synthesize peptides for application in physiological, chemical, physical, pharmacological, biochemical, and clinical studies. total of methods of peptide synthesis have been developed and improved: chemical synthesis, which uses chemical reagents to mediate peptide bond formation (andreu and rivas ) , enzymatic synthesis, in which the peptide bond formation is catalyzed by enzymes (bongers and heimer ; boeriu and others ) , and the dna recombinant technology synthesis, based on the use of cloning and ribosomal techniques from biological systems for peptide formation (sewald and jakubke ) . research on this synthesis method was first initiated more than y ago. however, the construction of peptides has recently become more accessible due to advances in process efficiency, including the development and use of fast coupling reagents, as well as the minimization of side reactions (borgia and fields ) . the main aspects of chemical synthesis are protection and activation. protection strategies are intended to provide chemical selectivity necessary for the construction of a particular peptide sequence. activation refers to the chemical coupling necessary to ensure quantitative formation of each peptide bond in the sequence (andreu and rivas ) . in chemical synthesis, chemical reagents are used to activate the carboxylic acid (rcooh) of the amino acid, which will donate the acyl group (r-co-) to form the peptide bond. the peptide bond presents a nucleophilic attack of the α-amino group by another amino acid (h n-r). in this synthesis, the reactive functional groups that are not directly involved in peptide bond formation receive prior protection (machado and others ) . there are types of chemical peptide synthesis, synthesis in solution (classical synthesis) and solid-phase synthesis. chemical synthesis in solution is performed with all reagents and reaction products dissolved in the medium (kent ) . in comparison, solid-phase synthesis (sps) is a simple procedure to produce peptides in large quantities on a solid support which remains insoluble in the reaction medium (shigeri and others ) . the solid support is a polymeric resin that has a functional group on its surface (linker) that allows it to form stable bonds in the peptide sequence to the reagent used for the de-protection of the n-amino group. peptide synthesis in the solid phase generally consists on the acylation of an amino acid to be linked to an insoluble support (resin) via a linker ( figure ). after that, the protecting group of the n-terminal is removed (the unprotecting step) to allow the next amino acid of the sequence to be attached to the complex "peptide-linker-resin." the unprotecting-coupling cycle is repeated until the desired sequence is complete. finally, the cleavage reagent is used to separate the complex "peptide-linker-resin." this reagent should also remove the protecting groups of side chains that are stable to unprotecting conditions of the n-terminal group (borgia and fields ) . peptide chemical synthesis can use protocols, boc (tertbutyloxycarbonyl) and fmoc ( -fluorenylmethyloxycarbonyl), named according to the type of protector of the reactive group of the amino acids (n-terminal) involved in the synthesis. the first protocol employs the tert-butyloxycarbonyl (boc) group for n-amino protection. this protocol is based on gradual differences in their sensitivity to acids. thus, the boc group is typically removed with trifluoroacetic acid (tfa), while the protecting groups of the lateral chains (ester, ether, and urethane derivatives based on benzyl alcohol) are specifically designed to be stable to repeated cycles of boc removal and are removed only with a specific reagent, a relatively stronger acid, usually hydrofluoric acid (borgia and fields ) . the second protocol uses a -fluorenylmethyloxycarbonyl (fmoc) as the n-amino protecting group. this protocol provides a greater degree of chemoselectivity than the boc protocol, since the fmoc group is removed under basic conditions (piperidine in n, n-methylpyrrolidone or dimethylformamide), without alteration of the acid-sensitive lateral chains (andreu and rivas ) . protection groups of lateral chains are compatible with the fmoc protection group; these are mainly ether, ester, and urethane derivatives based on t-butanol. protection groups of lateral chains are removed by the end of the synthesis using tfa (borgia and fields ) . in this method, the peptide bond formation is mediated by an enzyme (protease) in free or immobilized form. the enzymatic method is especially useful in the synthesis of very short peptides ( - oligomers) and in the condensation of large peptide fragments (so and others ) . proteolytic enzymes such as chymotrypsin, papain, pepsin, subtilisin, termolisin, trypsin, among others, have been used in the presence of organic solvents as catalysts for the synthesis of peptide bonds (ogino and others ) . the enzymatic synthesis of peptides has several advantages over chemical methods, including good stereoselectivity and regioselectivity. however, it has certain shortcomings, such as peptide synthesis being thermodynamically unfavorable in water, as well as the secondary hydrolysis of synthesized peptide chains, which hinders their use in peptide synthesis with long sequences (so and others ) . thus, the main practical obstacle to employment of a protease for peptide bond formation is finding suitable conditions to allow bond formation without mediating secondary hydrolysis of the peptide or peptide fragments used as reagents (bongers and heimer ) . the formation of a peptide bond by enzyme catalysis can occur through several mechanisms, including the reverse hydrolysis reaction of amides and transpeptidation (machado and others ; boeriu and others ) . the mechanism of the reverse hydrolysis reaction is based on the microscopic reversibility principle. this indicates that the peptide bond formation and hydrolysis reaction come from the same intermediate ( figure ). thus, the reaction conditions are manipulated to shift the equilibrium towards peptide bond formation. the transpeptidation mechanism occurs as a result of the break of a peptide bond, with the formation of an active acyl-enzyme intermediate ( figure ). this intermediate is attacked in the presence of a nucleophile (peptide or amino acid blocked in the α-carboxyl group) and consequently causes the formation of a new peptide bond. for both mechanisms, the equilibrium should shift to the synthesis reaction direction, requiring the use of protective groups of α-amino and carboxyl substrates, the addition of organic solvents to the media reaction, excess substrates, and the removal of products from the reaction medium (machado and others ) . this synthesis uses modern methods of cloning and gene expression in microorganisms, allowing the production of a recombinant peptide or several peptides simultaneously. bacteria are the expression system generally used, with e. coli being the most widely used host. since antimicrobial peptides present a natural destructive activity against the host and relative sensitivity to proteolytic degradation, peptides are often expressed as fusion proteins to neutralize their innate toxic activity and increase their expression levels (wang and others ) . compared with isolation from natural sources and the other synthesis methods, the recombinant approach offers the most costeffective alternative for large-scale peptide production (li ) . peptides are increasingly being produced for various purposes, and these may contain closely related impurities resulting from incomplete reactions or from several side reactions. peptides synthesized for therapeutic and clinical research, as well as for biological and structural studies to explore the structure-activity relationships must have % purity or greater (ridge and hettiarachchi ) . however, there are other applications where low values of purity, between % and %, are tolerable (table ) . peptide purification depends on a series of separation techniques. peptides made on a preparative scale (in gram amounts) can be obtained from a separation process, to isolate one or more individual components from a peptide mixture for future research, or on an analytical scale (about mg of peptide) to identify and determine the relative amounts of some or all components in the mixture. studies on an analytical scale are the first steps for improving separation conditions, which are developed prior to the execution of any preparative separation process (sewald and jakubke ) . after the synthesis process, peptides are submitted to a separation procedure consisting of centrifugation and washing to remove residues of the reagents used, as well as products of side reactions. subsequently, peptides are cleaved and subjected to filtration, as well as lyophilization (dagan and others ) . the most widely used methods used for the purification of peptides are reverse-phase high-performance liquid chromatography (rp-hplc), ion-exchange chromatography, size exclusion chromatography, affinity chromatography, and capillary electrophoresis ( table ) . the purity of a peptide must be verified by a method different from that used for purification, since the results of homogeneity derived from such a system can lead to misinformation and be misleading (ridge and hettiarachchi ) . thus, the characterization should be analyzed by different methods of mass spectrometry. mass spectrometry has different ionization methods, such as electrospray ionization mass spectrometry (esi-ms), fast atom bombardment mass spectrometry (fab-ms) or matrix-assisted laser desorption/ionization mass spectrome-try (maldi-ms) that can be used for peptide characterization ( table ) . the different mass spectrometry techniques are based on the accurate determination of the molecular mass-charge ratio of the peptide, as well as on a chemical structure determination, with high sensitivity and resolution (sewald and jakubke ) . the growing resistance of pathogens against many commonly used antibiotics has led to research of new compounds with the same functions. an interesting approach is the study of molecules of natural origin to replace antibiotics (bechinger and lohner ) . several studies in recent decades have shown that peptides have certain bioactive properties (agyei and danquah ). short peptides ( - amino acids) with cationic and hydrophobic properties are known to be potent defenses of the host organism, providing activity against a wide variety of pathogenic microorganisms such as gram-negative and gram-positive bacteria, fungi, viruses, and parasites (hancock and sahl ) . studies have shown remarkable results of peptide antitumor activity, observed mainly in cancer therapy (korhonen and pihlanto ) . although several peptides have biological activity, antimicrobial activity is one of the most studied. one of the most-used analytical techniques to determine peptide antimicrobial activity is the broth microdilution test. in this test, the microorganisms are cultured in titration microplates and the peptide to be tested is added to each well at different concentrations. the microorganism growth causes turbidity in the wells. however, when a certain concentration of the peptide tested inhibits bacterial growth no turbidity is observed. turbidity is usually read by spectrophotometry, with the greatest frequency at nm, but it can also be seen through visual inspection of the wells (otvos and cudic ) . the standard methods developed by the clinical and laboratory standards inst. (clsi) have been used to test the activity of antimicrobial peptides. among them are the standards for antimicrobial disk susceptibility tests (clsi ) , the method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically m -a (clsi ) , and the method for broth dilution antifungal susceptibility testing of yeast m -a (clsi ) , all of which have been widely used (jang and others ; rubinchik and others ; hwang and others ) . the most studied peptides are those with antimicrobial activity, characterized by their interaction with the cytoplasmic membrane of the microorganism regardless of the final target (powers and hancock ) . factors influencing the antibacterial activity are the electrostatic interactions between the peptide and positively charged and anionic lipids on the surface of the target microorganism. also, the hydrophobicity of the peptide (factor required for insertion into the membrane) and peptide flexibility allow peptide interaction with the microbial membrane (jenssen and others ) . although these characteristics are variable according to each peptide, all of them are essential to the function of peptides as antimicrobials. the exact mechanism of action of antibacterial peptides is not yet fully understood. however, there is a consensus among researchers regarding the first step in the initial interaction between peptide and the target cell (reddy and others ) . exclusion liquid chromatography based on separation process according to the size of the peptide relative to pore sizes in the stationary phase. used primarily in the early stages of purification of the peptide, when performed in multiple steps used to separate low-molecular-weight impurities from a mixture of peptides. however, the separation of the peptide of interest with other closely related peptides is virtually impossible affinity chromatography based on the biological specificity of the peptide. consists of a ligand (small specific biomolecule such as an antibody) that is immobilized in the column. the separation occurs because of highly specific biochemical interactions between the peptide and the ligand used when a high degree of specificity is required, for example, isolation of a target protein present in low concentration in a biological fluid or a cell extract capillary electrophoresis based on the migration of the peptide according to its charge in solution, depending on the application of an electric field. complementary technique to reversed-phase chromatography used for peptides and proteins table -ionization methods used in mass spectrometry. fundamental principle the ions are produced from a peptide contained in a solvent (for example, an organic compound such as methanol or acetonitrile) that is scattered in a fine aerosol fab-ms the peptide analyzed is mixed with a matrix, which is a non volatile reagent of protection (glycerol, diethanolamine, and triethanolamine, among others), and is bombarded with a beam of high-energy atoms ( to ev) in a vacuum. atoms are of an inert gas such as argon or xenon maldi-ms the peptide analyzed is bombarded by a laser beam (nitrogen), while a matrix (sinapinic acid) is used to protect the peptide. the matrix allows avoiding direct contact of the peptide with the beam, facilitating its vaporization, and ionization the initial attraction between the peptide and the target cell occurs via electrostatic binding between the cationic peptide and the components of the negatively-charged outer cell membrane, such as lipopolysaccharides in gram-negative bacteria or lipoteichoic acid on the surface of gram-positive bacteria (jenssen and others ) . this electrostatic interaction removes the native divalent cations (mg + , ca + ) from the cell surface, thus destabilizing the outer membrane and facilitating the entry of the peptide and subsequent peptide contact with the cytoplasmic membrane, a process known as autopromoted uptake (powers and hancock ) . after the peptide is bound to the target cell, an arrangement of the peptide occurs on the surface of the cytoplasmic membrane. this fact is of considerable debate, since several arrangement models have been proposed, such as the barrel-stave or the carpet model among others. depending on the model, the peptide can permeabilize the cytoplasmic membrane and/or translocate through it. thus, antimicrobial peptides can be classified into major groups, the first consisting of those peptides which act on the cytoplasmic membrane, and the second consisting of those which have no action on the cytoplasmic membrane of the target microorganism. this means that the peptide just moves into the cell without causing major disturbances in the membrane (powers and hancock ; jenssen and others ) . peptides acting on the bacterial membrane. several models have been proposed to explain how, after initial attachment, antibacterial peptides are distributed on the surface of the bacterial cytoplasmic membrane to form pores. pore formation results in membrane permeabilization, thereby affecting cellular respiration. it also deprives the microorganisms of their source of energy by interrupting the electrochemical gradient and causing an increase in the flow of water and ions across the membrane, thus leading to cell swelling followed by cellular lysis (bechinger and lohner ) . to explain the formation of pores, the aggregate toroidal pore, barrel-stave, and the carpet models have been proposed. the last models, the barrel-stave and carpet model, have been the most widely studied. barrel-stave model. this model describes the formation of a transmembrane channel (pore) through the binding of amphipathic α-helices. the hydrophobic surface of the peptide interacts with the lipid core of the membrane, while the hydrophilic surface of the peptide is oriented inside, producing an aqueous pore (figure ) . the progressive recruitment of additional peptides to the membrane surface increases the size of the pores, causing the loss of cell content and thus cell death (reddy and others ) . this model has been proposed to explain the activity of antimicrobial peptides, such as magainins (matsuzaki and others ) . carpet model. in this model, the peptide in high concentration is in contact with phospholipids located on the outer surface of the bacterial membrane, a fact that allows the peptide to permeate the membrane ( figure ). the peptides bind to the surface of the target membrane and cover it like a carpet. according to this model, the peptides exhibit a preferential binding for the phospholipid groups. the binding step is followed by the alignment of the peptide on the membrane surface so the hydrophilic surface is in contact with phospholipid or water molecules, causing a reorientation of hydrophilic residues and creating a hydrophobic core. finally, the peptide disintegrates the membrane by deformation of the membrane curvature (reddy and others ) . this model has been proposed to explain the action mechanism of dermaseptins (dagan and others ) . toroidal pore model. this model is considered a variant of the barrel-stave model. it is suggested that a perpendicular inclusion of the peptides to the membrane with their hydrophilic regions is associated with phospholipids, whereas their hydrophobic regions are associated with the lipid core. in this process, the membrane is bent inward so the pores are formed (jenssen and others ) . the main difference between this model and the barrel-stave model is the intercalation of the peptide with phospholipids to form the pore ( figure ). this model has been used to explain the mechanism of action of the peptide melittin (yang and others ) . aggregate model. this model, proposed by wu and others ( ) , has some similarity to the toroidal pore model. this model consists mainly of the arrangement of the peptide in the membrane forming an extension and developing micelle-like aggregate of peptides and lipids, but without adopting a particular orientation (jenssen and others ) . peptides with no activity on the bacterial membrane. these antimicrobial peptides have the ability to translocate into bacterial cells without causing membrane permeabilization. the peptide is accumulated within the cell where it reaches a variety of essential cellular processes that result in bacterial cell death (jenssen and others ) . the target process includes inhibitions of nucleic acid synthesis, protein synthesis, enzyme activity, and cell wall synthesis. peptides such as buforin ii and pleurocidin have shown this mechanism of action (park and others ; patrzykat and others ) . there is a growing need for new antifungal agents due to the increased resistance of molds to therapies with regularly used compounds (de lucca and walsh ) . peptides have emerged as alternative antifungal agents. initially, the antifungal mechanism of action was described as a result of fungal cell lysis or as a result of interferences in fungal cell wall synthesis. however, the discovery of new antifungal peptides in the last decade has led to the identification of new mechanisms of action, including membrane permeabilization, binding to ergosterol/cholesterol in the fungal membrane, the attack of mitochondria or other intracellular organelles, and the deformation of cell membrane structure (jenssen and others ) . according to de lucca and walsh ( ) , fungal peptides can be classified with respect to their mode of action into groups: peptides that act through cellular lysis; peptides that cross the fungal membrane and interact with the intracellular target; and peptides that act by forming pores. peptides acting by cellular lysis. these peptides are characterized by their amphipathic nature, being molecules with faces, one positively charged and the other neutral and hydrophobic. some of these peptides bind only to the membrane surface, damaging the membrane structure, and they may or may not pass through it. peptide smap- (a synthetic peptide derived from the sequence of cathelicidins) has shown antimicrobial activity against the fungus trichosporon beigelii by interaction, penetration, and subsequent damage to the cell membrane (lee and others ) . this result suggests that the main target of smap- peptide is the fungal plasmatic membrane. a similar mechanism was observed for the synthetic peptide ib-amps (an analogue sequence to peptides isolated from seeds of impatiens balsamina), showing antifungal activity by bonding the peptide to the fungal cell membrane and subsequent penetration (thevissen and others ) . peptides that pass into the membrane and interact with intracellular targets. these peptides interfere with cell wall synthesis or the synthesis of essential cellular components such as chitin or glucan. as such, the synthetic peptide omiganan (an indolicin analogue peptide, isolated from bovine neutrophils) has shown antifungal activity against candida albicans, and the main mechanism of action of this peptide is related to its activity in the cytoplasmic membrane, resulting in macromolecules synthesis inhibition of macromolecules and finally cell death (rubinchik and others ) . pore-forming peptides. these peptides are aggregated in a selective way to form pores of varying sizes, which then allow the passage of ions and other solutes. the synthetic peptide di-k hc (a halocidin analogue peptide, isolated from the invertebrate marine animal halocynthia aurantium known as sea peach), has shown antifungal activity against several strains of aspergillus and candida (jang and others ) . the activity of di-k hc results in the formation of pores on the surface of fungal membranes. moreover, these researchers pointed out the specific binding of di-k hc with b- , -glucan, a component of the cell wall of fungi. this mechanism has also been observed with the antifungal peptide psacotheasin, isolated from the yellow-spotted long-horned beetle (psacothea hilaris), which has shown activity against c. albicans (hwang and others ) . the researchers indicated that there was damage to the cell wall, membrane depolarization with the formation of pores ( . - . nm), as well as an increase in membrane permeability, all being responsible for the antifungal activity of this peptide. several studies have shown the ability of cationic peptides to inhibit viral infections. the peptide cecropin a has shown antiviral activity against junin virus (jv-which causes argentine hemorrhagic fever). the peptide melittin inhibited jv and herpes simplex virus (hsv- ) multiplication, as well as magainin i and ii, and has shown inhibitory activity against hsv- and hsv- (albiol-matanic and castilla ). antimicrobial peptides isolated from fish, such as tilapia hepcidin - , have shown activity against the nervous necrosis virus (nn virus), an infectious agent that causes mass mortality of several species of marine fish in the larval stage (chia and others ) . in addition, synthetic peptides consisting of arginine and tryptophan repetitions have shown activity against vaccinia virus (the cause of cowpox) (mohan and others ). the antiviral activity of peptides is often related to virus adsorption and its entry into the host cell or, in other cases, is the result of a direct effect on the viral envelope. thus, the antiviral activity of peptides may result from multiple mechanisms of action, the most important being blocking virus entry through interaction with the host cell and blocking viral entry through interaction with the virus. blocking viral entry through interaction with the host cell. peptides can interact directly with specific viral receptors on the host cell, thus preventing the virus from binding to the cell membrane or binding intracellularly (jenssen and others ) . proteoglycans are proteins found in all types of tissue, in intracellular granule secretions as well as in the extracellular matrix and cell surface. proteoglycans are covalently linked to one or more chains of glycosaminoglycans (gag), long polysaccharide unbranched structures, which have a sugar that contains nitrogen and are usually sulfated. gag chains are present on the surface of mammalian cells and their degree of sulfation makes these compounds more anionic. this network of strong negative charges allows gag to attract and bind to small cations, such as enzymes and proteins, and also pathogens such as viruses (spillmann ) . heparan sulfate, one type of gag chain, is one of the most important molecules related to viral binding (spillmann ) . thus, by blocking heparan sulfate molecules can be inhibited viral infection. jenssen and others ( ) have suggested that antimicrobial peptides which interact with heparan sulfate have the ability to block a number of viral infections. due to the large number of amino acid residues positively charged peptides can interact electrostatically with negatively charged heparan sulfate molecules on the cell surface. studies on lactoferrin (lf) have shown that this peptide prevents infection of the host cell rather than inhibiting virus replication after infection of the target cell. the interaction of lf with heparan c institute of food technologists ® vol. , r comprehensive reviews in food science and food safety sulfate molecules has been proposed as the mechanism responsible for lf antiviral activity (van der strate and others ). similarly, jenssen and others ( ) showed the antiviral activity of synthetic peptides (consisting of arginine and lysine residues) against herpes simplex virus and (hsv- and hsv- ). the peptides presented higher affinity in binding to heparan sulfate with an increasing number of cationic residues, thereby blocking the entry of hsv (- or - ). in addition, luganini and others ( ) reported the inhibition of cytomegalovirus by binding synthetic peptide dendrimers with molecules of heparan sulfate on the surface of fibroblasts and endothelial cells. thus, cytomegalovirus infection was blocked by the interaction of synthetic peptide binding sites with heparan sulfate. blocking viral entry through interaction with the virus. the interactions of peptides with the glycoproteins (gp) in the viral envelope have been proposed as another mechanism that influences the process of viral entry and virus inactivation. in this way, peptides generated from chemical modification of milk proteins, such as α-lactalbumin, β-lactoglobulin, and lysozyme with -hydroxyphthalic anhydride ( -hp) inhibited infection of vero cells with hsv- (oevermann and others ) . according to those researchers, the antiviral activity of these peptides is based on their direct interaction with viral glycoproteins (gb, gc, gd), which are responsible for adsorption and penetration of the virus into the host cell. similarly, lf has shown the ability to bind to the gp glycoprotein (a protein present in the outermost layer of the hiv virus) with antiviral effects, since the gp glycoprotein plays an important role in the adsorption and entry of hiv into target cells (van der strate and others ; pan and others ) . on the other hand, other peptides, such as magainins, have shown antiviral effects through direct interaction with virus cells. egal and others ( ) have indicated that the effect of magainins is the result of the peptide acting on the viral envelope. a similar mechanism was suggested for the activity of mucroporin-m , a defense cationic peptide present in scorpion venom, which has shown activity against the measles virus, the coronavirus that causes severe accurate respiratory syndrome (sars), and flu virus h n (better known as the bird flu virus) (li and others ) . the researchers have suggested that the antiviral activity of the peptide is the result of direct interaction with the virus envelope, thereby reducing viral activity in the host cell. cancer, also known as malignant neoplasm, is a general term that refers to more than different diseases affecting various tissues and different types of cells. all forms of cancer are characterized by abnormal cell growth, that is, they lack the mechanisms that control normal cell division. this lack of regulatory mechanisms is the result of a multistep process involving genetic mutations induced by inheritance or environmental changes (hütter and sinha ) . despite major advances in cancer therapy, there is considerable interest in the development of antitumor agents with a novel mode of action, since the cells have shown carcinogenic development of resistance to current chemotherapy (hoskin and ramamoorthy ) . carcinogenic cells often become resistant to chemotherapy. this mainly occurs as a result of increased expression of intracellular enzymes for the detoxification of antitumor agents, the correction of dna damage, generation of intracellular organelles with the ability to eliminate and/or transport the drugs out of the tumor, and irreversible defects in the cellular machinery that mediates apoptosis (hütter and sinha ) . thus, recent studies have shown peptides as an alternative to conventional cancer treatments. however, not all peptides have selective activity against carcinogenic cells. according to hoskin and ramamoorthy ( ) peptides that have antitumor activity can be classified into major groups: peptides with selective activity, and peptides with non selective activity, that is, those that have activity against bacteria, carcinogenic cells, and healthy cells. peptides with selective activity toward carcinogenic cells. these peptides have activity against bacteria and carcinogenic cells, but not against normal cells. several peptides, such as the cecropins, buforins, and magainins have shown antitumor activity without affecting normal eukaryotic cells (cruciani and others ; cho and others ) . studies with magainin ii have shown to inhibit the proliferation of carcinogenic cells (in bladder cancer) without any effect on normal cells (lehmann and others ) . similar results were observed by chen and others ( ) in the study of the synthetic peptide th - (isolated from tilapia and analogous to the peptide hepcidin), with antitumor activity shown primarily by direct interaction and lysis of target carcinogenic cells (human fibrosarcoma cells). these researchers indicated that the lytic activity of the peptide and proliferative cells were restricted mainly to carcinogenic cells, since normal cells showed no significant effects. likewise, the synthetic peptide th - (isolated from tilapia and an analogue to the peptide hepcidin) has shown antitumor activity against carcinogenic cells, due to interaction with and penetration of the membrane. this peptide has less toxicity toward normal cells supposedly because it can discriminate between healthy cells and carcinogenic ones (chang and others ) . researchers have also indicated that the interaction with the cell membrane and its subsequent damage is caused by the formation of pores on its surface. it has been suggested that the internalization of the peptide and the subsequent damage to the mitochondrial membrane activates apoptotic pathways (chang and others ) . according to hoskin and ramamoorthy ( ) there are fundamental differences between the membranes of malignant cells and normal cells which allow the selectivity of certain peptides to attack carcinogenic cells without affecting healthy cells. electrostatic interactions between cationic peptides and anionic components of the cell membrane have also been considered an important factor. carcinogenic cells typically have a negative charge due to a higher expression than normal of anionic molecules such as phosphatidylserine (ps) and mucin (glycoprotein) (oren and shai ) . however, normal cells are not affected, since these cells have a neutral surface charge, conferred by the zwitterionic nature of most membrane components such as phosphatidylethanolamine (also known as cephalin), phosphatidylcholine, and sphingomyelin (sok and others ) . membrane fluidity and the surface area of the cell are also considered factors that contribute to the selectivity of peptides for carcinogenic cells. the fluidity of carcinogenic cells is greater than that of normal cells, which may increase the activity of lytic peptides through the easy destabilization of the membrane. in addition, the carcinogenic cells have a higher surface area than healthy cells due to the presence of greater numbers of microvilli, which are small projections of the cell membrane, irregular in size and shape. the microvilli may allow the bonding between peptide and carcinogenic cells (hoskin and ramamoorthy ) . peptides with nonselective activity. this group is comprised of peptides with activity against bacteria, carcinogenic cells, and against normal eukaryotic cells (hoskin and ramamoorthy ) . according to papo and shai ( ) non selective activity of these antimicrobial peptides results from their ability to interact with and cause damage to negatively charged membranes and those of a zwitterionic nature. dathe and others ( ) have indicated that the hydrophobic moment of antimicrobial peptides exerts a substantial influence on the neutral lipidic membranes, although it has a small role in the permeabilization of highly charged lipid membranes. peptides of this group include melittin, isolated from bee venom; taquiplesin ii, isolated from the horseshoe crab; defensins, isolated from insects; and plantaricin, a bacteriocin isolated from lactobacillus plantarum (schweizer ). plantaricin has shown activity against carcinogenic cells and against normal lymphocytes and neuronal cells (sand and others ) . the mechanism of action of antitumor peptides consists of permeabilization of the cell membrane mediated by electrostatic interaction. the electrostatic interaction is generated by the negatively charged phospholipids in the cell membrane and the positively charged peptide (schweizer ). unlike carcinogenic cells, eukaryotic cells have most of their negatively charged phospholipids, particularly ps, in the inner membrane, while neutral lipids are positioned on the outside (zhao and others ) . however, the result obtained by sand and others ( ) suggests that in addition to the mechanism of action related to the electrostatic interaction, there is another mechanism which explains the sensitivity of normal eukaryotic cells to plactaricin. probably another negatively charged macromolecule present on the membrane surface of healthy cells is also involved in plantaricin activity. similar results were observed by nan and others ( ) in the study of synthetic peptides consisting of lysine or arginine enriched with tryptophan. the peptide with arginine residues showed higher toxicity against human erythrocytes and mammalian cells. the hydrophobicity of the peptides has been suggested as an important factor in the increase of hemolytic activity and cytotoxicity in mammalian cells, as hydrophobic regions are required for direct interaction between peptide with membrane lipid components. the peptide with arginine residues was slightly more hydrophobic than the peptide with lysine residues. thus, these researchers suggested that small differences in hydrophobicity of these peptides may be responsible for the cytotoxic activity of this peptide in mammalian cells. the growing problem of microorganism resistance to conventional antibiotics, as well as the need for new agents with antibiotic properties has stimulated interest in developing antimicrobial peptides aiming for their application in the medical field (zasloff ) . most of the studies are devoted to the development of topical agents with antibacterial and antifungal activities. also, due to their antiviral activity, antimicrobial peptides have also been proposed as chemical preservatives. in the food industry, antimicrobial peptides, especially those produced by bacteria, have been widely researched in recent years due to their potential use as natural preservatives (papagianni ; coma ; settanni and corsetti ) . the direct application of antimicrobial peptides in food preservation can be achieved by methods: the direct addition of peptide to the food matrix, or the inoculation of the food matrix with the bacteriocin producer strain under the conditions favorable for the in situ production of the antimicrobial peptide. bacteriocins can be obtained ex situ by the cultivation of the producer strain at an industrial scale in a food-grade substrate, followed by a series of separation and purification techniques. these ex situ bacteriocins are commercially available in concentrated form, such as alta tm or microgard tm , and can be added directly to the food matrix. the production of bacteriocins in the food matrix offers several legal and cost advantages. the use of bacteriocin producer strain requires careful selection depending on the particular food intended for inoculation to ensure the producer strains will produce bacteriocins in the necessary amounts to inhibit the target microorganism. in addition to the peptides being studied as antimicrobial agents for direct addition to foods, they also have shown potential for being incorporated into food preparation surfaces (such as cutting surfaces) and processing equipment, as well as in food packaging (appendini and hotchkiss ) . active packaging includes the incorporation of antimicrobial agents in the packaging material to control and extend the shelflife of food (soares and others a). these types of packaging are considered an innovative technology in food preservation, since they allow better antimicrobial efficiency on food surfaces, thus improving stability. the development of active packaging by incorporating antimicrobial peptides in food packaging material can be done either to prolong the life of the product or to reduce the microbial load of the packing before use (steven and hotchkiss ) . the development of active packaging with antimicrobial peptides can be accomplished by main methods of incorporation: direct peptide incorporation in the polymer; peptide coating on the polymeric surface; and peptide immobilization in the polymer. numerous studies have reported the incorporation of antimicrobial peptides directly in the polymeric material, especially bacteriocins. the peptides are relatively resistant to heat (appendini and hotchkiss ) . however, their antimicrobial activity may be greater when heat is not used in the incorporation process. moreover, bioactive peptides incorporated in polymer films must be able to diffuse to the package surface over time to be effective. thus, polymers such as cellulose acetate, alginate, chitosan, and soy protein, among others, have been widely used to develop films with direct incorporation of these antimicrobials (marcos and others ; pires and others ; sivarooban and others ; santiago-silva and others ). researchers have studied the antimicrobial activity of bacteriocins incorporated into polymeric materials in synergy with other antimicrobial agents. synergistic activity against staphylococcus aureus, listeria monocytogenes, and bacillus cereus has been observed for nisin with potassium sorbate and garlic oil when incorporated into chitosan films (pranoto and others ) . in addition, soy protein films incorporated with nisin, grape seed extract, and ethylenediaminetetraacetic acid (edta) have shown inhibitory synergistic activity against pathogenic microorganisms such as l. monocytogenes, e. coli o : h , and salmonella typhimurium (sivarooban and others ) . the activity of bacteriocins incorporated into polymeric materials in synergy with other conservation technologies has also been reported. films incorporated with enterocins a and b (bacteriocins produced by enterococcus faecium) have shown synergistic activity when used together with high-pressure processing. thus, the use of antimicrobial packaging developed in conjunction with the high-pressure process allowed the control of l. monocytogenes at below detectable levels after d of storage at • c (marcos and others ) . this is an alternative method when the polymer requires extreme processing conditions during packaging material manufacture, such as high pressure and temperature, which can result in inactivation of the antimicrobial agent (appendini and hotchkiss ) . in some cases, the antimicrobial coating is done by contacting the film with or immersing it in the peptide solution. in this way, linear low-density polyethylene (lldpe) has been coated with lactocin and lactocin al (both bacteriocins produced by lactobacillus curvatus crl ), by direct contact of the film with a bacteriocin solution, showing antimicrobial activity in vitro against lactobacillus plantarum crl and listeria innocua (massani and others ) . similarly, scannell and others ( ) used alternatively lacticin and nisin adsorbed on the surface of plastic bags (polyethylene/polyamide) through direct contact of the polymeric material with bacteriocin solution. the film coated with nisin showed inhibitory activity against l. innocua and s. aureus, maintaining its activity for mo either at room temperature or under refrigeration. however, the film coated with lacticin did not show antimicrobial activity. the researchers suggested that lacticin was not retained by the polymer (scannell and others ) . proper handling of solvents and polymeric structures has been suggested to increase the adsorption of the peptide into the polymer matrix (appendini and hotchkiss ) . for example, the polymeric surface can be coated by applying a filmogenic solution that can be deposited on the film surface by the casting method. accordingly, chollet and others ( ) developed a laminated film of polyethylene (pe) and polyamide (pa), with the structure pe/pa/pe, coated with a filmogenic solution of hydroxypropyl methyl cellulose (hpmc) and adsorbed with nisin. the developed film presented antimicrobial activity in vitro against kocuria rhizophila (chollet and others ) . peptides can be immobilized or attached to solid supports by physical methods, such as layer-by-layer assembly, or by chemical methods, such as covalent bonding (onaizi and leong ) . layer-by-layer assembly. in this process, the peptide is sandwiched between polyionic polymers and the number of peptides and polymers is flexible (figure ) . the effectiveness of the peptide depends on its relative mobility. the advantage of this method is that it allows the slow release of the peptide embedded in the surface of the polymer. however, a key drawback of this method is that the peptide immobilized in the layers closest to the solid support will not be in direct contact with the target surface, thus reducing peptide activity. this peptide must be able to diffuse through the different layers of the assembly to the interface (onaizi and leong ) , to ensure efficient release and consequent bioactivity. the diffusion process of the peptide in the different layers is more complex than its diffusion in solution, since additional factors such as tortuosity of the diffusion path, the number of layers, and the polymer-peptide interactions can affect the diffusion process (appendini and hotchkiss ; sukhishvili ) . covalent bonding. in this process, the antimicrobial peptide will react chemically with a given surface to form a stable bond, which results in the formation of an antimicrobial coating on the polymeric surface (haynie and others ) . covalent bonding offers several advantages, including a more stable attachment between the peptide and the polymer surface (goddard and hotchkiss ) . covalent bonding reduces attached peptide ability to destabilize and improves its bioactivity, by protecting it from denaturation. due to the inert nature of most polymers, they must be subjected to a functionalization process on the surface before bonding with the peptide. the polymers can be functionalized with different spacers, which are reactive functional groups that allow peptide attachment on the spacer surface (humblot and others ) . the quantity of reactive functional groups generated in the functionalization process results in a restricted number of covalent bondings, which limits the amount of peptide that can be attached to the packaging. according to goddard and hotchkiss ( ) , direct bioactive compound applications require small amounts to be effective. however, for the applications of peptides in polymeric matrixes it is necessary to maximize the amount of peptides per unit area. to accomplish this, the functionalization technique must be optimized with the objective of linking the desired type and quantity of reactive functional groups. stiff or flexible spacers have been used as reactive groups for the functionalization of polymers (figure ). stiff spacers, such as polymethyl-methacrylate (pmma) and polyvinyl chloride (pvc), restrict the lateral mobility of the peptide bond, keeping the peptide firmly in a specific orientation. on the other hand, flexible spacers, such as polyethylene glycol (sand and others ) allow lateral mobility of the peptide bound, which can result in different orientations of the peptide molecules at the interface (onaizi and leong ) . among the different types of spacers, polyethylene glycol (peg) is widely used in the immobilization of peptides. the use of peg has several advantages, such as rapid and free peptide orientation, promoting peptide-bacteria interactions (costa and others ) . although the potential penetration and translocation of peptides through the microorganism cytoplasmic membrane is low due to the covalent bond that attaches the peptide to the polymer, it has been reported that peptides have sustained their bioactivity after attachment to polymeric matrixes. the synthetic peptide k l (a peptide sequence derived from magainin) was covalently bound to polystyrene (ps) resin by functionalization with peg and showed antimicrobial activity against foodborne pathogenic microorganisms, including e. coli o : h , l. monocytogenes, and pseudomonas fluorescens (appendini and hotchkiss ) . similarly, the synthetic peptide e lkk was covalently immobilized in ldpe film after chromium oxidation and functionalization with peg. this active packaging had antimicrobial activity against e. coli showing a reduction of log cycles when compared with the control (steven and hotchkiss ) . the sustained bioactivity of attached peptides is caused by the presence of spacers which allow sufficient freedom of motion for the active portion of the peptide to contact microorganisms on the food surface (appendini and hotchkiss ) . haynie and others ( ) have previously demonstrated that peptide-bacteria interactions are sufficient for peptide bioactivity. moreover, costa and others ( ) have indicated that the efficacy of attached peptides could possibly result from a higher peptide-relative surface availability, contrary to the other methods of peptide applications in which peptide aggregation can occur, producing uneven distribution. on the other hand, the diffusion of attached peptides into the food surface is restricted due to the covalent bonding. however, diffusion to the food product can occur in extreme conditions, such as high temperatures, which can promote hydrolysis reactions. the characterization of active packaging involves processes: structural analysis and measurement of their properties (table ) . according to goddard and hotchkiss ( ) , the type of analytical tool used in the structural characterization of polymers depends on the kind of modification, the specificity required, and available resources. some of the techniques used in structural analysis of active packaging with antimicrobial peptides are the contact angle, x-ray photoelectron spectroscopy (xps), fourier transform infrared spectroscopy (ftir), and scanning electron microscopy (sem). steven and hotchkiss ( ) used the techniques of contact angle and xps to assess changes in the surface of ldpe films after treatment with chromic oxide and functionalization with peg as spacer, subsequently covalently binding a synthetic peptide. the contact angle for the film before being subjected to any process of functionalization showed values of • . however, after chromic oxidation and peg bonding, films presented values of • and • , respectively; which indicated that the film surface became hydrophilic. these researchers concluded that the decrease in the contact angle is the result of an increased ionization at the film surface after oxidation, due to the presence of functional groups, such as carboxylic acid (cooh); and a value even lower after functionalization was observed due to solubility of peg. the xps technique showed changes in chemical composition on the film surface, resulting in the detection of nitrogen ( . %) and an increased percentage of oxygen (initially from . % to . % after oxidation and . % after functionalization). the oxygen increase was due to the presence of carboxylic acid after chromic oxidation. also, this increase was the result of functionalization with peg due to the main presence of o in the peg chain backbone. in addition, the functionalization with peg also introduced nitrogen originating from its amino-terminal functions (nh -peg-nh ). the ftir spectroscopic technique has been used by pranoto and others ( ) to study the interactions between chitosan films and nisin. they observed an increase in the band of the amide i corresponding to the wave number cm − related to the increased concentration of nisin incorporated in the film. according to the researchers, this is probably due to the interaction between the amine functional groups of chitosan and functional groups of nisin, which resulted in covalent bonds, and consequently in a larger peak. microscopic techniques have also been widely used to evaluate morphological changes in the surface of films that have been incorporated with antimicrobial peptides. pires and others ( ) used sem and observed that cellulose-based films incorporated with nisin, or a mixture of nisin and nantamicin, showed crystals deposited on the surface. these results indicated a heterogeneous distribution of the peptide in cellulosic films, while the control film presented a homogeneous structure. similarly, santiago-silva and others ( ) used sem to observe changes in surface morphology of cellulose acetate film incorporated with pediocin. when the concentration of peptide was increased, the films incorporated with pediocin had a rough surface c institute of food technologists ® vol. , r comprehensive reviews in food science and food safety table -characterization techniques of packaging incorporated with antimicrobial peptides. technique factor studied structural analysis contact angle quantifies surface hydrophobicity by measuring how far a droplet of water spreads on a surface x-ray photoelectron spectroscopy (xps) determines the atomic composition of the top several nanometers of a solid. this technique can be used to quantify the percent atomic composition and stoichiometric ratios fourier transform infrared spectroscopy (ftir) detects and identifies the chemical functional groups present in the polymer scanning electron microscopy (sem) allows the characterization of the polymer surface morphology and the observation of the dispersion quality of the peptide in the polymeric matrix property measurements mechanical properties measurement of the mechanical performance of the polymer. generally according to the standard method astm d (astm a) barrier properties measurement of water vapor permeability. generally according to the standard method astm e /e m (astm b); astm f (astm ) due to large amounts of pediocin granules dispersed in the matrix. this resulted from the lack of peptide solubility. on the other hand, the control film showed a homogeneous and transparent surface. in addition to the analysis of structural changes and interactions between the peptide and the polymeric matrix, the study of packaging properties is important as well. these properties show the performance of the developed material and how it will relate to the primary functions of food packaging, such as physical integrity. thus, mechanical and barrier properties have become increasingly relevant and are more frequently studied. the mechanical properties of films incorporated with antimicrobial peptides serve as the basis for assessing the effects on the mechanical performance resulting from the modification made to the polymer (table ) . guiga and others ( ) investigated the effect of nisaplin ® ( . % purity of nisin) coating on the mechanical properties of laminated films (pe/pa/pe), studying the mechanical properties of elongation at break and young's modulus. their results showed a significant difference in mechanical properties between the films incorporated with the peptide and the control treatment; peptideincorporated films showed an increase of young's modulus and a decrease in elongation at break. the polymer-based coating (hpmc) applied in laminated film, as well as the interaction between proteins and salts present in nisaplin, may have modified the mechanical behavior of the manufactured packaging, thereby increasing the rigidity of the film with the consequent decrease in its elongation. a similar result was observed by santiago-silva and others ( ) in their study of cellulosic films incorporated with pediocin. the researchers indicated that the addition of % of the peptide increased the maximum load required for film rupture when compared to the control. the researchers pointed out that a possible interaction between the pediocin and the polymeric matrix allowed the development of a more resistant film. however, a significant drop in the maximum load value was observed at a % concentration. according to the researchers, there was an excessive amount of the peptide incorporated, which weakened the cellulose chains of the film and resulted in a reduction of film resistance. a decrease in the mechanical strength of films incorporated with antimicrobial peptides has also been observed. sivarooban and others ( ) reported a decrease in puncture resistance and tensile strength values of soy protein films incorporated with nisin. similarly, pires and others ( ) indicated that nisin incorporated into cellulose-based films affected the film structure, reducing maximum load and elongation values. these resulted from the heterogeneous distribution of the peptide in the polymeric matrix, which consequently lead to the formation of stress points and reduced film resistance. although several studies have indicated changes in film properties, in some cases peptide incorporation into polymeric matrices had no significant effects. massani and others ( ) reported no significant difference in tensile strength, elongation, and water vapor permeability of ldpe films coated with lac-tocin and lactocin al . similar results were observed by chollet and others ( ) who indicated that the incorporation of nisin into pe/pa/pe laminated films, by coating with hpmc, showed neither changes in tensile strength at break nor in water vapor permeability. similarly, guiga and others ( ) reported that the direct incorporation of nisin into multilayer films of ethyl cellulose (ec) and hpmc (ec/hpmc/ec) did not alter the properties of tensile strength, young's modulus, or elongation at break. barrier properties of packaging include resistance to water vapor or gases (o and co ). the water vapor barrier property of the packaging can be determined by calculating the water vapor permeability (wvp) or the water vapor transmission rate (wvtr). both parameters cover the determination of the passage of water vapor through a polymeric material. however, the wvp considers vapor pressure difference between specific surfaces (internal and external) of the analyzed packaging (astm b). both parameters, wvp as well as wvtr, have been used to indicate that the addition of nisin in different polymeric matrixes, such as ldpe film coated with a cellulose-based solution (grower and others ; massani and others ) , sodium caseinate films (kristo and others ) and pe-film coated with hpmc (chollet and others ), causes no significant changes in the water vapor barrier property. on the other hand, studies regarding gas permeability in active packaging have been limited due to their applications as films or coatings for food products. during the evaluation and characterization of antimicrobial packaging it is important to research the transference of antimicrobial substances from the packaging material into the food, since this information allows the determination of how the antimicrobial agent is released from the active packaging. the mass transfer can occur by the diffusion mass transfer mechanism or by the convective mass transfer mechanism. the convective mass transfer mechanism occurs in a moving fluid, known as natural convection, if the movement is caused by differences in the density, or as forced convection, if the movement is caused by external agents or when a fluid is flowing on the solid surface by forced movement. on the other hand, diffusion mass transfer consists of a random motion of individual molecules as a result of a concentration gradient (crank ; geankoplis ) . the migration of substances from packaging materials takes place through the diffusion mass transfer mechanism, since the active packaging and the food contain a concentration gradient for the antimicrobial agent incorporated in the packaging. unlike the research on the release of active substances from drugs or the release of solvents from polymers, the study of antimicrobial release from active packaging is still limited (buonocore and others ; bastarrachea and others ) . the knowledge of diffusion parameters allows an efficient design of active packaging. several factors must be considered when studying the migration from antimicrobial packaging, including the release rate of the antimicrobial molecules from the packaging. if this rate is high, the active packaging would release the antimicrobial rapidly, resulting in a large concentration at a determined time. however, the large concentration would not be maintained over time, depending on the solubility of the antimicrobial in the selected food. if the solubility is very high, the antimicrobial will migrate rapidly to the food matrix, and therefore result in a decreased concentration of the antimicrobial on the food's surface along time. on the other hand, if the release rate is low, the antimicrobial agent will be slowly released in a desired concentration and if it presents a low solubility in the selected food, the antimicrobial can accumulate on the food surface and slowly migrate into the food matrix. in this situation the release rate should not be slower than the microbial growth (bastarrachea and others ) . in either case, the release of the antimicrobial agent from the packing material is indicated by the diffusion coefficient (d). thus, the diffusion characteristics of the antimicrobial agent can be used to determine the amount needed to maintain the proper concentration on the food surface (buonocore and others ) . the literature reports a few migration studies of antimicrobial peptides incorporated in active packaging. most evaluate the migration of nisin, probably due to the fact that this is the only antimicrobial peptide substance indicated as generally recognized as safe (gras) for direct contact with food in the united states (fda ). nisin is also widely accepted as a food preservative in the european community where it is classified as a safe preservative for food contact, coded as e (fsa ), as well as in brazil where the use of nisin is permitted by brazilian law as a natural preservative for biological products (anvisa ) . diffusion of several antimicrobial agents, such as potassium sorbate or lysozyme incorporated in active packaging, has been explained by fick's second law (han and floros ; gemili and others ) : where, c is the diffusing substance concentration; d is the apparent diffusion coefficient; t is the diffusion time; and x the distance. depending on the conditions of the migration test, different analytical solutions have been applied to solve fick's second law and to calculate the d-value in the migration of the antimicrobial peptide nisin incorporated in different types of packaging materials (table ) . different analytical solutions of fick's second law have been used in previous studies to calculate the d-value of nisin at a specific temperature ( table ) . some of these studies were conducted at different temperatures to characterize the d-value as a function of this parameter. protein films (corn zein or wheat gluten) and poly(butylene adipate-coterephthalate) (pbta) films incorporated with nisin showed an increase in d-value with increasing temperature, indicating that the peptide concentration was higher in the simulant at equilibrium state with increasing temperature (teerakarn and others ; bastarrachea and others ) . similarly, at low temperatures, lower d-values of nisin diffusivity indicate that the film retains larger amounts of the peptide in the polymer matrix while in contact with the simulant. the arrhenius activation energy model (eq. ) has been shown to confirm the dependence of the diffusivity with respect to temperature. where, d is a constant; e a is the activation energy for the diffusion process (j/mol); r is the universal gas constant ( . j/mol k); and t abs is the absolute temperature (k (− ) n ierfc nh √ d t m , is the initial amount of nisin in the film; m t , released amount of nisin at time t; h, film thickness; d, diffusion coefficient; ierfc, associated function of the mathematical error function teerakarn and others ( ) paper coated with acrylic polymer and ethylene-vinyl acetate co-polymer (eva) is the amount of nisin released at time t; m ∞ , is the migration in a state of equilibrium; l p coating layer thickness, d, diffusion coefficient kim and others ( ) hydroxypropyl methyl cellulose (hpmc) films is the amount of nisin in the simulant at time t; m f, , amount of nisin in the film when t = ; α, mass ratio between the amount of nisin in the simulant and in the film at equilibrium; q n , is the ''n" root of tanq n = −αq n ; l, is a half of the film's thickness bastarrachea and others ( ) teerakarn and others ( ) indicated e a values of . and . kj/mol for corn zein and wheat gluten films, respectively, and bastarrachea and others ( ) obtained an e a value of . kj/mole for pbta film incorporated with nisin. the value of e a represents the degree of molecular interactions between the antimicrobial substance incorporated and the polymeric matrix. thus, higher e a values represent stronger antimicrobial-polymer interactions, which is reflected in a lower d-value due to the greater energy level required for antimicrobial release (bastarrachea and others ) . the relationship between temperature and diffusivity of the antimicrobial agent is the result of structural changes in the polymer matrix, since above the glass transition temperature (t g ) the molecular mobility in the system increases along with temperature, which leads to an increase in the ability of the packaging material to transport substances through its polymeric matrix (teerakarn and others ) . in addition to the interactions between polymer and antimicrobial agent, the d-value is also influenced by interactions between the antimicrobial and the food matrix. thus, the food nh -r-w-c-f-r-v-c-y-r-g-i-c-y-r-k-c-r-conh miyata and others ( ) * x = means that the specific identity of an amino acid cannot be determined unambiguously. * * dhb = (z)- , -didehydrobutyrine. * * * abu = -aminobutyric acid. composition, as well as the solubility of the antimicrobial in these components also affects the d coefficient. in the study of paper coated with eva incorporated with nisin, the d-values varied according to the composition of the food in contact with the active packaging (kim and others ) . the highest d-value was observed when the film was in contact with a % citric acid solution, and the lowest value was observed when in contact with a % nacl solution. characteristic parameters of each solution, such as ph and ionic strength, have been shown to influence nisin solubility. nisin has a high solubility (up to mg·ml − at ph ) at low ph, but at high concentrations of nacl (above m) nisin solubility dependence on ph almost disappears and the solubility decreases to values below mg·ml − at any ph (rollema and others ) . chollet and others ( ) also investigated the influence of food composition on the migration of nisin incorporated in pe/pa/pe films coated with hpmc by changing the fat percentage. they found that increasing the fat content in the food resulted in an increased d-value and, therefore, in a greater diffusion of incorporated nisin. in their experiment, nisin diffusion mechanism was governed by the fat content. the increase in fat content resulted in microstructural changes, such as enlargement of pore size in the food matrix, which favored nisin diffusion into it. consumer demand for minimally processed foods and additivefree products has led to the development of antimicrobial packaging. peptides have shown various bioproperties, among them antimicrobial activity, leading to the application of these compounds in the food preservation area by either direct addition or incorporation into packaging materials (table ) . active packaging materials incorporated with antimicrobial peptides have shown effectiveness in inhibiting pathogenic microorganisms, an improvement in food safety. moreover, antimicrobial peptides incorporated into the polymeric matrix may af-fect the engineering characteristics of the packaging material, and lead to differentiated diffusion performance. this review highlights the characteristics of pure peptides, as well as their incorporation into polymeric matrices. several studies have indicated significant changes in mechanical properties and surface morphology of the films incorporated with antimicrobial peptides. however, research related to the study of barrier properties to gases and water vapor is still limited. more studies on the release of other peptides, different from nisin, from food packaging materials are needed to better understand the mechanism of dissemination of antimicrobial agents. finally, in the years ahead, the advent of nanotechnology will lead to research on the synergistic effects of antimicrobial peptides and nanoparticles, such as metals, metal oxides, and nanoclays, with the objective being to improve the mechanical and barrier properties of antimicrobial packaging. bioactive peptides: synthesis, properties, and applications shortened cecropin a-melittin hybrids significant size reduction retains potent antibiotic activity uso da nisina com a função de conservador para queijos pasteurizados. portaria deten/ms n • , de de janeiro de surface modification of poly(styrene) by the attachment of an antimicrobial peptide review of antimicrobial food packaging in: standard test method for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor astm d in: standard test method for tensile properties of thin plastic sheeting astm e /e m in: standard test methods for water vapor transmission of materials biochemical and genetic characterization of enterocin a from enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins engineering properties of polymeric-based antimicrobial films for food packaging: a review release kinetics of nisin from biodegradable poly(butylene adipate-co-terephthalate) films into water detergent-like actions of linear amphipathic cationic antimicrobial peptides optimized enzymatic synthesis of c-terminal peptide amides using subtilisin a from bacillus licheniformis recent applications of enzymatic peptide synthesis chemical synthesis of proteins a general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications tilapia (oreochromis mossambicus) antimicrobial peptide, hepcidin - , shows antitumor activity in cancer cells a fish antimicrobial peptide, tilapia hepcidin th - , shows potent antitumor activity against human fibrosarcoma cells antimicrobial peptides (amp) with antiviral activity against fish nodavirus buforins: histone h a-derived antimicrobial peptides from toad stomach monitoring nisin desorption from a multi-layer polyethylene-based film coated with nisin-loaded hpmc film and diffusion in agarose gel by an immunoassay (elisa) method and a numerical modeling reference method for broth dilution antifungal susceptibility testing of yeast performance standards for antimicrobial disk susceptibility tests methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically bioactive packaging technologies for extended shelf life of meat-based products covalent immobilization of antimicrobial peptides (amps) onto biomaterial surfaces the mathematics of diffusion antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation in vitro antiplasmodium effects of dermaseptin s derivatives hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides antifungal peptides: novel therapeutic compounds against emerging pathogens antiviral effects of synthetic membrane-active peptides on herpes simplex virus, type the possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease direct food substances affirmed as gras -electronic code of the bactericidal activity of pediocin pa- is specifically inhibited by a -mer fragment that spans the bacteriocin from the center toward the c terminus food standards agency. current eu approved additives and their e numbers defensins: antimicrobial peptides of innate immunity immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins principles of mass transfer development of cellulose acetate-based antimicrobial food packaging materials for controlled release of lysozyme polymer surface modification for the attachment of bioactive compounds development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of listeria monocytogenes antimicrobial plastic film: physico-chemical characterization and nisin desorption modeling innovative multilayer antimicrobial films made with nisaplin ® or nisin and cellulosic ethers: physico-chemical characterization, bioactivity and nisin desorption kinetics simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films the role of cationic antimicrobial peptides in innate host defences antimicrobial and host-defense peptides as new anti-infective therapeutic strategies antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin a coating for use as an antimicrobial and antioxidative packaging material incorporating nisin and [alpha]-tocopherol studies on anticancer activities of antimicrobial peptides the antibacterial activity of magainin i immobilized onto mixed thiols self-assembled monolayers proteomics for studying cancer cells and the development of chemoresistance antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from psacothea hilaris antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, halocynthia aurantium peptide antimicrobial agents chemical synthesis of peptides and proteins properties of nisin-incorporated polymer coatings as antimicrobial packaging materials bioactive peptides: production and functionality thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on listeria monocytogenes antifungal mechanism of smap- ( - ) isolated from sheep myeloid mrna against trichosporon beigelii antitumor activity of the antimicrobial peptide magainin ii against bladder cancer cell lines virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses recombinant production of antimicrobial peptides in escherichia coli: a review peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate sínteses química e enzimática de peptídeos: princípios básicos e aplicações high-pressure processing and antimicrobial biodegradable packaging to control listeria monocytogenes during storage of cooked ham development and characterization of an active polyethylene film containing lactobacillus curvatus crl bacteriocins relationship of membrane curvature to the formation of pores by magainin antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin ii, and polyphemusins i and ii: chemical structures and biological activity antiviral activity of selected antimicrobial peptides against vaccinia virus mammalian cell toxicity and candidacidal mechanism of arg-or lys-containing trp-rich model antimicrobial peptides and their d-enantiomeric peptides purification and characterization of plantaricin a, a lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides the antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification peptide synthesis catalyzed by organic solvent-stable protease from pseudomonas aeruginosa pst- in monophasic aqueous-organic solvent systems tethering antimicrobial peptides: current status and potential challenges selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study broth microdilution antibacterial assay of peptides purification and amino acid sequence of lactocin , a bacteriocin produced by lactobacillus casei crl antiviral properties of milk proteins and peptides ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes mechanism of action of the antimicrobial peptide buforin ii: buforin ii kills microorganisms by penetrating the cell membrane and inhibiting cellular functions a novel antimicrobial peptide from bufo bufo gargarizans sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in escherichia coli development and evaluation of active packaging for sliced mozzarella preservation the relationship between peptide structure and antibacterial activity enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin applications of antimicrobial peptides from fish and perspectives for the future antimicrobial peptides: premises and promises peptide purity and counter ion determination of bradykinin by high-performance liquid chromatography and capillary electrophoresis improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering antimicrobial and antifungal activities of a novel cationic antimicrobial peptide, omiganan, in experimental skin colonisation models plantaricin a, a peptide pheromone produced by lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells antimicrobial efficiency of film incorporated with pediocin (alta ® ) on preservation of sliced ham antioxidative peptides from food proteins: a review development of bioactive food packaging materials using immobilised bacteriocins lacticin and nisaplin ® bioactive peptides: synthesis, properties, and applications health-related costs: from foodborne illness in the united states. produce safety project. available from: www.producesafetyproject.org. accessed cationic amphiphilic peptides with cancer-selective toxicity controlled diffusion of an antimicrobial peptide from a biopolymer film application of bacteriocins in vegetable food biopreservation synthesis and application of caged peptides and proteins physical and antimicrobial properties of grape seed extract, nisin, and edta incorporated soy protein edible films lipase-catalyzed synthesis of peptides containing d-amino acid: facts and artifacts recent patents on active packaging for food application active and intelligent packaging for milk and milk products membrane fluidity characteristics of human lung cancer heparan sulfate: anchor for viral intruders? immunomodulatory activity of small peptides covalent immobilization of an antimicrobial peptide on poly(ethylene) film responsive polymer films and capsules via layer-by-layer assembly nisin diffusion in protein films: effects of film type and temperature influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by lactococcus lactis antiviral activities of lactoferrin natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities expression and purification of antimicrobial peptide buforin iib in escherichia coli lantibiotics: peptides of diverse structure and function mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of escherichia coli barrel-stave model or toroidal model? a case study on melittin pores antimicrobial peptides of multicellular organisms interaction of the antimicrobial peptide pheromone plantaricin a with model membranes: implications for a novel mechanism of action the authors would like to thank to nicholas j. walker for providing language help and writing assistance. financial support for this research was provided by coordenação de aperfeiçoamento de pessoal de nível superior (capes) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq). key: cord- -ynti g j authors: nosonovsky, michael; roy, prosun title: scaling in colloidal and biological networks date: - - journal: entropy (basel) doi: . /e sha: doc_id: cord_uid: ynti g j scaling and dimensional analysis is applied to networks that describe various physical systems. some of these networks possess fractal, scale-free, and small-world properties. the amount of information contained in a network is found by calculating its shannon entropy. first, we consider networks arising from granular and colloidal systems (small colloidal and droplet clusters) due to pairwise interaction between the particles. many networks found in colloidal science possess self-organizing properties due to the effect of percolation and/or self-organized criticality. then, we discuss the allometric laws in branching vascular networks, artificial neural networks, cortical neural networks, as well as immune networks, which serve as a source of inspiration for both surface engineering and information technology. scaling relationships in complex networks of neurons, which are organized in the neocortex in a hierarchical manner, suggest that the characteristic time constant is independent of brain size when interspecies comparison is conducted. the information content, scaling, dimensional, and topological properties of these networks are discussed. scaling methods and dimensional analysis are widely used in various areas of physics. the concepts of fractals (scale-free objects), power exponents, and near-critical behavior are central to the study of scaling. one particularly interesting field of application is biophysical problems, where both experimental observations and theoretical explanations have been suggested of how various quantitative characteristics of a living organism (for example, the rate of metabolism) depend on its mass and linear size. the area is often referred to as allometry [ ] [ ] [ ] [ ] [ ] [ ] . one of the most widely known examples of an allometric scaling relationship is the empirical kleiber law, which is based on a comparison of the metabolism rates, b, of different species. kleiber [ ] established a power law dependency with the exponent of b upon the mass of an animal, b∝m / . the explanation of the value of the exponent was suggested in an influential paper by west et al. [ ] , who used a fractal model of branching of blood vessels serving a certain volume with the conservation of the cross-sectional area of the vessels and of the volume covered at every stage of branching. despite its shortcomings [ ] [ ] [ ] [ ] , the fractal theory by west et al. [ ] remains the main explanation of the allometric scaling exponents, and it can likely be expanded on such topics as the analysis of the ergodicity in the vascular network [ ] . besides the biophysical applications, scaling problems are also prominent in those areas of physics, where the so-called intermediate asymptotic behavior is found [ ] . a typical example of an intermediate asymptotic process would be a transition from a single object to a thermodynamic description of a continuum medium. the intermediate ensemble of a few natural or artificial objects, which bridges between a single particle and continuum matter, is often called a cluster, and it possesses many properties absent from both single-particle and continuum medium description [ ] . this includes clusters are defined as collections of somewhat similar, but not necessarily identical objects. in physics and chemistry, clusters are often built of liquid droplets or small solid particles, such as nanoparticles, and colloidal particles [ ] . from the physical point of view, they occupy an intermediate position between individual objects and bulk material consisting of a large number of objects, to which the methods of statistical physics and thermodynamics are often applied. since clusters bridge between individual particles and bulk matter, they may have certain unique collective properties absent in both individual objects and in bulk materials. given that there are interactions between particles in a cluster, the latter can be modeled by a clusters are defined as collections of somewhat similar, but not necessarily identical objects. in physics and chemistry, clusters are often built of liquid droplets or small solid particles, such as nanoparticles, and colloidal particles [ ] . from the physical point of view, they occupy an intermediate position between individual objects and bulk material consisting of a large number of objects, to which the methods of statistical physics and thermodynamics are often applied. since clusters bridge between individual particles and bulk matter, they may have certain unique collective properties absent in both individual objects and in bulk materials. given that there are interactions between particles in a cluster, the latter can be modeled by a network. a network is a graph, which consists of nodes (vertices) connected by edges. the clustering coefficient is defined as a ratio of the number of closed triplets to all triplets. this coefficient is a measure of the degree to which nodes in a graph tend to cluster together. it is used to quantify aggregation in granular media, indicating the degree of clustering of particles in the cluster. two physical concepts relevant to the self-organization of various networks are self-organized criticality (soc) and percolation [ ] [ ] [ ] , ] . there is a big class of dynamical systems that operate in such a manner that they always tune themselves to the critical state, where the stability of the system is lost. since the s, it has been suggested that a very specific type of self-organization, called soc, plays a role in diverse "avalanche-like" processes. typically, energy is accumulated in these systems until the critical state is reached, and then energy is suddenly released. examples are various avalanche systems, including those describing landslides, earthquakes, and frictional stick-slip. a random perturbation can trigger an avalanche (or a slip event) in such a system. the magnitude of the avalanche cannot be predicted in advance, because it is random. after the release, the system returns to the stable state for some time until the next event is triggered. the amplitudes of the events have statistical characteristics of critical behavior, such as universality, critical exponents, and fractality. a famous example is the power law, which relates the frequency and the magnitude of earthquakes, known as the gutenberg-richter law. for example, similar behavior is observed in frictional stick-slip systems and in many other systems [ ] . the best-studied example of soc is the "sandpile model", which represent a conical pile of sand with new grains of sand randomly placed into the pile (figure ). when the slope exceeds a threshold value (the critical slope angle is related to the coefficient of dry friction between the grains), a grain would move down the slope. placing a random grain at a particular site may have no effect, or it may trigger an avalanche that will affect many sites at the lattice. thus, the response does not depend on the details of the perturbation. it is worth mentioning that the scale of the avalanche may be much greater than the scale of the initial perturbation. entropy , , x for peer review of coefficient is defined as a ratio of the number of closed triplets to all triplets. this coefficient is a measure of the degree to which nodes in a graph tend to cluster together. it is used to quantify aggregation in granular media, indicating the degree of clustering of particles in the cluster. two physical concepts relevant to the self-organization of various networks are self-organized criticality (soc) and percolation [ ] [ ] [ ] , ] . there is a big class of dynamical systems that operate in such a manner that they always tune themselves to the critical state, where the stability of the system is lost. since the s, it has been suggested that a very specific type of self-organization, called soc, plays a role in diverse "avalanche-like" processes. typically, energy is accumulated in these systems until the critical state is reached, and then energy is suddenly released. examples are various avalanche systems, including those describing landslides, earthquakes, and frictional stickslip. a random perturbation can trigger an avalanche (or a slip event) in such a system. the magnitude of the avalanche cannot be predicted in advance, because it is random. after the release, the system returns to the stable state for some time until the next event is triggered. the amplitudes of the events have statistical characteristics of critical behavior, such as universality, critical exponents, and fractality. a famous example is the power law, which relates the frequency and the magnitude of earthquakes, known as the gutenberg-richter law. for example, similar behavior is observed in frictional stick-slip systems and in many other systems [ ] . the best-studied example of soc is the "sandpile model", which represent a conical pile of sand with new grains of sand randomly placed into the pile (figure ). when the slope exceeds a threshold value (the critical slope angle is related to the coefficient of dry friction between the grains), a grain would move down the slope. placing a random grain at a particular site may have no effect, or it may trigger an avalanche that will affect many sites at the lattice. thus, the response does not depend on the details of the perturbation. it is worth mentioning that the scale of the avalanche may be much greater than the scale of the initial perturbation. the sand-pile conceptual model of self-organized criticality (soc). the pile tends to have a slope angle defined by the friction between grains. adding one new grain to the pile may have no effect (grain is at rest) or it may cause an avalanche. the magnitude and frequency of avalanches are inversely related (based on [ ] ). the concept has been applied to such diverse fields as physics, cellular automata theory, biology, economics, sociology, linguistics, and others. there are typical external signs of an soc system, such as the power-law behavior (the magnitude distribution of the avalanches) and the 'one-overfrequency' noise distribution (the amplitude of random fluctuations is inversely proportional to the frequency). of course, not every system with a one-over-frequency spectrum is a soc system. the one-over-frequency noise (referred to also as the white noise) may be present also in non-soc systems. another important concept, which is related to soc, is the percolation [ , ] . typically, during percolation, a certain controlled parameter is slowly changed; for example, nodes are removed from the sand-pile conceptual model of self-organized criticality (soc). the pile tends to have a slope angle defined by the friction between grains. adding one new grain to the pile may have no effect (grain is at rest) or it may cause an avalanche. the magnitude and frequency of avalanches are inversely related (based on [ ] ). the concept has been applied to such diverse fields as physics, cellular automata theory, biology, economics, sociology, linguistics, and others. there are typical external signs of an soc system, such as the power-law behavior (the magnitude distribution of the avalanches) and the 'one-over-frequency' noise distribution (the amplitude of random fluctuations is inversely proportional entropy , , of to the frequency). of course, not every system with a one-over-frequency spectrum is a soc system. the one-over-frequency noise (referred to also as the white noise) may be present also in non-soc systems. another important concept, which is related to soc, is the percolation [ , ] . typically, during percolation, a certain controlled parameter is slowly changed; for example, nodes are removed from a network or conducting sites are added (figure a) , or shear force is increased in a system with friction. when a critical value of the controlled parameter is achieved, the avalanche can be triggered and a corresponding output parameter, such as the correlation length, may reach infinity (figure b ). for example, the correlation length can characterize the average size of black or white islands on a field of the opposite color, when random pixels are added (figure c ). at the critical point, the configuration is fractal (an infinite set of white islands on the black background forming larger black islands on the white background, and so on). entropy , and a corresponding output parameter, such as the correlation length, may reach infinity (figure b ). for example, the correlation length can characterize the average size of black or white islands on a field of the opposite color, when random pixels are added (figure c ). at the critical point, the configuration is fractal (an infinite set of white islands on the black background forming larger black islands on the white background, and so on). (a) (b) (c) showing that the first four iteration steps at the eighth step all sites become active [ ] . (b) a typical dependency of the correlation length on the shear load for an avalanche. at the critical value of the load, τ , the correlation length approaches the infinity. (c) with the increasing normal load, the size of slip zone spots (black) increases. a transition to the global sliding is expected when the correlation length approaches infinity. the topological concepts from the theory of networks turn out to be useful for physical characterization of packing of granular material, colloidal crystals, and clusters of droplets and colloidal particles. the so-called force network is important for the understanding of packing of granular material. figure shows a granular material (blue and black circles represent granules) flowing in a channel. some grains are tightly packed and apply a force upon their neighbors, while others are loose and do not transmit force. with increasing pressure in the channel, the number of force-transmitting grains (black) increases, and when the chain of black grains through the entire width of the channel, it is jammed, which is called the jamming transition. [ ] . (b) a typical dependency of the correlation length on the shear load for an avalanche. at the critical value of the load, τ , the correlation length approaches the infinity. (c) with the increasing normal load, the size of slip zone spots (black) increases. a transition to the global sliding is expected when the correlation length approaches infinity. the topological concepts from the theory of networks turn out to be useful for physical characterization of packing of granular material, colloidal crystals, and clusters of droplets and colloidal particles. the so-called force network is important for the understanding of packing of granular material. figure shows a granular material (blue and black circles represent granules) flowing in a channel. some grains are tightly packed and apply a force upon their neighbors, while others are loose and do not transmit force. with increasing pressure in the channel, the number of force-transmitting grains (black) increases, and when the chain of black grains through the entire width of the channel, it is jammed, which is called the jamming transition. the so-called force network is important for the understanding of packing of granular material. figure shows a granular material (blue and black circles represent granules) flowing in a channel. some grains are tightly packed and apply a force upon their neighbors, while others are loose and do not transmit force. with increasing pressure in the channel, the number of force-transmitting grains (black) increases, and when the chain of black grains through the entire width of the channel, it is jammed, which is called the jamming transition. [ , ] ). the force is transmitted through chains of connecting grains shown in black. [ , ] ). the force is transmitted through chains of connecting grains shown in black. the force network connects centers of mass of each pair of grains that have a force transmitting contact. such network presentation provides key insights for understanding the mechanical response of a soil or sand heap. moreover, percolation, i.e., the formation of a force-transmitting chain in such a network, corresponds to the jamming transition in the granular material [ , ] . the percolation phenomenon in application to networks will be discussed more in detail in the consequent section. near-percolation behavior is known to demonstrate scale-free features. in particular, the small-world effect and scale-free behavior were reported for packing problems related to aggregation of granular media, which employs the so-called "apollonian packing" [ ] . for the simple packing of identical (monodisperse) particles, the force networks do not possess self-similar, scale-free, or small-world properties. however, to achieve high packing densities, the apollonian construction can be employed ( figure ). such construction involves a multiscale set of circles with smaller circles fitting the space between larger ones. this may be needed for high-performance concrete (hpc) and certain ultra-strong ceramics. andrade et al. [ ] found that the force networks resulting from the apollonian construction, which they called apollonian networks (ans), have many special properties, including the scale-free and small-world effects. entropy , , x for peer review of the force network connects centers of mass of each pair of grains that have a force transmitting contact. such network presentation provides key insights for understanding the mechanical response of a soil or sand heap. moreover, percolation, i.e., the formation of a force-transmitting chain in such a network, corresponds to the jamming transition in the granular material [ , ] . the percolation phenomenon in application to networks will be discussed more in detail in the consequent section. near-percolation behavior is known to demonstrate scale-free features. in particular, the small-world effect and scale-free behavior were reported for packing problems related to aggregation of granular media, which employs the so-called "apollonian packing" [ ] . for the simple packing of identical (monodisperse) particles, the force networks do not possess selfsimilar, scale-free, or small-world properties. however, to achieve high packing densities, the apollonian construction can be employed ( figure ). such construction involves a multiscale set of circles with smaller circles fitting the space between larger ones. this may be needed for highperformance concrete (hpc) and certain ultra-strong ceramics. andrade et al. [ ] found that the force networks resulting from the apollonian construction, which they called apollonian networks (ans), have many special properties, including the scale-free and small-world effects. even more interesting phenomena where the theory of networks can be applied are small droplet clusters [ ] [ ] [ ] [ ] [ ] and small colloidal crystals. self-assembled clusters of condensed microdroplets (with the typical diameter of dozens of microns) are formed in an ascending flow of vapor and air above a locally heated thin (approximately mm) layer of water. the droplets form a d monolayer levitating at a low height (comparable with their radii) where their weight is equilibrated by the drag force from the ascending vapor flow. due to an aerodynamic interaction (repulsion) between the droplets and their migration toward the center of the heated spot, they tend to form an ordered structure ( figure ). even more interesting phenomena where the theory of networks can be applied are small droplet clusters [ ] [ ] [ ] [ ] [ ] and small colloidal crystals. self-assembled clusters of condensed microdroplets (with the typical diameter of dozens of microns) are formed in an ascending flow of vapor and air above a locally heated thin (approximately mm) layer of water. the droplets form a d monolayer levitating at a low height (comparable with their radii) where their weight is equilibrated by the drag force from the ascending vapor flow. due to an aerodynamic interaction (repulsion) between the droplets and their migration toward the center of the heated spot, they tend to form an ordered structure ( figure ). microdroplets (with the typical diameter of dozens of microns) are formed in an ascending flow of vapor and air above a locally heated thin (approximately mm) layer of water. the droplets form a d monolayer levitating at a low height (comparable with their radii) where their weight is equilibrated by the drag force from the ascending vapor flow. due to an aerodynamic interaction (repulsion) between the droplets and their migration toward the center of the heated spot, they tend to form an ordered structure ( figure ). for large clusters consisting of many dozens or hundreds of droplets, a hexagonally symmetric (honeycomb) structure is typically formed. however, for small clusters, more complex symmetric structures can form in comparison with those in large clusters. for example, the applicability of the mathematical ade-classification or the so-called simply laced dynkin diagrams has been suggested [ ] . small clusters can be used for the in situ tracking of bioaerosols and biomolecules [ ] . the method of voronoi entropy is applied to characterize quantitatively the degree of orderliness of the geometric arrangement of the droplet clusters. the voronoi entropy is calculated using the so-called voronoi tessellation, when an image is divided into a set of polygons. each polygon consists of all points closer to the center of a particular droplet than to any other droplet. the voronoi entropy is then calculated as s vor = − n p n ln p n , where p n is the fraction of polygons with n sides or edges [ ] . in general, three scaling laws relevant to the voronoi entropy in such colloidal systems are lewis' law, the desch law, and the aboav law. lewis observed a linear relationship between the average area of a typical n-gon and n for various random d cellular mosaics. the desch law states a linear relationship between the perimeter of polygons and the number of their edges, while the aboav law relates the average number sides of a voronoi cell neighboring an n-gon a + b/n, where a and b are constants [ ] . unlike liquid water droplets, colloidal particles are solid, and they can form small clusters, which can levitate due to acoustic waves or due to another mechanism, but they form a close packed structure. thus, perry et al. [ ] studied the structural rearrangement in a d levitating cluster of solid sulfate polystyrene spherical particles. individual particles in a particular configuration (an excited state) of the cluster may have bonds between them. for a system of six particles, perry et al. [ ] identified a number of seven-bond and eight-bond configurations. in similar systems, lim et al. [ ] reported the transitions from sticky to ergodic configurations in six-particle and seven-particle systems of hard . µm diameter spheres (figure ). the six-particle system can form various arrangements, with different probabilities of these arrangements. solid sulfate polystyrene spherical particles. individual particles in a particular configuration (an excited state) of the cluster may have bonds between them. for a system of six particles, perry et al. [ ] identified a number of seven-bond and eight-bond configurations. in similar systems, lim et al. [ ] reported the transitions from sticky to ergodic configurations in six-particle and seven-particle systems of hard . μm diameter spheres (figure ). the six-particle system can form various arrangements, with different probabilities of these arrangements. for many distributions of small elements (for example, the words in a language or letters in a text), an empirical power law, such as the zipf law, is found. the zipf law is given by the formula where p(k) is the frequency of an element of rank k, a is a power exponent, and the denominator is needed for normalization. a power law distribution may be expected for the probabilities of various excited states of the cluster forming a set of rearrangement configurations. it is instructive to investigate the probabilistic distributions and information content in graphs corresponding to small clusters. for the data by perry et al. [ ] , the probability distribution of various configurations was plotted in figure (the rank is a number of a given configuration). a calculation based on the empirical formula of the zipf law (equation ) using the experimental data points has been done to fit those experimental points. then, a fitted curve has also been plotted in figure with the data presented in figure . schematic of colloidal particles forming small clusters (concept based on [ ] ). for many distributions of small elements (for example, the words in a language or letters in a text), an empirical power law, such as the zipf law, is found. the zipf law is given by the formula where p(k) is the frequency of an element of rank k, a is a power exponent, and the denominator is needed for normalization. a power law distribution may be expected for the probabilities of various excited states of the cluster forming a set of rearrangement configurations. it is instructive to investigate the probabilistic distributions and information content in graphs corresponding to small clusters. for the data by perry et al. [ ] , the probability distribution of various configurations was plotted in figure (the rank is a number of a given configuration). a calculation based on the empirical formula of the zipf law (equation ( )) using the experimental data points has been done to fit those experimental points. then, a fitted curve has also been plotted in figure with the data presented in table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . entropy , , x for peer review of table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . structures. each point is representing each distinguished structure of a colloidal cluster (based on data from [ ] ). the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the each point is representing each distinguished structure of a colloidal cluster (based on data from [ ] ). table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . structures. each point is representing each distinguished structure of a colloidal cluster (based on data from [ ] ). the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. table . the value of the power exponent in the curve fitting equation is almost one, which indicates that the fitted curve is hyperbolic. see also a discussion of d colloidal clusters by janai et al. [ ] . the information content of a distribution is characterized by the shannon entropy, which is given by where pn is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scalefree networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. the information content of a distribution is characterized by the shannon entropy, which is given by where p n is the statistical probability of the n-th state and n is the total number of states. the shannon entropy is used in materials science, for example, as a surface roughness parameter characterizing informational content in the surface given by its profile [ ] . the shannon-entropy-based informational approach is also used for various other aspects of surface science, such as wetting transitions [ ] and stick-slip transition [ ] . using the data from figure and tables and , the following values were obtained. for the seven-bond configurations, the value of the shannon entropy s = . was obtained, while for the eight-bond configuration, the value of s = . was obtained. the shannon entropy provides an estimation of the information content in these configurations; in particular, one could expect that the seven-bond cluster is more random than the eight-bond cluster. table . structures of seven-bond colloidal clusters and their magnitudes of probability distribution and zipf-law distribution (data from [ ] we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer ( figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer ( figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer ( figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer ( figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer ( figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer ( figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . to conclude this section, methods of network science can be used for the analysis of various systems studied by physical chemistry and materials science. these include granular materials, colloidal crystals, and clusters made of small particles or droplets. many such systems form sets of configurations somewhat similar to the set of symbols (e.g., letters) and characterized by power-law statistical distributions typical for the latter. the power law distribution is also characteristic for scale-free networks, which will be discussed more in detail in the following chapter. the information content of these structures can be estimated using the shannon entropy approach. we conclude that using the network representation for colloidal systems, such as the granular material, colloidal crystals made of small rigid particles, and levitating droplet clusters, results in the scale-free behavior and in a number of important scaling relationships such as the zipf, lewis, desch, and aboav scaling laws. another area closely related to colloidal science is surface science. surface scientists and engineers often deal with parameters of surfaces such as surface roughness, surface free energy, and water contact angle. usually, these parameters are determined in an experimental manner, and they cannot be predicted from the first physical principles. a more applied branch of surface science, which deals with friction, roughness, lubrication, and adhesion is called tribology. tribology deals with such characteristics of contacting surfaces as the coefficient of friction and the wear rate. however, one of the challenges in the tribological studies is that while there is a big amount of experimental data about the frictional, wear, and surface properties of various materials, systems, and engineering components, this interdisciplinary area is highly empirical. tribology remains a data-driven inductive science. it has been recently suggested to apply machine learning techniques in order to predict surface wetting properties. kordijazi et. al. [ ] applied a multilayer perception neural network model to study the wetting properties of a ductile iron-graphite composite including complex dependencies between the contact angle, composition, surface roughness, and the time of exposure to liquid. understanding these correlations allows predicting water-repellent properties of metallic composite materials for the optimized design of novel hydrophobic and superhydrophobic materials. artificial neural networks (anns) are computer models somewhat resembling neural networks in the human brain. anns incorporate a series of functions to process the input data and convert them over several stages into the desired output. since ann models learn by examples and training, they are suited for storing and retrieving acquired knowledge. a typical ann model has interconnected nodes that model complex neurons and synapses in the human brain. the knowledge acquired during the training process is stored in the synaptic weights of the inter-nodal connections leading to the ability to represent complex input-output relationships. anns learn by examining individual records, generating the prediction for each record, comparing the result with the prediction, and making adjustments. training makes the network more accurate in predicting new outcomes. typically, a neural network has three parts or layers: the input, intermediate (hidden) layers, and output layer (figure ). the input layer with units representing the input data, such as the data about the material composition and surface roughness or conditions of the experiment, for example, the size of the droplets used in wetting experiments, and the time of exposure. one or more hidden layers connect the units with varying connection weights until the results are finally delivered to the units in the output layer. the units in the hidden layer compute their activations based on the data from the input layer and a non-linear transfer function and transmit it to the output layer [ ] . ann models and other machine learning techniques will likely become a popular tool for the analysis of properties of surface and colloidal systems. note that anns themselves do not possess any scaling properties relevant to the study of physical systems. however, anns represent a socalled biomimetic approach, because they attempt to mimic learning algorithms in human brains. therefore, it is of interest to investigate natural neural networks or cortical networks, and these will be discussed in the following section. let us start the discussion of scaling in biological objects from the scaling rules in branching networks, for which the typical example is the vascular system of mammals. according to the empirical allometric klieber law [ ] formulated in , metabolism rates, b, in various species are well approximated by a power-law scaling dependency on the mass of an animal, ∝ . . based on the metabolism rates, one can estimate other parameters in species including the lifespan. the value of the klieber law exponent, a = . , remained mysterious until the seminal paper by west et al. [ ] explained it using the fractal model of blood vessel branching. the blood vessels serve a certain volume with the simultaneous conservation of the cross-sectional area of the vessels and of the volume covered at every stage of branching. despite its shortcomings [ ] [ ] [ ] [ ] , this theory remains the main explanation of the allometric scaling exponents. according to west et al. [ ] , when a tube with the length lk and radius rk branches into n tubes with the lengths lk+ = γlk and radii rk+ = βrk (where γ and β are constants), the volume served by the next-generation tubes and their cross-section area should conserve, which leads to two separate scaling relationships for the constants ∝ / and ∝ / . these relationships are satisfied simultaneously [ ] . the area is preserved due to the constant rate of the fluid flow at different ann models and other machine learning techniques will likely become a popular tool for the analysis of properties of surface and colloidal systems. note that anns themselves do not possess any scaling properties relevant to the study of physical systems. however, anns represent a so-called biomimetic approach, because they attempt to mimic learning algorithms in human brains. therefore, it is of interest to investigate natural neural networks or cortical networks, and these will be discussed in the following section. let us start the discussion of scaling in biological objects from the scaling rules in branching networks, for which the typical example is the vascular system of mammals. according to the empirical allometric klieber law [ ] formulated in , metabolism rates, b, in various species are well approximated by a power-law scaling dependency on the mass of an animal, b ∝ m . . based on the metabolism rates, one can estimate other parameters in species including the lifespan. the value of the klieber law exponent, a = . , remained mysterious until the seminal paper by west et al. [ ] explained it using the fractal model of blood vessel branching. the blood vessels serve a certain volume with the simultaneous conservation of the cross-sectional area of the vessels and of the volume covered at every stage of branching. despite its shortcomings [ ] [ ] [ ] [ ] , this theory remains the main explanation of the allometric scaling exponents. according to west et al. [ ] , when a tube with the length l k and radius r k branches into n tubes with the lengths l k+ = γl k and radii r k+ = βr k (where γ and β are constants), the volume served by the next-generation tubes and their cross-section area should conserve, which leads to two separate scaling relationships for the constants γ ∝ n − / and β ∝ n − / . these relationships are satisfied simultaneously [ ] . the area is preserved due to the constant rate of the fluid flow at different hierarchical levels. the volume is preserved, assuming that the same volume in the organism is served by blood vessels of different hierarchical levels ( figure ). on the metabolism rates, one can estimate other parameters in species including the lifespan. the value of the klieber law exponent, a = . , remained mysterious until the seminal paper by west et al. [ ] explained it using the fractal model of blood vessel branching. the blood vessels serve a certain volume with the simultaneous conservation of the cross-sectional area of the vessels and of the volume covered at every stage of branching. despite its shortcomings [ ] [ ] [ ] [ ] , this theory remains the main explanation of the allometric scaling exponents. according to west et al. [ ] , when a tube with the length lk and radius rk branches into n tubes with the lengths lk+ = γlk and radii rk+ = βrk (where γ and β are constants), the volume served by the next-generation tubes and their cross-section area should conserve, which leads to two separate scaling relationships for the constants ∝ / and ∝ / . these relationships are satisfied simultaneously [ ] . the area is preserved due to the constant rate of the fluid flow at different hierarchical levels. the volume is preserved, assuming that the same volume in the organism is served by blood vessels of different hierarchical levels ( figure ). the total volume of fluid in the vascular system can be calculated as a sum at different levels of the hierarchy using the summation of the geometric series where v is a certain elementary volume (e.g., volume served by a capillary), and n is the total number of branch generations. therefore, the volume scales as v ∝ γβ −n . from this, the scaling dependency of the total number of thinnest capillaries as a function of volume is n n ∝ v a ∝ γβ −na ∝ n − / −na ∝ n na/ yielding a = / , the well-established empirical results known as the kleiber law, which is based on the assumption of a constant flow rate. the model by west et al. [ ] , while simplified, is important at the conceptual level. it was suggested that the fractal scaling of the vascular system may explain the non-ergodicity of the blood flow [ ] . branching in the vascular network provides a classical mechanism for estimating scaling, which can be applied to more complex neuron networks. in this section, we will review certain aspects of the current knowledge about the cortical networks in human and animal brains related to their scaling and self-organizing properties. this area of neuroscience is rapidly developing and intersects with the network science in many instances. since this area is less known to biophysicists, colloidal scientists, and engineers, we will introduce some concepts on the structure of cortical networks prior to discussing their scaling and self-organizing properties. let us start from the discussion of the architecture of the cortical network. neuron connections in the human brain constitute a very complex network of about neurons with more than synapses connecting between them. while it is extremely difficult to study such a complex network, a number of important insights have been achieved, particularly, since the early s. this knowledge was obtained due to novel methods of in vivo observation of neural activity, including the electroencephalography (eeg), functional magnetic resonance imaging (fmri), diffusion tensor imaging (dti), two-photon excitation microscopy (tpef or pef), and positron emission tomography (pet). many insights were also achieved using the comparison or analogy of the human brain with the neural system of much simpler organisms, such as the nematode caenorhabditis elegans (a tiny worm with the size of less than mm), which has only neurons and about synapses. since a complete connectome (a map of neuron connections) and genome have been obtained and published for c. elegans, in [ ] , this worm serves as a model organism for genetic and neurological research including, for example, d simulations of its motion and behavior [ ] . as far as the human brain, the higher-order brain functions, such as cognition, language, sensory perception, and the generation of motor commands are associated with the neocortex. the neocortex is an external part of the brain, which is - mm thick and with the surface area of . m . the neocortex is made of six distinct layers of neurons, and it consists of cortical mini-columns with the diameter of about - µm spanning through all six layers, with about neurons in each mini-column ( figure ). although the functionality of the microcolumns (and their very existence) is being debated by some researchers, the columnar structure of the neocortex is widely accepted by most neuroscientists. the microcolumns are combined into the large hyper-columns or macro-columns, which are - µm in diameter. the hyper-columns have a roughly hexagonal shape, and each column is surrounded by six other columns. each hyper-column, by some estimates, may include - microcolumns [ ] [ ] [ ] . while little is known about the processes inside the microcolumns, it is widely believed that a cortical column can process a number of inputs, and it converts them to a number of outputs using overlapping internal processing chains. each minicolumn is a complex local network that contains elements for redundancy and plasticity. the minicolumn unites vertical and horizontal cortical components, and its design has evolved specifically in the neocortex. although minicolumns are often considered highly repetitive clone-like units, they display considerable heterogeneity between cortex areas and sometimes even within a given macrocolumn. a comprehensive map of neural connections in the brain is called the connectome [ ] . a connectome of the c. elegans worm has been obtained in , while obtaining a human brain connectome remains a much more challenging task of the scientific discipline referred to as connectomics (compare with genome and genomics or proteome and proteomics). a much more complex connectome of the drosophila melanogaster fruit fly, a model insect used for various genetic research, whose brain contains about , neurons and synapses, has been presumably obtained by [ ] . it is believed that the human connectome can be studied at three distinct levels of structural organization: the microscale (connection of single neurons), mesoscale (cortical columns), and the macroscale (anatomical regions of interest in the brain). quantitative estimates of the brain network characteristics are remarkable. a measure of the diameter (largest geodesic distance) of the scale-free network of n nodes was suggested by bollobás and riordan [ ] as d=log(n)/log(logn). according to freeman and breakspear [ ] , the neocortical diameter of each hemisphere is close to d = , for neurons . × neurons and synapses per neuron yielding n = × . these numbers are consistent with the idea that at least a three-level while little is known about the processes inside the microcolumns, it is widely believed that a cortical column can process a number of inputs, and it converts them to a number of outputs using overlapping internal processing chains. each minicolumn is a complex local network that contains elements for redundancy and plasticity. the minicolumn unites vertical and horizontal cortical components, and its design has evolved specifically in the neocortex. although minicolumns are often considered highly repetitive clone-like units, they display considerable heterogeneity between cortex areas and sometimes even within a given macrocolumn. a comprehensive map of neural connections in the brain is called the connectome [ ] . a connectome of the c. elegans worm has been obtained in , while obtaining a human brain connectome remains a much more challenging task of the scientific discipline referred to as connectomics (compare with genome and genomics or proteome and proteomics). a much more complex connectome of the drosophila melanogaster fruit fly, a model insect used for various genetic research, whose brain contains about , neurons and synapses, has been presumably obtained by [ ] . it is believed that the human connectome can be studied at three distinct levels of structural organization: the microscale (connection of single neurons), mesoscale (cortical columns), and the macroscale (anatomical regions of interest in the brain). quantitative estimates of the brain network characteristics are remarkable. a measure of the diameter (largest geodesic distance) of the scale-free network of n nodes was suggested by bollobás and riordan [ ] as d = log(n)/log(logn). according to freeman and breakspear [ ] , the neocortical diameter of each hemisphere is close to d = , for neurons . × neurons and synapses per neuron yielding n = × . these numbers are consistent with the idea that at least a three-level hierarchy exists formed by nodes as neurons, hypercolumns, and modules. the reduction of neurons and synapses to a depth of three levels and a diameter of d = is viewed as a simplification [ ] . klimm et al. [ ] estimated the quantitative characteristics of the human brain network including the hierarchy of the network and its fractal topological dimension. the hierarchy of a network, β, is defined quantitatively by the presumed power law relationship between the node degree and the local clustering coefficient (the ratio of the triangle subgraphs to the number of node triples), after discussing the architectural structure of the cortical networks, let us briefly review what is known about the formation of such a complex network, from both an ontogenetic and phylogenetic point of view. the question of to what extent the brain wiring is coded in the dna remains controversial. the human brain cortex contains at least neurons linked by more than synaptic connections, while the number of base pairs in a human genome is only . × . therefore, it is impossible that the information about all synaptic connections is contained in the dna. currently, two concepts, namely, the protomap hypothesis and the radial unit hypothesis, which complement each other, are employed to explain the formation of the neo-cortex. both hypotheses were suggested by pasko rakic [ ] . the protomap is a term for the original molecular "map" of the mammalian cerebral cortex with its functional areas during early embryonic development when neural stem cells are still the dominant cell type. the protomap is patterned by a system of signaling centers in the embryo, which provide information to the cells about their position and development. this process is referred to as the "cortical patterning". mature functional areas of the cortex, such as the visual, somatosensory, and motor areas are developed through this process. during the mammalian evolution, the area of the cortical surface has increased by more than times, while its thickness did not change significantly. this is explained by the radial unit hypothesis of cerebral cortex development, which was first described by pasko rakic [ ] [ ] [ ] . according to this hypothesis, the cortical expansion is the result of the increasing number of radial columnar units. the increase occurs without a significant change in the number of neurons within each column. the cortex develops as an array of interacting cortical columns or the radial units during embryogenesis. each unit originates from a transient stem cell layer. the regulatory genes control the timing and ratio of cell divisions. as a result, an expanded cortical plate is created with the enhanced capacity for establishing new patterns of connectivity that are validated through natural selection [ ] . an interesting observation about the human connectome was made by kerepesi et al. [ ] . by analyzing the computer data of the "budapest reference connectome", which contains macroscale connectome data for individuals, they identified common parts of the connectome graphs between different individuals. it was observed that by decreasing the number of individuals possessing the common feature from down to , more graph edges appeared. however, these new appearing edges were not random, but rather similar to a growing "shrub". the authors called the effect the consensus connectome dynamics and hypothesized that this graph growth may copy the temporal development of the connections in the human brain, so that the older connections are present in a greater number of subjects [ ] . an important model was suggested recently by barabási and barabási [ ] , who attempted to explain the neuronal connectivity diagram of the c. elegans nematode worm by considering neuron pairs that are known to be connected by chemical or electrical synapses. since synaptic wiring in the c. elegans is mostly invariant between individual organisms, it is believed that this wiring is genetically encoded. however, identifying the genes that determine the synaptic connections is a major challenge. barabási and barabási [ ] identified a small set of transcription factors responsible for the synapses formation of specific types of neurons by studying bicliques in c. elegans' connectome. according to their model, a set of log (n) transcription factors is sufficient to encode the connection, if transcription factors are combined with what they called the biological operators. it was proposed that soc plays a role in the formation of the brain neural network [ , , ] . the neural connectivity is sparse at the embryonic stage. after the birth, the connectivity increases and ultimately reaches a certain critical level at which the neural activity becomes self-sustaining. the brain tissue as a collective system is at the edge of criticality. through the combination of structural properties and dynamical factors, such as noise level and input gain, the system may transit between subcritical, critical, and supercritical regimes. this mechanism is illustrated in figure a [ ] . the network evolves toward regions of criticality or edge-of-criticality. once critical regions are established, the connectivity structure remains essentially unchanged. however, by adjusting the noise and/or gain levels, the system can be steered toward or away from critical regions. according to freeman and breakspear [ ] , the power-law distribution of axonal length (figure b ) is the evidence of scale-free activity. to freeman and breakspear [ ] , the power-law distribution of axonal length (figure b ) is the evidence of scale-free activity. self-organizing critical behavior was reported by liu et al. [ ] for the organization of brain gabaa receptors (these are receptors of γ-aminobutyric acid or gaba, the major neurotransmitter). the mean size of receptor networks in a synapse followed a power-law distribution as a function of receptor concentration with the exponent . representing the fractal dimension of receptor networks. the results suggested that receptor networks tend to attract more receptors to grow into larger networks in a manner typical for soc systems that self-organize near critical states. an amazing feature of brain operations is that they are distributed, rather than localized at a particular neuron or a group of neurons. the distributed operations are performed by a collection of processing units that are spatially separate and communicate by exchanging messages. mountcastle [ ] formulated the following properties of such distributed systems: • signals from one location to another may follow any of a number of pathways in the system. this provides the redundancy and resilience. • actions may be initiated at various nodal loci within a distributed system rather than at one particular spot. local lesions within a distributed system usually may degrade a function, but not eliminate it completely. the nodes are open to both externally induced and internally generated signals. various aspects of scaling behavior have been studied for networks associated with the brain, including both special and temporal structures. several neuroscientists suggested in the s that self-organizing critical behavior was reported by liu et al. [ ] for the organization of brain gaba a receptors (these are receptors of γ-aminobutyric acid or gaba, the major neurotransmitter). the mean size of receptor networks in a synapse followed a power-law distribution as a function of receptor concentration with the exponent . representing the fractal dimension of receptor networks. the results suggested that receptor networks tend to attract more receptors to grow into larger networks in a manner typical for soc systems that self-organize near critical states. an amazing feature of brain operations is that they are distributed, rather than localized at a particular neuron or a group of neurons. the distributed operations are performed by a collection of processing units that are spatially separate and communicate by exchanging messages. mountcastle [ ] formulated the following properties of such distributed systems: • signals from one location to another may follow any of a number of pathways in the system. this provides the redundancy and resilience. • actions may be initiated at various nodal loci within a distributed system rather than at one particular spot. local lesions within a distributed system usually may degrade a function, but not eliminate it completely. the nodes are open to both externally induced and internally generated signals. various aspects of scaling behavior have been studied for networks associated with the brain, including both special and temporal structures. several neuroscientists suggested in the s that the human brain network is both scale-free and small-world, although the arguments and evidence for these hypotheses are indirect [ , ] , including power-law distributions of anatomical connectivity as well as the statistical properties of state transitions in the brain [ ] . freeman and breakspear [ ] suggested that if neocortical connectivity and dynamics are scale-free, hubs should exist for most cognitive functions, where activity and connections are at a maximum. these hubs organize brain functions at the microscopic and mesoscopic level. they are detectable by macroscopic imaging techniques such as fmri. therefore, these are hubs rather than localized functions, which are revealed by these imaging techniques. when connection density increases above a certain threshold, a scale-free network undergoes an avalanche-like abrupt transition and resynchronization almost instantaneously independent of its diameter. scale-free dynamics can explain how mammalian brains operate on the same time scales despite differences in size ranging to (mouse to whale), as it will be discussed in more detail in consequent sections. random removals of nodes from scale-free networks have negligible effects; however, lesions of hubs are catastrophic. examples in humans are coma and parkinson's disease from small brain stem lesions [ ] . avalanches are a common characteristic of brain signals, along with the so-called bursting [ ] [ ] [ ] . neural avalanches show such characteristics as power-law distributions, which is believed to be an indication of near critical behavior [ ] . thus figure a , redrawn from ref. [ ] , suggests that the actual size distribution of neuronal avalanches is a power law and it is different from the poisson model distribution of uncorrelated activities. it is further believed that whether the avalanche occurs depends on the branching regime during the neuron connection (figure b ) [ ] [ ] [ ] [ ] [ ] . increases above a certain threshold, a scale-free network undergoes an avalanche-like abrupt transition and resynchronization almost instantaneously independent of its diameter. scale-free dynamics can explain how mammalian brains operate on the same time scales despite differences in size ranging to (mouse to whale), as it will be discussed in more detail in consequent sections. random removals of nodes from scale-free networks have negligible effects; however, lesions of hubs are catastrophic. examples in humans are coma and parkinson's disease from small brain stem lesions [ ] . avalanches are a common characteristic of brain signals, along with the so-called bursting [ ] [ ] [ ] . neural avalanches show such characteristics as power-law distributions, which is believed to be an indication of near critical behavior [ ] . thus figure a , redrawn from ref. [ ] , suggests that the actual size distribution of neuronal avalanches is a power law and it is different from the poisson model distribution of uncorrelated activities. it is further believed that whether the avalanche occurs depends on the branching regime during the neuron connection (figure b ) [ ] [ ] [ ] [ ] [ ] . (a) (b) figure . (a) avalanche size (log-log scale) distributions in brain shows a power-law dependency [ ] (b) the activity may decrease, stay at the same level, or grow with time depending on the branching regime (based on [ ] ). speaking of the human brain operation, it would be disappointing to avoid such an intriguing topic as what is currently known about the possible connection of brain activities to cognition. while identifying parts of the brain responsible for higher order brain activity, such as cognition or speech, figure . (a) avalanche size (log-log scale) distributions in brain shows a power-law dependency [ ] (b) the activity may decrease, stay at the same level, or grow with time depending on the branching regime (based on [ ] ). speaking of the human brain operation, it would be disappointing to avoid such an intriguing topic as what is currently known about the possible connection of brain activities to cognition. while identifying parts of the brain responsible for higher order brain activity, such as cognition or speech, and understanding their mechanisms remains a remote (if at all solvable) task, a number of significant observations have been made in the past years. some of these observations are related to the temporal scales involved. in the s, b. biswal, a graduate student at the medical college of wisconsin (mcw), discovered that the human brain displays so-called "resting state connectivity", which is observable in the fmri scans [ ] . the phenomenon was later called the default mode network (dmn), and it describes brain functions of a resting state. the dmn is active when a person is not focused on the outside world, and the brain is at wakeful rest, such as during daydreaming and mind-wandering. it is also active when individuals are thinking about others, thinking about themselves, remembering the past, and planning for the future. the dmn has been shown to be negatively correlated with other networks in the brain such as attention networks, although the former can be active in certain goal-oriented tasks such as social working memory or autobiographical tasks. several recent studies have concentrated upon dmn's relationship to the perception of a temporal sequence of events, as well as to its role in speech and language-related cognition. these features are of particular interest to the philosophy of mind because language, the ability to plan activities, introspection, and understanding the perspective of another person are often described as distinct characteristic features of humans, which separate them from other mammals. konishi et al. [ ] investigated the hypothesis that the dmn allows cognition to be shaped by memory-stored information rather than by information in the immediate environment, or, in other words, by "past" rather than by "now". using the fmri technique, they investigated the role of the dmn when people made decisions about where a shape was, rather than where it is now. the study showed that dmn hubs are responsible for the cognition guided by information belonging to the past or to the future, instead of by immediate perceptual input. on the basis of these observations, konishi et al. [ ] suggested that the dmn is employed for higher order mental activities such as imagining the past or future and considering the perspective of another person. these complex introspective activities depend on the capacity for cognition to be shaped by representations that are not present in the current external environment. in a different study, lerner et al. [ ] investigated how human activities involving the integration of information on various time-scales is related to the dmn activation. during real-time lasting activities, such as watching a movie or engaging in conversation, the brain integrates information over multiple time scales. the temporal receptive window (the length of time before a response during which sensory information may affect that response) becomes larger when moving from low-level sensory to high-level perceptual and cognitive areas. lerner et al. [ ] showed that the temporal receptive window has a hierarchical organization with levels of response to the momentary input, to the information at the sentence time scale, and to the intact paragraphs that were heard in a meaningful sequence. the researchers further hypothesized that the processing time scale is a functional property that may provide a general organizing principle for the human cerebral cortex. in a neurolinguistics study, honey et al. [ ] performed fmri research to figure out whether different languages affect different patterns in neural response. they made bilingual listeners hear the same story in two different languages (english and russian). the story evoked similar brain responses, which were invariant to the structural changes across languages. this demonstrated that the human brain processes real-life information in a manner that is largely insensitive to the language in which that information is conveyed. simony et al. [ ] further investigated how the dmn reconfigures to encode information about the changing environment by conducting fmri while making subjects listen to a real-life auditory narrative and to its temporally scrambled versions. the momentary configurations of dmn predicted the memory of narrative segments. these are interesting studies that may provide insights into questions such as how the natural language is related to the hypothetical language of thought, which has been postulated by some cognitive scientists and philosophers of language. the suggestion that the brain's connectome as a network possesses topological properties of fractal, scale-free, or small-world networks brings a number of interesting questions. the hierarchically organized network with the fractal dimension of d (some estimates state the value of the fractal dimension d = . ± . [ ] ) is packed into the d cortex, forming a thin layer whose thickness is almost two orders of magnitude smaller than its two other dimensions. barabási and barabási [ ] noted that in order to store the information about the exact structure of the connectome of n neurons, a neuron with k links would need k·log (n) bits of information, with the total information in all neurons kn·log (n). for large organisms, this would significantly exceed the information contained in dna (table ) . thus, . · bits of information would be required to characterize the human brain; for comparison, some estimates indicate that human brain memory capacity is - bit, while the human genome has about pairs of nucleotides. to overcome this difficulty, barabási and barabási [ ] suggested a selective coding model. according to their model, the brain cannot encode each link at the genetic level. instead, selective operators are employed, which inspect the transcription factor signatures of two neurons (somewhat close in space) and facilitate or block the formation of a directed link between them. this can be achieved by external agents, such as glia cells, which select specific neurons and facilitate synapse formation between them or detect the combinatorial expression of surface proteins, whose protein-protein interactions catalyze synapse formation. the action of such selective operators is evidenced by the emergence of detectable network motifs in the connectome, namely, bicliques. the model by barabási and barabási [ ] could provide an insight into the question of how much of the information in the structure of the brain is contained in the dna and how much is generated during the embryonal and post-embryonal development by self-organizing processes. the lower boundary of genetic information can be estimated by the number of transcription factors, t, which encode the identity of a neuron times the total number of neurons. in this section, we will review current experimental data about the scaling properties of cortical networks related to their spatial and temporal organization and their informational content from the entropic viewpoint. there are several approaches to what constitutes a "time constant" for the brain and how this time constant (i.e., the rate of neural processes) scales with the size of an animal. brain networks show a number of remarkable properties. one important experimental observation is that despite the difference in their size by times from a mouse to a whale, mammalian brains tend to operate at almost the same time scales. this can be called the law of conservation of the characteristic time scale. there are two approaches to the characterization of the time scale of brain activity of different creatures: studying brain waves (rhythms of oscillation) and investigations of the critical flicker fusion (cff) thresholds. the cff is defined as the frequency of an intermittent light, at which the light appears steady to a human or animal observer (similar to frames in the cinema). it has been hypothesized that the ability of an animal to change their body position or orientation (manoeuvrability) is related to the ability to resolve temporal features of the environment and eventually to the cff [ ] . manoeuvrability usually decreases with increasing body mass. buzsáki et al. [ ] reviewed the temporal scaling properties of cortical networks by studying the hierarchical organization of brain rhythms in mammals. the brain is known to generate electromagnetic oscillations of different frequencies, which can be observed with the eeg. while the exact nature and function of these oscillations remains debatable, they are highly reproducible and classified by their frequencies: alpha waves ( - hz), beta waves ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , gamma waves (> hz), and others. the frequency of brain oscillations covers almost five orders of magnitude, from < . hz to hundreds of hz. the power distribution of brain oscillations tends to show the /f n (where f is the frequency and n is the power exponent) noise spectrum when measured at long-scale ranges. as we have discussed in the preceding sections, such a distribution spectrum is a signature of soc. however, when specific brain activities are considered, such as concentrating on particular features, moving or orienting in space or various cognitive functions, particular oscillation frequencies become dominant, and the spectrum deviates from the /f or /f n statistics, showing peaks at some characteristic frequencies. these frequencies of various rhythm classes do not vary significantly with the size of the brain [ ] . as far as modeling the origin of oscillations, several dynamic models have been employed to study brain rhythms at various scales and, in particular, at the mesoscale. thus, the so-called freeman's k-sets model follows the katzir-katchalsky suggested treatment of cell assemblies using network thermodynamics. hierarchical models of several layers of these sets, from k-i to k-v, provide a modeling tool to conduct analyses of the unifying actions of neocortex related to intentional and cognitive behaviors [ ] . the correlation of spatial and temporal brain activity organization was studied by honey et al. [ ] . they related the spontaneous cortical dynamics to the underlying anatomical connectivity at multiple temporal scales. structural (spatial) hubs corresponded to the hubs of the long-run (minutes) neural activity. for the activities with shorter characteristic time (seconds), significant deviations from special hubs were observed. at the shorter time scale (fraction of a second), individual episodes of interregional phase-locking were detected. the critical flicker fusion threshold is viewed by many researchers as the time scale (or frequency) at which the brain operates. the higher frequency of the cff threshold implies the brain's ability to discern signals and react faster. healy et al. [ ] studied the effect of body mass and metabolic rates of various species on their cff threshold ( figure ) . the comparative metabolic rates are determined separately by measuring oxygen consumption through ventilation [ ] . it is expected that smaller animals have higher temporal resolution. larger animals respond to a stimulus slower than the smaller animals. therefore, high temporal resolution in larger animals is unnecessary. on the other hand, faster and more manoeuvrable fly species have higher temporal resolutions [ ] , which makes it, for example, so difficult for a human to catch a fly. note that the mass-specific metabolic rates are almost constant (or, more accurate to say, lie within a certain relatively narrow range) for different life forms with orders of magnitude difference in the body mass [ ] . furthermore, the accuracy of the / -power allometric scaling kleiber law for metabolic rate is not considered universal by some scholars [ ] . range) for different life forms with orders of magnitude difference in the body mass [ ] . furthermore, the accuracy of the / -power allometric scaling kleiber law for metabolic rate is not considered universal by some scholars [ ] . we conclude that the characteristic frequencies of brain activity are either almost constant or slightly decrease with increasing body and brain size. (a) (b) figure . the effect of (a) body mass (gram) and (b) temperature-corrected mass-specific resting metabolic rate (qwg) on the critical flicker fusion (cff) shows that the cff increases with the metabolic rate but decreases with body mass (based on [ ] ). figure . the effect of (a) body mass (gram) and (b) temperature-corrected mass-specific resting metabolic rate (qwg) on the critical flicker fusion (cff) shows that the cff increases with the metabolic rate but decreases with body mass (based on [ ] ). we conclude that the characteristic frequencies of brain activity are either almost constant or slightly decrease with increasing body and brain size. the allometric approach can be applied to the inter-species analysis of brain activity frequencies and time scales in both humans and various species. the observation that typical frequencies of brain activities are often independent of brain size requires an explanation. when brains of various species are compared, the distance between homologous regions of the brain increases with the growing size of the brain. moreover, the number and length of axons increase even more rapidly than the number of neurons with the growing brain size. consequently, the average number of synaptic connections in the shortest path between two neurons (the "synaptic path length") also grows in a very fast manner. assuming that the cortical network is a scale-free and/or a small-world network would decrease the path length; however, it does not eliminate completely the scale dependency. the mechanisms that facilitate the increase of signal speed in larger species should be investigated. such an investigation was suggested by buzsáki et al. [ ] . they used experimental data about the scaling of the brain, in particular, those presented by wang et al. [ ] on the size of axons and the amount of white matter in the brain. these are parameters that affect the signal speed. the experimental observations indicate that the increase in axon caliber (size) and their insulation (myelination) compensates for the increased time of signal transfer. the volume of the myelin or white matter in relation to the gray matter (neurons) in the brain scales as power / with the size. for instance, while the white matter constitutes % of the neocortical volume in hedgehogs, in humans, it exceeds % of brain volume. this is because a larger brain requires faster signal propagation, and the degree of myelination in larger brains increases. the velocity of propagation of the neural signal is another significant parameter. buzsáki et al. [ ] noted that for the phase synchronization of gamma waves in a mouse (brain size approximately - mm), the speed of conduction of m/s is sufficient, while for humans ( - mm) , much larger conduction speeds are needed [ ] . while an increase in conduction velocity can be achieved by both the increase of the volume fraction of white matter and of the axon size, there are several problems associated with this approach. an increase of axon diameter proportional to the size of the brain would enormously increase the brain volume. experimental observation suggests that, apparently, only a small fraction of all axons have large diameters. particularly, the largest axons scale linearly with the brain size (figure ), and they result in an increase of the connection speed [ , ] . at the same time, the required increase of the white matter volume in larger brains does not lead to an unreasonable growth of the volume, because only a small fraction of all axons is large. consequently, despite a , -fold difference in mammalian brain volumes, the oscillation rhythms with their typical time scales independent of the brain size are supported by the same mechanisms, and they still have the same typical frequencies. = const ( ) which can be presented as = = * ln( ) . equation relates the ratio of white-to-gray matter to the characteristic size of the brain. the relationship is plotted in figure for the value of d = . , for several values of the exponent d. the experimental data, based on [ ] , for several animals are also presented. it is seen that the best agreement with equation is for < d < , which is consistent with the concept that the growth of the white matter content compensates for the increasing linear size of the brain. (a) (b) (c) (d) figure . interspecies scaling relations in the brain (based on [ ] and [ ] ). (a) cross-brain conduction times for myelinated axons; (b) the fraction of myelinated axons; (c) the fraction of volume filled by axons; (d) distribution of axon densities. figure . scaling relationship between the brain diameter (cm) and the ratio of white and gray matter. an interesting spin-off of the network approach to the neural science has been developed in the field of immunology, where niels jerne [ ] and geoffrey hoffmann [ ] have suggested the socalled immune network theory (int). according to this theory, the immune system of a human or of an animal is a network of lymphocytes (immune cells) and molecules (antibodies, such as immunoglobulins), which interact with each other. an invasion of a foreign antigen a (which may be a virus, microbe, protein, or even an inorganic compound) activates immune cells and molecules the number of nodes, n, scales proportionally to the power d of the characteristic length size of the brain, l, as n ∼ l d where d is the fractal dimension of the network. the velocity of neural signal propagation is dependent on the ratio w = w/g of the volume fractions of the white matter, w, to the gray matter, g, as a power function, as we have discussed in the preceding sections, in small-world networks, the distance between two random nodes is proportional to the logarithm of the total number of nodes, l ∼ ln(n) ∼ d· ln(l). the rate of neural processes, i.e., the time scale of the brain activity is related to the size of an animal. however, given the experimental observation that the rate is almost a constant independent of the size of the brain, one can assume that the volume fraction of white-to-gray matter affects the velocity of the signal from equation ( ), it follows immediately that d· ln(l) which can be presented as equation ( ) relates the ratio of white-to-gray matter to the characteristic size of the brain. the relationship is plotted in figure for the value of d = . , for several values of the exponent d. the experimental data, based on [ ] , for several animals are also presented. it is seen that the best agreement with equation ( ) is for < d < , which is consistent with the concept that the growth of the white matter content compensates for the increasing linear size of the brain. (a) (b) (c) (d) figure . interspecies scaling relations in the brain (based on [ ] and [ ] ). (a) cross-brain conduction times for myelinated axons; (b) the fraction of myelinated axons; (c) the fraction of volume filled by axons; (d) distribution of axon densities. scaling relationship between the brain diameter (cm) and the ratio of white and gray matter. an interesting spin-off of the network approach to the neural science has been developed in the field of immunology, where niels jerne [ ] and geoffrey hoffmann [ ] have suggested the socalled immune network theory (int). according to this theory, the immune system of a human or of an animal is a network of lymphocytes (immune cells) and molecules (antibodies, such as immunoglobulins), which interact with each other. an invasion of a foreign antigen a (which may be a virus, microbe, protein, or even an inorganic compound) activates immune cells and molecules figure . scaling relationship between the brain diameter (cm) and the ratio of white and gray matter. an interesting spin-off of the network approach to the neural science has been developed in the field of immunology, where niels jerne [ ] and geoffrey hoffmann [ ] have suggested the so-called immune network theory (int). according to this theory, the immune system of a human or of an animal is a network of lymphocytes (immune cells) and molecules (antibodies, such as immunoglobulins), which interact with each other. an invasion of a foreign antigen a (which may be a virus, microbe, protein, or even an inorganic compound) activates immune cells and molecules anti-a, which, in turn, activate anti-anti-a, and so on. the nodes of the network are immune cells, antibodies, and antigens, while the edges are interactions between them. therefore, the reaction of the immune system on the antigen is somewhat similar to the reaction of a network upon a stimulus applied to one of its nodes: it may be a local perturbation or an avalanche-type response affecting many nodes of the system. according to jerne, there are many similarities between the immune system and the central nervous system. the numbers of cells in both systems are of the same order, - . the mass of immune cells in a human is on the order of kg, which is somewhat comparable with the brain mass, at least, by the order of magnitude. both the immune and nervous systems respond to external stimuli, and they both possess memory [ ] . the int theory was suggested in the s. although jerne became a winner of the nobel prize for his work in immunology, the int remains a hypothesis that may require further experimental validation. jerne apparently sought an analogy of the int not only with the principals of cortical network organization, but also with the rules, which govern human language, such as the chomskian concept of the generative grammar. jerne's nobel lecture is entitled "the generative grammar of the immune system" [ ] . since the turn of the st century, there have been attempts to investigate the scaling properties of the immune network, including their scale-free and fractal properties [ , ] . this is often conducted in the context of a broader area of the protein networks, since the interactions between antigens and antibodies or immune cells are bio-specific (ligand-receptor) protein-protein interactions [ ] . conceptually, these efforts are supposed to give insights, for example, on autoimmune diseases. one can hypothesize that, similarly to the soc sand-pile model, where the addition of a sand grain usually has a local effect, but sometimes it can cause avalanches, the reaction of the immune network to an external stimulus (a pathogenic antigen) can be an immune response or a catastrophic series of events leading to a disease. however, the int still largely misses a connection with experimental science. the term "immunome" (and therefore, the area of "immunomics") has already been suggested [ ] as an analogy for the genome (and genomics), proteome (and proteomics), and connectome (and connectomics). as far as the protein networks, it has been shown that hydrophobic interactions during protein folding result in the soc mechanisms that can explain scaling properties and power-law exponents (e.g., size-volume dependencies) in proteomics [ ] . the relation of hydrophobic interactions and soc has been established [ ] , and similar considerations have been used in materials science, in particular, for the design of anti-icing coatings [ ] . networks of cortical neurons in the brain are a source of inspiration for the area of biomimetic ann. computational algorithms inspired by the int have been suggested as well [ ] . according to the clonal selection theory of immunity, when a pathogen invades the organism, immune cells (lymphocytes) that recognize these pathogens would proliferate yielding effector cells with secrete antibodies (immunoglobulin proteins) and memory cells. the multitude of available immune cells is explained as a result of somatic mutations with high rates, while pathogens drive their selection force. a rough model of this process is used as a basis for genetic computational algorithms called the artificial immune systems (ais) algorithms [ ] . methods of the network science and information theory can be used for the analysis of diverse types of physicochemical and biological systems. the systems reviewed in this article include granular materials, droplet clusters, colloidal crystals, artificial neural networks, biological vascular networks, cortical networks of neurons connected by synapses, and immune networks. scaling, topology, and dimensional analysis can apply to networks that describe these different physical and biophysical systems. some of these networks possess fractal, scale-free, and small-world scaling properties. others exhibit different types of scaling relationships often involving power laws. the amount of information contained in a network can be found by calculating its shannon entropy. we discussed the properties of colloidal networks arising from small granular and colloidal particles and from droplet clusters due to pairwise interaction between the particles. many networks found in colloidal science possess self-organizing properties due to the effect of percolation. the force networks in packed granular material leading to the jamming transition are a typical example. other systems may exhibit self-organized criticality. they are brought to a critical point by a combination of slow motion and a dissipation mechanism, which can balance out. these systems have critical states, which have distinct signatures of fractal dimensions and power laws (often one-over-frequency spectrum), although, of course, not every system with a one-over-frequency spectrum is an soc system. colloidal systems exhibit various scaling relationships including the fractal (scale-free), zipf, lewis, desch, and aboav scaling laws. branching vascular systems demonstrate the allometric power exponents of . this power exponent in such systems is explained by a fractal model of branching with the simultaneous conservation of the volume served by blood vessels at different levels and the flow rate. then, we discussed much more complex networks of neurons, which are organized in the neocortex in a hierarchical manner, forming micro-and macro-columns. the scaling relationships in these networks suggest that the characteristic time constant is independent of brain size when interspecies comparison is conducted. this is because the increased diameter of the network is compensated by the increasing velocity of the signal due to myelination (the insulation of neurons by the white matter). the characteristic time constant can be defined in terms of the frequency of different types of brain waves or as the cff threshold. the brain networks possess many characteristics typical to other networks, including the one-over-frequency and power-law activities, avalanches, small-world, scale-free, and fractal topography. it is particularly interesting to look for the correlation between the spatial distribution (for example, hubs) and temporal organization (frequency spectrum) of human brain cognitive activities. such research is being conducted by many groups, for example, the study of the dmn during such activities as the comprehension of a text in a natural language versus contemplating it (the "language of thought"). the information content of the neural networks can be studied using the standard characteristics of the information theory, such as the shannon entropy. it may provide ways to distinguish between dna-encoded information and information generated during the embryonal and post-embryonal development, which may be driven by the self-organizing process. for engineers and computer scientists, neural networks serve as a source of inspiration for artificial neural networks, which can serve as a means of machine learning and establishing correlations in data-reach areas, such as surface engineering. cortical networks also served as a source of inspiration for the concept of the immune network, which, in turn, became an inspiration for artificial immune systems algorithms in computer science. both network science and brain physiology are dynamic, rapidly expanding fields. new approaches are likely to emerge. for example, in the study of small droplet clusters, unusual properties, such as the applicability of the dynkin diagrams for colloidal studies have been suggested. a huge amount of information has been obtained in recent studies about both the structure and properties of biological and artificial networks. there are still many questions and problems remaining, such as obtaining connectomes of various species, understanding the relation between the genomic information and organization of cortical networks, and the internal organization of the latter. body size and metabolism a general model for the origin of allometric scaling laws in biology is west, brown and enquist's model of allometric scaling mathematically correct and biologically relevant? beyond the " / -power law": variation in the intra-and interspecific scaling of metabolic rate in animals demystifying the west, brown & enquist model of the allometry of metabolism a general basis for quarter-power scaling in animals allometric scaling law and ergodicity breaking in the vascular system self-similarity, and intermediate asymptotics clustering and self-organization in small-scale natural and artificial systems statistical mechanics of complex networks emergence of scaling in random networks on random graphs the physics of networks do hierarchical mechanisms of superhydrophobicity lead to self-organized criticality? friction-induced vibrations and self-organization: mechanics and non-equilibrium thermodynamics of sliding contact resilience of the internet to random breakdowns collective dynamics of 'small-world' networks network robustness and fragility: percolation on random graphs contact force measurements and stress-induced anisotropy in granular materials what determines the static force chains in stressed granular media? apollonian networks: simultaneously scale-free, small world, euclidean, space filling, and with matching graphs small levitating ordered droplet clusters: stability, symmetry, and voronoi entropy langevin approach to modeling of small levitating ordered droplet clusters droplet clusters: nature-inspired biological reactors and aerosols characterization of self-assembled d patterns with voronoi entropy symmetry of small clusters of levitating water droplets two-dimensional clusters of colloidal spheres: ground states, excited states, and structural rearrangements cluster formation by acoustic forces and active fluctuations in levitated granular matter non-crystalline colloidal clusters in two dimensions: size distributions and shapes logical and information aspects in surface science: friction, capillarity, and superhydrophobicity ternary logic of motion to resolve kinematic frictional paradoxes machine learning methods to predict wetting properties of iron-based composites the structure of the nervous system of the nematode caenorhabditis elegans: the mind of a worm three-dimensional simulation of the caenorhabditis elegans body and muscle cells in liquid and gel environments for behavioural analysis the columnar organization of the neocortex the minicolumn hypothesis in neuroscience the cortical column: a structure without a function ome sweet ome: what can the genome tell us about the connectome? the diameter of the scale-free random graph scale-free neocortical dynamics resolving structural variability in network models and the brain specification of cerebral cortical areas a small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution evolution of the neocortex: a perspective from developmental biology how to direct the edges of the connectomes: dynamics of the consensus connectomes and the development of the connections in the human brain the robustness and the doubly preferential attachment simulation of the consensus connectome dynamics of the human brain a genetic model of the connectome neuro percolation: a random cellular automata approach to spatio-temporal neuro dynamics phase transitions in the neuro percolation model of neural populations with mixed local and non-local interactions mesophasic organization of gabaa receptors in hippocampal inhibitory synapse the small world of the cerebral cortex fine spatiotemporal structure of phase in human intracranial eeg neuronal avalanches in neocortical circuits neuronal avalanche phase transitions in scale-free neural networks: departure from the standard mean-field universality class using expression profiles of caenorhabditis elegans neurons to identify genes that mediate synaptic connectivity dynamics of a neural system with a multiscale architecture statistics and geometry of neuronal connectivity functional connectivity in the motor cortex of resting human brain using echoplanar mri shaped by the past: the default mode network supports cognition that is independent of immediate perceptual input topographic mapping of a hierarchy of temporal receptive windows using a narrated story not lost in translation: neural responses shared across languages dynamic reconfiguration of the default mode network during narrative comprehension metabolic rate and body size are linked with perception of temporal information scaling brain size, keeping timing: evolutionary preservation of brain rhythms mean mass-specific metabolic rates are strikingly similar across life's major domains: evidence for life's metabolic optimum photomechanical responses in drosophila photoreceptors functional trade-offs in white matter axonal scaling the generative grammar of the immune system nobel lecture the fractal immune network fractal immunology and immune patterning: potential tools for immune protection and optimization fractal proteins studying the human immunome: the complexity of comprehensive leukocyte immunophenotyping hydropathic self-organized criticality: a magic wand for protein physics anti-icing superhydrophobic surfaces: controlling entropic molecular interactions to design novel icephobic concrete a neural network model based on the analogy with the immune system an introduction to artificial immune systems: a new computational intelligence paradigm key: cord- -cu cylyi authors: leung, w.k.; lau, a.p.s.; yeung, k.l. title: bactericidal and sporicidal performance of a polymer‐encapsulated chlorine dioxide‐coated surface date: - - journal: j appl microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: cu cylyi aims: to investigate the physical characteristics and the bactericidal and sporicidal potential of a polymer‐encapsulated clo( ) coating. methods and results: an antimicrobial coating based on polymer‐encapsulated clo( ) was developed. a low viscosity, water/oil/water double emulsion coating was formulated for easy on‐site application. escherichia coli, pseudomonas aeruginosa, bacillus subtilis and staphylococcus aureus were applied onto the coating to study the bactericidal capabilities of the coating. the bactericidal performance of the coating increased when the contact time with the tested bacteria increased. over % of the e. coli, ps. aeruginosa, b. subtilis were killed with a contact time of min. although endospores of b. subtilis are more resistant, about % of the spores were killed after h on the coating. moreover, a sustained release of gaseous clo( ) was achieved to maintain about % removal of b. subtilis with a ‐min contact time during a ‐day study period. the coating also exhibits antiadhesive properties against bacteria. conclusions: a polymer‐encapsulated clo( ) coating with sustained release of clo( ) and promising bactericidal and sporicidal features was tested for days. significance and impact of the study: this study provides a new direction for developing polymer‐encapsulated clo( ) coatings that possess persistent bactericidal and sporicidal properties. micro-organisms are ubiquitous in our environment. although many are harmless and even beneficial, some are well-known pathogens while others can elicit allergic responses in humans. casual contact with surfaces and objects contaminated with infectious droplets (i.e. fomites) is an established route for transmission of many diseases (pruss et al. ) . maintaining clean and pathogen-free surfaces is necessary in controlling the spread of infectious diseases. metal surfaces containing silver, copper and brass possess intrinsic germicidal properties that can kill many pathogenic micro-organisms upon contact. they are commonly known as 'contact-killing' surfaces (fang ; block ) . similarly, chromium and nickel in stainless steel and zinc in galvanized iron are known to inhibit microbial growth (fang ) . nanosilvers (fu et al. ; yu et al. ) , photocatalytic tio (sunada et al. ; liu et al. ) and surface-tethered bactericides (e.g. quarternary ammonium compounds, phosphonium salts) (isquith et al. ; cen et al. ; popa et al. ) are among the new generation of contactkilling, antimicrobial surface coatings developed in recent years. nonmetals such as ceramics and enamels whose surfaces are physically nonadhesive and resist biofilm formation are also extensively used in sanitary settings. microbes adhere poorly to ultra-hydrophobic surfaces and bacteria-repelling poly(ethylene glycol) (desai et al. ) . however, the antimicrobial properties of these materials diminish rapidly when the surface is fouled by dirt and contaminants, thus requiring frequent cleaning to maintain their effectiveness. it is possible to store biocides (e.g. phenols, halogens) (chung et al. ) and metals (e.g. silver ions) (klueh et al. ) in bulk materials and coatings for slow and constant release into the environment to provide sustained 'release-killing' until the biocide content is exhausted. for example, a chlorine dioxide (clo ) biocide is generated from the reaction between stored sodium chlorite salt and acids released from a polymer matrix by either hydrolysis or photolysis reactions (callerame ; wellinghoff ) . the latest developments in new antimicrobial coatings are oriented towards multiple mechanistic approaches for surface disinfection. cohen's group (li et al. ) employed layer-by-layer, self-assembly methods to produce antibacterial coatings capable of both release-killing and contact-killing based on stored silver salts and surfacegrafted quarternary ammonium. ho et al. ( ) used a polymer film to immobilize nanosilver to achieve both contact-and release-killing effects, while a grafted layer of polyethylene glycol repelled the adhesion of bacteria. chlorine dioxide is a fast-acting biocide that is effective against a broad spectrum of micro-organisms including, bacteria, fungi, spores, mold and viruses (huang et al. ; young and setlow ; sy et al. ; simonet and gantzer ) . released as a gas, the antimicrobial action of clo is less affected by dirt and surface fouling. the gaseous clo was originally used for large-scale disinfection by bottling companies and as a replacement for the more toxic chlorine in drinking water treatment. the successful stabilization of clo in liquid form has resulted in broad applications, including the use of a clo fumigant to disinfect the air ducts in the us capitol following the anthrax episode in (gordon and rosenblatt ) . clo is safe compared with other oxidizing disinfectants (e.g. chlorine and ozone) and is approved by us fda for disinfection of poultry and beef products, fruits and vegetables. the aim of this work was to develop a polymer microencapsulating liquid clo coating that could provide long-term surface disinfection through the sustained release of gaseous clo from encapsulated liquid clo . this study describes: (i) the physical characteristics of the polymer-encapsulated clo coating, (ii) the bactericidal performance of the coating against gram-positive and gram-negative bacteria, (iii) the sporicidal performance of the coating against the endospores of the b. subtilis and (iv) the potential antiadhesive property of the coating against bacteria. preparation and coating of polymer-encapsulated clo the basic rules for preparing stable water-in-oil-in-water (w ⁄ o ⁄ w) emulsions described by ficheux et al. ( ) were followed. the stabilized clo aqueous solution (united laboratories inc., il, usa) was encapsulated by a water-in-oil-in-water (w ⁄ o ⁄ w) emulsion method (pays et al. ) , in which ml of % (v ⁄ v) clo was suspended in lemon oil (dreamworld, guangzhou, china) and ml of % (w ⁄ v) pluronic p surfactant solution (eo po eo , mw g mol ) , with a critical micelle concentration of ae wt% at °c, basf) was added by gentle stirring. the resulting emulsion was then added to an aqueous suspension of pluronic f (eo po eo , mw g mol ) , critical micelle concentration ae wt% at °c, basf) obtained by dissolving ae g of f in ml of deionized water. this formulation had a storage capacity of % (w ⁄ w) or mg clo per gram of solid. the emulsion was stored at °c before use, and as the antimicrobial coating was not immediately apparent to the eye, lemon oil was added to provide an olfactory cue. glass was chosen as the carrier substrate for the study. rigorous screening was carried out because different components of the manufactured glass (e.g. zno and tio ) might have intrinsic bactericidal properties that could interfere with the measurements. glass manufactured by sail brand, china was selected for its inertness and availability. the glass was cut into ae · ae cm pieces and prepared according to the association of official analytical chemists (aoac) guideline ( a). any defective glass was eliminated and the remaining substrates were rinsed once with distilled water and thrice with ethanol ( ae %; merck) followed by another rinse with excess amounts of distilled water. the glasses were then sterilized in an autoclave (hirayama, ha- p) at °c for min. next, ll of the formulation was coated on the cleaned glass to give a coating of about mg clo cm ) loading. the uniformity of the coating was ensured by the good wettability of the p ⁄ f surfactant polymers used in the formulation. cleaned glass pieces without any coating (uncoated) served as the control. the coated glass samples were examined under a jeol scanning electron microscope (jeol ltd, tokyo, japan) at an acceleration voltage of - kv. the samples were mounted on aluminum stubs using a conducting carbon tape and sputter-coated with a thin layer of gold (c. nm) to prevent sample charging. the scanning electron micrographs provided information about the uniformity of the coating and the morphology of the deposited polymer microcapsules. the composition of the coating surface was analysed by time-of-flight secondary ion mass spectrometry (tof-sims) (physical electronics phi ). a finely focused ion beam ( ions per cm ) was scanned across the sample and the ionized molecular fragments from the surface were collected and analysed by the spectrometer. in assessing the amount of clo released from the encapsulated coatings, a batch of the prepared coatings was exposed in ambient room conditions ( ± °c, % r.h.) for a period of days. five coatings were collected randomly immediately after they were prepared (day ), and at h, day, , , , and days after the coating preparation to quantify the amount of clo that remained in the coatings by a titration method. the sampled coatings were sonicated in ml deionized distilled water to dissolve the coatings. an excess amount of ae mol l ) potassium iodide (ki, bdh) was added and iodometric titration was carried out in an acidic medium. the free iodine (i ), the product of the oxidation of ki by clo , was titrated with ae mol l ) sodium thiosulfate (na s o , rdh) using starch as the indicator. the titration results were summarized as follows: the amount of clo that remained in the coating was calculated with a molar ratio of ½ of the free i . the amount of clo released from the coating on the day it was sampled was then calculated from the difference between the residual amount of clo in the coating and that of the freshly prepared coating (day ). preparation of bacteria cells stock bacillus subtilis (carolina - a), escherichia coli k (carolina - a), pseudomonas aeruginosa (carolina - a) and staphlococcus aureus cells (department of biology, hong kong university of science and technology) were kept on tryptone soya agar (tsa, oxoid) plates (difco) and stored at °c. the bacteria were activated by subculturing a loopful of inoculum in ml of oxoid nutrient broth in a culture tube and gently shaking the tube in an incubator (gallenkamp) at ± ae °c and rev min ) for h. the viable cell concentration was determined by the plate counting technique on tsa plates after serial dilution. the nutrient broth and tsa plates were sterilized in an autoclave at °c for min. the endospores of b. subtilis (carolina - a) were prepared with some modification from the reported procedure (riesenman and nicholson ) . the bacteria were cultivated on tsa plates at ± ae °c for days to obtain a high spore yield. one or two bacterial colonies were harvested from the plates and transferred to a -ml centrifuge tube containing ml sterilized deionized water. the suspension was mixed well with a vortex. the spores were purified by centrifugation and water washing. two millilitres of the suspension was transferred to an eppendorf tube. the suspension was centrifuged at g for min at °c. the supernatant was decanted and ml of cold sterilized deionized water ( °c) was added and the sample was resuspended at °c. an aliquot of the suspension was examined under a phase contrast microscope. the centrifugation and washing steps were repeated until more than % free spores were obtained as indicated by phase contrast microscopy. the purified spores were suspended in a phosphate-buffered saline (ph ae ) solution and stored in the dark at °c for no more than days. the concentration of the viable spores in the suspension was determined by the plate counting technique on tsa plates following serial dilution of an aliquot of the suspension. testing the bactericidal properties of the polymerencapsulated clo coating the tests followed the standard operating procedure of aoac international ( b,c) and were conducted in a sterilized biological safety cabinet (nuaire, nu- - e) under ambient conditions ( ± °c, % r.h.). to investigate the bactericidal performance of the coatings, a suspension of ll of cell ml ) bacteria was spread evenly on the coated and uncoated (control) glass pieces for a specific contact time, which was , , and min, respectively. at the end of each designated contact time, the amount of viable bacteria that remained on the coating or control surface was enumerated. the tested glass pieces were firstly immersed into a culture tube containing ml neutralizer for min to stabilize and wash off the still surviving bacteria from the surface. the sterile neutralizer solution was freshly prepared by adding % (v ⁄ v) ae mol l ) na s o to ml of ae % (w ⁄ v) normal saline (nacl, rdh) solution containing ae % (v ⁄ v) (final concentration) of polyoxyethylenesorbitan monooleate (tween ) followed by autoclaving at °c for min. the glass pieces were then drip-dried and transferred to a second culture tube containing ml of sterile nutrient broth (nutrient broth no. , oxoid) for min to collect the residual bacteria, if any, on the surface after bathing in the neutralizer solution. a ll aliquot of the neutralizer solution and nutrient broth collecting the residual bacteria were spread onto separate tsa plates for viable culturing. the plates were incubated at ± ae °c for h. the viable bacteria were enumerated from the number of colonies formed and the concentration was calculated by normalizing with the volume ( ll) applied. three runs with five coating samples in each run were carried out for each contact time. to investigate the persistency in the bactericidal performance of the coating, a batch of the coatings was prepared and left under ambient room conditions for a period of days. five coatings were randomly selected after and min and on day , , , , and after the coating preparation for the bactericidal test on b. subtilis with a standard contact time of min. testing the sporicidal properties of the polymer-encapsulated clo coating endospores of b. subtilis (carolina - a) were used as a model to study the sporicidal capability of the coating. the testing procedures were similar to those for vegetative cells according to the standard operating procedures of aoac international ( ) to test sporicidal activity. in view of the resistant nature of the spores, longer contact times of the spores on the coatings were allowed from ae to , , , and h. the initial spore concentration was spore ml ) . the viability of the spores at the end of each contact time was enumerated by the plate-spreading method. testing the antiadhesive properties of the polymerencapsulant matrix the adhesion of e. coli k (carolina - a) and b. subtilis (carolina - a) on clean glass and glass coated with placebo encapsulants (polymer encapsulating sterilized distilled water) was examined. the coated glass was prepared by the same procedure described above, but the clo solution was replaced with sterilized, distilled water to eliminate possible bactericidal effects. to intensify the possible adhesion effect, a -fold higher bacterial cell concentration was used. hence, ll of the cell suspension with cell ml ) e. coli and b. subtilis was uniformly spread on the coated and uncoated glass surfaces, and incubated at °c for h without shaking to allow the bacteria cells to settle on the surface. the samples were then washed gently with sterile distilled water to remove any nonadherent bacteria (veyries et al. ) . gram staining was performed on the coated and uncoated glass pieces and images taken by an optical microscope (magnification of ·) were recorded to reveal the qualitative degree of bacterial adhesion on the coated and uncoated surfaces. the water-in-oil-in-water double emulsion approach that encapsulates clo in the triblock copolymers pluronic p and f results in a uniform, transparent and tactilely smooth coating as shown in fig. a . the coating on the glass was allowed to dry under ambient conditions (i.e., ± °c, % r.h.) for h. syneresis or liquid bleeding from the polymer was not observed. a scanning electron micrograph of the coating was taken at x magnification and it is shown in fig. b . it is apparent that the film displays uniform features that are similar to those of deposited microcapsules. the tof-sims mapping (fig. c) detected only organic molecular fragments originating from the polymer on the surface of the coating and none of the nonvolatile salts found in encapsulated clo solution, suggesting that the encapsulation of clo in the polymer matrix was successful. bactericidal and sporicidal potential of the polymerencapsulated coating the bactericidal potential of the polymer-encapsulated clo coating is summarized in fig. . different bacteria have different susceptibilities to the coating, yet, in general, the reduction profiles increase with the duration of the contact time of the tested bacteria on the coating. with a -min contact time, about ae -log, ae -log, ae log and > -log reduction of staph. aureus, ps. aerugenos, b. subtilis, and e. coli were recorded, respectively. when the contact time was increased to min, the reduction further increased to ae -log, -log, ae -log and over -log reduction, correspondingly. over ae % reduction of ps. aerugenosa and e. coli, b. subtilis and % reduction of staph. aureus were achieved when the contact time increased to min. the sporicidal potential of the coating on the endospores of b. subtilis is summarized in fig. . the plain glass surface (control) showed no sporicidal effect; on the contrary, the endospores on the glass started to germinate after h as reflected in the increase in the viable count. however, with the polymer encapsulated clo coating, there is a persistent reduction in the viability of the endospores, from ae -log reduction at h to about ae log reduction ( %) at h. the persistence of antibacterial activities is shown in the sustained release of gaseous clo from the coated glass surface and its sustained bactericidal potential (fig. ) . over the study period of days ( weeks), about % of the stored clo , equivalent to c. lg clo .g ) day ) , were released (fig. a) . this amount is sufficient to reduce microbial growth. a parallel bactericidal test on b. subtilis showed that a sustained ae -log reduction was achieved after a -min contact time over the day study period (fig. b) . this is equivalent to the disinfecting power of a ppm clo solution. by extrapolating the rate of release from the -day study, we estimated that the stored clo in the reported preparation would last for at least months with sustained bactericidal potential. members of the pluronic family have been reported to exhibit antiadhesive properties against micro-organisms such as ps. aeruginosa, staph. aureus and staph. epidermidis and are used as detergents in contact lens cleaning solutions (portole¢s et al. ) . water-containing emulsion microcapsules were prepared using the same procedure, with the clo solution replaced with sterilized, distilled water. this 'placebo' emulsion was coated on glass pieces and aliquots of e. coli and b. subtilis were added. it can be seen from the microscope images - · - · - · - · - · · · · · · · time (h) log reduction figure the reduction profile of the tested endospores of bacillus subtilis with respect to the contact time on the polymer coating with encapsulated clo ( mg cm ) ) (solid circles) compared with those on glass without the coating (open circles). each data point represents the mean of triplicate sets of five samples with the standard error bar. ( fig. a,c) that the amount of e. coli and b. subtilis on the coated glass was significantly less (i.e. < %) compared with that on the uncoated glass (fig. b,d) . this indicates that the polymers used for encapsulation prevented the adhesion of e. coli and b. subtilis on the glass surface. the world health organization has reported that the most common route for transmission of infectious diseases is by indirect contact with surfaces contaminated by infectious droplets produced by an infected person's coughing, sneezing or talking (pruss et al. ). bellamy et al. ( found amylase from saliva on close to % of exposed surfaces in domestic households. human expiratory droplets and hence any inherent pathogens or embedded bacteria can settle on surfaces for days. fomites, inanimate surfaces capable carrying germs, had been associated with contact transmission of viral, bacterial and fungal pathogens. regular surface cleaning and disinfection are therefore important and recommended for breaking the chain of transmission. chemical disinfectants are commonly employed for maintaining the cleanliness of surfaces. the oxidizing biocide, clo , is a common disinfectant. it is safe (mcdonnel and russell ) and effective against a broad spectrum of bacteria, spores and viruses over a wide ph range from to (huang et al. ; young and setlow ; sy et al. ; simonet and gantzer ) . the disinfection ability of clo has also been reported for b. anthrax cells and spores (canter et al. ) , the severe acute respiratory syndrome-associated coronavirus (wang et al. ) and the influenza a virus (h n ) (ogata and shibata ) . clo is a very reactive free radical molecule. owing to its unique oneelectron transfer reaction mechanism, it is also a highly selective oxidant (gordon and rosenblatt ) . it attacks electron-rich centres in organic molecules (gordon and rosenblatt ) and kills micro-organisms through oxidizing their cell membranes (berg et al. ) and denaturing their proteins (ogata ) . it breaks the inner membrane of spores preventing their proper germination (young and setlow ) and reacts with the viral envelope to cause irreparable damage and inactivation (ogata and shibata ) . clo solution can be applied or sprayed directly onto the target surface for action. the disinfection ability, however, diminishes with time in terms of minutes as the clo vaporizes. in enabling a longer lasting disinfection performance, clo gas was generated from sodium chlorite salts stored in a polymer matrix through a reaction with an acid (callerame ; wellinghoff ) , and the acid was either stored with the salt or formed as byproduct of polymer decomposition. this process is inevitably slow as the generation of clo gas depends on the release or production of acid and its subsequent reaction with sodium chlorite. the polymer-encapsulated clo coating system described in this study has promising antibacterial potential. a stabilized clo solution was microencapsulated in the active triblock copolymers pluronic p and f to form a stable water-in-oil-in-water (w ⁄ o ⁄ w) double emulsion (fig. ). this process does not require any acid activation to generate the clo biocide, which eliminates the need for acid storage and subsequent reactions. the pluronic family of triblock copolymers was selected because it is safe and widely used in medicines and cosmetics (thies ; morishita et al. ) . also, their responsiveness to different environmental cues such as temperature, ph and moisture has been amply demonstrated for controlled drug delivery (tarcha ; sosnik and cohn ) . the emulsion was readily sprayable and with the pluronic polymers acting as a surfactant. the emulsion droplets deposited uniformly on surfaces as shown by the sem picture in fig. b . the chemical composition of the surface indicated that the clo was completely encapsulated in the polymer matrix instead of residing on the surface layer (fig. c) . the experimental results in figs and indicated that clo remained encapsulated after surface coating. a slow but sustained release of clo at about lg clo g ) day ) from a coating area of mg cm ) loaded with mg clo per gram of polymer was reported in this study. assuming that there is a constant rate of clo release from the polymer, the coating prepared in this study can optimally last for months. the p polymer does not favour a fast transport route via inverted micelles of encapsulates in the oil phase (matsumoto and kang ; sela et al. ) . studies showed that molecules can migrate through the oil phase without affecting the double emulsion stability (bibette et al. ) . the clo encapsulated in this study is likely transported from the interior of the polymer to the peripheral surfaces via diffusion, resulting in a sustained release of clo over the -day study period. the coating also demonstrates consistent bactericidal potential. the clo released from the coating killed over % of the e. coli, ps. aerugenosa, b. subtilis, and staph. aureus and about % of b. subtilis endospores with a contact time of min. the amount of b. subtilis killed was comparable with that killed after exposure to ppm clo solution. the relative susceptibility of b. subtilis spores and the range of bacteria killed followed with the trend reported by russell ( ) . gram-negative e. coli was most susceptible and endospores of b. subtilis were the least susceptible (figs and ) . staphylococcus aureus was the least susceptible to clo among the tested vegetative species. staphylococcus aureus also exhibited similar low susceptibility to a hypochlorite (i.e. bleach) solution (data not shown). this may be related to the presence of carotenoid pigments in staph. aureus that are antioxidative and provide the bacteria with some degree of protection from oxidizing biocides (clauditz et al. ) . despite the different susceptibilities of the bacteria to polymer-encapsulated clo , the bactericidal and sporicidal performance generally increased as the contact time was prolonged. this trend favours the use of the encapsulated coating as an antimicrobial surface. under ambient conditions, droplets or fomites from whatever source will be deposited onto surfaces and remain on the site until they are transferred to other surfaces or objects after contact. this means that there is ample time for the clo to act on the species resting on the coating. we speculate that the bactericidal or sporicidal activity is caused by multiple actions of the coating. primarily, both dissolved and gaseous clo is released through diffusion from the clo encapsulated in the polymer matrix. in addition, the coating is rehydrated by droplets or fomites upon deposition. this rehydration destabilizes the outermost membrane film of the coating, resulting in its rupture and rapid flooding of the contact area with the encapsulated clo . the release is sustained by the difference in the osmotic pressures between the exterior and interior water phases of the polymer formulation. the rehydration thus initiates the sporadic release of clo in addition to the usual release-killing mechanism employed by antimicrobial systems using clo (callerame ; wellinghoff ) . this sporadic release potential gives the coating the advantage of self-disinfection of areas contaminated by droplets or fomites. formation of biofilms is detrimental to the coating surface. phenotypic tolerance to oxidizing biocide can arise from biofilm formation via consumption of the biocide by the organic constituents of the biofilm (chen and stewart ) , or by a 'population-based' resistance strategy (cochran et al. ) . no biofilm was observed in this reported polymeric matrix coating. the active biocide, clo , used in the formulation prevents indiscriminate reactions and rapid consumption of clo by the biofilm because of rapid diffusion (hosni et al. ) and greater reaction selectivity (gordon and rosenblatt ) . the absence of a biofilm may also be due to the antiadhesive properties of the p and p polymers that prevent the adhesion both of the gram-positive and gram-negative bacteria (e.g. b. subtilis and e. coli) on its surface (fig. ) . the chance of seeding a biofilm on the coating is also reduced. the antiadhesive property of the polymers is probably related to the anchorage of the hydrophobic core of the triblock polymers on the material surface, while its hydrophilic chains form sterically stabilized barriers against adhesion in the periphery (bridgett et al. ) . the antiadhesive property of the coating surface reduces the chance of the coating degeneration as a result of biofilm formation and allows the coating surface to be a self-maintaining bactericidal system. in conclusion, a prototype of an antimicrobial coating was prepared with polymer-encapsulated clo through the double w ⁄ o ⁄ w emulsion system for general surface application. the coating is easily sprayable because of its low viscosity. a sustained release of gaseous clo at a maintenance level was demonstrated for a prolonged period (i.e. days). a surface concentration of about ppm clo was obtained to prevent the bacteria or endospores from attaching on the coating. contamination by droplets or fomites triggers an increased release of biocide in the affected area, resulting in rapid disinfection. the polymer-encapsulated clo coating also exhibited promising bactericidal potential against a spectrum of bacterial species and sporicidal potential against bacillus endospores over a contact time of min. emulsions: basic principles disinfection, sterilization and preservation, th edn control of staphylococcal adhesion to polystyrene surfaces by polymer surface modification with surfactants process for the production of chlorine dioxide. us patent , us patent and trademark office remediation of bacillus anthracis contamination in the u.s. department of justice mail facility surface functionalization technique for conferring antibacterial properties to polymeric and cellulosic surfaces chlorine penetration into artificial biofilm is limited by a reaction-diffusion interaction evaluation of a polymer coating containing triclosan as the antimicrobial layer for a packaging film staphyloxanthin plays a role in the fitness of staphyloccus aureus and its ability to cope with oxidative stress reduced susceptibility of thin pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine surfaceimmobilized polyethylene oxide for bacterial repellance inhibition of bioactivity of uasb biogranules by electroplating metals some stability criteria for double emulsion construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosan-silver ions complex chlorine dioxide: the current state of the art nanoseparated polymeric networks with multiple antimicrobial properties diffusion of chlorine dioxide through aqueous and oil films disinfection effect of chlorine dioxide on bacteria in water surface-bonded antimicrobial activity of an organosilicon quaternary ammonium chloride efficacy of silver-coated fabric to prevent bacterial colonization and subsequent device-based biofilm formation two-level antibacterial coating with both releasekilling and contact-killing capabilities non-uv based germicidal activity of metal-doped tio coating on solid surface formation and applications of multiple emulsions antiseptics and disinfectants: activity, action and resistance pluronic f- gels incorporating highly purified unsaturated fatty acids for buccal delivery of insulin denaturation of protein by chlorine dioxide: oxidative modification of tryptophan and tyrosine residues protective effect of lowconcentration chlorine dioxide gas against influenza a virus infection double emulsions: how does release occur? study of quaternary 'onium' salts grafted on polymers: antibacterial activity of quaternary phosphonium salts grafted on 'gel-type' styrene-divinylbenzene copolymers poloxamer as a bacterial abhesive for hydrogel contact lenses safe management of wastes from health-care activities role of the spore coatlayers in bacillus subtilis spore resistance to hydrogen peroxide, artificial uv-c, uv-b, and solar radiation similarities and differences in the responses of microorganisms to biocides release of markers from the inner water phase of w ⁄ o ⁄ w emulsions stabilized by silicone-based polymeric surfactants degradation of the poliovirus genome by chlorine dioxide ethoxysilane-capped peo-ppo-peo triblocks: a new family of reverse thermo-responsive polymers studies on photokilling of bacteria on tio thin film evaluation of gaseous chlorine dioxide as a sanitizer for killing salmonella, escherichia coli o : h , listeria monocytogenes, and yeasts and molds on fresh and fresh-cut produce polymers for controlled drug delivery microcapsules for cosmetic applications control of staphylococcal adhesion to polymethylmethacrylate and enhancement of susceptibility to antibiotics by poloxamer study on the resistance of severe acute respiratory syndrome-associated coronavirus methods of making sustained release biocidal composition. us patent , us patent and trademark office mechanisms of killing of bacillus subtilis spores by hypochlorite and chlorine dioxide preparation and antibacterial effects of pva-pvp hydrogels containing silver nanoparticles the authors are grateful to the institute for the environment (ienv) and the materials characterization and preparation facility (mcpf) for use of their equipment. we also thank basf and orkney environmental technology for supplying the pluronic polymers and stabilized clo solution free of charge. key: cord- -ac txr h authors: grichko, varvara p.; shenderova, olga a. title: nanodiamond designing the bio-platform date: - - journal: ultrananocrystalline diamond doi: . /b - - . - sha: doc_id: cord_uid: ac txr h publisher summary this chapter discusses various methods of surface modification for the development of functionalized diamond nanoparticles for biomedical applications. to be used in biomedical applications, nanoparticles must be biocompatible, non-toxic, non-detective by immune systems, and should not induce side effects. size control of particles is a prerequisite for biomedical applications. carbon nanostructures span the same length scale as bio-compounds, ranging from subnanometer-size nucleotides to tens and hundreds of nanometer-sized organelles and viruses, and up to micron-sized cell sizes. the chapter also summarizes different approaches to the surface functionalization of nanodiamonds (nd) particles—that is, the key in successful biomedical applications followed by a discussion of modification of diamond surfaces with nucleic acids and proteins. both current and potential applications of diamond films and particles in the area of biosensing are addressed. all major forms of carbon at the nanoscale -fullerenes, nanotubes, and nanodiamond (nd), the last in the forms of both particulate and filmsappear to be valuable materials for biomedical applications (freitas, ; . importantly, carbon nanostructures span the same length scale as bio-compounds ( fig. . ) , ranging from subnanometer-size nucleotides, to tens and hundreds of nanometer-sized organelles and viruses, and up to micron-sized cell sizes. in the mid s it was discovered that fullerene compounds have biological activity, and their potential as therapeutic agents for the treatment of several diseases was demonstrated. as a result, a private biopharmaceutical company, c sixty inc., has been established with a primary focus on the discovery and development of novel fullerene-based therapeutics. at . Å in diameter, c is similar in size to steroid hormones and peptide alpha-helices, and, thus, fullerene compounds are ideal molecules to serve as ligands for enzymes and receptors (wilson, ) . within the last few years, a number of useful fullerene-based therapeutic applications have been developed, including as antiviral agents and anticancer drugs (www.csixty.com) and biosensors for diagnostic applications (anonymous, ) ; a protective agent against iron-induced oxidative stress (lin et al., ) ; and an in vitro antibacterial agent (da ros et al., ) . the exploration of buckytubes in biomedical applications is also underway. multiwall carbon nanotubes have been used for immobilization of proteins, enzymes, and oligonucleotides (lin et al., ) . significant progress has been made within the last few years in an effort to overcome some of the fundamental and technical barriers toward bioapplications of carbon nanotubes, especially on issues concerning solubility in water, biocompatibility, modifications of carbon nanotubes with various biological and biologically active compounds, and both design and fabrication of biosensor prototypes (lin et al., ) . nanodiamond particles and their derivatives, diamondoids and their derivatives, and ultrananocrystalline diamond (uncd) films all have high potential in biotechnological and biomedical applications. uncd films have been suggested to be the ideal platforms for future biochips and biosensors because of their superior mechanical, thermal, and chemical properties as compared to those of glass, silicon, and gold surfaces (yang et al., ) . uncd particles had been probed in the separation of proteins (bondar and puzr, ) , fabrication of integrated biochips and sensors (puzyr et al., a) , and even in anticancer applications (dolmatov, ) . in general, multifunctional hierarchical structures consisting of nanoparticle derivatives of organic and inorganic molecular entities began to play increasingly important roles in a variety of applications in nanobiotechnology (niemeyer, ) , genomics (wengel, ) , drug discovery (ozkan, ) , and nanomedicine (freitas, ) . for example, zero-dimensional nanostructures reported for medical applications include gold and magnetic nanoparticles, semiconductor quantum dots, a wide variety of polymer-based nanoparticles, nanoshells consisting of metals and dielectrics, and many others. nanoparticles act as potential carries for several classes of drugs such as anticancer agents, antihypertensive agents, immunomodulators, and hormones; and macromolecules such as nucleic acids, proteins, peptides, and antibodies. an absence of narrow fractions of nanodiamond particulate on the market hindered their biomedical applications. recently, however, production of narrow fractions of nd ( nm sized particle suspensions; several fractions within the - nm size range as well as fractions above nm) has been achieved in research laboratories (chapter of this book and private communications). in combination with the advances in the production of high-purity particles and the fact that uncd particles of detonation origin are relatively inexpensive (compared to fullerenes, pure carbon nanotube (cnt), and gold particles, for example), a fast growth of bioapplications of nd particulate is expected. this chapter will be organized as follows: in the next section different approaches to the surface functionalization of nd particles, that is, the key in successful biomedical applications, will be summarized, followed by a discussion of modification of diamond surfaces with nucleic acids and proteins. after that both current and potential applications of diamond films and particles in the areas of biosensing and medicine will be addressed. the concluding section will summarize results reported on the biocompatibility of nd, the paramount property in the biomedical applications of artificial nanostructures. diamond possesses a number of distinct properties that make it an attractive biotechnological material. although natural diamond is highly hydrophobic, hydrophilic surface groups, important for bioapplications, can be generated on a diamond surface by heating to high temperature in an oxygen atmosphere and using ion bombardment and other rather aggressive treatments. nd particles synthesized by detonation have numerous oxygen-containing chemical groups on the surface and, thus, are intrinsically hydrophilic. the detonation nd particles are - nm round-shaped monocrystals that form tightly and loosely bond aggregates of about nm and - nm in diameter, correspondingly. detonation nd possesses a very large specific surface area ( - m g − ) (dolmatov, ) , which is an important factor for bioapplications. detonation nd is likely to have a significant number of unpaired electrons that make it an efficient free radical scavenger and opens up the opportunity for its use in medicine (kulakova et al., ; nachalnaya et al., ) . in general, the size, purity, and surface chemistry of detonation nd varies considerably from one manufacturer to another. the composition of the explosive mixture, coolant media, chamber size, and consequent purification of detonation soot are all important factors in the manufacture of uncd particles with the desirable physical and chemical properties of their surface (donnet et al., ) . the methods of extraction and purification of nd from the detonation product may include ozone treatment, oxidation by different reagents with/without catalysts, treatment with acids, modification of the nd surface in gaseous and liquid media (kulakova, ) , selective inhibition of nd oxidation (chiganov, ) , and many others. disaggregating of detonation nd can be achieved, for example, by its graphitization in a n atmosphere at °c followed by oxidation in air at °c in order to remove the surface graphite layer. in this way, the average size of nd aggregates can be reduced to nm or less with a very high yield (xu and xue, ) . dispersion efficiency and stability of colloidal solutions of nd in water is important for nd applications in nanobiotechnology and medicine, and is determined by the electrostatic, hydration, and hydrophobic (the tendency of lypophilic nd to form aggregates in an aqueous solution) interactions among the particles (xu et al., a) . within the last few years a number of new methods have been introduced to control nd solubility in water by biological and chemical modifications of the nd surface. to obtain stable suspensions of well-dispersed nd particles in water the mechanochemical procedure has been developed, which employs a set of different mechanical treatments (high-power sonification or vibration milling techniques) along with the application of surfactant agents (xu et al., b; c) . the mechanochemical modification with anionic surface modifiers increased the zeta potential and lowered the amount of hydroxyl groups and the size of individual particles . the mechanochemical processes were also successfully applied for the preparation of stable highly dispersed nd suspensions in non-polar solvents : an oil suspension of nd particles with an average size of nm was prepared and stored for months without any visible signs of sedimentation. there has been steady progress in the development of chemical methods of nd surface modification as well. a summary of methods of altering the nd surface chemistry with the final purpose of biofunctionalization is illustrated in fig. . . various organic molecules can be attached to polycrystalline diamond films when under irradiation with uv light. prior to exposure to uv light the hydrogen-terminated diamond surface must be coated with a thin film of the solution containing organic molecules in order to be immobilized. by attaching molecules with specific protecting groups and removing protecting groups after the attachment, it is possible to obtain diamond surfaces functionalized with carboxyl or primary amine groups that may facilitate further steps in chemical modification of diamond surfaces such as dna and protein immobilization (strother et al., ) . to increase nd solubility in water and prepare the nd surface for biomolecule attachment, oxidation of the nd surface is frequently required (fig. . ) . under mild conditions the diamond surface can be directly functionalized with carboxylic acids by initiating radical reactions (tsubota et al., ) . however, the number of carboxylic acid residues introduced on the diamond surface may be low in some cases. indeed, oxidation of nd does not proceed easily under the mild conditions. in an aqueous slurry, nd of nm size may react with ozone, though a very long time is needed in order to functionalize the surface of nd with the ketonic groups (cataldo and koscheev, ) . in air nd may suddenly explode : designing the bio-platform, grichko and shenderova above a temperature of °c. the functionalization of nds at elevated temperature affects both their size and surface chemistry. the weight of nd particles was reported to decline by . % as a result of heating at °c in an inert atmosphere (cataldo and koscheev, ) . spherical diamond powder with a particle size ranging from to nm can be oxidized in an oxygen atmosphere at - °c (lee et al., ) . the oxidized diamond powders were further successfully functionalized by reacting with -(trifluoromethyl)benzylamine containing the reactive amino group . under certain conditions the high rate of diamond oxidation may result in an explosive process. ida et al. ( ) have investigated the reactivity of hydrogenated diamond surfaces with peroxide radical initiators such as benzoyl peroxide, lauroyl peroxide, dicumyl peroxide, and di-t-butyl peroxide. with benzoyl peroxide, they detected ir peaks that were then assigned to the aromatic c-h and c=o stretching vibrations. the c=o stretching vibration was observed after the diamond was exposed to lauroyl peroxide. the areas under the peak of spectra determined with fourier transform infrared spectroscopy (ftir) increased with reaction time and amount of reagent. dicumyl peroxide and di-t-butyl peroxide did not react with diamond surfaces. while the yield of reaction of diamond surface with radical species generated from benzoyl peroxide depends on the organic solvent used, the same functional groups were synthesized in toluene, tetrahydrofuran, n,ndimethylformamide, cyclohexane, and hexane (tsubota et al., a) . the hydroxyl groups located on the surface of diamond powders, which was treated either with sulfuric acid alone or with a mixture of sulfuric and nitric acids, can be reacted with metoxy groups of silanecoupling reagents ( -aminopropyltrimethoxysilane, -mercaptopropyltrimethoxysilane, or n-octyltrimethoxysilane) with the formation of stable modified diamonds (tsubota et al., b) . the silane-modified nds can be used for surface synthesis of dna and protein immobilization. the substantial increase in the number of surface c-h groups was achieved by the gas treatment of nd powder with h , plasma-ionized hydrogen, n , methane and air, and in vacuum at different temperatures (jiang et al., ) . surface decomposition, decarbonylation, and decarboxylation were likely to be the main reactions. the h-terminated nd surfaces can be further reacted photochemically (λ = nm) with longchain ω-unsaturated amines to produce a homogeneous layer of amine groups for consequent dna attachment (yang et al., ) . nd purification and functionalization can also be carried out using gas and vapor reactive media ( fig. . ). the chlorinated diamond was pre-pared by sotowa et al. ( ) by irradiating hydrogenated diamond with uv light in the presence of elemental chlorine. the formation of chlorinated diamond was confirmed by diffuse reflectance ftir, which revealed a strong peak corresponding to the c-cl stretching. the researchers then treated the chlorinated diamond surface further with ammonia and found that diamond amination is a temperature-dependent process and results in the formation of nh + ; c=n and nh ; and imines at room temperature, °c, and °c, respectively. high-temperature treatment of detonation nd with hydrogen, ccl , or nh has been studied recently by spitsyn et al. ( ) . nd, which was annealed in hydrogen flow at °c for h, possessed about cal g − lower combustion heat than non-modified nd powder. moreover, the initial dangling bonds density of ∼ . × spin/cm was reduced . times after the treatment with hydrogen. after treatment in a ccl /ar mixture at °c for . - h the hydrophilicity of nd was changed significantly: the atmospheric water vapor readsorbance was at least times lower than in the nd samples treated in pure ar. after treatment in a nh flow at °c for min the number of oxygen-containing groups had decreased and atmospheric water vapor readsorbance was four times lower than in the initial nd samples. a number of characterization methods such as chemical analysis, raman, ftir, electron spin resonance (esr), and chromatomass spectrometry confirmed the possibility of extensive modification and controlled functionalization of the nd using gas treatment (in terms of hydrophilic/ hydrophobic or acidic/basic nd termination) (spitsyn et al., ) . this prevents agglomeration of hydrophilic and hydrophobic detonation nd in polar and non-polar solvents, correspondingly. fluorination is another efficient method for nd chemical modification that enables a variety of applications in engineering and biological sciences ( fig. . ). treatment of detonation uncd powder ( - µm sized particles composed of . - . nm diamond nanocrystals, > % purity) with a f /h mixture at - °c resulted in the formation of fluorinated nds with . at.% fluorine . the fluorinated nd material was then used as a precursor for preparation of alkyl-, amino-, and amino acid-functionalized nds that showed an increased solubility in polar solvents and reduced particle agglomeration . application of fluorinated nd has been found in cost-effective synthesis of diamond coatings covalently bonded to glass surfaces (liu et al., ) . before that the production of diamond thin films could only be achieved by chemical vapor deposition that requires heating to °c. liu et al. ( ) applied a silane coupling agent, -aminopropyltriethoxysilane, to attach fluoro-nd to the glass slide surface, which was preliminary func-tionalized with terminal amino groups. using atomic force microscopy (afm), sem, and x-ray photoelectron spectroscopy (xps) analysis it was established that surface-bonded fluoro-nd particles were closely packed and had an average size of - nm. the fluoro-nd, which is covalently attached to the surface of the glass slide, can be further modified by chemical substitution of residual fluorine. nanoparticles are valuable platforms in controlled drug delivery and can be administered via most routes to carry various therapeutics, anticancer, antiviral, antibacterial, and antihypertensive agents, immunomodulators, hormones, antibodies, proteins, peptides, and nucleic acids to isolated cells, tissues, and organs (bala et al., ; ozkan, ) . successful design of highly efficient drug delivery systems may solve many problems faced by present-day medical sciences. in gene therapy, genes are delivered to the cell nucleus allowing cells to produce therapeutic proteins. gene delivery is achieved using either viral or non-viral vectors. viral vectors are genetically engineered adenoviruses, retroviruses, and other viruses that are very efficient when used for gene transfer in vivo. methods of non-viral gene delivery, including nanoparticle carriers, represent a small fraction of all methods used for transfection in vivo. however, they have gradually become as popular as the viral vector-based technology. in the development of non-viral technologies, the main goal is to have a transfection method that is efficient, reproducible, non-toxic, and allows the prolonged gene expression to occur. efficiency of targeted delivery of nanoparticles loaded with biologically active molecules is affected by many factors including particle size, surface charge, and chemistry, and mechanism of target recognition. over the years, a number of natural and synthetic materials have been used to prepare nanoparticles, and their stability, biocompatibility, and biodegradability were investigated (bala et al., ) . in some cases nanocarriers protect naked dna from nucleases while allowing dna plasmids to pass the cell membrane and get into the nucleus (kneuer et al., ; roy et al., ) . to optically monitor intracellular trafficking and gene transfection events roy et al. ( ) first prepared fluorescently labeled organically modified silica nanoparticles for use in non-viral gene delivery and biophotonics applications and then showed that these nanoparticles can serve as a delivery platform with superior efficacy in targeted drug therapy and as the realtime monitoring of drug action. the highly monodispersed stable water suspensions of the organically modified silica nanoparticles, which were labeled with the fluorescent dyes and functionalized by amino groups, were prepared using micelle chemistry. the nanoparticles efficiently bound dna due to positively charged amino groups and protected it from digestion by dnase i. imaging by fluorescence confocal microscopy confirmed that in vitro cells efficiently took up the nanoparticles in the cytoplasm, and the nanoparticles delivered dna to the nucleus. very recently bejjani et al. ( ) reported the breakthrough discovery that polylactic nanoparticles enable in vivo gene transfer and expression with a high efficiency. solid nanoparticles can be used to deliver drugs and biologically active molecules to any body organ including the brain, because they can cross the blood-brain barrier (lockman et al., ; koziara et al., ) . the nanoparticles that are biodegradable in a controlled way are likely to become the carriers of choice for in vivo non-viral gene delivery. surfacemodified and appropriately labeled nd particles may also serve as an efficient platform for in vivo transfection. however, because of the very low, if any, biodegradability of nd, nd nanoparticles, when applied in vivo, must be of a size small enough to allow their excretion by the kidney or applied cutaneously. immobilization of dna on diamond surfaces via covalent bonding has been explored intensively. for example, ushizawa et al. ( ) first modified diamond powder with particle sizes of - µm (fig. . ) by oxidation in a heated mixture of sulfuric acid and nitric acid and then converted it in chlorocarbonyl-diamond by reacting with thionyl chloride at °c for day. chlorocarbonyl-diamond was then reacted with thymidine in the presence of -dimethylaminopyridine. the dna was attached to the ′-end of diamond-attached thymidine by ′-end phosphatization. the formation of ester bonds was confirmed by diffuse reflectance ftir spectroscopic analysis. preparation of dna-modified diamond films for use in hybridization has received increased attention. the chemical stability of diamond surfaces is substantially greater than that of gold or silicon surfaces (lu et al., ) , and the dna molecules attached to the diamond surfaces are easily accessible to enzymes. nanocrystalline diamond thin films covalently modified with dna oligonucleotides following the photochemical modification of h-terminated surfaces with amine groups provide a very stable and highly selective platform for the surface hybridization reaction (yang et al., ) . after linking dna to the amine groups, hybridization reactions with fluorescently tagged complementary and non-complementary oligonucleotides did not reveal any non-specific adsorption, with extremely good selectivity between matched and mismatched sequences (yang et al., ) . in a similar manner, hydrogen-terminated diamond substrates were photochemically converted into amine-terminated surfaces following by linking to thiol-terminated dna oligonucleotides reported by knickerbocker et al. ( ) . the dna hybridization on dna-modified polycrystalline diamond is highly specific and when compared to the hybridization on dna-modified surfaces of crystalline silicon shows that the diamond surface exhibits superior chemical stability (knickerbocker et al., ) . dna-modified diamond surfaces are particularly suitable for invasive cleavage reactions, in which introduction of target dna to solution results in the specific cleavage of surface-bound probe oligonucleotides, permitting snp (single nucleotide polymorphisms) detection. the sensitivity of the analysis can be improved times by replacing the dna-modified gold surface with a more stable dna-modified diamond surface (lu et al., ) . cvd diamond has also found some applications for dna immobilization. using thymidine as a linker molecule, the fragment of the human pku gene was covalently bound to the cvd diamond film by wenmackers et al. ( ) . boron-doped diamond (bdd) thin films with enhanced conductivity offer a substantial advantage for use in dna hybridization analysis. for example, gu et al. ( ) electropolymerized a thin layer of polyaniline/poly(acrylic acid) onto the diamond surface. the carboxylic acid residues in the polymer film enhanced the electron transfer between dna and a bdd surface and acted as the binding sites for dna attachment. both fluorescence microscopy and cyclic voltammograms indicated that the polymer-modified bdd did not show significant non-specific dna adsorption, while providing a stable transduction platform for dna detection by hybridization. the absence of efficient technology for in vivo delivery of oligonucleoticles limits many therapeutic applications. for successful application in vivo, in most cases in vitro delivery platforms should be extensively modified in order to provide targeted drug delivery. the adamantanebased materials have found application in the preparation of carriers for in vivo nucleic acid delivery because they can be modified by using cyclodextrin/adamantane host/guest interactions to provide the particles suitable for systemic application (pun and davis, ) . transferrinmodified nanoparticles containing dnazymes (dna enzymes that are rna-cleaving phosphodiester-linked dna-based enzymes, which cleave their target mrna in a gene-specific fashion) for targeting tumors were prepared by using conjugates of adamantane with poly(ethylene glycol) and administered to tumor-bearing nude mice by intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. dnazymes packaged in polyplex formulations were concentrated and retained in tumor tissue, whereas unformulated dnazyme was eliminated from the body within hours after administration . in wound healing therapy, the localized delivery of growth factors is achieved by gene transfer to the wound site. synthetic biocompatible materials prepared with a linear, beta-cyclodextrin-containing polymer and an adamantane-based crosslinking polymer are very suitable for in vivo gene delivery to fibroblasts via the inclusion of adenoviral vectors in the synthetic construct . gene-deleted adenoviral vectors were originally developed as delivery vehicles for use in gene therapy trials and are currently being developed as hiv vaccines and in other medical applications. from a different perspective, in biological materials science and in rapidly emerging angstrom-scale chemical engineering the nucleic acids are thought to find many applications in the fabrication of self-assembling, multi-dimensional materials (wengel, ) . nd and diamondoid structures can be valuable candidates for crosslinking of oligonucleatides in carbon-based systems. both selective adsorption of proteins and their immobilization onto surfaces of nd particles (fig. . ) may be advantageous in nanobiotechnological applications and medicine. fibrinogen is widely accepted as an indicator in a biocompatibility test and material-caused inflammation. the adsorption of human fibrinogen on the surface of chemical-vapor-deposited diamond has been studied by tang et al. ( ) and was the first report on the diamond interaction with choi, .) a protein and its biocompatibility. the cvd diamond was found to be biologically compatible to the same extent as titanium and stainless steel. in kossovsky et al. demonstrated that surface-modified nd particles with a size ranging from to nm provided both conformational stabilization and a high degree of surface exposure to protein antigens and used them to generate antibodies. recently huang and chang ( ) developed the universal procedure for protein immobilization onto the surface of nm nd particles. it starts with nd particles with strong acids followed by modifying their surface with poly-l-lysine. covalent attachment of proteins is then carried out by activating the amine-terminated nd to react with the heterobifunctional linker ssmcc followed by mixing with the protein. the researchers successfully immobilized both alexa fluor dye and yeast cytochrome c using the free sh group for linkage. more information on the application of protein-modified nd in biosensing and biotechnology can be found in section . . hollow nanoparticles are thought to be the most suitable carriers of proteins and peptides susceptible to degradation. many nanoparticles can be made hollow by heating at high temperature, treating with strong acids, alkali, and organic solvents, or using gentle chemical treatment to encapsulate susceptible organic molecules such as proteins, peptides, and enzymes or others inside the cavity of hollow nanoparticles (sharma et al., ) . nd particles produced by detonation of the mixture of trinitrotoluene and cyclomethylenetrinitramine have a tetragonal structure and according to vereshchagin and yurjev ( ) are hollow particles with an inner diameter of . Å and outer diameter of . Å. yurjev et al. ( ) used synchrotron x-ray diffraction to characterize these spherical hollow detonation nds that can be modified even further by grinding in a planetary mill. the surface-modified nds and diamondoids are expected to find vast application in the development of a new generation of protein delivery platforms and antigen carriers because they can be readily modified to both carry and stabilize biologically active molecules, proteins, and enzymes. in addition, these particles are rigid, biocompatible, available in different shapes, and span the subcellular size range (fig. . ) . a biosensor (fig. . ) generally comprises biomolecules sensing an analyte, e.g., dna, antibody, receptor, or enzyme, and the electrochemical, optical, calorimetric, or piezoelectric transducer that detects an attach-ment of an analyte to the biorecognition layer (deisingh and thompson, ) . the physiochemical properties that determine the analytical characteristics of biosensing devices are surface characteristics of the sensing area and its size, as well as intrinsic properties of the material. in the past few years, significant advances have been made toward the development of both biosensors and biochips for single-cell analysis, detecting of pathogens and toxins (vo-dinh et al., ) , and other biological and medical applications. diamond films occupy a special place as an electrode material in biosensors. when made sufficiently electrically conducting by boron doping, thin-film and free-standing diamond electrodes exhibit remarkable chemical resistance to etching, a wide potential window, low background current responses, mechanical stability toward ultrasound-induced interfacial cavitation, a low stickiness in adsorption processes, and a high degree of tunability of the surface properties (reviewed by compton et al., ) . tatsuma et al. ( ) have examined direct electron transfer from bdd electrodes to heme peptide and horseradish peroxidase for the application to h o biosensors. diamond electrodes exhibit low sensitivity to interfering agents and may become a capable h o biosensor. nanocrystalline diamond films can be both a platform for biofunctionalization and serve as an electrode in biosensors based on electrochemical reactions. different proteins can be covalently attached to the hydrogen-terminated nanocrystalline diamond films modified with amino groups and remain fully functional. hartl et al. ( ) functionalized nanocrystalline diamond electrodes with catalase and detected a direct electron transfer between the redox centre of the enzyme and the diamond electrode. also, the electrode was found to be sensitive to hydrogen peroxide. hydrogenated diamond can gain surface conductivity after exposure to air and by transfer doping with c resulting in a significant rise in two-dimensional conductivity (strobel et al., ) . the fully hydrogen terminated diamond surfaces are not ph sensitive but can gain this sensitivity after a mild surface oxidation by ozone (garrido et al., ) . the difference in dna adsorption on the h-and o-terminated diamond surfaces may be useful in the nanofabrication of biosensors . compared to multiwalled carbon nanotube-based electrodes, bdd electrodes exhibit less selective voltammetric responses to the different biomolecules and slower electron-transfer kinetics. bdd electrodes have no intrinsic selective response to l-ascorbic acid, and surface modification by anodic polarization is required to resolve l-ascorbic acid and dopamine (poh et al., ) . highly conductive bdd electrodes are especially suited for electrochemical detection of nucleic acids in aqueous solutions. their distinctive features are high reproducibility, small background currents at high positive potentials, and robustness under extreme conditions (prado et al., ) . importantly, bdd electrodes can be used in analysis involving a heating step and ultrasonic treatment. well-defined peaks, observed with trna, single-and double-stranded dna, and ′deoxyguanosine ′-monophosphate were directly assignable to the electrooxidation of deoxyguanosine monophosphate (prado et al., ) . polycrystalline diamond films deposited by microwave plasma cvd were incorporated in the design of four different glucose sensors by troupe et al. ( ) . while a diamond-platinum-glucose oxidase sensor was affected by the presence of electroactive chemicals in blood, usage of bdd as a conducting electrode in place of the platinum provided a strong and repeatable response to glucose. the sensor that was fabricated with the surface-modified diamond allowed attachment of biologically active molecules and electron transfer from glucose oxidase to the electrode. this approach received further development in a study by . however, a , ′-diaminobenzidine-electropolymerized carbon nanotube-based electrode outperformed the diamond one in terms of selectivity and sensitivity. in another attempt to develop a nd-based biosensor immobilized polyclonal antibodies against salmonella typhimurium and staphylococcus aureus on nanocrystalline diamond films and evaluated the efficacy of immobilization by enzymelinked immunosorbent assay (elisa). the immobilization of antibodies and attachment of bacteria measured by sem were more efficient on a surface of air plasma-treated diamond than on a surface of diamond treated preliminarily with the hydrogen plasma. the plasma-oxidized surface of nd was more hydrophilic and was terminated with hydroxyl and carbonyl groups. modified nd particles and films have been used in heterogeneous and electrochemical oxidation catalyses and related applications, and in electrochemical analysis (bogatyreva et al., ; song et al., ; yang et al., ) . using the new amperometric biosensors designed with monocrystalline diamond and l-and d-amino acid oxidases, stefan et al. ( ) conducted differential pulse voltammetric assay of l-pipecolic and d-pipecolic acids in serum samples with a low limit of detection. the impedance of the diamond film can be affected by dna hybridization at the interface that induces a field effect in the diamond space-charge layer. by identifying a range of impedances, where the impedance is dominated by the diamond space-charge layer, and measuring the interfacial impedance, it is possible to directly monitor dna hybridization. no dna labeling is required. frequency-dependent interfacial electrical properties of nanocrystalline diamond films that were covalently linked to dna oligonucleotides were changed significantly in the presence of complementary dna oligonucleotides, with only minimal changes due to the presence of non-complementary dna oligonucleotides . the electrolyte-solution-gate field-effect transistors with hterminated polycrystalline diamond surface were shown to be sensitive to cl − and br − and can find application in cystic fibrosis tests . the capability for biomolecular recognition was provided to the highly sensitive field-effect transistor (bio-fet) made of a nanocrystalline diamond thin film by linking human immunoglobulin g to the diamond surface. electrical measurements showed that the bio-fet responded specifically to the anti-igg antibody . puzyr et al. ( a) have recently developed a prototype nd-based biochip for use in bioluminescent analysis. the biochip incorporates aluminum oxide film-adhesive layer-deposited nd particulate-luciferase, which retained substantial enzymatic activity. it is also worth mentioning here that diamond sensor applications include high-sensitivity detection of charged particles such as protons and pions that are possible due to diamond's high radiation tolerance. a cvd diamond strip detector that can be used for tracking and detection of charged particles has been recently introduced on the market. fast charged particles create charge carriers in the irradiated diamond followed by induction of an electric charge on the strips. similarly, detonation nd and monocrystal cvd diamonds may be used for detecting both intermediate and high-energy heavy ions and in other instrumental analysis (adam et al., ; berdermann et al., ) . because both carboxylated and oxidized nd exhibit a remarkably high affinity for proteins, proteins in dilute solutions can be easily captured by nds with a size of nm, separated by centrifugation, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms) without any need for pre-separation of the adsorbed proteins from nd (kong et al., ) . with the dilute mixed solution of cytochrome c, myoglobin, and albumin that preferentially adsorbs on a hydrophilic surface, the developed method offered significantly higher sensitivity than conventional maldi-tof-ms and had a limit of detection of pm for a ml sample solution. the potential of nd-assisted maldi-tof-ms for use in clinical proteomics was demonstrated with human blood serum analysis (kong et al., ) . if necessary, surfaces of nd particles can be modified to decrease non-specific protein adsorption and optimize their use for bioanalysis (lasseter et al., ) . application of detonation nds facilitated separation of obelia longissima apoobelin from the recombinant e. coli cell extract and made possible the preparation of purified protein with a yield of up to % . within the nd family, to a large extent medical applications have been developed for diamondoids. diamondoids such as adamantane derivatives (single-molecule unit of diamond with the formula c h ) have been used in pharmacology, clinical medicine, and biosensing (freitas, ) . until recently the cholinesterase inhibitors were the only available drugs for the treatment of alzheimer's disease, which is one of the leading causes of death for people over years of age. unfortunately, the cholinesterase inhibitors cannot stop the process of neurodegeneration and just symptomatically enhance the cognitive state to some degree (sonkusare et al., ) . in alzheimer's disease, neuronal death is caused by glutamate excitotoxicity mediated through the n-methyl-d-aspartate (nmda) receptors making them an excellent target for preventing the disease. excitotoxicity is excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. activity of the nmda receptor is also essential for normal neuronal function, and neuroprotective agents that fully block nmda receptor activity will have severe side effects (lipton, ) . memantine ( -amino adamantane derivative) is the potent nmda-receptor antagonist that blocks excessive nmda receptor activity without disrupting normal activity through its action as an uncompetitive, low-affinity, open-channel blocker. it also has beneficial effects in parkinson's disease, stroke, epilepsy, central nervous system (cns) trauma, amyotrophic lateral sclerosis, drug dependence, chronic pain, depression, glaucoma, and severe neuropathic pain. memantine is available in europe and has been recently approved for treatment of dementia in the usa. at the present time the second-generation memantine derivatives, which take advantage of the additional modulatory sites in the nmda receptor that could also be used for clinical intervention, are under development (lipton, ; sonkusare et al., ) . many adamantane derivatives possess antibacterial, antiviral, and antifungal activity (wang et al., ; el-sherbeny, ; orzeszko et al., ; el-emam et al., ) . for example, -(iadamantyl)- -substituted thio- , , -oxadiazoles and -( -adamantyl)- substituted aminomethyl- , , -oxadiazoline- -thiones exhibit substantial antimicrobial activity against gram-positive bacteria and the antiviral activity against hiv- significantly reducing viral replication at - µg ml − concentrations (el-emam et al., ) . adamantane-based drugs, namely, amantadine and tromantadine, are excreted unaltered in the urine and are not susceptible to hydroxylation (koppel and tenczer, ) . in , hodek et al. discovered that adamantane, diamantane, triamantane, -isopropenyl- -methyladamantane, and -isopropenyl- methyldiamantane inhibited cytochromes p of subfamily iib by binding to the active site and, thus, could be potent inhibitors for hepatic oxidative drug metabolism in humans. theoretical and experimental values of the dissociation constant of cytochrome p complexes with the diamondoids were in good agreement and confirmed the high potency of identified inhibitors. bananins, the antiviral agents that possess a trioxaadamantane moiety attached to a pyridoxal derivative, were shown to be potent inhibitors of the sars (severe acute respiratory syndrome) coronavirus helicase and can prevent replication of animal scv (sars-related coronavirus) (tanner et al., ) . recently, the adamantane derivatives have been identified as the potential drugs acting on the p x( ) receptor, which is involved in signaling in many inflammatory processes (cytokine release, no generation, cytotoxicity, killing of intracellular pathogens) (baraldi et al., ; romagnoli et al., ) . because nd is not mutagenic or toxic (oral ld value for rats is g kg − ), can neutralize free radicals, and possesses a very large surface area, it was suggested that detonation nd particles may have some antitumor activity (dolmatov and kostrova, ; see also chapter ). indeed, mice with erlich ascetic carcinoma that were given supplements with the nd suspensions were more active and lived almost % longer than non-treated animals. in , dolmatov reported the results of a new medical study of the possible oral administration of water suspensions of nds to terminally ill cancer patients. the oral administrations of nd did not result in any side effects and, moreover, were moderately beneficial in some cases. diamond has the outstanding reputation of a chemically inert and uniquely biologically compatible material that has found a number of applications in medicine (freitas, ) . diamond is biocompatible in both bulk and particulate forms. in orthopedic surgery the use of a diamond coating on the metallic components reduces generation of macrophages and improves the wearability of devices (santavirta et al., ) . in addition, nanocrystalline diamond films show an excellent resistance to bacterial colonization (jakubowski et al., ) . the diamond-like carbon coating is also very inert and particularly suitable for use in orthopedic implants. in an early, pioneering study thomson et al. ( ) grew mouse peritoneal macrophages and fibroblasts on tissue culture plates coated with . µm of an amorphous diamond-like carbon layer and assessed the biocompatibility both biochemically and morphologically. the diamond-like carbon coating caused no adverse effects on cells in the culture. recently diamond-like carbon films have been reexamined and it was reported that they have good biocompatibility and high corrosion resistance (kim et al., ) . additional evidence of diamond biocompatibility came from zheng et al. ( ) who fabricated nanocrystalline diamond films (ndfs) on optical glass using microwave plasma assisted cvd and used osteoblast cell cultures and platelet adhesion tests for in vitro evaluation of biocompatibility of ndfs. their results indicated that the diamond films exhibit good tissue compatibility and hemocompatibility, which makes them very suitable for biomedical applications. the excellent chemical inertness and smoothness of ndfs made them a promising material for medical implants, cardiovascular surgery, and coating of artificial heart valves (mitura et al., ; ) . in specht et al. were able to demonstrate the ordered growth of mammalian neurons on diamond. to accomplish this the researchers patterned proteins on diamond surfaces by micro-contact printing and cultured mouse cortical neurons on these substrates. the diamond biocompatibility and the suitability of neuron interfacing with the surface make this an interesting approach for implant engineering. the high biocompatibility, corrosion resistance, chemical inertness, low friction coefficient, electrical insulation, and excellent mechanical characteristics of cvd diamond suggested that diamond-coated materials may find numerous applications in medicine (tang et al., ) . in biomaterials research, it is known that the biocompatibility of a bulk material is not necessarily the same as the biocompatibility of fine parti-cles of the same material, which may penetrate inside live cells and their organelles (freitas, ) . the main threat to cell viability comes from possible mechanical damage to cellular organelles and membranes. foreign intracellular particles with a diameter of - nm do not damage cells mechanically (lu and rosenzweig, ) . detonation particles possess a rounded shape, superior lubricity characteristics, hardness, and wear resistance. the fine diamond particles and diamond-like carbon coatings were always found to be very inert and non-inflammatory (tse and phelps, ; hedenborg and klockars, ; swan et al., ; grill, ) . when higson and jones ( ) treated both pig and horse neutrophils with diamond crystals, no induction of peroxide and superoxide generation was observed. the interaction of leucocytes with diamonds with a size of - µm in . % diamond suspension did not cause any cell damage. no increase in degranulation and production of cell motility factors, and cell death, was observed (swan et al., ) . compared particles of diamond, sic, and hydroxyapatite in serum-free cultures of human monocytes and found that all particles were phagocytozed, and monocyte morphology changed except after the ingestion of diamond. it was concluded that diamond particles were inert in a serum-free human monocyte culture, while both sic and hydroxyapatite had a stimulatory effect comparable to that of polymethylmethacrylate. when suspensions of phagocytosable particles of diamond and sic in hyaluronan were introduced into a canal traversing the bone implant in rabbits neither the diamond nor the sic particles caused any decrease in bone formation. it confirmed that particles of diamond and sic are harmless . human blood cells' lack of adherence to the cvd diamond substrates, and blood clotting on diamond, produced a less rough surface than blood clotting on glass (baranauskas et al., ) . recent reports on possible nd-induced damage to both white and red human blood cells in vitro (puzyr et al., ; b; raised the question about the mechanism promoting these phenomena. prior to use, nds are usually modified to gain high stability in water colloids , and it is not clear whether bacterial contamination, unidentified impurities, defects (shames et al., ) , and surfactants were not the factors involved. indeed, in those studies, the total content of nondiamond carbon, non-combustible residue, and volatile compounds reached % in some samples (puzyr et al., ) . in contrast to these observations, dion et al. ( ) did not notice any in vitro hemolysis when % blood solutions were treated with . g cm − diamond powder produced by de beers industrial diamond division, and the early reports on crystalline diamond-induced damage to cells were never confirmed (freitas, ) . rodil et al. ( ) reported that the diamond-like coating did not have any toxic effect on human osteoblasts cells in vitro. interestingly, monteiro-riviere et al. ( ) found that carbon nanotubes, which were grown using a microwave plasma enhanced cvd system, accumulated within cytoplasmic vacuoles of the human epidermal keratinocytes and stimulated release of the proinflammatory cytokine interleukin . there are no reports on site-specific intracellular accumulation of nd particles by live cells. as was mentioned in the previous section, administration of detonation nd particles to both erlich ascetic carcinoma mice and terminally ill cancer patients did not result in any side effects. moreover, it was beneficial in some cases (dolmatov and kostrova, ; dolmatov, ) . however, . - . % nd suspensions administered orally to white mice over the course of months did cause a substantial increase in leukocyte content in the blood of animals (puzyr et al., c) . the treatment did not affect the weight of the experimental mice though. very unexpectedly, the intravenous administration of . ml of sterile colloids of % modified nds in % glucose to rats and dogs did not result in sickness or premature death of the animals (puzyr et al., b) . when ml of % nd was administered to a dog followed by the second administration week later, the ecg tests revealed substantial changes in heart activity immediately after each treatment. however, the cardiac condition became normal again within day. the safety and effectiveness of nanosystems and platforms made of nds will fully depend on their compatibility with human organs, tissues, cells, and cellular organelles. extensive thorough research has yet to be done to clearly establish physiological, immunological, and cytological responses of the human body to nd particles and films. in this review, methods of surface modification were discussed for the development of functionalized diamond nanoparticles for biomedical applications. to be used in biomedical applications, nanoparticles must be biocompatible, non-toxic, non-detective by immune systems, and should not induce side effects. size control of particles is a prerequisite for biomedical applications. to meet all these criteria, the diameter of particles should be less than nm and their surfaces should be modified by hydrophilic moieties. such nanoparticles are likely to avoid uptake by the reticuloendothelial system and remain in blood at a high enough concen-tration to reach the target organs, tissues, or cells. the surface of nanoparticles has to be preliminarily modified by functional ligands with a high affinity to a disease site to achieve site-specific delivery. in many cases, nanoparticles may encapsulate and protect therapeutic agents against enzymes and hydrolysis; it is not clear yet how nanodiamond particles can satisfy this requirement. it is obvious from the reviewed literature that both nanodiamond particulates and films have become very popular objects in biomedical research. performance of irradiated cvd diamond micro-strip sensors nanotechnology in biology: the good of small things. the economist benign response to particles of diamond and sic: bone chamber studies of new joint replacement coating materials in rabbits plga nanoparticles in drug delivery: the state of the art agonists and antagonists acting at p x( ) receptor analysis of the coagulation of human blood cells on diamond surfaces by atomic force microscopy behar-cohen f ( ) nanoparticles for gene delivery to retinal pigment epithelial cells synthetic biocompatible cyclodextrin-based constructs for local gene delivery to improve cutaneous wound healing charged particle detectors made of singlecrystal diamond application of modified nanodiamonds as catalysts of heterogeneous and electrochemical catalyses nanodiamonds for biological investigations applications of nanodiamonds for separation and purification of proteins a study on the action of ozone and on the thermal stability of nanodiamond selective inhibition of the oxidation of nanodiamonds for their cleaning progress in enzyme-based biosensors using optical transducers electroanalysis at diamondlike and doped-diamond electrodes easy access to watersoluble fullerene derivatives via , -dipolar cycloadditions of azomethine ylides to c biosensors for the detection of bacteria detonation synthesis ultradispersed diamonds: properties and applications detonation-synthesized nanodiamonds and the possibility to develop a new generation of medicines detonation and shock synthesis of nanodiamonds b synthesis, antimicrobial, and anti-hiv- activity of certain -(i-adamantyl)- -substituted thio- , , -oxadiazoles and -( -adamantyl)- -substituted aminomethyl- , , -oxadiazoline- -thiones synthesis, antitumor activity, and anti-hiv- testing of certain heterocyclic systems containing an adamantane nucleus nanomedicine, volume i: basic capabilities ph sensors based on hydrogenated diamond surfaces diamond-like carbon coatings as biocompatible materials-an overview conductive polymer-modified boron-doped diamond for dna hybridization analysis protein-modified nanocrystalline diamond thin films for biosensor applications quartz-dust-induced production of reactive oxygen metabolites by human-granulocytes oxygen radical production by horse and pig neutrophils induced by a range of crystals structural requirements for inhibitors of cytochromes p b: assessment of the enzyme interaction with diamondoids adsorption and immobilization of cytochrome c on nanodiamonds immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications chemical reaction of hydrogenated diamond surface with peroxide radical initiators nanocrystalline diamond surface is resistant to bacterial colonization ftir studies on the spectral changes of the surface functional groups of ultradispersed diamond powder synthesized by explosive detonation after treatment in hydrogen, nitrogen, methane and air at different temperatures electrochemical behavior of diamond-like carbon films for biomedical applications a nonviral dna delivery system based on surface modified silicananoparticles can efficiently transfect cells in vitro dna-modified diamond surfaces highaffinity capture of proteins by diamond nanoparticles for mass spectrometric analysis a revision of the metabolic disposition of amantadine surface-modified diamond nanoparticles as antigen delivery vehicles situ blood-brain barrier transport of nanoparticles surface chemistry of nanodiamonds chemical properties of detonation-synthesized ultradispersed diamond covalently modified silicon and diamond surfaces: resistance to nonspecific protein adsorption and optimization for biosensing oxidation of cvd diamond powders reactions of amines with cvd diamond nanopowders carboxyfullerene prevents iron-induced oxidative stress in rat brain advances toward bioapplications of carbon nanotubes paradigm shift in nmda receptor antagonist drug development: molecular mechanism of uncompetitive inhibition by memantine in the treatment of alzheimer's disease and other neurologic disorders functionalization of nanoscale diamond powder: fluoro-, alkyl-, amino-, and amino acidnanodiamond derivatives fluorinated nanodiamond as a wet chemistry precursor for diamond coatings covalently bonded to glass surface nanoparticle technology for drug delivery across the blood-brain barrier diamond and carbon nanotube glucose sensors based on electropolymerization nanoscale fluorescent sensors for intracellular analysis invasive cleavage reactions on dna-modified diamond surfaces influence of carbon coatings origin on the properties important for biomedical application nanocrystalline diamond coatings multi-walled carbon nanotube interactions with human epidermal keratinocytes special features of structure and physico-mechanical properties of natural diamonds of ukraine nanoparticles, proteins, and nucleic acids: biotechnology meets materials science human monocytes stimulation by particles of hydroxyapatite, silicon carbide and diamond: in vitro studies of new prosthesis coatings synthesis and antimicrobial activity of new adamantane derivatives iii quantum dots and other nanoparticles: what can they offer to drug discovery? biosensing properties of diamond and carbon nanotubes electrochemical analysis of nucleic acids at boron-doped diamond electrodes development of a nonviral gene delivery vehicle for systemic application targeted delivery of rna-cleaving dna enzyme (dnazyme) to tumor tissue by transferrinmodified, cyclodextrin-based particles damaging effect of detonation diamonds on human white and red blood cells in vitro design of a luminescent biochip with nanodiamonds and bacterial luciferase destruction of human blood cells in interaction with detonation nanodiamonds in experiments in vitro dynamics of the selected physiological responses in laboratory mice under the prolonged oral administration of nanodiamond suspentions destruction of human blood cells upon interaction with detonation nanodiamonds in experiments in vitro a possibility of using of intravenous administration of sterile colloids of modified nanodiamonds in vitro cytotoxicity of amorphous carbon films recent progress in the discovery of antagonists acting at p x( ) receptor optical tracking of organically modified silica nanoparticles as dna carriers: a nonviral, nanomedicine approach for gene delivery studies of host response to orthopedic implants and biomaterials defects and impurities in nanodiamonds: epr, nmr and tem study enzymes in the cavity of hollow silica nanoparticles carbon nanostructures cl-sensitive biosensor used electrolyte-solution-gate diamond fets surface-modified diamond field-effect transistors for enzyme-immobilized biosensors dementia of alzheimer's disease and other neurodegenerative disorders-memantine, a new hope effect of treatment temperature on the amination of chlorinated diamond ordered growth of neurons on diamond purification and modification of nanodiamond new amperometric biosensors based on diamond paste for the assay of l-and d-pipecolic acids in serum samples surface transfer doping of diamond photochemical functionalization of diamond films a comparison of the effects of urate, hydroxyapatite and diamond crystals on polymorphonuclear cells-relationship of mediator release to the surface-area and adsorptive capacity of different particles diamond nanofabrication and characterization for biosensing application biocompatibility of chemical-vapor-deposited diamond the adamantane-derived bananins are potent inhibitors of the helicase activities and replication of sars coronavirus electron transfer from diamond electrodes to heme peptide and peroxidase biocompatibility of diamondlike carbon coating diamond-based glucose sensors polymorphonuclear leukocyte motility in-vitro. . release of chemotactic activity following phagocytosis of calcium pyrophosphate crystals, diamond dust, and urate crystals abstraction of hydrogen atoms on diamond surface using benzoyl peroxide as a radical initiator surface reforming of the oxidized diamond surface with silane coupling reagents chemical modification of diamond surface with various carboxylic acids by radical reaction in liquid phase covalent immobilization of dna on diamond and its verification by diffuse reflectance infrared spectroscopy structure of detonation diamond nanoparticles nanosensors and biochips: frontiers in biomolecular diagnostics in vitro and in vivo growth inhibition of cancer cells by adamantylmaleimide derivatives nucleic acid nanotechnology -towards angstrom-scale engineering covalent immobilization of dna on cvd diamond films biological aspects of fullerenes a new method for deaggregation of nanodiamond from explosive detonation: graphitization-oxidation method dispersion and stability of nanodiamond in clean oil effect of sodium oleate adsorption on the colloidal stability and zeta potential of detonation synthesized diamond particles in aqueous solutions mechanochemical dispersion of nanodiamond aggregates in aqueous media influence of surface modification adopting thermal treatments on dispersion of detonation nanodiamond fabrication and characterization of a biologically sensitive field-effect transistor using a nanocrystalline diamond thin film dna-modified nanocrystalline diamond thin-films as stable, biologically active substrates interfacial electrical properties of dna-modified diamond thin films: intrinsic response and hybridization-induced field effects electrically addressable biomolecular functionalization of conductive nanocrystalline diamond thin films structural study of detonation nanodiamonds mpacvd nanocrystalline diamond for biomedical applications. high performance ceram chemical mechanical modification of nanodiamond in an aqueous system key: cord- - kwny authors: mariani, stefano; minunni, maria title: surface plasmon resonance applications in clinical analysis date: - - journal: anal bioanal chem doi: . /s - - - sha: doc_id: cord_uid: kwny in the last years, surface plasmon resonance (spr) and its advancement with imaging (spri) emerged as a suitable and reliable platform in clinical analysis for label-free, sensitive, and real-time monitoring of biomolecular interactions. thus, we report in this review the state of the art of clinical target detection with spr-based biosensors in complex matrices (e.g., serum, saliva, blood, and urine) as well as in standard solution when innovative approaches or advanced instrumentations were employed for improved detection. the principles of spr-based biosensors are summarized first, focusing on the physical properties of the transducer, on the assays design, on the immobilization chemistry, and on new trends for implementing system analytical performances (e.g., coupling with nanoparticles (nps). then we critically review the detection of analytes of interest in molecular diagnostics, such as hormones (relevant also for anti-doping control) and biomarkers of interest in inflammatory, cancer, and heart failure diseases. antibody detection is reported in relation to immune disorder diagnostics. subsequently, nucleic acid targets are considered for revealing genetic diseases (e.g., point mutation and single nucleotides polymorphism, snps) as well as new emerging clinical markers (microrna) and for pathogen detection. finally, examples of pathogen detection by immunosensing were also analyzed. a parallel comparison with the reference methods was duly made, indicating the progress brought about by spr technologies in clinical routine analysis. surface plasmon resonance (spr) appeared as revolutionary technology almost years ago, when the first commercial instrumentation was launched on the market by pharmacia biosensors ab, a swedish company, derived from pharmacia ab. the company developed an innovative technology by the joint effort of physicists, chemists, biologist, engineers, and computer scientists. since then, many scientists joined the spr to test new applications in various analytical fields, such as food safety [ ] (e.g., mycotoxins [ ] , genetically modified organism (gmo) [ ] ), microbial contamination such as escherichia coli [ ] , doping analysis [ ] , laboratory medicine [ , ] , proteomics [ , ] , bacteria detection [ ] , and also environmental monitoring [ ] . among them, assuredly, clinical analysis has also been explored as a fruitful application field. the advantages brought about by current spr technology include real-time monitoring of the analyte/molecular markers, label free and parallel analysis (with spri), minimal sample pretreatment, quantitative response, and very good sensitivity and reproducibility, (reported detection limits are in atto-or femtomolar ranges and coefficient of variations below %). these features, coupled to miniaturization, make spr suitable for point of care (poc) diagnostics [ ] , where fast analysis and multi-analyte detection are mandatory. in this review, we focused on the analysis of target of interest in molecular diagnostics in complex and real matrices (e.g., serum, saliva, blood, and urine) but also in standard solution when the detection strategy is innovative and involves the improvement of analytical performances. so the panel of revised analytes includes hormones (steroids and peptides), protein clinical markers, antibodies involved in immune disorders, nucleic acids for genetic disease and as clinical markers (i.e., mirna), bacterial cells, and viruses for pathogens detection. so far, most of the research articles come from academic exercises but we are more and more confident that the application of spr to the clinical and medical analysis will gain momentum in the next future. the physical principles and the state of the art of surface plasmon resonance [ ] [ ] [ ] [ ] [ ] and surface plasmon resonance imaging-based [ ] [ ] [ ] [ ] biosensors were reviewed in many excellent works. at the beginning of the th century ( ), wood was the first scientist who described the inhomogeneous distribution of light in a diffraction grating spectrum caused by surface plasmon wave (spw) [ ] . sixty-six years later ( ), kretschmann [ ] and otto [ ] rigorously demonstrated the optical excitation of surface plasmons (sps) with the method of attenuated total reflection (atr). spr is defined as a charge-density oscillation at the interface between two media, with dielectric constants of opposite signs (e.g., metal and a dielectric), which generate a surface plasmon wave (spw) with a propagation constant β, expressed by the following equation [ ] : where ω is the angular frequency, c is speed of light in vacuum, and ε d ε m are the dielectric constants of dielectric and metal, respectively. at the metal-dielectric interface the electromagnetic field of the spw has as a maximum intensity that exponentially decreases (evanescent wave, ew) into both media with a variable penetration from to nm (for vis and nir wavelengths) [ ] . prism couplers, grating couplers, or metal-dielectric waveguides are the configuration used for the excitation of surface plasmons but the first one represents the most common approach for the plasmon excitation via the attenuated total reflection method (atr) with kretschmann geometry becoming also the most suitable for sensing and biosensing applications. in the kretschmann geometry, the light wave is totally reflected at the boundary between a thin metal layer (typically gold or silver with nm of thickness) and a high refractive prism coupler (typically in glass). the reflected light excites the surface plasmons of the metal film generating an ew (or spw) penetrating the metal layer (fig. ) . spr based biosensor belongs to refractometric devices, since the propagation of the spw is sensitive to changes in the refractive index of the dielectric. the binding between analytes and bioreceptors immobilized on the sensing surface causes a local change in refractive index (fig. ) and the variation of the propagation constant β, generating a real time signal in label-free way, since no labels have to be added in solution for the development of the analytical signal. classic spr spectroscopy is based on the monitoring of coupling angle, coupling wavelength, intensity, phase [ ] , whereas spr imaging (spri) is based on the measurement of reflectivity of monochromatic incident light at a fixed angle by means of a ccd camera. compared with traditional spr, spri offers main breakthroughs such as multiplexed detection (by designing -d patterned microarrays of molecular probes) and the real-time visualization of the whole biochip surface to monitor simultaneously multiple bioreceptor/analyte bindings with the parallel support of a digital image, representing the intensity of binding in a color scale [ ] . on the other side, spri suffers from order of magnitude worse resolution than classical spr ( − versus − riu) [ ] . biosensor selectivity depends on the bioreceptors immobilized on the sensing surface. here we will present and discuss works that appeared in the last years aiming at developing robust and reliable spr assays in clinical diagnostics. among bioreceptors, we can enumerate antibodies, nucleic acid probes or synthetic receptors (molecular imprinted polymers (mips), aptamers, or artificial dna (xna). accordingly, it is customary to talk about immunosensors, nucleic acid sensor, aptasensor, and so on. an immunosensor uses the affinity interaction between an antigen and an antibody [ , ] . nucleic acid (na) sensors are based on oligonucleotidic probes and na hybridization reaction [ , ] . in na sensors, artificial nucleosides (xna) oligomers have also been employed as bioreceptor for clinical applications. xnas increase sensor stability by avoiding biodegradation or exploiting improved affinity of xna versus the target sequence compared with conventional dna probes. d'agata et al. reviewed works dealing with the great potential of artificial dna (pna, lna, hna, and morf) in nucleic acid spr-based sensing, exhibiting higher selectivity and sensitivity in detecting complementary or mismatching nucleic acid targets [ ] . behind that, aptasensors emerged in the last years as an interesting alternative to immunosensing. aptamers are na molecules that can bind to predetermined targets (small molecules as well as proteins and peptides) with high affinity and specificity [ , ] . these nucleic acid aptamers are engineered through repeated rounds of in vitro selection or equivalently with systematic evolution of ligands by exponential enrichment (selex) [ , ] . finally, mip sensors take advantage of totally synthetic molecular recognition elements, constituted by highly crosslinked polymers engineered to bind one target compound or fig. in a spr biosensor based on a prism coupler and working in kretschmann geometry with the atr method, light is totally reflected by a thin gold layer ( nm) that excites the surface plasmons (sps) and generates a spw (a). the affinity binding between bioreceptors and analytes causes a change of refractive index vs. time (b) entailing a shift of the reflectivity curve vs wavelength of source light or angle of reflected light (c) a class of structurally related target compounds with high selectivity [ , ] . interesting applications to clinical diagnostics have recently been reported for synthetic receptor such as xna, but still very few are using aptamers and mips instead. bioreceptors have first to be immobilized on the sensing surface and for this purpose, different approaches were reported. furthermore, the suitable assay format has to be applied depending on the clinical application. both aspects will be discussed in the next paragraphs. in spr-based biosensor development, the immobilization of the biomolecule on the sensing surface is a key step essential for the success of the analysis. in particular the features to be considered for the optimal bioreceptors immobilization procedure deal with the retentions of biological activity for the analyte after immobilization, the compatibility between binding activity and range of analyte detection, amount of immobilized bioreceptor (to avoid steric hindrance), reproducibility of immobilization (to assure reproducibility of results), and finally the regenerability of the biosensing surface (receptor-analyte dissociation without compromising the biological activity of the receptor). for the sensor applicability to real matrices, the immobilization chemistry should be optimized to also prevent unspecific interaction on the surface arising from matrix components (i.e., proteins in blood, serum, etc.). the binding of biomolecules to gold-sensing surface can be achieved through many suitable immobilization approaches, such as physical absorption, hydrophobic (via lipid layer), electrostatic interactions, covalent coupling (coupling of nucleophiles to carboxylic groups, to thiol groups or to aldehyde groups), and coupling of native and tagged molecules (avidin/ biotin) [ ] . among them, the last two approaches are surely the most exploited in clinical diagnosis application for cheapness, ease of realization, stability, and robustness. in particular, nucleotides are efficiently immobilized after modification with the thiol group, exploiting the high affinity between sulphur and gold surface or, alternatively, with biotin labeling via affinity interaction with avidin or streptavidin coated chip surface. in both strategies, the formation of a self-assembled monolayer (sam) increases the degrees of freedom of the bioreceptor and, consequently, the bindings with analytes. proteins (e.g., antibodies) and other peptides are widely immobilized by covalent amine coupling between amino groups of proteins and activated (with nhs/edc solutions) carboxylic linkers of alkanethiols (or disulfide) in sam format or carboxymethylated dextran ( d matrix immobilization). these approaches are rapid, simple, and inexpensive but bring about random orientation of bioreceptors (attributable to multiple functional groups on the protein), which could decrease the conformational flexibility, hindering the bioreceptors/analytes interaction and thus lowering the sensor sensitivity. an alternative strategy for a controlled orientation is, instead, based on the affinity reaction between biotin-conjugated protein and (strept)avidin-coated chip. in addition, the interaction between fc and protein g or a could be also exploited for the abs immobilization. preventing nonspecific adsorption of biomolecules (e.g., protein) on the spr sensing surface is another key-step for the development of specific biosensor, with real application to clinical diagnostics where complex matrices (such as serum, blood, and urine) are analyzed. a strategy frequently adopted is the fuctionalization of surface with non-fouling materials resistant to nonspecific interaction such as glycol-derivate oligo/polymers: oligo(ethylene glycol) (oeg), poly(ethylene glycol)(peg), eg -cooh, eg -oh but also poly(l-lysine) grafted with poly(ethylene glycol) (pll-g-peg) [ ] . the success of protection from nonspecific interaction is due to steric-entropic barrier properties coupled with a high degree of hydration of peg molecules as reported by blättler et al. [ ] . two assay formats, direct and indirect, were mostly adopted on spr biosensor for the detection of analyte in clinical diagnosis (schemes in fig. ). the assay selection is based on the mw analyte, on the bioreceptors/analyte affinity, and on the matrices under investigation. in the direct assay, bioreceptors are immobilized on the sensing surface and analytes are added in solution. the affinity interaction causes a refractive index variation, providing analytical signals proportional to analyte concentration. this strategy is usually adopted for analyte with mw greater than da, sufficient to develop a refractive index variation and resulting in detectable signal. moreover with a multi-step direct assay (fig. ) dls can be further lowered using a secondary bioreceptor (specific for analyte) in a sandwich format. labeled secondary bioreceptors (with biomolecules or nanoparticles) are often employed to increase the assay sensitivity. indirect assays are preferred for low mw analytes ( x da). here, purified analytes (or conjugates) are immobilized on the spr surface, while specific bioreceptors are incubated with the samples to be analyzed and injected to sensing surface; when the solution reaches the surface, only remaining free bioreceptors bind to immobilized analyte, providing a signal inversely proportional to its concentration in the sample. besides, secondary bioreceptors (labeled or not) can be added in solution to further improve sensitivity and lowering dls with multi-step assays. recently, many developments in spr technology were performed for the improvement of sensor analytical performances in terms of sensitivity, specificity and detection in real matrixes [ ] . a strategy was offered by nanoparticles coupled to plasmonic sensors in miniaturized and low cost systems [ ] . in , gao et al. examined recent works in engineering hybrid spr interfaces such as oxide-based hybrid spr interfaces and nanocomposite films, analyzing the optical properties and the suitable applications in sensing [ ] . more recently ( ), bedford et al. have specifically reviewed the recent trend of incorporation of gnps within the sensing surface itself or as tagging molecules in spr biosensor. significant sensitivity increases and dls decreases were described for a variety of analytes [ ] . as reported by minunni and spoto, the increase of sensitivity is explainable by both the mass enhancement of the nps in the ew as well as by the optical coupling between surface plasmons (sps) and electric fields of localized surface plasmons (lsps) of nps when they are close to the metallic surface [ ] . lsps are defined as collective electron oscillations confined in the mnp surface and promoted by interaction with the light. if the mnp dimension is much smaller than the wavelength of the excitation light, the local electromagnetic fields are strongly enhanced. a further contribution to the increase of sensitivity is also expected if nps clustered since it was proven that clusters of spherical metal and dielectric colloids show strong electric, magnetic, and fano-like resonances from the electromagnetic coupling between closely spaced particles [ ] . zanoli et al. reviewed the employment of functionalized gold nanoparticles for ultrasensitive dna detection on different transduction systems (including spr), useful to bypass pcr amplification steps [ ] . since we have previously defined the spr-based biosensor as a label-free system, for the sake of clarity we specify that the addition of bioreceptors labeled with nanoparticles is not employed for signal development itself but for the improvement of system analytical performance (sensitivity increases and dls decreases), really essential in clinical diagnostics where analytes are in complex matrices at very low concentrations. some drawbacks of nps uses could be found in the nps synthetic step, in tuning dimensions, and absorption wavelengths (for coupling between plasmons), since these steps are time-and reagent-consuming. furthermore, it seems constrictive to employ nps in reusable sensors since surface poisoning could occur, compromising serial measurements on the same chip: no data of regeneration are available in literature. very recently, graphene-based surface plasmon resonance interfaces were also studied for suitable application in spr sensor. the functionalized surfaces showed several advantages such as high surface-to-volume ratio, adsorption of organic or biological molecules (pi stacking), passivation against oxidation, and controlling the number of graphene layers [ ] . other strategies, different from nps-based approach, have been also adopted in this last decade for the improvement of spr based sensor. in , abbas et al. collected the last forefront tendencies in spr instrumentation with advanced performances identified in: new excitation schemes and optical configurations, liquid handling by microfluidics, and microsystems for integrated spr chips generally based on glass or poly(dimethylsiloxane) (pdms). new material such conducting metal oxides zno and zno/au in the adhesion layer to replace cr or ti were also discussed as well as forefront spr-related optoelectronic components (light source and detector) and finally the spr coupling with other analytical techniques (hyphenation approach with scanning probe microscopy, electrochemistry, fluorescence and raman spectroscopy) were also evaluated for the achievement of higher sensitivity and resolution [ ] . after focusing on different features of the spr technology (physical principle and instrumental details with the most recent trends) on bioreceptors immobilization approaches and on assay designs (indirect and direct, simple, multistep), we move on to review the applications of these strategies to real cases of interest in clinical diagnostics. hormones and small peptides analysis is a key area in diagnostic and, more recently, also in anti-doping controls. endocrine diseases diagnosis may be difficult, and it requires to directly assaying hormone levels in blood or eventually making indirect measurements. for example, diabetes mellitus is diagnosed via measurements of blood glucose rather than direct assays of plasma insulin. some interesting examples of hormones detection are provided by different authors, demonstrating the ability of spr to provide interesting solutions also in this clinical area. frasconi et al. developed a spr immunosensor for the detection of cortisol and cortisone levels in urine and saliva samples with an eco chemie autolab spr system. the measurement of free cortisol level in these matrices was an indicator for adrenal or pituitary gland disorder, and in doping analysis a marker of glucocorticoids illicit use. urine samples ( ml) were hydrolyzed by an incubation for h at °c with ml of pbs (ph . ), μl of α-methyltestosterone (internal standard), and μl of β-glucuronidase from escherichia coli. saliva samples were analyzed without pretreatment. specific antibodies were immobilized on polycarboxylate-hydrogel-based coatings, and the proposed method resulted to be simple, inexpensive, reproducible (rsd% < %), and sensitive, with detection limits less than μg /l (∼ . nm). a linear detection range from to μg/l for cortisol and - μg/l for cortisone was found and a very good correlation with the reference lc/ ms-ms method was proven, confirming the potential utility of spr for clinical, pharmacological and anti-doping application [ ] . estriol- -glucuronide, a hormone for the monitoring of ovarian function, was detected by jiang et al. with biacore x- system in urine samples from nonpregnant and pregnant subjects. the authors developed an inhibition (indirect) immunoassay by the steroid immobilization on the sensor surface, through estriol- -glucuronide-ovalbumin conjugate with an oligoethylene glycol (oeg) as linker. au nanoparticle-ab conjugate (au-ab) was used to enhance the sensitivity of the spr assay. a detection limit of . μg/l (∼ pm) in urine diluted with tm buffer ( : , : and : ) was rapidly achieved in min without complicated sample pretreatment and with a good reproducibility (cv% < . %). the assay was fast and inexpensive compared with traditional method based on ria, hplc-ms, or hplc with uv and fluorescence detection. it required less clean-up steps, resulting an efficient way to monitor e - g production rates in urinary samples of a normal menstrual cycle [ ] . besides the analysis of very low mw hormones, spr sensing has been also applied to the detection of peptide hormones such as insulin (mw da), which regulates carbohydrate metabolism. insulin sensing in serum is greatly important for clinical diagnostics and to follow-up patients affected by various types of diabetes but also for doping control. recently ( ) frasconi et al. detected insulin with indirect immunoassay in human serum samples from healthy and diabetic patients using eco chemie autolab spr system. hydroxyl/lc-pdp-functionalized g -pamam dendrimerencapsulating gnp was covalently bound by amino coupling to an alkanethiolates (carboxylated) sam-modified gold surface. then insulin was covalently immobilized on the functionalized surface exploiting the amino-coupling chemistry for the immunoassay (fig. ) . the innovative surface chemistry reduced nonspecific surface interactions and the effect of coupling between localized plasmon of the nps and surface plasmon lowered the dl down to . pm (∼ . ng/l). ten-fold diluted sera were analyzed in the - pm linear concentration range and radioimmunoassay reference method confirmed the reliability of the analytical device; indeed the biological level for insulin in the diabetic patients is - pm, corresponding to a range . - pm in a -fold diluted sample. moreover, the sensor provided reproducible results (cvs ranged between . % and . %) and it was reusable up to times [ ] . other high mw peptide hormones, such as human pituitary hormones, were recently detected with spr. in particular, lechuga et al. developed binding inhibition (indirect) immunoassays on a spr from sensia sl, where hormones were immobilized on the sensor surface by amino-coupling and working conditions (assay buffer and regeneration solution) were optimized. they detected first hgh (growth hormone) in human serum samples (mixed : in pbst), essential for development and normal growth [ ] . then they also performed, using the same strategy, the single and multi-analyte determination of two other gonadotropic hormones in urine and serum sample (both mixed : in pbst): follicle stimulating hormone (hfsh) and luteinizing hormone (hlh) involved in the development and function of the reproductive system [ ] . finally they detected hgh, hlh, hfsh, and htsh (thyroid gland stimulation for thyroxine production) in urine and serum sample (again both mixed : in pbst) using an indirect immunoassay coupled to multi-analyte sensor. all biosensors resulted sensitive (about μg/l (∼ pm) as dl for each analyte), reusable from to consecutive assay cycles and specific for each analyte. the spr based immunoassays resulted reproducible with mean intra-and inter-day cvs about %, appearing as a highly reliable tool for endocrine real-time and daily monitoring in clinical laboratory compared to official methods as ria, ima or elisa [ ] . as reported by segura in , there are many areas of common interest and overlaps between clinical analysis and anti-doping monitoring such as methodological similarities, analysis of human biological matrices, and detection of the same analytes (e.g., hormone). the author was confident that the mutual interaction between the fields would be able to bring mutual improvements in terms of technological solutions and innovative strategy [ ] . in the anti-doping code, the list of prohibited substances, under the category peptide hormones, growth factors and related substances, a variety of hormones and their releasing factors can be found: ( ) erythropoiesis-stimulating agents [e.g., erythropoietin (epo), darbepoetin (depo), hypoxiainducible factor (hif) stabilizers, methoxy polyethylene glycol-epoetin beta (cera), peginesatide (hematide)]; ( ) chorionic gonadotrophin (cg) and luteinizing hormone (lh) and their releasing factors, in males; ( ) corticotrophins and their releasing factors; ( ) growth hormone (gh) and its releasing factors and insulin-like growth factor- (igf- ). in addition, several growth factors are also banned [ ] . an excellent review on the potential use of spr in anti-doping analysis is provided by the segura group where it is highlighted how conventional analytical methods for detecting human biotics as doping agents (i.e., recombinant products), may hardly distinguish between endogenous and exogenous origin. for larger molecules (i.e., protein hormones) the innate structural complexity, the heterogeneous nature, and the extremely low levels in biological fluids made the analytical procedures heavily dependent from the immunological approaches [ ] . a protein biomarker is an indicator of a specific biological stage used in clinical diagnosis to monitor the stage of related diseases, to guide molecularly targeted therapy, and to evaluate a therapeutic response [ ] . the increasing number of clinically relevant protein biomarkers and the advances of proteomics techniques indicate the need of reliable methods for their detection in complex matrices. at this purpose, spr and spri provide a suitable platform for daily routine clinical analysis thanks to real-time and label-free detection [ ] . in this section, we focused on the most recent assays based on spr biosensor for the detection of protein biomarkers for multiple diseases first and then specific for cancer and cardiac diseases. the c-reactive protein (crp, kda) is an important blood serum marker for inflammatory processes (e.g., cardiovascular disease (cvd), inflammatory bowel diseases), currently detected by latex-enhanced turbidimetric immunoassay. jung et al. developed a label-free array system using amidelinked (al) nhs-dextran biacore cm chip for crp detection in human sera. after afm studies, they found this surface [ ] more suitable for proteins immobilization than a normal epoxide-linked carboxymethyl-dextran layer. monoclonal anti-crp antibodies were immobilized onto the surface and rapid analysis of crp was performed down to μg/l (∼ . nm) concentration in human sera diluted ( : in pbs, ph . ); results showed a good correlation with the turbidimetry reference immunoassay [ ] . bini et al. immobilized a biotinylated mer rna aptamer for crp on a carboxylated dextran-streptavidin-modified cm chip of a biacore x. in the assay optimization they evaluated the effect of different buffers, the effect of ca + ion on the interaction between the aptamer and crp, and finally the specificity of the aptasensor against putative interfering biomolecules. the highest spr signals and the lowest detection limit ( μg/l, ∼ . nm, suitable for clinical applications) were recorded with the following experimental condition: serum diluted : with hepes buffer at ph . and mm ca + concentration. cv% was estimated to be about % and a liner correlation was found within - μg/l range of concentration. experiments in serum solution showed an interfering effect caused by igg, suggesting the need of sample pretreatment [ ] . the two assays described above were suitable for clinical monitoring of crp because the protein is about . mg/l in adult serum (above the achieved dls) [ ] . circulating annexin a (anxa , kda), a biomarker related to various diseases (acute myocardial infarction, trauma, thrombosis, inflammation, and cancer) was rapidly and inexpensively detected in human blood samples (diluted : in hbs-ep) by trouvé et al. using a biacore . they immobilized anxa antibody on a carboxymethylated dextran cm sensor chip by amino coupling, and they found for the first time that the level of circulating anxa was higher in a male . (± . ) μg/l than in a female . (± . ) μg/l, indicating a difference by gender. a good linearity was observed between . and . μg/l with a good correlation (r = . ) and cv% below %. these results proved that the label-free spr biosensor was a very good alternative to the label-based conventional elisa immunoassay with advantages such as very good sensitivity, high reproducibility, and fast responses (in few min) [ ] . the long-term outcome of cancer therapy depends on early diagnosis and the response to therapy. an important aspect of cancer management includes careful monitoring of cancer biomarkers in physiological fluids [ ] . vaisocherová et al. used a home-built spr instrument for the immunodetection in human serum of alcam [ ] (activated leukocyte cell adhesion molecule/cd ), a - kd transmembrane glycoprotein biomarker of pancreatic cancer, typically with a concentration of μg/l in human serum [ ] . anti-alcam was immobilized via physical adsorption on positively charged amine-terminated alkanethiol sam surface. despite nonspecific binding recorded, a sufficiently low dl was achieved ( μg/l, ∼ pm in serum diluted : in pbs) for the discrimination of alcam levels in cancer cases from control sera. a good reproducibility was also confirmed by the cv% less than %. the direct detection (without signal amplification) showed also an excellent correlation with elisa reference method [ ] . in the same year, ladd et al. developed an immunosensor for the simultaneous and specific detection of alcam and transgelin- (tagln ), a -kda protein that has been a biomarker of breast and colorectal carcinoma). specific antibodies were immobilized by amino-coupling on cooh-oeg-coated gold sensor chip of a home-built spri platform. dls for alcam and tagln in pbs buffer were μg/l (∼ pm) and μg/l (∼ . nm), respectively. no crossreactivity was recorded but high levels of nonspecific adsorption were found with serum solution ( : in pbs), indicating the need of highly non-fouling surfaces in sensor applications [ ] . in , piliarik et al. combined high-resolution homebuilt spri sensor and high-density protein array with lowfouling background for the parallel detection of protein cancer biomarkers alcam and hcg (human chorionic gonadotropin) in diluted blood plasma samples ( % in te buffer). specific antibodies were immobilized by amino coupling on a low-fouling chip surface (bsa immobilized on carboxylterminated thiols). the biosensor showed a very low nonspecific protein adsorption (less than ng/cm ), and a linear dependence between response and analyte concentration was found (below μg/l for hcg and below μg/l for alcam). the following dls were reached: μg/l ( . nm) for alcam and μg/l ( . nm) for hcg and a very good reproducibility was confirmed by the cvs% less than % [ ] . in , kazuno et al. developed a novel spr biosensor based method for multi-sequential detection of sna- , aal, and pha-l lectins to estimate the glycosylation of haptoglobin (a complementary marker to ca in ovarian cancer) in sera of patients with prostate cancer disease. the method involved anti-haptoglobin immobilization by amino-coupling, sera dilution : in hbs-p buffer, filtration, and finally detection of the sugar chain by lectin solution. a calibration curve for haptoglobin was obtained in - mg/ml concentration range. multi-sequential analysis of sna- and haptoglobin represented an accurate diagnosistic approach for prostate cancer. the label-free spr biosensor was less complex and time-consuming than conventional elisa, where labeling step with enzymes, radioactive isotopes, or fluorescein are required [ ] . gill et al. quantified p αmap kinase, a prognostic marker in head and neck squamous cell carcinoma (hnscc), by an immunosensor (biacore ). they investigated the correlation between p α and hnscc in diluted serum ( : in hbs-ep) of patients undergoing radiation therapy (rt). they immobilized anti-p α abs by amino-coupling on a cm sensor chip and used western blot and elisa as reference methods. patients with hnscc showed -fold increase in p α levels ( . mg/l∼ nm) compared with control sample while values during-rt and post-rt treatments decreased to . mg/l (∼ . nm) and . mg/l (∼ . nm), respectively, showing a reduction attributable to a clinical tumor regression by rt. these results confirmed the suitability of p α as a serum marker in hnscc, and that if coupled to spr sensing offers higher sensitivity than traditional antibody-based methods such as elisa and western blotting [ ] . pimková et al. developed spr biosensor with dispersionless microfluidics (home-built) for the detection of svegfr- (soluble vascular endothelial growth factor receptor), a biomarker abnormally produced from cancer cells in myelodysplastic syndromes (mds). vegf-a (homodimeric glycoprotein) was covalently immobilized on the sensor surface by amino coupling, allowing the detection of svegfr- in human blood ( % in pbs) down to a dl of μg/l ( . nm) and with a good reproducibility between % and %. the approach suggested a model for future protein multi-array for mds diagnosis based on protein-protein interactions. the assay resulted in being less sensitive than conventional detection of svegfr- based on elisa, capable of detecting . μg/l and up to . μg/l, respectively, for healthy individuals and mds patients [ ] . an innovative approach for the same biomarker detection was presented by liu et al. where vegf was released in solution by living skov- ovarian cells (i.e., cancer cell) (fig. ) . anti-vegf abs were immobilized on a protein gcoated chip surface, and the analyte produced in the flow chamber (in pdms) was directly monitored. the linearity of the response in the . - . mg/l vegf concentration range and reproducibility (inter-assay rsds% less than . %) were assessed in pbs buffer. further, the cell viability was demonstrated and then the in vivo microenvironment of the vegf signaling pathways was mimicked. from the quantification of vegf released by cells, the carcinoma cell number was accurately predicted, showing the suitability of an innovative strategy for the real-time monitoring of biomarker expressed from living cells [ ] . we underline here vegf is also a target molecule in anti-doping analysis, present in the wada list of prohibited substances [ ]. battaglia et al. developed a fiberoptic spr biosensor for the simultaneous detection of three cytokines related to chronic wound healing, interleukin- (il- ), interleukin- (il- ), and tumor necrosis factor-r (tnf-r), reaching a detection limit below μg/l in hbs buffer and in spiked cell culture medium (ccm). specific antibodies were immobilized on the spr fiberoptics by aminocoupling, and no nonspecific bindings from the cell culture medium proteins were observed on the biosensor surface. the sensor emerged as a reliable device for multiple and specific biomarker detection since the il- and tnf-r concentrations are ∼ μg/l in normally healing wounds and and μg/l, respectively, in chronic wounds. moreover, the il- levels are∼ . μg/l (not detectable) in a normally healing wound and∼ ng/ml (detectable) in not healing wounds [ ] . cardiac markers are proteins released by injured myocardial cells into the bloodstream during cardiovascular diseases (cvds) and so are useful to better diagnose cvd conditions promptly and efficiently [ ] . in particular, brain natriuretic peptide (bnp, . kda) is a hormone secreted mainly from the cardiac ventricle into the blood, frequently monitored as marker in cardiac failure [ ] . teramura et al. developed a sandwich-type immunosensor for bnp detection in donor plasma (not pretreated) using nanobeads for signal amplification (fig. ) . primary anti-bnp abs were immobilized on cooh-sam chip surface (amino coupling) of a home-built spr instrument, and a sandwich assay was performed with a secondary biotinylated antibody after immunoaffinity interaction with bnp. finally, streptavidin-conjugated nanobeads ( nm in diameter) were added for signal amplification, enhancing sensitivity, and lowering dl from μg/l ( . nm) to ng/l ( . pm). linearity was observed in the - ng/l bnp concentration range, confirming the suitability of this immunosensor for clinical necessities since the normal level of bnp in plasma is about ng/l but it increases approximately to μg/l during acute or chronic cardiac failure. sensitivity was comparable to commercially available reference assays for bnp (ria and chemiluminescent reactions ( - ng/l) [ ] . reproduced by permission of the royal society of chemistry [ ] in addition, an indirect immunoassay coupled to portable spr instrumentation (with developed microfluidic) was also reported for bnp detection in human serum ( : in . m pbs). samples were incubated with acetylcholine esteraselabeled antibodies and then introduced into the microchannel of the device so that only free-bnp conjugate bound to bnp previously immobilized on the surface (by amino-coupling). then acetylthiocholine was added as substrate and hydrolyzed by acetylcholine esterase. the thiol compound (thiocholine) produced covalently bound to a thin gold layer located downstream in the microchannel, generating a spr angle shift monitored as analytical signal. a concentration range from to ng/l was examined and trace levels of analyte ( fg) were detected in about min. cv% for five measurements of ng/l bnp was . % and although this value was high compared with a commercial immunosensing system, the authors considered the rsd% acceptable for clinical diagnosis since bnp concentration significantly increases in heart failure patients [ ] , as mentioned above. two other proteic myocardial infarction biomarkers, myoglobin (mg) and cardiac troponin i (ctni), were quantified at biological levels in undiluted serum and without any sample pretreatment, using a fiberoptic spr immunosensor (jobin-yvon spex m spectrometer). specific antibodies were immobilized via amino coupling on a -mercaptohexadecanoic sam, minimizing the nonspecific signal the serum proteins. dls were . μg/l ( pm) for mg and . μg/l ( pm) for ctni, well below threshold limits to detect myocardial infarction disease (approximately - μg/l for mg and - μg/l for ctni). a good reproducibly (cv% < %) and linear correspondences between signals and concentrations up to μg/l were also assessed for both analytes [ ] . liu et al. designed a spr biosensor with high anti-fouling ability for the detection of the cardiac marker troponin t in d-pbs buffer. anti-troponin t antibodies were immobilized by amino-coupling on the chip surface (navi spr) coated with a homogeneous sam of oligo(ethylene glycol) (oeg)terminated alkanethiolate and mercaptohexadecanoic acid (mhda). the system showed interesting high anti-fouling properties (high resistance to nonspecific adsorption of hsa) and high affinity and specificity for the detection of troponin t. a good linear correlation (r= . ) below μg/l and a dl of μg/l ( . nm) were also confirmed [ ] . biosensing plays a crucial role in the analysis of autoantibodies in human serum for the real-time monitoring of autoimmune diseases. an increasing number of researches were available in literature. schlichtiger et al. recently reviewed different biosensor-based approaches in this field. authors also emphasized the main advantage of spr for monitoring bindings in real-time and for multiplexed analyses of autoantibodies by use of microarrays (e.g., spri) [ ] . the detection of pathogenic anti-dsdna abs in sera of patients affected by lupus erythematosus (sle) was reported buhl et al. an antigenic construct was formed as follow: a synthetic oligonucleotide ′-aldehyde-modified (cho) was coupled to biotinylated human transferrin using -hydrazinonicotinate acetone hydrazone (sanh) as covalent linker; then it was hybridized with the complementary antistrand and ligated with a human recombinant dsdna fragment bp in length. the conjugate was finally immobilized on a gold surface coated with carboxymethyldextran functionalized with streptavidin, and diluted sera ( : in diluents from sle patients and healthy donors) were analyzed with the spr biosensor system (biacore x). as a result, authors confirmed sle in patients with . % specificity at a sensitivity fig. schematic illustration of spr-based sandwich-type immunoassay for bnp and amplification of spr signal by accumulation of streptavidin nanobeads. reprinted with permission from elsevier [ ] of . % compared with positive results in the farr ria assay with . % specificity at a sensitivity of . %. moreover, a very good reproducibility was confirmed by intra-assay imprecision ( . %- . %) and day-to-day variation ( . %), making the spr biosensor really suited for anti-dsdna abs detection in sle disease. in addition, the device provided information about binding kinetics and affinities of the specific autoantibodies [ ] . the same group also studied interactions between dsdna and anti-dsdna autoantibodies from sle sera of patients, again with biacore x and with the same dsdna immobilization strategy previously described. they characterized kinetics, off-rates, and functional affinities (avidities) for three anti-dsdna mabs, confirming the importance of kinetic properties for the explanation of behavior of mabs in traditional methods such as the farr ria and elisa [ ] . metzger et al. developed a biosensor for the diagnosis of antiphospholipid syndrome (aps) through the discrimination of disease-relevant autoantibodies (anti-β -gpi) from crossreactive antibodies developed in other infections. human β -gpi (β -glycoprotein i) was covalently linked to a cm chip surface coated with n-alkanethiol sam (biacore x). sera ( : in hbs) from aps patients or patients with sle, syphilis, parvovirus b , and healthy donors were analyzed and the results were compared with those from elisa test. no significant antibody bindings (signal < ru) were recorded in samples from healthy individuals or patients with other infections, whereas response levels in the range of - ru were recorded from positive aps patient sera, with a correlation coefficient of . with reference elisa test. the spr-based assay was also reproducible since no significant loss of activity (< %) was recorded after measurement cycles of patient sera [ ] . in a later study, müller et al. used the same instrumental setup to prove the correlation between the affinity of anti-β -gpi in patient sera ( : in hbs) and the antigen β -gpi preparations (manufacturer, origin, and purification method). the study explained the inter-assay differences of anti-β -gpi elisas, confirming that only one common β -gpi preparation would have improved the inter-assay comparability [ ] . in the same year, the authors also demonstrated that the employment of β -gpi-derived domain-specific peptides would have offered diagnostic advantages in primary autoantibody screening for aps and in discrimination of sera of aps patients from sera of healthy patients [ ] . schlichtiger et al. detected antiphospholipid antibodies (apl, a serological indicators of the disease) with an immunosensor developed on biacore x platform. in particular, they detected cardiolipin antibodies (acl) in sera ( : in hepes) from healthy donors and aps patients. cardiolipin was immobilized by amino coupling (after activation with edc/nhs) on -mercaptoundecanoic acid sam-coated chip surface. the binding ag/ab was monitored and the assay confirmed aps with % diagnostic specificity, equal to the standard elisa method used in routine diagnostics but showing more sensitivity than elisa ( % versus . %). moreover, the chip was regenerable (up to measurements) with excellent intra-chip reproducibility (rsd%= . %) and chip-to-chip reproducibility (rsd%= . %) [ ] . rutgers et al. reported kinetic analysis of anti-gbm autoantibodies, from sera of nephritic patients, performed with biacore instrumentation. purified bovine collagen α (iv)nc (control) and α (iv)nc (ag) were immobilized onto a carboxymethylated dextran hydrogel surface after edc/nhs activation. autoantibodies from patient sera bound to α (iv)nc whereas no binding was recorded to α (iv)nc (control). from the estimation of the dissociation and association constants, they demonstrated the high affinity ab/ag, the high velocity of association and the slowness of dissociation, explaining the rapid course of the disease as well as the resistance to therapy (persistent ab glomerular aggregation could generate potentially incessant inflammation) [ ] . alaedini et al. detected autoantibodies against the monosialotetrahexosyl ganglioside gm (anti-gm ) related to acute and chronic motor neuropathies. gm (ag) and gm (control) gangliosides were absorbed on a methyl dextran layer of a chip and the discrimination between patients sera and healthy controls was possible with sensitivity and specificity comparable to classic elisa tests [ ] . antiglutamic acid decarboxylase (gad) autoantibody detection, an indicator of type i diabetes mellitus, was developed by lee et al. with biacore . the ratio between -mpa and -mua ( : ) thiols was first optimized for covalent binding of biotinylated gad via streptavidin immobilization (after nhs/edc activation). anti-gad abs were analyzed in hbs buffer within . - . μm concentration range and the interference with other biomolecules (bsa as control) was found to be negligible [ ] . carlsson et al. detected insulin autoantibodies (iaa) in serum samples from individuals at high risk of developing type diabetes (t d). they designed an indirect competitive immunoassay to bypass nonspecific adsorption of matrix proteins that could mask the analytical signal. insulin was immobilized on cm chip of a biacore x after activation with nhs/edc and the sensor was calibrated with iaa ( - u/ml) in sera derived from pooled nondiabetic serum spiked with pooled high iaa-positive serum to obtain identical matrix composition. excellent assay performances were reported since analysis time was -fold reduced from days to min, and the sensitivity was comparable to that offered by ria; the cut-off for positivity was . u/ml (in healthy swedish children) [ ] . a parallelized immunoassays on spr microarray imaging (ibis technologies) was used by lokate et al. to detect anti-citrullinated protein antibodies (acpa) in sera of patients ( : in pbst) affected by rheumatoid arthritis (ra). carboxylated xantec hc nm sensor chips were activated with nhs/edc and then two different linear citrullinated peptides (cita and citb) were immobilized on the surface; arga and argb (with arginine instead of citrulline) were spotted as corresponding control peptides. interactions between citrullinated peptides and serum autoantibodies of ra patients and controls were monitored with an experimental dl of . pm. although the sensitivity was slightly lower than that of the reference elisa test, the spr system presented the advantage of automation and surface regeneration by repetitive incubations with mm glycine•hcl for s, making the test really suitable for daily routine clinical control [ ] . three years later, a similar approach was applied by van beers et al. with ibis-ispr (imaging) for the analysis of the autoantibodies against peptide fractions in ra patients diluted sera ( -fold in pbst). peptides obtained from citrullinated human fibrinogen were immobilized on a dextran hydrogel surface (after activation with nhs/edc) and three major citrullinated epitope were identified. the multi-array enabled the simultaneous detection of autoantibodies/peptides interactions with a significant reduction of analysis time, resulting in less time-consuming and more accurate than reference elisa screening methods [ ] . spr imaging-based sensing was also used by scarano et al. for anti-bovine igg detection in untreated human serum and milk since high levels of these antibodies are related to type diabetes in serum of children. bovine iggs were immobilized in microarray-mode (spri lab + , horiba) by amino coupling on mua-coated gold chip surface. the nonspecific adsorption and the matrix effect were evaluated by a dedicate software [ ] aimed to optimize a guided automated selection of best-reacting spot (fig. ). anti-bovine igg was detected in a range of concentrations from . to . mg/l in real diluted matrices ( / for serum and / for milk in hepes buffer) with the standard addition method. a good linear response (r = . ), a good reproducibility among experiments (cv%= . %), and a dl of . mg/l ( nm) were achieved in serum while in milk the equally good performances were evaluated as r = . , cv%= . %, and dl= mg/l ( nm). the assay opened new horizons for the detection of protein in complex real matrices with spri-based biosensing, since clinical data (e.g., in serum) displayed anti-bovine igg values up to mg/l with median value of mg/l [ ] . carcinoembryonic antigen (cea) autoantibodies (specific for cea, a biomarker associated to colorectal, gastric, and pancreatic cancer) were monitored in cancerous human serum samples by ladd et al. with a home-built spr instrument. cea was immobilized on a carboxylated gold surface by aminocoupling and autoantibodies were detected by a sandwich assay adding a secondary antibody (goat anti-human igg-hrp conjugate). sera were diluted in pbsa buffer ( : , : , and : ) and tested; cea autoantibody concentrations were around mg/l, resulting∼ . μg/l after dilution ( : ). autoantibodies were distinguishable in cancerous sera samples from the healthy sample, using elisa as reference assay. this finding could be used as a diagnostic criterion for early cancer detection as well as for the diagnosis of disease progression [ ] . recently, two excellent reviews summarized the progresses made by spr-and spri-based nucleic acid (na) sensing [ , ] and their employment in clinical analysis. many oligonucleotidic analytes of interest in medical diagnostics have been detected by na spr based sensing. here we reviewed the na assays in two main classes: assay for the detection of chromosome anomalies (i.e., point mutations and snps) and assay for the detection of genetic material as biomarkers (i.e., micrornas and biomarkers of pathogens). as for the other analytes, we focused only on na assays applied to real matrices with the exception of those developed in standard solution that in our opinion improved on the analytical approach or the system analytical performance. the study of chromosome anomalies by spr in real samples deals with the detection of point mutation and single nucleotide polymorphisms (snps), which are point mutations that occur with frequencies > %. both mutations may have remarkable involvements in clinical diagnosis if they occur in a specific coding region of chromosome-related to common diseases [ , ] . in particular, spr was used as a tool for the detection of point mutation related to tumor suppressor genes (e.g., tp [ ] [ ] [ ] [ ] and k-ras [ ] ), cancer disease [ , ] , and hereditary disease [ ] [ ] [ ] [ ] [ ] [ ] . the tumor suppressor gene tp is the mostly mutated gene in presence of human cancers [ ] and, for this reason, it is one of the most studied genes in cancer research also with assays-based on spr transduction systems. in , jiang et al. detected tp point mutation with a dna spr-based sensing using portable instrumentation (a texas instruments spreeta). synthetic thiolated oligonucleotides ( mer) were immobilized on the gold chip surface as capturing probe for the snp analysis of real dna samples (extracted and amplified at a . μm concentration) from both normal wild-type (jurkat) and mutated (molt ) cell lines. the biosensor distinguished between sequences differing by only one base in the snp position (signal lowered by % for the molt ) in only min with a very low nonspecific response and with the ability to regenerate the sensor up to times. these findings coupled to the good reproducibility (cv%= %) proved that the method has potential for routine clinical analysis [ ] . in the same year, wilson et al. reported the use of the muts, a natural protein able to recognize selectively only mismatching sequences, for the detection of snp in μm synthetic oligonucleotides (tp analogue) with the same instrument. the prior injection of ssb (single strand binding) protein prevented the nonspecific interaction between muts and thiolated ssdna immobilized on the sensor. the sensor was regenerable for times (at least) making the whole approach really inexpensive for the detection of clinically relevant mutations [ ] . sipova et al. developed a sandwich-like assay for the detection of snp (codon ) in a short ( mer) tp synthetic analogue with a four-channel spr biosensor developed at the institute of photonics and electronics (prague). immobilized thiolated probes hybridized the target while streptavidin-oligonucleotide (son) complexes investigated the polymorphic site with a secondary hybridization (enhancement). a good sensitivity down to pm and a good specificity were achieved in the detection [ ] . with the beginning of the nanotech era, metal noble nanoparticles assumed a central role in the development of more sensitive assays for point mutation discrimination. yao et al. developed a method based on sandwich-like assay coupled to nanoparticles, for the detection of snps again in tp gene (excised from the pc -sn plasmid and bp) with a home-built spr. thiolated oligonucleotides were linked to gnps (signal enhancer) for the snp discrimination down to . f. target concentration in μl of sample. the method was adequately reproducible (rsd% < %) and specific thanks to the carboxylated dextran film that prevented the nonspecific adsorption of nucleotide-capped gnps. therefore, the spr-based test was attractive when analyte concentrations were very low and the sample availability was very limited [ ] . another original approach, based again on nps coupled to spr imaging dna sensor (spr imager, gwc technologies), was developed by the corn group for the high sensitive detection of snps of clinical interest. in particular, picomolar detection of snp in brca gene, associated with breast cancer, was reached in non-amplified human dna samples by measurements of surface enzymatic ligation reactions enhanced by gold nanoparticles. the coupling of spri and thiolated dna microarrays for snp genotyping was very attractive because it could be multiplexed for the simultaneous analysis of multiple snps [ ] . the same instrument was used by spoto and coworkers to further enhance snp detection with the employment of streptavin-coated gnps linked to biotinylated oligonucleotide and pna probes immobilized on a gold surface previously functionalized with dithiobis(n-succinimidylpropionate) (dtps). femtomolar sensitivity was reached with synthetic dna target in pbs buffer, making the strategy suitable for detecting dna samples without pcr amplification [ ] . indeed, years later the same approach (fig. ) was applied for the snp discrimination in the human β globin gene involved in several type of β thalassemia, directly in nonamplified genetic material (isolated from leucocytes in peripheral blood) and down to attomolar concentration bypassing the pcr step [ ] . as mentioned in the introduction, the labeling with nps was demonstrated to be useful for the improvements of analytical performances such as increase of sensitivity and lowering of dls. with this strategy, genomic dna was directly detected without any pcr amplification step but, on the other side, synthesis of nps was time-and reagent-consuming. interesting examples of the combined use of artificial dna and spr, able to enhance sensitivity of diagnostic snp investigations, were eventually reported in literature [ ] . in , feriotto et al. reported the first application of pnas and spr to human hereditary mutations. in particular, these authors detected the w x snp on cf gene, associated to cystic fibrosis, with biacore spr. pcr-amplified samples were biotinylated and immobilized on the sensor surface with streptavidin-biotin chemistry while pna probes were hybridized on the immobilized pcr products for the recognition of polymorphism. the reported results suggested that spr was an easy, fast (few min), and automatable platform for detecting mutations with real-time monitoring of hybridization, unlike other methods based on gel electrophoresis and/or dot-spot analysis [ ] . three years later, corradini et al. used biacore to discriminate the same snp in cf gene ( μm) with chiral chains modified pna (based on d-lisine), improving the recognition ability compared to simple pna [ ] . in other approaches reviewed below, the snp discrimination was performed without the employment of enhancing factor (i.e., protein or nanoparticles) or artificial dna, speeding the assays and lowering costs. recently ( ), ermini et al. have developed a very sensitive ( am) and label-free dna sensor for the specific detection of non-amplified abcb gene fragment (extracted from human lymphocytes) on spri lab + platform (horiba scientific) [ ] . abcb is a key gene in pharmacogenomics, with many snps implicated in a common set of problems such as altered drug levels and susceptibility to diseases (e.g., inflammatory bowel disease, parkinson's disease, refractory seizures, and cd cell recovery during human immunodeficiency virus therapy) [ ] . the sandwich assay was based on the optimization of sample pretreatment (fragmentation and denaturation) coupled to a rational design of very selective and performing probes. in particular, thiolated capturing probes, immobilized on the sensor surface, hybridized the target (abcb gene fragment) while a secondary probe hybridization confirmed the selectivity of the capturing [ ] . in the same year, the assay described before was optimized (secondary probes labeling and length) and successfully applied for the detection of rs snp (in the abcb gene) related to susceptibility of many diseases such as colorectal [ ] , breast, and renal cancer [ ] . snp discrimination was performed in genomic dna extracted from human lymphocytes and randomly enriched at mg/l (∼ . fm) by whole genome amplification (wga). the measures were reproducible (cv% < %) and the sensor was reusable for up to times [ ] . micrornas (mirnas) are important gene regulatory nucleic acids (about mer) in humans, affecting transcriptional and post-transcriptional regulation of gene expression [ ] . mirna also has a key role as marker of serious disease (i.e., cancer [ , ] , heart disease [ , ] , diabetes [ ] , nervous system diseases [ ] , and liver disease [ ] , included from high femtomolar to low nanomolar range of concentration in human blood [ , ] . however, mirnas are not yet mentioned in literature coupled to spr for clinical routine analysis; below, we reviewed some academic researches for the detection of mir- , the most abundant mirna in [ ] hepatocytes with an achievable future key role in clinical diagnosis as a predictive marker for viral, alcohol, and chemical-induced liver disease [ ] . a fast and simple assay was recently ( ) described by zhang et al. for the detection of mir- extracted from human breast tumor cells. the detection was based on a simple and hybrid sandwich-like assay: mir- was hybridized from a fully complementary thiolated dna probe immobilized on the sensor surface using a biacore x. a secondary biotinylated dna probe (linked to streptavidin) was hybridized to the target, enhancing sensitivity and lowering the detection limit down to pm [ ] . in addition, the assay was pcr-free and showed a good reproducibility (cv%= . %). corn and coworkers described an innovative approach for the detection of mirna, extracted from mouse liver tissue, based on thiolated lna microarray immobilized on the gold chip surface (spr imager, gwc technologies). simultaneous detection of mir- , mir- b, and mir- b from mouse liver tissue, was achieved down to fm, enhancing spri transduction with poly-(a)enzyme chemistry and t -coated au nanoparticles (fig. ) . despite the high sensitivity and specificity, the method required an elaborate multistep protocol [ ] . a portable na sensor based on sprcd (coupler and dispenser) with a special diffraction grating was developed by sipova et al. nonamplified mir- extracted from mouse liver tissue was captured by thiolated dna immobilized on the sensor surface. a specific antibody recognized dna/ rna-formed hybrid, enhancing the response and allowing the detection in less than min and down to pm (results that were in good agreement with those obtained using qpcr) [ ] . although the two described assays were developed for the detection of mirna from mouse liver, they represented excellent model systems for the improvement of analytical performance (e.g., dls lowering) suitable for next applications to detection of human mirna samples. since current methods for the detection of pathogens are expensive, time consuming, and involve culture-based techniques [ ] [ ] [ ] , spr platforms were also employed for the survey of bacteria (e.g., escherichia coli) in environmental monitoring and in clinical diagnostics, through the detection of bacterial genetic material as biomarker [ ] . kai et al. developed a method for the rapid detection of verotoxin-producing escherichia coli o :h isolated from stools, amplified by pcr, and using te as sample buffer. biotinylated pnas (bioreceptors) were immobilized on a streptavidin-coated sensorchip sa of biacore . a good correlation was found with positive results for pcr ( cfu per . g of stool) samples from patients and healthy carriers. the sensor was reusable for times after regeneration with a washing solution ( mm naoh) [ ] . wang et al. detected simultaneously four pathogenic microorganisms (pseudomonas aeruginosa, staphylococcus aureus, clostridium tetani, and clostridium perfringens). dna was extracted from the four pathogens, amplified simultaneously by universal primers ( s rna) and then hybridized on a spr-based multichannel sensor with specific dna probes immobilized by thiol chemistry. the assay was specific, fast, and with dls down to pm (for c. tetani). in addition, a good linear dependence (r > . ) was recorded between signals and pcr products concentrations ( . - nm). although the dna was extracted from commercial bacterial strains and not from human matrices, the study clearly showed potential of multi array for high analytical productivity in clinical diagnostics ( min for four specific interactions) [ ] . finally, in zhang et al. developed a label-free and sensitive method for the detection of inva gene, extracted from bacterial culture of salmonella and amplified by pcr. biotinylated ssdna probes were immobilized on streptavidin-coated dextran chip surface of a biacore x. the sensor was previously calibrated with synthetic target dna and a good linearity from o nm and a detection limit of . nm were assessed. detection of salmonella was possible as low as cfu/ml within . h and the excellent regeneration of sensor surface (up to cycles binding/ regeneration) allowed sensor reuse with cost reduction [ ] . beyond the identification of pathogens via na detection, other approaches based on the developing of different spr immunosensors were reported. in particular, we reviewed the pathogens detection in real matrices, and examples in buffer solution were reported only when these approaches were found very promising. viruses spr-based detection of antibodies against viral pathogens in medical diagnostics was previously reviewed by homola in . in particular, the review focused on antibodies detection for hepatitis g and c, hepatitis b, herpes simplex virus type (hsv- ) and type (hsv- ), epstein-barr virus, varicella-zolter virus, human respiratory syncytial virus, and adenovirus [ ] . antibodies for herpes simplex virus type (hsv- ) and type (hsv- ) were detected with biacore x in diluted human serum ( : in hbs buffer). biotinylated peptides, from corresponding segments of the n-terminus of hsv- and hsv- glycoprotein b (gb), were immobilized as antigen on chips coated with streptavidin. spr biosensor discriminated specific ab/ag binding better than conventional reference method (elisa), evaluating low-avidity background reactivity recorded frequently in human sera [ ] . abad et al. detected anti-adenoviral antibodies (rad/p ) in diluted sera of patients ( : in hepes buffer) after dosing with an adenoviral-based gene therapy vector. dextran and amino-coupling chemistry were used for the immobilization of intact virus on chip surface of a biacore . the assay was useful for designing more efficient dosing schedules automated and requiring less analysis set-up time than reference elisa method [ ] . antibodies detection against hepatitis g in patient serum was performed by rojo et al. with synthetic peptide (antigens) immobilized on biacore via dextran and aminocoupling chemistry, finding a good correlation with the elisa reference test [ ] . the same chemistry of immobilization was used by mcgill et al. for the detection of human respiratory syncytial virus (rsv) in diluted serum ( : in hbs buffer) with biacore . antibodies against virus glycoproteins (f and g) were covalently immobilized on dextran for the discrimination of the antigenic differences between the two virus genotypes (f-and g-glycoproteins) [ ] . an indirect assay was described by chung et al. for the detection of antibodies against human hepatitis b virus (hhbv) in diluted patient serum ( % in pbs). hhbvantigens were immobilized (via aminocoupling) on the spreeta surface platform and dls of . nm or . nm (after amplification with avidin-biotinylated secondary antibodies) were achieved, with very similar results of the reference elisa kit for the diagnosis of hepatitis [ ] . regnault et al. developed an assay for the detection of antiprotein s antibodies, expressed after varicella-zolter viral diseases in diluted plasma ( % in hbs buffer) of infected patients. a dextran layer was employed for the immobilization of protein s via amine coupling on the surface (with biacore x). high binding ( ru) was observed in serum of patients whereas low signals ranging from . to ru were recorded in plasma from healthy donors [ ] . vaisocherová et al. described the detection of antibodies for epstein-barr virus (anti-ena) in diluted human serum ( % in pbs buffer) with a home-built spr sensor platform based on the wavelength division multiplexing (wdm). synthetic peptides (bioreceptors) were immobilized on the surface via hydrophobic and electrostatic interaction and anti-ebna antibodies were detected down to . μg/l (∼ pm). inter-chips rsds% of the sensor response was estimated to be within the %- % range whereas intra-chip rsd% was found to be lower (∼ %), showing a good reproducibility in both situations. in summary, the analytical performances were comparable with that of a conventional peptide-based immunoassay (elisa) [ ] . kumbhat et al. developed an immunosensor for the detection of dengue virus specific igm antibodies in infected patients serum (different dilutions in pbs), using an autolab model springle. dengue antigen-bsa conjugates were immobilized as bioreceptor via aminocoupling on gold sensing surface functionalized with a mercaptoundecanoic sam. in addition, the sensor was regenerable with pepsin solution in glycin-hcl buffer (ph . ), and the results were comparable with those obtained by mac-elisa [ ] . nilsson et al. designed an inhibition assay (indirect) for the quantification of hemagglutinin (ha), a glycoprotein on the capsid of the seasonal influenza viruses. recombinant ha proteic ags (a/h n , a/h n , and b) were immobilized via aminocoupling on the dextran matrix of a biacore t chip while specific antibodies were mixed in serum solution of virus antigen (fig. ). the detection limit was . mg/l and the method resulted in more precise (intra-cv= %) and faster ( h) than single-radial immune-diffusion (srid) reference method (intra-cv= %; analysis time= h) [ ] . park et al. reported a preliminary study in pbs buffer for the detection of sars coronaviral envelope protein (scvme) in the diagnosis of severe acute respiratory syndrome (sars). scvme was anchored to gold chip surface (spri, k-mac) by gold binding protein (gbp) and selective and sensitive detections of anti-scvme antibodies were performed down to μg/l (dl) in min. afm and spr imaging analyses confirmed the anti-scvme-specific capturing by antigen domain with an appropriate orientation [ ] . wang et al. employed high-resolution spr microscopy (sprm) for the label-free and imaging detection of single viruses particles (h n influenza a/pr/ / and hcmv) in pbs buffer down to ∼ . fg/mm (mass dl). anti-influenza a antibodies were covalently immobilized on peg/peg-cooh coated gold chip surface via aminocoupling. this study demonstrated the suitability of the method to assess masses and binding activity of individual viral particles [ ] . in this section detections of pathogenic bacteria or other microorganisms were reviewed focusing on immunological assays for direct pathogen or anti-pathogen antibodies detection. salmonella, a frequent gastrointestinal pathogen involved in many diseases such as diarrhea, gastrointestinal inflammation, and life-threatening typhoid fever [ ] , has been widely reported in literature coupled to spr-based sensing. a spr-based sandwich immunoassay was developed in pbs buffer by muzumdar et al. for serotyping of salmonella b, c, d. polyclonal antibody for salmonella, immobilized on the spr chip (plasmonic biosensoren ag), captured the salmonella bacterial cells further probed with antigenspecific antibodies for serotyping. the method was rapid and quantitative with a detection limit of about cell/ml, and the spr immunosensor provided more comprehensive and quantitative information than conventional slide agglutination test (sat) [ ] . more recently ( ), gupta et al. designed an immunosensor for salmonella typhi antibody detection in pbs buffer and then in patients sera. salmonella typhi antigen was immobilized on a -mercaptobenzoic acid ( -mba) modified gold surface chip of an electrochemical surface plasmon resonance system (autolab esprit). the discrimination between positive (widal positive) and negative sera (widal negative) was achieved in less than min. experimental dls were, respectively, : dilution for mab and : dilution for salmonella typhi antibodies, and a good reproducibility ( measures) was recorded (cv%= . %) [ ] . boer et al. detected glycan-specific serum antibodies, biomarkers of exogenous pathogens, in human sera infected by the parasite schistosoma mansoni. sera were diluted ( : in pbst) while glycans samples were printed on an epoxide-activated chip (biacore flexchip). different serum antibody profiles between infected sera and control sera were revealed in real-time and without fluorescent labeling [ ] . up to now, isi web of knowledge reported almost , publications related to spr sensing and, if we analyze the distribution of the topics, the technology impacts significantly on clinical chemistry. however, studies demonstrate proofs of principle and only very recently, reports on applications to real matrices have appeared. in this review, we considered a panel of analytes of interest for clinical diagnostics, also focusing on emerging technological innovations such as coupling between spr and nanoparticles, microfluidics, or new chip design, for improving the analytical performance. the detection of hormones related to endocrine disorders, protein biomarkers in relation to various conditions (inflammation states or traumas) or, more specifically, for cancer and overlay plot showing sensorgrams of injected serum mixed with a concentration series of virus standard. report points (marked as x) are taken before and after injection and response levels are measured between those (arrow). after each injection, the surface is regenerated in preparation for a new injection. reprinted with permission from elsevier [ ] cardiac diseases were discussed as case studies. antibody detection was also reported in relation to diagnosis of infections and immune system diseases. in addition, we reviewed nucleic acid sensing for the detection of genetic disorders, but also for recognizing specific sequences for pathogens (virus, bacteria) detection, or for mirna as emerging biomarkers. finally, we discussed immunosensing approaches for pathogens detection. in our selection, we privileged studies conducted on real matrices (blood, serum, and urine) with a few exceptions, when the analytical strategies appeared especially original and innovative. the development of innovative chip chemistry and antifouling strategies guarantees reduced nonspecific binding, which is essential for complex biological fluids analysis. amine coupling allows for covalent immobilization of receptors, ensuring repeated measurement on the same chip, after a simple dissociation of the receptor-ligand complex with chaotropic reagents. interestingly, sample pretreatment is reduced to a minimum: dilution, sometimes a short heating or filtration. in summary, spr-based biosensors offer analytical performances (sensitivity, dls, and reproducibility) comparable to conventional methods employed in clinical analysis (electrophoresis, chromatography, elisa, ria, etc.); in addition, they ensure real-time monitoring, label-free solutions, parallel analysis (spri), high throughput, little sample pretreatment, fast responses, and cheapness. for all these reasons, we believe that in the near future, spr will emerge as an efficient, powerful, and alternative tool for daily routine clinical analysis, opening also new horizons for future developments in personalized medicine and in point of care diagnostics. advances in surface plasmon resonance biosensor technology towards high-throughput, food-safety analysis recent developments and applications of surface plasmon resonance biosensors for the detection of mycotoxins in foodstuffs surface plasmon resonance for detection of genetically modified organisms in the food supply direct detection of e. coli o :h in selected food systems by a surface plasmon resonance biosensor surface plasmon resonance in doping analysis realtime and label-free bio-sensing of molecular interactions by surface plasmon resonance: a laboratory medicine perspective cancer biomarker detection by surface plasmon resonance biosensors proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays surface plasmon resonance mass spectrometry in proteomics rapid and label-free bacteria detection by surface plasmon resonance (spr) biosensors recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest emerging optofluidic technologies for point-of-care genetic analysis systems: a review surface plasmon resonance sensors: review advances in surface plasmon resonance biosensor analysis present and future of surface plasmon resonance biosensors recent advances in surface plasmon resonance based techniques for bioanalysis surface plasmon resonance sensors for detection of chemical and biological species surface plasmon resonance imaging surface plasmon resonance imaging for affinity-based biosensors surface plasmon resonance imaging measurements of ultrathin organic films surface plasmon resonance imaging as a tool to monitor biomolecular interactions in an array based format on a remarkable case of uneven distribution of light in a diffraction grating spectrum radiative decay of non-radiative surface plasmons excited by light excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection surface plasmon resonance sensing of nucleic acids: a review immunosensors-principles and applications to clinical chemistry surface plasmon resonancebased immunoassays surface plasmon resonance imaging for nucleic acid detection artificial dna and surface plasmon resonance new trends in affinity sensing: aptamers for ligand binding aptamer-based molecular recognition for biosensor development from oligonucleotide shapes to genomic selex: novel biological regulatory loops methods developed for selex advances in the manufacture of mip nanoparticles molecularly imprinted polymers and their use in biomimetic sensors the art of immobilization for spr sensors. in: homola j (ed) surface plasmon resonance-based sensors se- high salt stability and protein resistance of poly(l-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates surface plasmon resonance imaging: what next? nanostructured plasmonic sensors recent developments and applications of hybrid surface plasmon resonance interfaces in optical sensing surface plasmon resonance biosensors incorporating gold nanoparticles functionalized gold nanoparticles for ultrasensitive dna detection recent advances in the development of graphene-based surface plasmon resonance (spr) interfaces new trends in instrumental design for surface plasmon resonance-based biosensors surface plasmon resonance immunosensor for cortisol and cortisone determination determination of estriol -glucuronide in human urine with surface plasmon resonance and lateral flow immunoassays multifunctional au nanoparticle dendrimer-based surface plasmon resonance biosensor and its application for improved insulin detection determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay single-and multi-analyte determination of gonadotropic hormones in urine by surface plasmon resonance immunoassay surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples is anti-doping analysis so far from clinical, legal or forensic targets? the added value of close relationships between related disciplines protein biomarker discovery and validation: the long and uncertain path to clinical utility multiplexed surface plasmon resonance imaging for protein biomarker analysis analysis of c-reactive protein on amide-linked n-hydroxysuccinimide-dextran arrays with a spectral surface plasmon resonance biosensor for serodiagnosis development of an optical rna-based aptasensor for c-reactive protein surface plasmon resonance shows a gender difference in circulating annexin a in human multi-sequential surface plasmon resonance analysis of haptoglobin-lectin complex in sera of patients with malignant and benign prostate diseases comparative study of spr and elisa methods based on analysis of cd /alcam levels in cancer and control human sera label-free detection of cancer biomarker candidates using surface plasmon resonance imaging surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma quantification of p α map kinase: a prognostic marker in hnscc with respect to radiation therapy surface plasmon resonance biosensor for the detection of vegfr- -a protein marker of myelodysplastic syndromes real-time monitoring biomarker expression of carcinoma cells by surface plasmon resonance biosensors quantification of cytokines involved in wound healing using surface plasmon resonance brain natriuretic peptide as a novel cardiac hormone in humans: evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide, and brain natriuretic peptide surface plasmon resonancebased highly sensitive immunosensing for brain natriuretic peptide using nanobeads for signal amplification on-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system quantitative measurement of cardiac markers in undiluted serum surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin t biosensor approaches for the detection of autoantibodies in human serum novel biosensor-based analytic device for the detection of anti-double-stranded dna antibodies optical biosensor-based characterization of antidouble-stranded dna monoclonal antibodies as possible new standards for laboratory tests biosensor analysis of beta -glycoprotein ireactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies standardized antigen preparation to achieve comparability of anti-β -glycoprotein i assays -glycoprotein i-derived peptides as antigenic structures for the detection of antiphospholipid antibodies covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum high affinity of anti-gbm antibodies from goodpasture and transplanted alport patients to alpha (iv)nc collagen a surface plasmon resonance biosensor assay for measurement of anti-gm( ) antibodies in neuropathy characterization of a selfassembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-gad antibody an indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging mapping of citrullinated fibrinogen b-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance surface plasmon resonance imaging (spri)-based sensing: a new approach in signal sampling and management surface plasmon resonance imaging-based sensing for anti-bovine immunoglobulins detection in human milk and serum direct detection of carcinoembryonic antigen autoantibodies in clinical human serum samples using a surface plasmon resonance sensor large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome characterization of single-nucleotide polymorphisms in coding regions of human genes detection of tp mutation using a portable surface plasmon resonance dna-based biosensor a novel optical biosensor format for the detection of clinically relevant tp mutations streptavidin-enhanced assay for sensitive and specific detection of single nucleotide polymorphism in tp sub-attomole oligonucleotide and p cdna determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification detection of a k-ras point mutation employing peptide nucleic acid at the surface of a spr biosensor single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions single nucleotide polymorphism detection by optical dnabased sensing coupled with whole genomic amplification direct detection of point mutations in nonamplified human genomic dna biosensor technology for real-time detection of the cystic fibrosis w x mutation in cftr enhanced recognition of cystic fibrosis w x dna point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor point mutation detection by surface plasmon resonance imaging coupled with a temperature scan method in a model system surface plasmon resonance imaging as a multidimensional surface characterization instrument-application to biochip genotyping surface plasmon resonance imaging (spri) system and real-time monitoring of dna biochip for human genetic mutation diagnosis of dna amplified samples the iarc tp database: new online mutation analysis and recommendations to users ultrasensitive detection of dna by pna and nanoparticleenhanced surface plasmon resonance imaging direct detection of genomic dna by surface plasmon resonance imaging: an optimized approach polymorphisms in human mdr (p-glycoprotein): recent advances and clinical relevance a comprehensive investigation on common polymorphisms in the mdr /abcb transporter gene and susceptibility to colorectal cancer mdr gene c t polymorphism and cancer risk: a meta-analysis of case-control studies micrornas: genomics, biogenesis, mechanism, and function oncomirs: micrornas with a role in cancer microrna expression profiles classify human cancers micrornas: novel regulators in cardiac development and disease microrna regulatory networks in cardiovascular development plasma microrna profiling reveals loss of endothelial mir- and other micrornas in type diabetes a microrna feedback circuit in midbrain dopamine neurons plasma microrna- as a biomarker for viral-, alcohol-, and chemical-related hepatic diseases multiplexed detection methods for profiling microrna expression in biological samples streptavidin-enhanced surface plasmon resonance biosensor for highly sensitive and specific detection of microrna attomole microarray detection of micrornas by nanoparticle-amplified spr imaging measurements of surface polyadenylation reactions surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level a review on viral biosensors to detect human pathogens biosensors as innovative tools for the detection of food borne pathogens review of biosensors for foodborne pathogens and toxins label-free detection of s ribosomal rna hybridization on reusable dna arrays using surface plasmon resonance imaging detection of pcr products of escherichia coli o : h in human stool samples using surface plasmon resonance (spr) rapid labelfree identification of mixed bacterial infections by surface plasmon resonance label-free and high-sensitive detection of salmonella using a surface plasmon resonance dna-based biosensor detection of human serum antibodies against type-specifically reactive peptides from the n-terminus of glycoprotein b of herpes simplex virus type and type by surface plasmon resonance development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors gb virus c (gbv-c)/hepatitis g virus (hgv): towards the design of synthetic peptides-based biosensors for immunodiagnosis of gbv-c/hgv infection detection of human respiratory syncytial virus genotype specific antibody responses in infants application of spr biosensor for medical diagnostics of human hepatitis b virus ( hhbv ) anti-protein s antibodies following a varicella infection: detection, characterization and influence on thrombin generation surface plasmon resonance biosensor for direct detection of antibody against epstein-barr virus surface plasmon resonance based immunosensor for serological diagnosis of dengue virus infection a novel assay for influenza virus quantification using surface plasmon resonance protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner labelfree imaging, detection, and mass measurement of single viruses by surface plasmon resonance cellular aspects of immunity to intracellular salmonella enterica surface plasmon resonance (spr) as a rapid tool for serotyping of salmonella surface plasmon resonance immunosensor for the detection of salmonella typhi antibodies in buffer and patient serum serum antibody screening by surface plasmon resonance using a natural glycan microarray acknowledgments the authors acknowledge fondazione arpa progetto dolore and miur -prin biosensori realizzati con nanomateriali per una rapida identificazione di biomarcatori tumorali for financial support. key: cord- - qx b authors: garrett, j. hudson title: the importance of the clinical environment in the transmission of health care-associated infections date: - - journal: journal of the association for vascular access doi: . /j.java. . . sha: doc_id: cord_uid: qx b nan c ollaboration is a critical component in the prevention of health care-associated infections (hais) in today's health care environment. now more than ever, a clean and sanitary patient environment is being measured as a component of the infection prevention and control process. in addition, outcome measures such as patient satisfaction and the cleanliness of the environment are common metrics in this era of continual health care reform. payers such as the centers for medicare and medicaid services correlate hospital reimbursement with many of these measures and metrics, resulting in financial effects low-performing facilities. the cleanliness of medical equipment such as a portable ultrasound machines, patient care surfaces, and environment surfaces are all included; thus, an impeccably clean environment is a shared goal between environmental services workers and vascular access professionals. patients, visitors, and health care providers routinely contaminate health care environments through daily activities, and this can increase the risk for infection transmission. transmission can result from contact with either contaminated hands or environment surfaces, and also a patient's own skin flora. one of the most critical interventions that can be routinely performed to decrease the risk for crosstransmission and development of hais is routine cleaning and disinfection of the environment. this includes both medical equipment and environment surfaces. high-touch items, such as those used between patients regularly, should be disinfected between each single use to minimize the risk for contamination. recent expert opinions have posited that it is actually environmental surfaces professionals who spend the most time with patients in hospital rooms. this creates an interesting opportunity to utilize environmental services professionals as a part of infection prevention advocacy plans. facilities should include environmental services team members in infection prevention unit-based education, and also engage them in an active and personal role in preventing infections among the patients that they serve. this concept was demonstrated by researchers at john hopkins through the comprehensive unit-based safety program initiative. environmental services and vascular access professionals can also serve as educators by informing patients of the steps being taken to mitigate the risk for infection, such as daily and terminal cleaning and use of alcohol-based handrubs, and encouraging family members to follow isolation precautions as appropriate. there is typically a positive correlation between enhanced patient satisfaction and increased interaction with members of health care delivery teams, which most certainly includes environmental services professionals. environmental services professionals are not only experts in maintaining the environment, but also in serving as patient safety advocates who reduce the incidence of hais. surface disinfection is an important factor in the prevention of hais. many surfaces in health care settings are considered noncritical and therefore require cleaning with a low-level disinfectant. however cross-contamination can and does occur in a variety of ways, most often when a surface becomes contaminated and then serves as a reservoir for microbial growth. consider a situation wherein the hands of either a health care provider or patient come in contact with a contaminated surface, then hand contact is made with another device or surface, contaminating it as well. thus the chain of infection transmission begins. unfortunately, all too often high-touch surfaces are not properly cleaned and disinfected routinely due to a variety of reasons. the ability of microorganisms to survive and reproduce on environment surfaces has never been greater. organisms such as methicillin-resistant staphylococcus aureus, escherichia coli, clostridium difficile, and mycobacterium tuberculosis can survive on surfaces for several months. because of the resilience of these microorganisms, it is important to routinely disinfect all potentially contaminated surfaces to reduce the risk of transmission. emergent threats such as middle east respiratory syndrome coronavirus and carbapenem-resistant enterobacteriaceae continue to plague the health care delivery system. although environmental services professionals are certainly experts in the maintenance of a clean environment, clinical nursing teams have accountability for items such as ventilators, intravenous pumps, and other medical devices. family members visiting patients can serve as an extension of the teams by either cleaning certain soiled surfaces themselves, or promptly notifying staff members when surfaces become soiled. before effective disinfection can occur, it is important to thoroughly clean visibly soiled surfaces to allow for the full efficacy of the chosen disinfectant product. the latest centers for disease control and prevention guideline for disinfection and sterilization in healthcare facilities (released in ) describes cleaning as: the removal of foreign material (eg, soil and organic matter) from objects, and is normally accomplished using water with detergents or enzymatic products. thorough cleaning is essential before highlevel disinfection and sterilization because inorganic and organic materials that remain on the surfaces of instruments interfere with the effectiveness of these processes. cleaning removes bioburden from an affected surface by reducing the number of microorganisms that must be inactivated. removing bioburden from a surface before application of a disinfectant solution will result in increased disinfectant efficacy. it is also important to also apply friction to the area being cleaned and disinfected to remove more resistant forms of microorganisms such as spores (eg, c difficile spores) from surfaces that may not be readily inactivated by the disinfectant. this will decrease the risk for development of multidrugresistant organisms. the selection of the appropriate disinfectant product is crucial. always choose a disinfectant that is approved by the environmental protection agency and that has established efficacy claims. in addition, infection prevention specialists should refer to their facility's risk assessment document and ensure that the disinfectant selected has efficacy claims for microorganisms that are routinely found within the facility. efficacy claims are readily available through product manufacturers and should be carefully reviewed before introduction of a product into the facility. the environmental services team should work carefully with colleagues in infection prevention and vascular access to ensure that the disinfectant selected meets the efficacy needs for infection prevention but also is safe for the staff to use and also for use around patients. high-touch surfaces such as blood pressure cuffs, stethoscopes, and glucometers require frequent disinfection to prevent cross-transmission between patients. the physical number of microorganisms present on any given surface is influenced by a number of factors, including the amount of moisture present on the surface, the amount of activity taking place in the immediate environment, the number of people who have contact with the environment, and the type of surfaces present and their ability to support the growth of microorganisms. high-touch surfaces are contaminated continually throughout the day; therefore, it is critical to have an understanding between nursing personnel and environmental services professionals regarding the frequency of cleaning necessary and also the ownership for each item. this methodical approach to environmental hygiene will produce meaningful and sustainable results. the primary focus of a thorough environment disinfection program should be on those items that are used with multiple patients and/or procedures. spaulding created a standardized approach to disinfection in the health care environment that consists of categories: critical, semicritical, and noncritical. noncritical items such as wheelchairs and bedside tables are those that have contact with intact skin, but not sterile body tissues or mucous membranes. these items require the use of a low-level disinfectant. with the recent move of most acute care facilities to use of electronic medical records, disinfection of noncritical items such as computer keyboards is of high importance to reduce transmission of microorganisms throughout the entire environment. hand hygiene is still the most critical intervention to break the chain of infection, but routine disinfection of these potential reservoirs for microbial growth is a key component as well. it is critical to have a complete set of policies and procedures identifying each individual's and department's responsibility in the cleaning and disinfection process. careful collaboration with the environmental services team is necessary to ensure that all surfaces are routinely disinfected by the appropriate personnel. education programs are available through organizations such as the association for the healthcare environment. also, a collaborative partnership with the facility's health care engineering team is critically important in executing infection prevention/vascular access and environmental services projects. the association for the healthcare environment publishes the only evidence-based practice guidance resource (www.ahe.org) specifically for environment cleaning. it is a tool that can be referenced for managing the disinfection needs specific to vascular access professionals and the equipment used in their daily roles. education of health care staff members is key to minimizing risks associated with using any disinfectant product. staff members should be educated on the appropriate indications for use for each product, the instructions for use, including total overall contact time required to effectively inactivate the microorganisms according to the product's efficacy label, the product's material safety data sheet, and also the appropriate use of personal protective equipment as required by the occupational safety and health administration bloodborne pathogens standard. in addition, steps must be taken to ensure that infection prevention in vascular access is addressed across the entire continuum of care, including inpatient and outpatient settings. by educating the appropriate staff members on the appropriate use of the chosen product, the manufacturer's documented product efficacy will be achieved and end users of the product will be protected from adverse reactions. a thorough cleaning and disinfection program combined with careful selection of the most appropriate hospital-grade disinfectants will dramatically improve health care professionals' daily fight against hais. appropriate product use combined with education of the users will give health care facilities the greatest opportunity to reduce contamination within the facility. with the continual development of new surface disinfection technologies, it is also crucial for health care providers to evaluate these new technologies carefully and review the supporting efficacy data thoroughly before changing processes already in place. hand hygiene combined with disinfection of the patient environment will significantly reduce the risk for cross-transmission. it is only through a comprehensive and collaborative effort between vascular access and environmental services teams that facilities can target zero hais. it is time for perfect care every time, for every patient. dr. garrett is the vice president of clinical affairs for pdi, inc., atlanta, ga and also the industry liaison for the board of directors for the association for the healthcare environment (ahe). correspondence concerning this article should be addressed to hudsongarrettjr@gmail.com. hai transmission in the health care environment. health facil manage healthcare infection control practices advisory committee. guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) a review of the cdc recommendations for prevention of hais in outpatient settings key: cord- - ixbqlb authors: carroll, gregory t.; wang, denong; turro, nicholas j.; koberstein, jeffrey t. title: photons to illuminate the universe of sugar diversity through bioarrays date: - - journal: glycoconj j doi: . /s - - - sha: doc_id: cord_uid: ixbqlb in this mini-review, we summarize the photochemical approaches for developing high-throughput carbohydrate microarray technologies. newly established methods for photo-immobilizing unmodified monosaccharides, oligosaccharides and polysaccharides onto photoactive surfaces and coupling of photoactive carbohydrates onto polymer surfaces are reviewed. sugar chains in living organisms are structurally diverse and characteristically suitable for storing and presenting bio-signals for specific molecular recognition [ ] [ ] [ ] . in many physiological and pathophysiological conditions, expression of cellular glycans, in the form of either glycoproteins or glycolipids, is differentially regulated. characteristic patterns of complex carbohydrates are frequently associated with the stages or steps of embryonic development and cell differentiation, as well as transformation of normal cells to abnormally differentiated tumor or cancer cells [ ] [ ] [ ] [ ] [ ] . sugar moieties are also abundantly expressed on the outer surfaces of the majority of viral, bacterial, protozoan and fungal pathogens. many sugar structures are pathogen-specific, which makes them important molecular targets for pathogen recognition, diagnosis of infectious diseases, and vaccine development [ ] [ ] [ ] [ ] . developing microarray-based high-throughput technologies for structural and immunological characterization of carbohydrates has been one of the focused efforts in the areas of post genomics research and technology development [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the past few years, relatively nascent carbohydrate microarray technologies began to show their potential in biomedical applications. this is highlighted by rapid identification and characterization of immunogenic sugar moieties of sars-cov [ ] , hiv- [ ] and anthrax spores [ ] , as well as fine specificity studies of carbohydrateprotein interactions of biomedical importance [ , , ] . recently, photochemical methods have shown great potential in the field of carbohydrate microarrays as they have produced some of the earliest examples of covalently bound microarrays that do not require chemical modification of the carbohydrates. photochemical methods are an important emerging route to patterning substrates for various materials or biomaterials applications as photochemical surface modification can be spatially controlled using a photomask [ , ] . such photolithographic processes allow micro-and nanometer sized shapes to be patterned on a surface directly without the requirement of mechanical processes such as stamping [ ] . photochemical immobilization and patterning of biological molecules on a surface is well-known [ ] , although few reports have focused on carbohydrates. aside from being amenable to standard patterning techniques, photochemistry also provides a clean method of surface derivatization in that many reactions require only photons as a reagent. in addition, the absorption of photons allows for chemicals that are normally not reactive to form new carbon-carbon bonds. surfaces functionalized with aromatic ketones have been used to photoimmobilize a variety of biological molecules. ketones are well-known chromophores that can participate in hydrogen abstraction reactions [ ] . when an appropriate ketone is irradiated with uv light, the excited n−π * state of the ketone intersystem crosses to the triplet state, from which it can abstract a hydrogen atom from a nearby donor to create radicals as shown in fig. . note that other processes can compete with hydrogen abstraction including phosphorescence, internal conversion and other photochemical reactions including electron transfer, addition to a double bond, α-cleavage and β-cleavage. the photogenerated radical can recombine to form various products. when the ketone is bound to a surface, recombination of a given radical with the ketone will covalently bind the molecule to the surface. although direct proof of covalent bond formation is difficult to prove unequivocally due to the low number of molecules on the surface and limited number of surface spectroscopic techniques for characterizing the formation of carbon-carbon bonds, substrates composed of aromatic ketones have been shown to stabilize a variety of biological and synthetic polymers [ ] [ ] [ ] . recently, photoactive surfaces that incorporate surfacebound phthalimide chromophores have been applied to fabricate carbohydrate microarrays. phthalimide derivatives are a class of aromatic ketones that are known to undergo all the major photochemical reactions of ketones [ ] . their salts can be easily incorporated into halogenated molecules and the commercial availability of halogenated silanes allows for a one-step synthesis of an appropriate heterobifunctional molecule for self-assembly onto an appropriate substrate as shown in fig. a . self-assembled phthalimide monolayers have been prepared on glass, si and quartz substrates [ ] . irradiation of spin-coated films of polysaccharides, disaccharides and monosaccharides on the phthalimide monolayer results in a stable binding of the carbohydrates presumably by a covalent bond formed by hydrogen abstraction followed by radical recombination. microarrays can be constructed by using a spotter to deliver carbohydrates to the photoactive surface followed by uv illumination. the propensity for physisorption and subsequently the yield of the photo-immobilization reaction can be increased substantially by modifying the surface energy of the substrate such that the capillary interactions between the spotter containing aqueous carbohydrate solution and the photoactive surface favor wetting of the solution. incorporation of aminopropyltrimethoxy silane into a phthalimide-derivatized surface (fig. b) provides a more favorable substrate for spotting carbohydrates and results in a stable photo-immobilization of the carbohydrates onto the chip after irradiation. photo-immobilized α - dextran polysaccharides were shown to retain their antigenic reactivity toward an anti-dextran monoclonal antibody ( . . e), which is specific for the terminal non-reducing ends of the polysaccharide [ ] . however, spotted mono-and oligosac- fig. upon absorption of a photon, ketones can abstract a hydrogen atom from an appropriate donor, forming radicals that can recombine to form carbon-carbon bonds charide arrays showed differential activities with the lectin concanavalin a (con a). this lectin is man-and/or glcspecific and requires the c- , c- , and c- hydroxyl groups of the man or glc ring for binding. photo-coupled oligosaccharides with three (im ), five (im ), and seven glucoses (im ) are reactive to con a on the photoactive surface but not on a nitrocellulose-coated slide. by contrast, none of the spotted monosaccharides were reactive to the lectin on these surfaces. given that this method of photocoupling can target any ch-group on the sugar rings with varying specificity depending on the structure of the ring [ , ] , there is a possibility that the site of covalent attachment may interfere significantly with lectin binding to the monosaccharides man and glc. the limited specificity of the reaction and the lesser amount of saccharide epitopes present for smaller carbohydrates reduces the probability that a biologically active epitope presents itself at the air-monolayer interface. it is interesting to note that another method that allows for underivatized carbohydrates to be covalently immobilized via a chemical reaction with a hydrazide monolayer similarly was unable to properly display some monosaccharides for lectin recognition [ ] . for example, con a was not able to recognize mannose, glucose and n-acetylglucosamine. the lack of biological activity was attributed to an improper β-configuration at the anomeric position. although the degree of selectivity of the surface photoimmobilization reaction is unknown, studies of hydrogen abstraction towards carbohydrates in solution have been performed and provide insight into what may occur on the surface. care should be exercised in applying such information to surface reactions however, since surface effects such as the orientation of reactants can affect reactions. using model compounds, such as tetrahydrofuran (thf), it has been shown that hydrogen abstraction reactions occur preferentially at the anomeric center [ ] . the selectivity of the reaction is due to stabilization of the resulting radical by the electron donating oxygen atom. when actual carbohydrates are used, the degree of selectivity has in some cases been found to be dependent on substituents. time resolved epr experiments have shown that the photo-excited triplet of acetone attacks hydrogens in the c -c positions preferentially on glucose, galactose, xylose and maltose [ ] . the reaction of hydroxyl radicals with carbohydrates has also been studied using epr [ ] . although hydroxyl radicals may show different reactivity than photogenerated triplets in a given system, they do provide insight into radical reactivity towards the various carbohydrate c-h groups. for example, hydrogen abstraction from carbons in the pyranose ring and linked by glycosidic bonds in dextran polysaccharides was found to be inhibited. additionally, abstraction from galacturonan and d-galacturonic acid were found to occur dominantly on the carbon atom adjacent to the carboxyl group. based on these studies the excited phthalimide is expected to attack the accessible c-h groups of an adsorbed carbohydrate indiscriminately, if the only other substituents are oh groups. addition of other substituents may favor selectivity. the type of reaction occurring may also change when electron-donating heteroatoms are introduced into the carbohydrate. for example, amine-containing glycans may undergo an electron transfer reaction with a phthalimide. note that phthalimide electron transfer reactions have been shown to render regioselective product formation for some systems [ ] . the affect of the surface on reaction products and selectivity is currently unknown. additionally, the aminopropyl silanes mixed into the surface may also affect the reaction. for example, a fig. photoactive surfaces allow for adsorbed carbohydrates to bind to the surface. a a phthalimide self-assembled monolayer can bind underivatized carbohydrates to the surface most likely by hydrogen abstraction followed by radical recombination. b a phthalimideamine mixed surface enhances carbohydrate adsorption to the surface when a robotic spotter is used. subsequent irradiation binds the sugars to the surface. c an aziridine derivatized polysaccharide film can bind spotted carbohydrates to the surface after irradiation by forming a carbene that reacts with adsorbed sugars. carbohydrates immobilized on surfaces (b and c) have been shown to recognize appropriate antibodies and lectins competing primary process may be electron abstraction from an amine. in any case, the phthalimide-amine derivatized surface was found to immobilize carbohydrates on the surface after irradiation with uv light and the larger immobilized carbohydrates retained their biorecognition properties. the phthalimide-derivatized surface has been used to characterize the immunogenic sugar moieties of bacillus anthracis [ ] . a tetrasaccharide composed of a trisaccharide of rhamnopyranosyl units attached to a terminal residue given the name anthrose is o-linked to the glycoprotein bcla expressed on the surface of anthrax spores [ ] . the tetrasaccharide and components of the tetrasaccharide were synthesized and photoimmobilized on the phthalimide-amine-coated substrates. the surface-bound tetrasaccharide was found to be specifically reactive with antibodies elicited by anthrax spore immunization indicating that the tetrasaccharide forms an antigenic determinant on the glycoprotein. inhibition studies showed that an anthrose monosaccharide inhibits reactivity of the antibody towards the tetrasaccharide and other rhamnopyranosyl oligosaccharides containing anthrose, showing that the anthrose residue contributes significantly to the antigenic determinant of the tetrasaccharide. this study illustrates the feasibility of using photogenerated microarrays for studying the immunogenic properties of carbohydrates. other carbohydrate microarrays have been prepared by photogenerating carbenes and nitrenes. carbenes and nitrenes can be generated from diazo compounds and azides by photo-elimination of n as shown in fig. [ ] . the kinds of reactions these intermediates undergo will depend on whether the reaction is initiated from the singlet or triplet state, however both can result in carbon-carbon bond formation. singlets, which contain more zwitterionic character, can undergo , sigmatropic shifts, stereospecific insertion into sigma bonds, stereospecific insertion into pi bonds and addition of a nucleophile or electrophile. triplets contain more diradicaloid character and can undergo atom abstraction reactions, nonstereospecific addition to pi bonds and addition of radicals. often compounds incorporating caged carbenes and nitrenes contain fluorine substituents which help prevent autoreactivity. an added benefit in terms of surface reactions is that c-f substituents are low energy functional groups that tend to migrate to the solid-air interface in order to decrease interfacial tension, inadvertently making the photoactive group more accessible to an adsorbate [ ] . in general this is more of a potential problem for photoactive polymer films rather than more constrained end-functionalized self-assembled monolayers. sigrist and colleagues [ ] developed aziridine derivatized polysaccharide films for photoimmobilizing underivatized carbohydrates as shown in fig. c [ ] . upon absorption of a photon, the aziridine group loses n to form a highly reactive carbene that can react with the spotted sugars to form a covalent bond. bacterial exopolysaccharides were photoimmobilized and positively stained by appropriate lectins. similarly, glycoproteins, neoglycoproteins and cell extracts were immobilized. oligosaccharide immobilization required conjugation to fig. irradiation of diazo compounds and azides results in the loss of n and formation of carbene and nitrene intermediates fig. adsorption of photoactive monosaccharides onto polymer films followed by irradiation produces a stable linkage to the surface a protein prior to deposition. the authors point out that the hydrophilic polysaccharide surface resists non-specific protein binding. in addition the polymer film may provide a larger amount of surface area for adsorption of greater amounts of carbohydrates relative to the oftenutilized "two-dimensional" self-assembled monolayer. the above methods allow for underivatized carbohydrates or carbohydrates lacking an appropriate functional group to be immobilized on a chip. however, the photocoupling sites on the carbohydrates are not specifically defined. for polysaccharides this is less of a concern since they can contain many epitopes. as a sugar decreases in size, the probability of inactivating the functional part of the carbohydrate increases. statistically one may expect to get a mixture of inactivated and activated sugars in a single spot. for very small sugars that are susceptible to secondary processes that degrade the sugar ring all activity may be annihilated. as has been described above, the photoactive surfaces have been shown to be functional for many carbohydrates, suggesting that at least some of the photoimmobilized carbohydrates within a given microspot retain their activity. regardless of the percentage of biologically active carbohydrates, a discernable fluorescence signal can be detected after staining with antibodies. by chemically derivatizing a sugar with photoactive groups, carbohydrates can be site-specifically photo-immobilized. for example, diazirine derivatized mono-and disaccharides (fig. ) have been photochemically immobilized on diamond [ ] and poly(styrene) [ ] . similarly, mono-and disaccharides derivatized with perfluorophenylazide (pfpa) have been photo-immobilized on peo films that were photo-immobilized on pfpa monolayers [ ] . pfpa loses n upon irradiation to produce a nitrene, which is isoelectronic with carbenes and undergoes similar reactions. mono-and disaccharides immobilized in this fashion are able to distinguish between different lectins. although this approach allows a variety of monosaccharides to be immobilized that have discernable signals after staining with lectins, one of the disadvantages of this approach is the need for derivatizing the carbohydrates prior to immobilization. one could imagine combining this approach with one of the above by incorporating photoactive molecules into a polymer. in this way monosaccharides derivatized with photoactive groups could be spotted along with underivatized sugars, resulting in the photogeneration of a large array of active carbohydrates using only photons as reagents. in principle a photomask could be used in conjunction with a spotter to break an individual spot into many very small spots. in summary, photochemical methods for immobilizing carbohydrate microarrays on a surface allow for a clean and stable binding that can be patterned using either a photomask or robotic spotter. methods that have been demonstrated include the spotting and immobilization of underivatized sugars and the use of derivatized, photoactive carbohydrates for polymer-coated substrates. the latter has the advantage of site-selective immobilization, but has the disadvantage in that each carbohydrate in the array must be chemically modified prior to deposition. regardless of the method used to construct the arrays, it is critical to validate whether the biological reactivities of the carbohydrates are preserved on the array substrates. probing the antigenic diversity of sugar chains carbohydrate antigens (polysaccharides) carbohydrate recognition systems: functional triads in cell-cell interactions the antigens ii, ssea- and abh are in interrelated system of carbohydrate differentiation antigens expressed on glycosphingolipids and glycoproteins progress in deciphering the information content of the 'glycome'-a crescendo in the closing years of the millennium carbohydrate-mediated sperm-egg interaction and species specificity: a clue from the unio elongatulus model aberrant glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives the elaboration of specific soluble substance by pneumococcus during growth the soluble specific substance of pneumococcus polysaccharide-protein conjugates: a new generation of vaccines t cell-independent antigens type sugar-coated microarrays: a novel slide surface for the high-throughput analysis of glycans carbohydrate microarrays oligosaccharide and glycoprotein microarrays as tools in hiv glycobiology glycan-dependent gp /protein interactions synthesis of sugar arrays in microtiter plate oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions carbohydrate arrays for the evaluation of protein binding and enzymatic modification fabrication of carbohydrate chips for studying protein-carbohydrate interactions carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells glycan arrays lead to the discovery of autoimmunogenic activity of sars-cov photogenerated glycan arrays identify immunogenic sugar moieties of bacillus anthracis exosporium glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities light-directed, spatially addressable parallel chemical synthesis photolithographic technique for direct photochemical modification and chemical micropatterning of surfaces lithographic imaging techniques for the formation of nanoscopic features photoimmobilization for microarrays modern molecular photochemistry immobilization of antibodies on a photoactive self-assembled monolayer on gold photolithographic immobilization of biopolymers on solid supports a facile photochemical surface modification technique for the generation of microstructured fluorinated surfaces photoreactions of cyclic imides. examples of synthetic organic photochemistry photochemical micropatterning of carbohydrates on a surface variable region cdna sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. t-dependent analogues of alpha( , )dextran time-resolved, electronspin resonance study of radical species derived from naturally occurring carbohydrates the oxidation of some polysaccharides by the hydroxyl radical: an e.s.r. investigation facile preparation of carbohydrate microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides carbohydrate photochemistry the synthetic potential of phthalimide set photochemistry novel oligosaccharide side chains of the collagen-like region of bcla, the major glycoprotein of the bacillus anthracis exosporium molecular design of functional polymer surfaces glycoprofiling with micro-arrays of glycoconjugates and lectins synthesis and characterization of a photoactivatable glycoaryldiazirine for surface glycoengineering immobilisation on polystyrene of diazirine derivatives of mono-and disaccharides: biological activities of modified surfaces photogenerated carbohydrate microarrays key: cord- -otsrexqu authors: goel, saurav; hawi, sara; goel, gaurav; thakur, vijay kumar; pearce, oliver; hoskins, clare; hussain, tanvir; agrawal, anupam; upadhyaya, hari m.; cross, graham; barber, asa h. title: resilient and agile engineering solutions to address societal challenges such as coronavirus pandemic date: - - journal: mater today chem doi: . /j.mtchem. . sha: doc_id: cord_uid: otsrexqu the world is witnessing tumultuous times as major economic powers including the us, uk, russia, india, and most of europe continue to be in a state of lockdown. the worst-hit sectors due to this lockdown are sales, production (manufacturing), transport (aerospace and automotive) and tourism. lockdowns became necessary as a preventive measure to avoid the spread of the contagious and infectious “coronavirus disease ” (covid- ). this newly identified disease is caused by a new strain of the virus being referred to as severe acute respiratory syndrome coronavirus (sars cov- ; formerly called -ncov). we review the current medical and manufacturing response to covid- , including advances in instrumentation, sensing, use of lasers, fumigation chambers and development of novel tools such as lab-on-the-chip using combinatorial additive and subtractive manufacturing techniques and use of molecular modelling and molecular docking in drug and vaccine discovery. we also offer perspectives on future considerations on climate change, outsourced versus indigenous manufacturing, automation, and antimicrobial resistance. overall, this paper attempts to identify key areas where manufacturing can be employed to address societal challenges such as covid- . coronaviruses (covs) belong to the family coronaviridae which includes four genera: α, β, γ and δ as well as several subgenera and species [ ] . sars cov- is a β-coronavirus with a single-stranded rna genome of ~ kb [ ] . recent topical research has revealed several new covs (three α-coronaviruses, three new βcoronaviruses, and one previously described α-coronavirus) from bats captured from myanmar and future emergence of new diseases caused by these covs due to change of land use has been speculated [ ] . furthermore, newer mutations of the virus that originally spread from wuhan were confirmed as deadlier in some countries compared to others, which has led to added confusion and concern [ ] . sars cov- was first identified from the outbreak of respiratory illness cases in wuhan city in the hubei province of china. initial reports of the virus were made to the world health organisation (who) on december , . this was followed by the who declaring covid- as a global health emergency on january , due to rapid spreading, and a later pandemic declaration on march , . the disease has quickly engulfed most of the world and has caused severe infection to populations across numerous countries as shown in figure . covid- is shorthand for "coronavirus disease " which is caused by the sars cov- virus, also called coronavirus in typical usage. viruses cause disease by binding to receptors on cells in the human body and then replicating at a rapid rate which triggers a variety of pathogenic processes. in the case of covid- , the structures on the surface of the virus bind to receptors in the airway or the lungs of human beings. the lungs may become inflamed, making breathing more difficult. for some people, the infection becomes severe and leads to critical care requirements. the anatomy of sars cov- , including its internal biological structure of spike protein legs, envelope protein, and membrane protein, surrounding the genomic rna is shown in figure (a). the virus shown in figure (a) highlights a large diameter of about . µm with variations in sizes reported by different researchers. the spike-shaped protein legs that make up a portion of the outer capsule of the virus create the crown-like or corona-like appearance. thus, the name coronavirus. the mechanism of infection due to the surface interactions between the spike protein and the lung cells is depicted in figure (b). this understanding was gathered from the genetic analysis which revealed that the mutations located in the spike surface glycoprotein might induce conformal changes and play an essential role in binding to receptors on the host cell (lungs) and determine host tropisms by leading to possible changing antigenicity [ ] . viruses are fundamentally different to bacteria so the classically developed work on nano-structuring and biomimicking surfaces, such as the cicada wing, primarily targeting bacterial killing, should not be confused as a readily available knowledge to kill sars cov- . physically, a bacterium is larger than a virus (the biggest size of a virus is smaller than the smallest known bacteria). bacteria are living cells which are ± nm capable of prolific reproduction independently, whereas viruses are non-living particles, requiring a host cell for replication. also, bacteria possess a cell-wall engulfing a chromosome, whereas viruses consist of genetic material, either dna or rna, covered by a protein coating. bacterial infections can therefore be treated with high success using antibiotic drugs. although some antiviral medicines are now available, the susceptibility of viruses to react to the treatment rate of medicines is significantly reduced as they are non-living. hence, the only long-term option to eradicate viruses is the development of vaccines that stimulate the natural production of antibodies. studies suggest that the source of sars cov- could either be ratg from horseshoe bats (rhinolophus affinis), pangolins (manis javanica), or a mix of both. their transmission has occurred by zoonotic mechanisms [ , ] . however, the coronavirus isolated from pangolins is % similar in a specific region of the spike protein, which corresponds to the amino acids involved in the angiotensin-converting enzyme (ace ) receptor binding domain, which allows the virus to enter human cells to infect them as shown in figure (b). the virus ratg isolated from rhinolophus affinis bats is highly divergent in this specific region (only % similarity). this observation indicates that the coronavirus isolated from pangolins can enter human cells, whereas coronavirus isolated from rhinolophus affinis bats is unable to enter human cells. the sars cov- has a high transmissible efficiency and covid- has high morbidity and mortality. a popular but possibly flawed measure for assessing fatality of disease is the use of deaths/case counts. this measure would yield a fatality rate of . % for covid- as of th may [ ] . the problem with this measure is that case counts reflect the number of tests that were done rather than infections, and the deaths lag the cases because fatality (if observed) may happen several days after the case is identified. a lag in reporting case numbers and incorrect tests may also occur. an alternative measure is the case fatality rate (cfr), which is the ratio of deaths / (deaths + recovered cases). this measure would yield a fatality rate of . % [ ] . the consensus is that the covid- disease has high fatality and can exceed the fatality ratio of the century-old "spanish flu", which had a % cfr [ ] . however, the data analysis of callaway et al. [ ] shown in figure suggests that covid- 's cfr is lower than that of mers and ebola and that its infection rate (r -the expected number of cases directly generated by one case in a population where all individuals are susceptible to infection) suggests that the infection can spread more easily than other diseases, including seasonal influenza. a further comparison of three different episodes of epidemics and pandemics caused by the family of coronavirus, namely, sars cov- , mers and sars cov- , is depicted in table . the exact mechanism of these drugs towards the virus is yet to be completely understood. however, the mechanism where a drug affects the replication of viruses in-vivo is a broadly accepted concept. in general, viruses replicate via protein processing using endosomes within golgi bodies. a drug such as hcq increases the ph of the golgi bodies making them more basic, thus disrupting the integrity of the internal nucleocapsid protein (see figure a ). this denatures the protein of the coronavirus rendering it dysfunctional. therefore, the rationale for using hcq is built on the premise that the change in ph brought about by the drug inhibits the endosomal transport necessary to spread the infection, hence the patient recovers. another short-term treatment being implemented by some hospitals across the world is to infuse patients with the antibody-rich blood plasma of people who have recovered from covid- [ ] . this approach has been used during disease outbreaks for over a century. however, the approach carries significant risk in terms of the already immunocompromised patients' immune response after administration, and results may vary between patients. as an early effort of investigation of the problem from scratch, researchers at toho university in japan used high-sensitivity cameras and laser beam guidance experiments to deduce that saliva spray during a sneeze (potentially containing thousands of viruses) can be classified into large vs small droplets or droplets vs aerosols. the droplets, due to their heavier weight, fall off, whereas aerosols remain airborne for a few hours due to their relatively small size. prima facie evidence suggests that the coronavirus has escalated to a pandemic due to the high contagion occurring via this 'airborne' spread model. recently, the possibility of asymptomatic or oligosymptomatic infection has also been highlighted [ ] . the aerosols may circulate near an infected patient in an airborne condition depending on the local conditions (airflow rate, humidity, dryness) for up to a few hours, even after the infected person has left the location. hence, the chances of contracting coronavirus are relatively high by merely occupying the same vicinity where an infected person has been or passed through. researchers have experimentally evaluated the stability of sars cov- recently, and these comparisons are shown in figure [ , ] . it is noteworthy that sars cov- was stable in the aerosol (airborne) form for up to hours. as opposed to this, the virus appears to be stable on surgical masks or stainless-steel surfaces for up to days and for up to days on smooth surfaces such as glass or currency notes. the same research also shows that the application of hand soap does not immediately deactivate the virus but rather takes up to minutes. therefore, a -minute waiting period after a handwash is required before bringing hands in contact with face, mouth or nose. covid- is not only present in the airway secretions of infected patients when they sneeze, but also when they simply breathe out or speak. studies have shown that covid- is also present in other body fluids of infected people, such as faeces, blood [ ], oral fluids, anal secretions [ ] , tears [ ] , and urine [ ] . the virus primarily attacks the respiratory system, however, new evidence shows the virus is not filtered by the kidneys, as traces of virus can be seen in sewage systems [ ] . therefore, as a mitigation strategy, testing of these fluids, which can be available and abundant in the wider population can be done for identifying the most effective areas. overall, when people are in close contact with one another, then transmission is more likely. most sources for infection worldwide have happened in an enclosed space, including homes, offices, public transport and restaurants. a second spreading pathway for the virus is through touching a surface or object that has the virus on it and then touching one's own mouth, nose, or eyes. if one is in a wellventilated space with fewer people, even for a longer period, the risk of infection is low. sustained contact with an infected person, even for a short period, even without a cough or sneeze, can spread the infection. by the virtue of disturbing the cell programming, covid- possesses the capability to cause a surprise attack on almost any part of the body ranging from lungs, heart, blood vessels, brain, eyes, nose, liver, kidneys and intestine. a schematic representation of this adverse impact is shown in figure (a) [ ] . recent literature suggests that apart from the airway, the human brain may also be targeted by the virus -see figure (b) and figure [ ] . patients have reported having mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if true, it makes sars cov- more lethal. [ ] (b) tissue distribution of ace receptors in humans [ ] (c) possible neuro disorders due to sars cov- [ ] and (d) life cycle of sars cov- in host cells [ ] . (figures reprinted with permission) our nasal lining tissue contains a rich number of cell receptors called angiotensinconverting enzyme (ace ), which are favourable sites for the sars cov- to attach its spiked protein to, thus paving way for the entrance of the virus inside the body. once attached to the cell, sars cov- can change cell programming, replicate itself, and attack new cells at a very rapid pace. within a week of entrance into the body, the virus exhibits effects. during this time, the person may show early symptoms such as fever, dry cough, sore throat, loss of smell and taste, or head and body aches. at this point, the failure of the immunity in defeating the virus means giving the virus a way forward to enter into the respiratory system, where the lungs are attacked. the lungs also have a cell lining rich in ace [ ] . as the immune system continues to fight the sars cov- , the supply of healthy oxygen is disrupted. as the disease progresses inside the body, the white blood cells release inflammatory molecules or chemokines, which attack and kill the virusinfected immune cells, leaving the dead cells and pus behind. this affects healthy oxygen transfer in the lungs and is the stage where the patient starts to complain of pneumonia, coughing, fever, and rapid, shallow respiration. acute respiratory distress syndrome may develop. at this stage, the lungs start to be filled with opaque black spots signifying closure of the air pores, and such patients require ventilator support. the survivors of this severity of infection can end up having long-term complications. a few hospitals have successfully applied artificial intelligence to monitor the recovery rate by monitoring the coronascorea general term used to quantify the extent of the blocked pores (in cm ) [ ] long-term research measures, advanced manufacturing and metrology have pivotal roles to play in containing a pandemic. specifically, the development of engineering innovations is timely for the control of the covid- pandemic. for diagnostic testing to be useful for informing doctors and governments about the true incidence at any one time of coronavirus in the population, it needs to be inexpensive, high in sensitivity and specificity (sensitivity refers to the ability of a test to correctly identify those with the disease or true positive rate, whereas specificity refers to the ability of a test to correctly identify those without the disease or true negative rate) and easy to use for non-experts. as doctors are treating infected patients in this emergent time in an unprepared state, emergent manufacturing measures can help design better testing equipment and thus help in both short and long term to tackle this problem. the most significant challenge now lies in probing early-stage symptoms of covid- . for a relatively late stage detection in severe cases, chest computer tomography (ct) using x-ray probes (providing > % sensitivity) has proved to be a more reliable test assay in comparison to the reverse-transcription polymerasechain-reaction (rt-pcr) test and other sensor-based detection methods currently being pursued [ ] . since the nervous system and respiratory dysregulation are both likely to co-exist, covid- testing should be done by combining brain mri and rt-pcr tests as shown in figure (a) [ ] . however, caution is required in the analysis of results, such that covid- should not be confused with drug-resistant tuberculosis (tb) as both would show symptoms of damages in the lungs. in an attempt to boost the testing in a populous country such as india, the indian council of medical research (icmr) has approved the use of diagnostic machines used for testing drug-resistant tuberculosis under the guidance that the throat/nasal swabs are collected in the viral transport medium [ ] . interestingly, the pcr test cannot identify asymptomatic infections or those people who were exposed to or infected with covid- in the past and did not suffer from the disease or have recovered from covid- and may still be spreading the virus (carriers). therefore, different tests may be required to collate vital information required by government bodies to carry out a risk-benefit analysis to relax lockdown measures to protect their economies. immunodiagnostic kits such as a lab-on-a-chip are being developed for asymptomatic detection of covid- [ ] . a lab-on-chip (figure (b) works on the principle of detecting the presence of patient-generated antibodies against the virus that causes a specific disease, in this case, covid- . the test uses a lateral flow immunoassay to assess the presence of an analyte from a patient sample or specimen and detects two types of antibody isotypes: igm and igg. igm antibodies are the first antibodies to appear in response to a novel antigen and imply a more recently initiated infection while igg antibodies are generated later in the course of infection and possess a higher affinity for the target antigen, meaning they are more specifically able to bind the substance which caused the immune response. a test is declared positive if either one or both antibodies are detected during the test, similar to widely used pregnancy tests. the test consists of an anti-human igg coating in the g test line region and an igm coating the m test line region (see figure (b). during testing, the sample (blood, urine etc) reacts with sars-cov- antigen-coated gold nanoparticles (aunp) in the conjugation pad of the test cassette. any antibody in the patient sample that recognises the sars-cov- antigen binds to the antigen-aunp complex. the mixture then migrates laterally across the membrane by capillary action/lateral flow. as these human antibody/antigen/aunp complexes move across the test lines, they are captured at the anti-human igm 'm' line, the anti-human igg 'g' line, or both, depending on the antibody contents of the specimen. the sample first reaches the anti-human igm antibodies which coat the m line. if the specimen contains igm antibodies to sars-cov- , a coloured line will appear in the m test line region. next, the sample reaches the anti-human igg antibodies which coat the g line. if a specimen contains igg antibodies to sars-cov- , the conjugate-specimen complex reacts with anti-human igg. a coloured line appears in the g test line region as a result. the rabbit igg-aunp complexes are captured by the control line (which contains anti-rabbit-igg). this visible line indicates that there has been successful lateral flow across the detection strip. the last check ensures that the sample had enough volume to move across the entirety of the test cassette. only human antibody/sars-cov- antigen/aunp complexes will produce a visible red or pink line at the m or g line. other antibodies produce no colour. the accuracy of these lab-on-chip tests is still being debated as it depends on two key parameters, sensitivity and specificity. to date, igg related sensitivity and specificity has been found to be higher than that of igm. a schematic diagram in figure (c) and figure (d) shows that unlike currently available diagnostic methods, field-effect transistor (fet)-based biosensing devices may have several advantages, including the ability to make highly sensitive and instantaneous measurements using small amounts of analytes [ ] . the fet sensor shown in figure (d) makes use of a graphene sheet since graphene-based fet biosensors can detect surrounding changes on their surface and provide an optimal sensing environment for ultrasensitive and low-noise detection. from this standpoint, graphene-based fet technology is attractive for applications related to the sensitive immunological diagnosis. graphene as a sensing material is selected, and sars-cov- spike antibody is conjugated onto the graphene sheet via -pyrenebutyric acid n-hydroxysuccinimide ester [ ] (figures reprinted with permission) a group of researchers at mit and harvard are developing coronavirus-identifying sensor embedded face masks. this ongoing work is an extension to their previous work where they developed a low-cost method to detect a zika virus [ ] . in this technique, the sensor is made by using genetic material such as the rna or the dna which binds to the viruses. the researchers used a lyophilizer to freeze-dry the genetic material onto the fabric of the mask. the material deposited onto the fabric remains stable for many months (at room temperature) and the detection process starts merely by the presence of moisture (such as saliva). the detection signal is small (in terms of voltage) and can be detected by an additional fluorimeter device that can quantify this signal and emit in form of a fluorescent light. thus, one can expect to see light glowing masks to detect coronavirus in the future. fumigation chambers allow quick disinfection of a person while visiting areas such as hospitals, universities or airports. the aim is to use tubes releasing hydrogen peroxide in a chamber designed to be five-feet wide and seven-feet tall. the fumigation chamber usually seals automatically after the person's entry to avoid any leakage outside of the chamber and has designated sensors facilitating the chamber entry. the disinfection lasts for five seconds. it is to be noted here that hydrogen peroxide has also been recommended by the food and drug administration (fda) to be used as a sterilisation material to decontaminate n or n -equivalent respirators for reuse by health care workers in hospital settings [ ] . a populist view is that sunlight and high temperature during peak summers kills the coronavirus, which would help containment of its spread. it is reported that sars cov- does not survive beyond min after being exposed to ºc [ ] . however, it has yet to be ascertained as to how long does sunlight take to deactivate sars cov- and at what intensity. a more specific question is whether ultra-violet (uv) light can kill coronavirus? sunlight usually contains three types of uv: uva ( - nm), uvb ( - nm), and uvc ( - nm). uva and uvb can both cause sunburn, however, uvc is shorter and a more energetic wavelength of light. while uvc is effective at destroying genetic material, whether in humans or viral particles, it is filtered by the ozone layer and does not reach the earth's surface. preliminary findings from the national biodefense analysis and countermeasures centre of the usa (which houses a bsl- level lab) indicate that "sunlight seems to be very detrimental to the virus… within minutes, the majority of the virus is inactivated on surfaces and in the air in direct sunlight." [ ] . research on the use of uv as a treatment is still evolving and the behaviour of sars cov- under uv light is unknown. results were relatively favourable for sars cov- treatment with uvc [ , ] . sars cov- was efficiently inactivated after minutes of uvc exposure (at about a wavelength of nm), whereas addition of psoralen (a light-sensitive drug) to uva enhances inactivation of the sars cov- . popular press reports suggest that uv light booths are capable of deactivating coronavirus without any human contact and are proposed to be deployed ( figure ). one must be cautious, since uvc can be dangerous to the skin, causing burns within seconds and harmful to the eyes if observed directly. therefore, risk assessment and associated precautions would need to be deployed that may render this strategy difficult in the short-term. . . . antimicrobial coatings chouirfa et al. [ ] summarised nanomaterials based coatings for antibacterial applications and xing et al. [ ] have summarised the potential of natural polymer chitosan as an antimicrobial agent ( figure ). their research on antimicrobial properties of a nanocomposite coating formed by polysaccharide -deoxylactit- -yl chitosan (chitlac) and silver nanoparticles (nag) on methacrylate thermosets showed satisfactory results. figure : antimicrobial mechanism of chitosan-based coating with antimicrobial agents. reprinted with permission from [ ] . antimicrobial surfaces are based on three main mechanisms (see figure ): the anti-biofouling mechanism which repels microbes and prevents them from adhering to the surface, the release-killing mechanism where microbes are killed in the nearsurface environment with a release of antimicrobial agents and the contact-killing mechanism where microbes adhere and are killed on the surface [ , ] . researchers have also experimented with developing long-lasting antimicrobial surfaces to stop spreading pathogenic microbes through commonly touched surfaces or at-risk surfaces and have focused on the use of antimicrobial/antiviral materials such as copper. experimental trials for copper oxide impregnated respiratory protective facemasks have yielded % efficiency in eradicating the infectivity of human and avian influenza a virus in simulated breathing conditions [ ] . warnes et al. [ ] have shown that the surfaces of dry copper alloys are lethal to viruses such as mnv- at room temperature, with higher copper content being more time-efficient. generally, the antimicrobial activity of copper is attributed to oxidative behaviour of copper and the solubility properties of copper oxides [ ] . more recently, surface texturing of copper via cold spray methods has shown enhanced promise as an antiviral agent [ ] . combining the aforementioned antimicrobial contact-killing properties of copper, the anti-adhesion properties of polymeric micelles and the release-kill abilities of chlorine dioxide (clo ), li et al. [ ] developed a multifunctional coating viricidal for influenza virus h n . this multifunctional coating is composed of clo encapsulated in polymeric micelles (with slow on-demand release) on which copper nanoparticles were covalently tethered. other studies have investigated hydrophobic polycationic coatings as antimicrobial coatings [ ] [ ] [ ] . hsu et al. [ ] investigated the mechanism by which the n,n-dodecyl,methyl-polyethelenimine (pei) coated surfaces killed the influenza a virus. it was concluded that upon contact with the coated surface, the virus adheres irreversibly and through hydrophobic and electrostatic interactions, the virus' disintegration is then initiated resulting in rna leakage into the solution. the incorporation of pei into protective mask textiles has also been investigated with nearly a -fold improvement in the capture of the t d bacteriophage virus of escherichia coli b [ ] . finally, the direct mechanical action of sharp nanostructures such as darts, blades and spikes as a kind "mechano-cide" has been shown to have some success for bacteria, but remains largely unexplored for viruses [ ] . from a contact mechanics perspective, the combined effect of sharp ( somewhat surprisingly, while researchers have investigated bactericidal properties of nanoparticles of silver, yet the current understanding of the interaction of these nanoparticles with viruses is limited. however, some positive results are reported and, based on these reports, this direction of manufacturing research holds a good promise to tackle sars cov- . previous studies showed that the size of nanoparticle is critical to manifest a viricidal effect. for example, a nanoparticle size of less than nm was found to be effective against tacaribe virus [ ] , while a particle size of between to nm worked well against herpes simplex virus (hsv) type / and human parainfluenza virus type- [ ] . nakamura et al. [ ] reported that materials with immobilised silver nanoparticles possess enhanced microbicidal activities against the virus. these researchers developed a material using silver nanoparticle absorbed on a chitin sheet having a nanoscale fibre-like surface structure shown in figure and obtained favourable results against h n influenza a virus. figure : the mechanism of viricidal activity of silver (ag) nanoparticles (np) chitin nanofiber sheets showing strong antimicrobial activity via reactive oxygen species (ros) and silver ions on the substrate. reprinted with permission from [ ] . moreover, the unique characteristics of metallic nanoparticles such as high surface to volume ratio, surface-enhanced raman scattering and localized surface plasmon resonance can be utilised for virus detection and therapy via destruction through laser-induced localised hypothermia as well. some of the candidate nanoparticles are metallic and high entropy alloy nanoparticles (agauptpdcu high entropy nanoparticles, feo nps, ag nps, tio nps, au nps, ag-au core-shell nps) [ ] . for this purpose, uv-visible spectroscopy can be deployed to study the surface plasmon resonance, refractive index, and fluorescence change when nanoparticles interact with virus particles. nanoparticle-based investigation can help detect and inactivate viruses (figure ). specific to coronavirus, graphene oxide sheets with silver nanoparticles (go-ag nps) were reported to inhibit the growth of feline coronavirus (fcov) by up to %, in comparison to pure go that inhibited it only up to % [ ] . additionally, chiral biosensor with self-assembled chiral gold nanohybrids (cau nps) on account of multiple plasmonic scattering showed better detection performance on coronavirus [ ] . figure : nanoparticle based therapy for sars cov- . reprinted with permission from [ ] as with all fundamental and translation nanomaterial research, progress is not immediate. caution is required with nanomaterial technologies, as the long-term impact on human health and the environment of free nanoparticles has not been ascertained, and this may ultimately pose greater risk, particularly in the liver and respiratory tract of human beings and in ecosystems. lack of governmental regulation strategies for nanomaterials can also hinder the speed of approval and ultimately the availability in the marketplace. nevertheless, a call has gone out to nanomedicine researchers to utilise their existing knowledge base and translate their technologies towards covid- , should the outbreak last more than months [ ] . filtration efficiency is a strong measure of penetration prevention of aerosols through mask filters and is usually required to be above % for surgical face masks [ ] . ultrasonic welding is usually deployed to produce filters with sufficient filtration efficiency. both polymer and textile-based masks are popular and can benefit from the adoption of sequential micromachining techniques [ ] . a surgical mask (procedure mask) is worn by health professionals during surgery to avoid exposure to aerosols. such masks are not designed to protect the wearer from inhaling airborne bacteria or virus particles and are less effective than respirators, such as n or niosh masks which provide better protection due to their material, shape and tight seal. the who laboratory biosafety manual necessitates biosafety level (bsl- ) requirements for non-propagative diagnostic laboratories and bsl- for laboratories handling high concentrations of live sars-cov- . according to the manual and the who biosafety guidance for sars-cov- , the exhaust air from such laboratories should be discharged through high-efficiency particulate air (hepa) filters. it is worth mentioning that particle collection efficiency of such mechanical filtering methods decreases to about % at particle sizes of . µm due to diffusion and diffusioninterception regimes of particles with sizes in the range from . up to µm [ ] . therefore, hepa filters are not ideal in screening the viruses like sars cov- which has a typical diameter from nm to nm [ ] . mass production of appropriate filters to screen the virus entry is a micromanufacturing challenge. this could potentially involve technologies such as direct laser micromachining or punch-based microstamping methods. for this purpose, a plasma spraying, or laser micromachining method may be used to obtain a well-suited punch which can be used to stamp (pierce) polymer sheets (e.g., pet), to achieve appropriate filter sizes. another candidate process of subtractive manufacturing is metal anisotropic reactive ion etching with oxidation (mario) [ ] . an illustration of how the proposed micromachining strategies can be deployed for scalable fabrication of filters to screen virus scale particles is shown in figure . while self-assembly techniques such as block copolymers have shown promise filtering small viral particles such as human rhinovirus [ ] , direct printing methods allow greater engineering control over pore size and spacing, critical to tuning membrane efficiency, fluid resistance and mechanical strength. protection of surrounding or nearby people is important and an infected patient can help to control the spread by wearing a mask, leading to the concept of social distancing suggested by various governments. the safe distance guideline is an important concept especially in populous countries where an assembly of people on the streets can cause the spread to grow exponentially. keeping this in mind, the who urged people to maintain a safe social physical distance of about ft (~ m), while the centre of disease control and prevention recommends this to be ft (~ m). these limits are now challenged by recent research that suggests this safe distance should be to ft ( to m) [ ] . the same study also suggested that the currently available commercial masks need to be redesigned. it was suggested that the masks available at present are not suitable for containment of sprayed aerosols (during a sneeze) travelling at the velocity of kmph [ ] , as this increases the possibility of escape of viruses through contaminated droplets from edges of the mask making nearby people more vulnerable to contracting the coronavirus. also, chin et al. [ ] tested the strength of the virus and suggested that the virus is highly stable at ºc as well as in the ph range of to . the virus was found to be detectable on a mask surface even after days. these findings suggest a product design strategy to develop more appropriate masks for handling exhalations travelling at high speeds by considering human ergonomics, material aspects, antimicrobial nature and structural aspect. while the design and strategies for developing these new types of masks are underway, a possible technique for deploying the extant fabrication methods would be to merge the two approaches, namely, additive and subtractive manufacturing methods described above. this development would mean that even in the current landscape, a new mask-making strategy would provide more protection than the currently available masks. astm f - is the relevant international standard that details specifications of materials to be used in medical face masks. however, the current standards do not capture the techniques required to make the mask reusable especially as the current materials are yet to be tested against sars cov- . as of now, curad antiviral isolation masks making use of a cocktail coating (citric acid, zinc and copper) are available as an option to disinfecting the outer surface of the mask. these are useful for medical professionals as they can come in contact with the virus infected aerosols more frequently. another concept is that of a 'germ trap' surface [ ] , which is akin to fooling a virus. the team at the university of manchester, uk identified specific glycoproteins that have carbohydrates attached to their surface, similar to those seen on the surface of the cell of the nasal passages. this study suggests that a glycoprotein biocoating on a snood surface [ ] denatures a virus by causing the same interfacial chemical reaction between the spike proteins and carbohydrate cell surface as the one when the coronavirus attaches to the ace receptors, but due to unavailability of any physical cells to invade on the snood surface, the virus decomposes and becomes deactivated immediately after landing on the textile surface (see figure ). the efficiency of germ trap coating in achieving its goal is indicated to be %. based upon the existing literature, an immediate measure to functionalise the personal protective equipment's (ppes) could be to introduce the use of antimicrobial coatings comprising of proven materials (e.g., zinc and sodium [ , ] based compounds) on the surface of ppes, especially the shields and visors. such coatings can be deposited by additive methods such as spraying (in d) or via roll to roll (r r) manufacturing. in terms of processing techniques, one limitation of thermal spraying is that it requires heat resistant feedstock materials, limiting the use of most of the chemical species traditionally used to functionalise surfaces, such as polyethylene glycol (peg) [ ] . in view of this limitation, a possible research avenue is to develop functional coatings with viricidal properties such that the coating ingredients do not degrade at high temperatures, thus providing a controlled release over time. with this goal in mind, a suitable processing and material matrix availability is crucial to obtain a coating to possess desirable advantages in the medical field. on the other hand, the use of thermal sprayed hydroxyapatite (ha) coatings on orthopaedic implants has been widely adopted in the medical field since the late s [ ] . as reported in the early s [ ] , the use of thermal sprayed ha coatings on metal implants presents several advantages and a positive potential for its use in the medical field [ ] . taking into consideration the status of ha as the standard in thermal sprayed coatings and the decades of research on its behaviour and response to living tissue [ ] , the choice of ha as a base material for the development of anti-microbial functional coatings has been most popular to date. at a personal level, deploying smart ppe incorporating viral detection capability into masks, gloves or wearing "viral dosimeter" badges might help monitor the strength of direct transmission vectors as well as environmental exposure rates and accumulation. conversely, masks could collect exhaled viral particles for early detection of infection [ ] . detection could be implemented as microfluidic channels functionalized with bioelectronic miniaturized detection schemes [ , ] with specific virus sensitivity [ , ] . local area smart sensing filter textiles incorporated into mobile or in-place ventilation systems designed to both filter and detect could also help provide early alert alarms and protection in buildings and other areas of restricted air replacement. the importance of wearing a mask by the infected person and by a healthy person coming in the vicinity of the infected person is ranked on the priority order shown by the cartoon model in figure . safe disposal of materials used in the treatment of infected patients such as gloves, masks, test samples, clothes or the human waste, especially in icu patients who may be closer to death is a challenging task. in addition to the possibility of an infection, this waste could lead to other secondary issues including emergence of a new category of virus/bacteria. hence, safe disposal of biomedical waste is very important. additionally, gaining support from members of the public to adequately dispose of their used personal masks etc. is essential. recently, an absorbent gel with embedded disinfecting material has been developed at sree chitra tirunal institute for medical sciences and technology, india for liquid respiratory and other body fluid solidification and disinfection for the safe management of infected respiratory secretions. this material helps to solidify the liquid respiratory secretions from icu patients or those with copious secretions treated in the wards. the material so collected becomes fit for disposal through the usual incineration system for biomedical wastes. newer guidelines are required to refine the existing risk assessments procedures [ ] . big data analytics and extant research utilizes accelerated development of techniques for data mining, machine learning and use of artificial intelligence (ai) that shows promise in the treatment of sars cov- . as a direct benefit to this approach, a digitalised shadow of the data to study plausible scenarios of certainty of an event becomes easier. as for sars cov- , genome data now exist offering scope for soft-computing application researchers as well as those involved in ai based research to offer new insights into the area. as an example, the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org), made available in march , contains a compilation of over , sars-cov- complete and partial genomes from all over the globe since december [ ] . recently, researchers from cambridge used phylogenetic network on complete genomes of sars cov- [ ] to predict probable mutation and migration paths of the coronavirus. atomistic modelling and molecular docking [ ] can hold the key to a scientific breakthrough to address questions surrounding sars cov- . the development of vaccines, antibodies and diagnostics is dependent in a large measure on our understanding of the interfacial science between the spike (s) glycoprotein of sars cov- and ace receptor in human lung cell (as shown earlier in figure ). wrapp et al. [ ] obtained a cryo-electron microscopy structure of the sars cov- s trimer in the metastable prefusion conformation, showing it undergoes a transient (hide and active states) structural rearrangement. their results relying on surface plasmonic resonance elucidated a stronger affinity of sars cov- with ace compared to sars cov- , and shed light on two states, namely, a "down state" or receptorinaccessible and an "up state" or receptor-accessible state shown in figure in yet another interesting study [ ] , computer-aided drug screening was used to run a test assay over , compounds as inhibitors to a key cov enzyme, m pro . ebselen was found to exhibit antiviral activity and illustrate a way forward use of this potentially promising modelling strategy to discover targeted drug and vaccine down: transient hidden state of rbd up: active binding state of rbd development. a molecular docking example shown in figure (b) is yet another effective example of accelerating the drug discovery [ ] . recently, liu et al. [ ] carried out an aerodynamic analysis by measurement of the rna of sars cov- . they used pre-sterilized gelatin filters (pore size of μm) to collect the aerosols from different areas of two wuhan hospitals. this study classified the sampling location in three areas: ( surprisingly, the researchers detected maximum stains of sars cov- in patients' toilet areas, while the levels were low in the isolation wards and ventilated patient rooms. also, the medical staff areas showed peaks of high concentrations with aerosol size ranging from . micrometres to . micrometres and they were neutralised after rigorous sanitisation procedures. the study suggests that there is a likelihood of resuspension of virus-laden aerosols from the surface of medical staff's protective equipment including surgical masks during their removal while having lunch, visiting toilets or breaks etc. the source of these virus suspensions was speculated to be from the direct deposition of patient's respiratory droplets or airborne sars-cov- onto the protective apparel. the researchers also found that the sars-cov- in aerosol forms had relatively longer residence time, implying the infection remains for a relatively longer time causing further transmissions. this study serves as a practice guide on the requirements and specification after the lockdown procedures are eased to avoid recurrent infection. the exit-strategy after the lockdown must consider carefully the ventilation of offices/classrooms, maximum use of open space (preferable sunlight), regular sanitisation of clothes, hairs and hands, and proper use and disinfection of toilet areas. the data obtained by toho university (figure ) suggests that laser technology can be employed efficiently to monitor the streamlines and microdroplets movements from a sneeze. this monitoring could be a highly effective lab-scale activity to not just guide the futuristic cfd models but also to efficiently design new masks effective for preventing diseases caused by high velocity spray caused by a sneeze. while engineering and manufacturing will undoubtedly play their part over the coming years, the interventions proposed herein can help avoid the recurrent spread and emergence of new infections. the patients who have survived while battling against covid- showed that they have developed antibodies to the virus. antibodies are virus-specific proteins, which have a memory of the exposure to the virus and will recognize the virus on a second exposure. antibodies help prevent future infections by detecting the virus and binding to their surfaces signalling the body's immune system to destroy such viruses or virus-infected cells. while reasons are not clear, common wisdom is that the human body does not have an adequate immune response to hiv. for covid- , an adequate immune response does exist. therefore, a vaccine is more likely to be created successfully for sars cov- . additionally, the observation that a large majority of people recover, either without any symptoms or with minimal symptoms such as fever and body ache, also bodes well for the development of a vaccine. three major scenarios are currently being pursued or envisaged in tackling the disease. (i) research and trials on vaccines: clinical trials are being conducted across the globe into novel vaccines and fast-tracking approval processes are being deployed. however, scientists fear that the virus itself, like the flu, can mutate and form different strains. therefore, immunity after immunisation or contracting and surviving covid- may only last as short as two years. (ii) therapeutic trials using available drugs: attention is being focused to drug repurposing and advanced formulations, including antimalarial, hiv, and other antiviral and immune-modulating drugs and biomolecules. (iii) nanotechnology: study of molecules that show promise but exhibit poor physiochemical properties or toxicity is currently being formulated across the globe into advanced nanomedicine platforms, translating the knowledge gained in drug targeting and efficacy enhancement from conditions such as cancer and cardiovascular disease. such technologies aim to provide better treatment prognosis for those patients who fall ill with coronavirus related diseases. attention to immunemodulating molecules is growing, as patients who contracted severe forms of covid- were reported to experience notorious cytokine storms which ultimately m m lead to their death. ability to suppress such immunological responses could be one key to controlling the progression of the virus from mild to severe infection. lessons learned during this pandemic will also impact the outlook for other viruses. these include governmental pandemic funding allocation, stockpiling of medical protective equipment and emergency aid strategies within and between countries across the globe. as the coronavirus problem increased across the globe in , many questions have been raised regarding what more governments could have done, why they did not act more quickly, and what were the fundamental failures within their policy. over time, addressing these issues will pave the way for a more streamlined and effective future response. in addition to this, contact tracing technology is being heavily invested in across the globe, with the intention that citizens would be able to download mobile applications, which could inform them (and the authorities) if they had been exposed to another person who tested positive to covid- . whilst early trials seem positive, the longer-term question remains over privacy and ownership of personal data collected by such applications. access to death records inside countries and across continents will allow identification of high-risk population categories which will aid risk prevention. the positive news is that this pandemic has cemented the importance of scientific integrity, the inclusion of scientific advisors into government, and the role of science in society. while this may offer little solace at such an upsetting time, yet, with the power of science, this disease will be managed unlike many other viral threats over the years. currently, vaccine trials are ongoing at various phases of progress in different countries (see table ) and good news may be expected soon. [ ] . the world has seen a surge in the use of digital tools being growingly used for teaching, research, consultation, banking and day to day activities. digital health innovation has been a prime example. these approaches culminated in the development of telemedicine consultation. the current situation has also driven many changes in work-life routines such as less travel and digital technology-based remote work. the coronavirus pandemic has brought significant disruption and caused severe emotional, sentimental and financial losses. there is even a chance that life on the planet will never be the same again and many stringent safety measures will continue to be followed in the time to come while using public transport, air travel, or while visiting touristic places. the continued extension of lockdowns (e.g., lockdown . to lockdown . in india) has resulted in making people restless when staying indoors. countries who were not very resilient in adapting to the situation even saw disrupted supply chains, disturbed migrant workers, lack of food and other supplies and unavailability of essential items -a situation comparable to a natural disaster. however, every disaster brings forth a new cycle of life and the episode of covid- did bring some good things. countries facing severe pollution in air, water, and land saw a life not seen in the last many decades. social websites and digital tools were used heavily to share the beauty of nature. rivers got cleaner, the air became more breathable, and the sky got clearer [ ] . as a result of this transition, even climate change and transition in weather were observed in many places and it will be befitting to say that we witnessed how "nature self-heals and mends itself". a prime example of climate change as a result of lockdown can be seen in china, europe and in india (see figure ). india has faced two major challenges for many years in the wake of rapid urbanisation: alarming levels of air pollution and a rapid decrease in the availability of clean water, especially from rivers like the ganga. the government of india had earlier set up a mega project called "namami gange" [ ] for freeing the rivers from industrial wastes. the project is of such importance that when addressing the indian community at madison square garden in new york in , the prime minister of india mr narendra modi had said, "if we can clean the ganga, it will be a huge help for % population of the country." covid- has rejuvenated the ganga and many other rivers around the world, making them cleaner to a point that their water was reported to be potable [ ] . the learning from the episode of covid- has been that resources offered by mother nature are meant to be used and not to be "exploited". if this simple rule is forgotten, then the world may continue to witness a regular self-healing cycle. on this occasion it was covid- but the next time, it could be readjustments in the ecosystem caused by a surge in the carbon emissions. researchers are reporting a decrease in solar activity leading to an increased flux of energetic particles in our galaxy [ ] . before it is too late to realize, and the entire human race wipes-off the face of the earth due to the climate change, we must take this pandemic as a wake-up call towards being considerate to the environment. what has apparently come clearer though is that problems like biohazards primarily emerge due to our lifestyle (e.g., consumption patterns including eating habits) or mishandling of biowastes (see figure ). there is an absolute necessity to pay utmost attention to biosafety and implementation of effective iso standards worldwide. health of people is also closely connected to the health of animals and our shared environment. this is because people, animals, plants, and our environment are interdependent [ ] and this direction of research considers the concept of "one health". in the wake of the covid- pandemic, the world witnessed an all-time-high demand of ventilators, ppe's, masks, bed liners, other essential health supplies and medicines. the pandemic struck everywhere, including the largest manufacturing economiesand pretty much all at once. as a result, most nations experienced manufacturing shocks. the two shocks that have impacted manufacturing worldwide are: • supply shock: the containment measures (such as lockdowns and social distancing) kept the workers away and resulted in reduced productivity and output. • demand shock: customer consumption patterns started changing (e.g., movement away from "non-essentials") and this affected the demand for a large variety of manufactured goods and associated services. as the pandemic spread, a few countries were resilient enough to cope with the pace of transformed supply chain requirements while many others who had earlier (in pre-covid- era) aligned themselves to outsourced or cloud-based manufacturing, primarily driven by low-cost, became hugely dependent for their essential supplies on other countries (primarily china the manufacturing related lesson learned from the covid- episode is that pandemics can affect manufacturing in several geographical locations simultaneously and therefore can affect an individual nation's surge capacity to deliver essential supplies such as diagnostics, drugs and vaccines to its population. going forward, this lesson will critically affect thoughts on manufacturing strategy and low-cost based outsourcing, and we may see a sustained push towards development of resilient indigenous manufacturing capabilities and a decreasing reliance on low-cost based outsourced manufacturing. a second trend that we perceive is an increasing focus on digitised automation and use of automated production systems including the use of robotics in day to day life as well as deployment of embedded robotics in manufacturing operations. an example of a futuristic automation capability was displayed at the indian institute of technology guwahati who developed remote controlled food delivery robot for covid- isolation wards (see figure ) . the robot was designed for hours run when fully charged. antimicrobial resistance (amr) refers to the resistance of microbes to antimicrobial drugs or "microbial immunity". the who as well as several national health organizations have identified antibiotic resistance as one of the greatest threats to global public health, economic growth, agriculture, economic security and national security. around the globe, people are being admitted to hospitals with infections that do not respond to antibiotic treatment. the problem of amr is rooted in the fact that when microbes are repeatedly exposed to antibiotics, they mutate to produce strains that are resistant to the antibiotic and are therefore able to resist the effects of medication that could successfully treat the microbe. in , lord jim o'neill and his team published a review commissioned by the united kingdom government entitled, "antimicrobial resistance: tackling a crisis for the health and wealth of nations" (the amr review) which is in line to the question that de kraker et al. [ ] asked "will million people die a year due to amr by ?" the problem of amr is connected to that of covid- with respect to the emergence of zoonotic diseases. the trend of overuse and/or misuse of antimicrobials, excess use of certain antibiotics in animals, and pharmaceutical industry pollution can lead to continuous emergence of zoonotic diseases. excessive use of antimicrobials stresses the naturally occurring microbiome and allows for resistant bacteria to become dominant. indeed, research has suggested that amr might spread to humans through food products of animal origin, the environment, and by direct contact in the case of agricultural workers. addressing the issue of amr as well as others shown in figure is thus a global priority as its impact on claiming lives is no less than that of covid- [ ]. the rapid emergence of the covid- pandemic provided minimal time for welldirected resource mobilization, and almost every country was tested for its resilience in its medical and manufacturing abilities during the first half of . the number of tragic health cases pushed organizations such as the fda to allow emergency use authorisation of various drugs without a systematic testing protocol, and among the drugs tried, favipiravir and tocilizumab were rated most effective. amidst the chaotic situation, the growing use of digital tools for professional communications and artificial intelligence to monitor the recovery of covid- patients showed some positive outcomes. a major question remains open to date, "how can the symptoms of covid- be detected in their early stages?" while more research is needed on the instrumentation and manufacturing sides, there are clear opportunities identified from the available scientific knowledgebase. in relatively advanced stages of the symptoms, rapid measurements by combining chest computer tomography (ct) and reverse-transcription polymerase-chain-reaction (rt-pcr) tests seem to provide a high confidence level in the screening. more work is needed on the sensing side for detection of the virus in the primitive stages. definitive promise has been exhibited using laser technology to monitor the aerosol spread and to detect the rna of the virus and gain a deeper understanding of the aerodynamic properties of emergent viruses. further development of fumigation chambers and ultra-violet chambers seem to hold promise as precautionary measures that can be implemented in public places like malls, airports, theatres and train stations etc. in this paper, we have also highlighted the possibilities of developing novel tools such as lab-on-the-chip, in addition to developing advanced personal protective equipment (ppe) using combinatorial additive and subtractive manufacturing techniques such as roll-to-roll manufacturing and thermal spray. these technologies allow the capability to filter the coronavirus, which has a diameter in the range of ± nm. it is envisioned that in future, wearing masks and social distancing would be mandated to avoid the recurrent spread of the virus. in the long term, micro-manufacturers, medical professionals, metrology engineers, material scientists and virologists will need to work in a more interdisciplinary way to develop modelling informed fabrication strategies. accelerated use of molecular modelling approaches -like molecular dynamics and molecular dockingalso seems to hold promise in drug and vaccine discovery to end this pandemic from its root. as society progresses to meet challenges such as covid- , many overarching issues will become important. strong measures will need to be implemented to ensure careful handling of biowastes, indigenous capability-based manufacturing focus will become stronger and issues such as antimicrobial resistance will demand increased attention. our hope is that the post-pandemic society will better heed the warning signs from nature and work on making manufacturing systems resilient to address emerging threats. . figure : airborne spread tracked by the laser experiments at toho university, japan laboratory diagnosis of emerging human coronavirus infections-the state of the art the architecture of sars-cov- transcriptome detection of novel coronaviruses in bats in myanmar patient-derived mutations impact pathogenicity of sars-cov- . medrxiv the proximal origin of sars-cov- methods for estimating the case fatality ratio for a novel, emerging infectious disease how blood from coronavirus survivors might save lives virological assessment of hospitalized patients with covid- aerosol and surface stability of sars-cov- as compared with sars-cov- stability of sars-cov- in different environmental conditions. medrxiv molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes. emerging microbes & infections evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov- infection novel coronavirus can be detected in urine, blood, anal swabs and oropharyngeal swabs samples. medrxiv a rampage through the body. neurological insights of covid- pandemic evidence of the covid- virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms remdesivir: a review of its discovery and development leading to emergency use authorization for treatment of covid- correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases updates on what acs reported: emerging evidences of covid- with nervous system involvement rapid detection of covid- causative virus (sars-cov- ) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor diagnosing covid- : the disease and tools for detection rapid, low-cost detection of zika virus using programmable biomolecular components issues-second-emergency-use-authorization-decontaminate-n inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products review of titanium surface modification techniques and coatings for antibacterial applications chitosan-based coating with antimicrobial agents: preparation, property, mechanism, and application effectiveness on fruits and vegetables antibacterial polymeric membranes: a short review surface modification of polyurethane with a hydrophilic, antibacterial polymer for improved antifouling and antibacterial function a novel anti-influenza copper oxide containing respiratory face mask inactivation of norovirus on dry copper alloy surfaces contact killing and antimicrobial properties of copper effectiveness of nanomaterial copper cold spray surfaces on inactivation of influenza a virus a smart multi-functional coating based on anti-pathogen micelles tethered with copper nanoparticles via a biosynthesis method using l-vitamin c polymeric coatings that inactivate both influenza virus and pathogenic bacteria hydrophobic polycationic coatings inactivate wild-type and zanamivir-and/or oseltamivir-resistant human and avian influenza viruses preparation, application and testing of permanent antibacterial and antiviral coatings mechanism of inactivation of influenza viruses by immobilized hydrophobic polycations a new material for airborne virus filtration mechano-bactericidal nanostructures and where to find them interaction of silver nanoparticles with tacaribe virus antiviral activity of mycosynthesized silver nanoparticles against herpes simplex virus and human parainfluenza virus type . international journal of nanomedicine synthesis and application of silver nanoparticles (ag nps) for the prevention of infection in healthcare workers nano-based approach to combat emerging viral (nipah virus) infection antiviral activity of graphene-silver nanocomposites against non-enveloped and enveloped viruses self-assembled star-shaped chiroplasmonic gold nanoparticles for an ultrasensitive chiro-immunosensor for viruses nano research for covid- surgical face masks: manufacturing methods and classification current trends and future of sequential micromachining processes on a single machine tool high-aspect-ratio bulk micromachining of titanium virus filtration membranes prepared from nanoporous block copolymers with good dimensional stability under high pressures and excellent solvent resistance the production of nanostructures by mechanical forming a direct-write, resistless hard mask for rapid nanoscale patterning of diamond. diamond and related materials turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid- respiratory virus shedding in exhaled breath and efficacy of face masks structural basis for human coronavirus attachment to sialic acid receptors zn + inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture universal and reusable virus deactivation system for respiratory protection. scientific reports thermal/oxidative degradation and stabilization of polyethylene glycol addressing processing problems associated with plasma spraying of hydroxyapatite coatings material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: a review the thermal spray roadmap chemical implant fixation using hydroxyl-apatite coatings: the development of a human total hip prosthesis for chemical fixation to bone using hydroxyl-apatite coatings on titanium substrates organic field-effect transistor sensors: a tutorial review interfacial electronic effects in functional biolayers integrated into organic field-effect transistors human influenza virus detection using sialyllactose-functionalized organic electrochemical transistors functional sensing interfaces of pedot: pss organic electrochemical transistors for chemical and biological sensors: a mini review covid- : a risk assessment perspective phylogenetic network analysis of sars-cov- genomes anti-hiv drug repurposing against sars-cov- cryo-em structure of the -ncov spike in the prefusion conformation fast identification of possible drug treatment of coronavirus disease- (covid- ) through computational drug repurposing study structure of mpro from covid- virus and discovery of its inhibitors aerodynamic analysis of sars-cov- in two wuhan hospitals covid- : a new digital dawn? covid- , climate change, and renewable energy research: we are all in this together, and the time to act is now pandemic spotlight on urban water quality. ecological processes update on galactic cosmic ray integral flux measurements in lunar orbit with crater. space weather biosafety materials: an emerging new research direction of materials science from covid- outbreak will million people die a year due to antimicrobial resistance by ? plos medicine authors are very much thankful to the research support provided by the ukri via grants no.: (ep/k / , ep/l / , ep/s / , ep/t / , ep/s / and ep/t / ) as well as the gcrf/ epsrc supported sunrise program (ep/p / ). additionally, we acknowledge the support received from h (cost actions (ca , ca , ca and ca ) and euramet empir a ( )), royal academy of engineering grant no. iapp - \ (indo-uk partnership), royal academy of engineering grant no. tsp (south africa-uk partnership) and newton fellowship award from the royal society (nif\r \ ). also, numerical calculations performed on the isambard bristol, uk and archer hpc were made available by the epsrc resource allocation panel (rap). all data in the manuscript will be available through cranfield university open repository. key: cord- -hkh grys authors: turnage, nicole l.; gibson, kristen e. title: sampling methods for recovery of human enteric viruses from environmental surfaces date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: hkh grys acute gastroenteritis causes the second highest infectious disease burden worldwide. human enteric viruses have been identified as leading causative agents of acute gastroenteritis as well as foodborne illnesses in the u.s. and are generally transmitted by fecal-oral contamination. there is growing evidence of transmission occurring via contaminated fomite including food contact surfaces. additionally, human enteric viruses have been shown to remain infectious on fomites over prolonged periods of time. to better understand viral persistence, there is a need for more studies to investigate this phenomenon. therefore, optimization of surface sampling methods is essential to aid in understanding environmental contamination to ensure proper preventative measures are being applied. in general, surface sampling studies are limited and highly variable among recovery efficiencies and research parameters used (e.g., virus type/density, surface type, elution buffers, tools). this review aims to discuss the various factors impacting surface sampling of viruses from fomites and to explore how researchers could move towards a more sensitive and standard sampling method. acute gastroenteritis causes the second highest infectious disease burden worldwide with an estimated . million deaths per year (ahmed et al., ) . in the united states alone, acute gastroenteritis causes . million illnesses, , hospitalizations, and deaths (scallan et al., ) . there are approximately major pathogenic agents known to cause acute gastroenteritis and/or foodborne illness including human enteric viruses such as astrovirus, rotavirus, hepatitis a virus (hav), and human norovirus (hnov) (scallan et al., ) . the most common enteric viruses that cause foodborne illnesses are hnovs and hav (cliver, ; koopmans and duizer, ) . generally, viral acute gastroenteritis is transmitted through food and water contamination, contaminated environmental surfaces, direct person-to-person contact, and other unknown sources (wikswo et al., ) . furthermore, enteric viruses are spread by fecal-oral contamination, and there is growing evidence of viral transmission occurring through contaminated fomites in a variety of ways and settings including food preparation environments (boone and gerba, ; rzezutka and cook, ) . enteric viruses have been shown to maintain infectivity on fomites over prolonged periods of time (escudero et al., ) . for instance, seminal research by kiseleva ( ) reported on the survival of echovirus, coxsackievirus, and poliovirus on representative surfaces (painted wood, glass, cotton fabric) in households and showed that these viruses maintained infectivity for two to more than days. human norovirus survival for up to days has also been reported on carpets subject to vomiting episodes after an initial outbreak in a hospital ward (cheesbrough et al., ) . there are some studies focusing on the role of fomites and environmental contamination in the transmission of enteric viruses however this specific route of transmission is difficult to determine during outbreaks (rzezutka and cook, ) . to better understand the role of environmental surface transmission during outbreaks due to human enteric viruses, the persistence of viruses on various surface types must be investigated. to do this, a surface sampling method must be applied for recovery of viruses. for instance, understanding the persistence of human enteric viruses on inanimate fomite surfaces in relation to various environmental conditions could provide insight on ways to limit and prevent virus transmission and subsequent outbreaks. however, studies on surface sampling techniques are typically limited to swabs for application in environmental sampling during foodborne outbreaks or for investigation of baseline virus prevalence. as a result, information is lacking on evaluating tools used in laboratory sampling studies for the optimal recovery of viruses. thus, this review aims to: ( ) discuss and compare evaluations of surface sampling methods for optimal recovery of human enteric viruses from inanimate fomite surfaces and ( ) explore how researchers could move towards one standard methodology for surface sampling of human enteric viruses and their surrogates. the most common foodborne viruses are categorized based on the type of disease they cause: ( ) gastroenteritis (e.g. rotavirus, hnov, aichi virus a, coronavirus, and others), ( ) enterically transmitted hepatitis viruses (e.g. hepatitis e and a), and ( ) viruses that replicate in the human gut then migrate to other organs to cause disease (e.g. poliovirus) (koopmans and duizer, ) . enteric viruses are typically spread by vomiting or shedding into the stool and have a greater chance of transmission the longer the virus is able to survive outside the host. this survival is impacted by various environmental conditions such as ph, moisture, and temperature (koopmans and duizer, ; rzezutka and cook, ) . as indicated previously, enteric viruses have been shown to maintain infectivity on surfaces over prolonged periods. human noroviruses have been detected on a variety of surfaces including cellular phones, public phones, televisions, chairs, keyboards, microwave ovens, bathroom light switches, various handles and knobs of kitchen and bathroom items, bed frames, and chairs (boxman et al., ; gallimore et al., gallimore et al., , . boxman et al. ( ) reported year round prevalence of hnovs on environmental surfaces of catering facilities even without a recently reported outbreak of acute gastroenteritis. the authors reported that hnov was recovered from . % of catering settings with recent outbreaks in contrast to only . % of catering settings without a recent outbreak. elderly homes and pension/hotels catering company types had the highest prevalence of positive swab samples for hnovs (boxman et al., ) . moreover, multiple studies have shown institutional settings such as cafeterias and long-term facilities are more likely to have hnovs on surfaces compared to food service settings (boxman et al., ; hall et al., ; verhoef et al., ) . for environmental surface sampling, the international organization of standardization ( ) recommends swabbing with a sterile cotton swab presoaked in pbs followed by rna extraction and reverse transcription, real time pcr (rt-qpcr) analysis for hav and hnov sampling and detection on nonporous fcs. in the u.s., there is not a standardized method available. however, the centers for disease control and prevention (cdc, ) does recommend the use of swabs for obtaining norovirus from environmental surfaces; however, the cdc has also reported that swabbing is highly variable and that the interpretation of results should be conducted with caution. currently, hnovs are most often detected by rt-qpcr due to its high sensitivity and low detection limits using measurements such as pcr amplifiable units (pcru/ml). these pcrus are determined by a standard curve produced from a -fold dilution series of the virus where one pcru corresponds to the highest dilution with a quantifiable rt-qpcr value (or cycle threshold [c t ] value) (knight et al., ; tung et al., ) . however, knight et al. ( ) pointed out that the determination of pcrus in correspondence to specific c t values is dependent on the sample matrix and the standard used. moreover, the cutoff c t values (i.e. endpoint of detection) for hnovs also vary across studies ranging from to (knight et al., ) . the presence of inhibitory components within some sample matrices could impact amplification efficiencies especially in contaminated food and environmental samples that typically have low viral loads (knight et al., ; sair et al., ) . regardless, rt-qpcr is primarily chosen for the analysis of viruses in environmental and food samples to allow for increased sensitivity to detect low viral concentrations that are typically present (knight et al., ) . however, as the authors of the review indicated, this method cannot determine infectivity since it may recognize intact or degraded viral nucleic acid, nonviable viruses, or defective viral particles (knight et al., ) . consequently, the use of surrogates and other infectivity assays remain important in investigating enteric viral viability and infectivity in lab-based studies as further discussed in section . . . virus density, the rate of positive environmental samples of total samples collected, and exposure magnitude provide information about virus contamination on surfaces (julian et al., ) . however, these factors are impacted by the surface sampling method and detection assay selected. subsection . . to . . will examine the variability among the many factors impacting recovery of viruses from surfaces, specifically surface type, virus type/density, drying time, elution buffers, and implement/recovery tool selection. fomites are generally categorized as either nonporous or porous. examples of nonporous surfaces are ceramic, glass, acrylic, and stainless steel, and examples of porous surfaces include carpets, lettuce, deli meats, wood, latex, and fruits. surface type has been shown to have some effect on surface sampling recovery efficiencies (table ) . tung-thompson et al. ( ) swabbed foods (cheese, apple, green pepper, tomato) and hard surfaces (stainless steel and ceramic) with wipes that were inoculated with μl of varying pcr-units (pcru)/ml of hnov gii. . the study obtained a mean range recovery efficiency of % to approximately % for all surfaces except for cheese, which was significantly different from the other surfaces with % to % recovery for high inoculum levels ( to pcru) and no detection at low inoculum levels ( to pcru) (tung-thompson et al., ) . the authors were not able to determine if the lipid content of the cheese contributed to the possible absorption and recovery of the virus samples even though a previous study suggested this possibility for hnovs (fumian et al., ; tung-thompson et al., ) . furthermore, surface properties can also impact recovery efficiencies in a variety of ways. for instance, stainless steel is a hydrophilic (contact angle of . °in water, surface energy of . mj/m ) and negatively charged surface in which microorganisms have been shown to develop irreversible attachment within one minute potentially making surface recovery more difficult (mafu et al., ; mafu et al., ) . the orientation of a surface could interfere with adequate surface sampling and collection as seen in a study involving vertical and horizontal stainless steel surfaces. taku et al. ( ) determined that greater recovery efficiency could be obtained by allowing the elution buffer to sit on the surface for min-something that cannot be performed on a vertical surface. the mean recovery for horizontal surfaces and sinks using the cell scraper-aspiration method ranged from % to % while vertical stainless steel surfaces only obtained a mean recovery of % since the buffer was not in contact with the surface long enough to facilitate virus recovery (taku et al., ) . scherer et al. ( ) suggested physical properties of nonporous and porous could reduce virus recovery via trapping virus particles within the matrix/ crevices or facilitate enhanced virus recovery by smooth/porous surfaces. mattison et al. ( ) suggested the low mean recovery of feline calicivirus (fcv) from strawberries might be due to its surface texture and how the crevices may shield viruses against environmental conditions. furthermore, the authors observed a ph change in the elution buffer from . to . when strawberries were immersed, which could impact virus recovery by either partial viral inactivation or interference with fcv recovery (mattison et al., ) . overall, physical and chemical properties of nonporous and porous food and food contact surfaces could impact recovery efficiencies of enteric viruses. this review will focus on surface sampling techniques for enteric viruses from nonporous, inanimate surfaces. (rönnqvist and maunula ). there has not been an in vitro cell culture system for hnovs available until recently (ettayebi et al., ) , and until reproducible and readily available infectivity assays are developed, surrogates still provide much needed information on infectivity of hnovs. multiple surrogates are important for understanding infectivity due to variations in their genetic relatedness to hnovs and the diversity among hnov genotypes. other cultivable viruses utilized in environmental persistence research include aichi virus a (aiv) and hav-both known human enteric pathogens (cannon et al., ; koopmans and duizer, , yeargin et al., cannon et al., cannon et al., duizer, , yeargin et al., ) . diversity among hnov genotypes could impact the recovery efficiency from surfaces; however, studies focus mainly on hnov gii. (table ). this focus is a result of gii. being the pandemic genotype of hnov and accounting for over % of all hnov outbreaks in the u.s. since (glass et al., ). surrogates provide essential information on hnov infectivity in relation to viral persistence on food contact surfaces (fcs), and numerous studies have shown fcv, mnv, and tuv to remain infectious on multiple surfaces for at least days or more (arthur and gibson ; fallahi and mattison ; mattison et al., ) . some studies have compared the recovery efficiency between different types of enteric viruses. scherer et al. ( ) compared hnov gii. and rotavirus recovery efficiencies using a cotton swab from various porous and nonporous fcs. table shows the recovery varied between virus types for a given surface. for instance, scherer et al. ( ) reported the highest percentage of hnov was recovered on ceramic ( - %) while rotavirus was recovered at a slightly higher percentage ( - %) on the same surface. the authors suggested the varying recovery rates observed between the two enteric viruses may be due to the abilities of the different viruses to adhere to the various surfaces as well as differences in virus properties affecting attachment (scherer et al., ) . a greater variety of surrogates and enteric viruses need to be evaluated for surface sampling to ensure accurate prevention and detection methods are being implemented. virus density could also impact the amount of virus recovered from a given surface. in general, higher starting densities of viruses equal greater recovery efficiencies-primarily due to the limit of detection of the downstream assay. tung-thompson et al. ( ) reported recovery efficiency variability by virus density when using wipes on food and nonporous food contact surfaces. the authors showed that recovery was consistent at high inoculum levels ( - pcru/ml) of gii. while more variability was observed at lower inoculum levels ( − pcru/ml). in contrast, rönnqvist et al. ( ) also reported variability among lower concentrations of gii. with higher mean recoveries for hnov gii. at pcru than pcru when evaluating four different swabs on environmental surfaces. for pcru of gii. , there was no significance difference for recovery efficiency among the swabs evaluated except on latex surfaces with polyester swabs regardless of buffer type. meanwhile, microfiber swabs combined with glycine buffer for elution was found to be a significantly better recovery method for pcru of gii. on all the surfaces (rönnqvist et al., ) . scherer et al. ( ) reported that the mean recovery efficiencies for rotavirus and hnov gii. were higher from various nonporous and porous surfaces using a cotton swab-rinse method at higher inoculum levels ( × pcru for hnov; × pcru for rotavirus) than lower inoculum levels ( × pcru for hnov; × pcru for rotavirus). the authors also mentioned how reverse transcription became less efficient at low inoculum levels resulting in an increase in statistical errors. overall, the higher the inoculum level for all enteric viruses, the higher the mean recovery rate regardless of the variability among methods, pa = plaque assay; pbs = phosphate buffered saline; pbst = pbs + . % tween ; pcru = polymerase chain reaction units; pe = polyethylene; pf = porous formic; pfu = plaque forming units; rh = relative humidity; rb = rubberized surface; rt-qpcr = reverse transcription quantitative pcr; rt = room temperature; ss = stainless steel. virus type, and high standard deviations of the mean recovery rates. additionally, organic matter such as coagulated food and other debris while on environmental surfaces may impact the effect of virus density on recovery efficiency. for instance, fatty foods such as cheese have been known to contribute to absorption and recovery of virus samples for hnovs due to lipid content (fumian et al., ) . furthermore, abad et al. ( ) studied the effect of fecal matter on the persistence of enteric viruses and reported varying results between virus types and fomites. the authors found no effect on the persistence of hav and human rotavirus with the exception of longer persistence of hav on latex surfaces. overall, abad et al. ( ) observed longer persistence for adenovirus and poliovirus on nonporous fomites (china, glazed tile, aluminum, and latex), and a decrease in persistence of adenovirus and poliovirus on porous fomites (cotton cloth and paper). for hnovs, the preparation of stool samples (i.e. because hnov does not have a routine culture method) is not always specifically stated in studies on virus persistence and recovery from surfaces. for example, park et al. ( ) include a clarification step-a brief centrifugation to separate the large particulates from the viruses in % fecal suspensions-while others (de keuckelaere et al., ; ronnqvist et al., ) use hnovs in the original % fecal suspension for their studies. the presence or absence of organic matter can certainly impact both virus persistence and recovery; however, it should also be noted that the presence of organic matter could also impact downstream analysis such as rt-qpcr via inhibition (wilson ) , also indicated in section . . even though virus persistence and recovery from food matrices are not within the scope of this review, enteric virus recovery from nonporous environmental surfaces as a function of particle association (e.g., food and debris) is lacking and does need further study. drying time for enteric virus surface sampling is highly variable and dependent on factors including volume of virus suspension and desiccation (table ) . drying times range from min to overnight at ambient conditions with volumes ranging from μl to μl. drying time impacts the recovery efficiencies of surface sampling methods, and generally, the longer a virus is on a surface, the harder it is to recover the virus from the surface. mattison et al. ( ) tested recovery of fcv from stainless steel surfaces using vortexing at min post inoculation versus immediate recovery after inoculation of . × fcv in μl. the difference in recovery between elution immediately following and after min of drying was and %, respectively-a three-fold difference. while this review is focused on fcs and not food, the authors did note that the difference between viral recovery from lettuce and stainless steel may be due to viruses being more influenced by the effects of air drying when on a flat nonporous surface. park et al. ( ) observed a reduction in the recovery efficiency of hnov gii. from stainless steel and toilet representative surfaces as a function of drying time. on stainless steel surfaces using macrofoam swabs, the recovery efficiency was . % ± . % without drying, . % ± . % at h, . % to . % ≤ h, and . % ± . % after h (park et al., ) . based on the evidence presented above, there is a need for uniformity among studies and standardization in drying time and inoculum amount in order to properly evaluate virus recovery and surface sampling methods. the recovery efficiencies for the numerous eluent-tool combinations are variable and often impacted by both intrinsic factors related to the actual tool and eluent types as well as the extrinsic factors already introduced (sections . . - . . ). the differences in eluent formulations such as ph, salinity, and use of a surfactant can impact the recovery efficiency of viruses from surfaces. ionic strength and ph of eluents have been known to impact the net charge of viral particles (gerba, ) . rönnqvist et al. ( ) obtained slightly higher recovery efficiencies using an alkaline glycine buffer (ph . ) than eluting with pbs (ph . ). conversely, taku et al. ( ) recovered more fcv from stainless steel surfaces using a slightly acidic glycine buffer (ph . ) with a mean recovery of % compared to and % recovery using glycine buffer (ph . ) or culture medium (ph . ), respectively. surfactants are another common component added to elution buffers. these are known to increase the water content of the surface, assist in solubilization of proteins and cells from the surface, and can disrupt hydrophobic interactions between charged viruses and surfaces thus enhancing virus recovery (farrah ; lukasik et al., ; moore and griffith ) . park et al. ( ) suggested that adding a surfactant ( . % tween ) to the pbs elution buffer of a swab rinse protocol enhanced viral recovery efficiency of hnov gii. even though no significance was observed. meanwhile, another study found higher recovery of hnov gii. and mengovirus from laminated wooden surfaces when using lysis buffer compared to mm tris-hcl − mm glycine − . % beef extract (tgbe, ph . ); however, again no significance difference was observed (ibfelt et al., ) . for ms recovery, two separate studies found the eluent type to not be significantly different (casanova et al., ; julian et al., ) . furthermore, eluent type for ms recovery was suggested to be selected based on experimental design such as considering eluents compatible with nucleic acid extraction for molecular detection-based sampling studies or with tissue culture for infectivity-based studies (julian et al., ) . moreover, rönnqvist et al. ( ) suggested an elution buffer be selected based on the specific situation with the consideration of factors such as the time elapsed between swabbing and sample analysis. overall, eluent type can impact viral recovery, and thus eluent-tool combinations must be chosen with consideration of surface, virus, and eluent interactions for efficient surface sampling and recovery. therefore, a matrix of elution buffers and when to apply given a certain situation or parameters would be a valuable resource. the majority of tools used in laboratory-based studies for evaluation of surface sampling methods have focused on various types of swabs (table ). this finding comes as no surprise since swabbing is known as the gold standard for hnov sampling and detection on fcs (iso, ). evaluation of swabs has shown varying recovery rates for enteric viruses; however, while the swab itself may be the primary driver in recovery, numerous other factors can play a role as indicated previously. more specifically, the material and properties of the recovery tool can impact recovery efficiencies. for example, the dying process of microfiber cloths can change its net surface charge, which could impact viral attachment and detachment from surfaces (rönnqvist et al., ) . taku et al. ( ) suggested the selection of swabs are due to the ease of operation over small surface areas even though swabs yield consistently poor results in comparison to other methods evaluated, possibly due to surface area of the swab head and smearing virus over surfaces. macrofoam, polyester-tipped, and/or cotton swabs have been shown to be more efficient among swabs tested in viral recovery from fomites depending on a given study's conditions and parameters (ibfelt et al., ; julian et al., ; scherer et al., ) . for instance, julian et al. ( ) reported that polyester-tipped swabs recovered a greater amount of infectious ms than antistatic cloths. however, as indicated in section . . , the elution buffer and tool combination complicates matters. for instance, rönnqvist et al. ( ) reported that elution buffer type only impacted the recovery efficiency of microfiber cloths composed of polyester and polyamide materials where mm glycine buffer (ph . ) performed better than pbs. additionally, the authors reported better recovery of low inoculum hnov gii. on latex surfaces when using polyester swabs, though it is unclear why. unfortunately, it is difficult to compare swab types across studies due to differences among surface types, virus types, virus volume, and virus concentrations used for the evaluations of the swab sampling protocols. as evidenced by table , surface sampling methods used in the recovery of enteric viruses are highly variable and diverse. a majority of studies focus on swabbing for a variety of reasons. in fact, the international organization of standardization ( ) recommends hnov sampling and detection on nonporous fcs to be collected with a cotton swab moistened with pbs followed by rna extraction and reverse transcriptionquantitative pcr (rt-qpcr) analysis. other tools and methods such as repeated pipetting, cell scraping, and sonication/stomaching have been used for viral persistence and disinfection studies (arthur and gibson ; mattison, ; yeargin et al., ) . studies involving environmental surface sampling for applications in detecting viruses during outbreaks can be used as a baseline for standard surface sampling techniques for enteric viruses. swabbing is the technique typically used for enteric virus studies involving applications in detection of viruses during outbreaks. thus, studies have focused on evaluating swab protocols on surfaces associated with outbreaks such as on cruise ships and fcs (table ) . rönnqvist et al. ( ) evaluated four swab types (e.g. flocked nylon, cotton wool, microfiber, and polyester) in either pbs or glycine buffer at ph . for collecting hnov gii. from stainless steel and plastic surfaces. park et al. ( ) evaluated five swab types (e.g. cotton, rayon, polyester, antistatic cloth, and macrofoam) using hnov gii. from stainless steel and toilet representative surfaces with macrofoam swabs producing the highest recovery efficiencies. during comparison of these two studies, microfiber performed better than macrofoam swabs with . % ± . % and . % ± . % recovery efficiency, respectively, when elution buffer (glycine buffer) and surface type (stainless steel) were the same. however, the amount and concentration of hnov gii. varies between the two studies, and this could also impact recovery efficiencies as reviewed in section . . . rönnqvist et al. ( ) also provides information on using swabs on plastic surfaces. overall, there is a need for more studies involving more viruses and nonporous surfaces to properly determine a standardized approach for surface sampling of enteric viruses during outbreaks. several different methods have been used to optimize recovery of enteric viruses from inanimate fomites in laboratory-based persistence studies. furthermore, differences among the studies include virus types, volume and concentration of virus as well as tools, fcs, and type of analysis. in this subsection, we will further examine these differences and how they could contribute to the varying results of surface sampling method evaluation studies. summaries of these studies are available in table . as stated in section . , swabbing has traditionally been the focus in studies on virus detection and persistence (table ) . a few studies focused on evaluating one swab implement for use in recovering enteric viruses from a variety of surface types and virus inoculum levels. scherer et al. ( ) evaluated a cotton swab with pbs (ph . ) elution buffer for collecting hnov gii. and rotavirus from different fcs (i.e. stainless steel, ceramic, high-density polyethylene, and wooden chopping board) with recovery efficiencies ranging from . ± . % (wood, pcru) to . ± . % (ceramic, pcru) for gii. and . ± . % (wood, tcid ) to . ± . % (ceramic, t-cid ) for rotavirus. the authors found recoveries for both hnov and rotavirus to be higher from fcs than food surfaces at both inoculum concentrations (scherer et al., ). additionally, ganime et al., ( ) evaluated the recovery rates of mnv- and bacteriophage pp from porous formic, non-porous formic, and rubberized surfaces using a rayon swab with culture media with recovery efficiencies ranging from . to . % (pp ) and . - . % (mnv- ). while these two studies evaluate how one particular swab performs, other studies expand their evaluations to provide a better comparison of different swabs and tools and their recovery of particular enteric viruses. for example, ibfelt et al. ( ) evaluated three different swabs (i.e. cotton, foamed cotton, and polyester) and two elution buffers (i.e. direct lysis or alkaline tgbe − ph . ) for recovery of hnov gii. and mengovirus from cm laminated wooden surfaces. the authors found a significantly better virus recovery using polyester swabs with the direct lysis in comparison to other combinations tested; however, recovery efficiencies were ≤ % for all combinations. ibfelt and others ( ) suggested their low recovery rates may be due to the size of the surface or differences in experimental design in comparison to other swab studies. furthermore, julian et al. ( ) also recommended the use of polyester swabs pre-moistened in either ringer's or . % saline solution for ms recovery from plastic and stainless steel surfaces following evaluation of three tools (cotton swab, polyester swab, and antistatic cloth) and four elution buffers (saline, ringer's solution, viral transport media, and acid/base). based on a meta-analysis of ms surface sampling, the authors noted that polyester swabs obtained significantly higher positive ms rates in comparison to rayon and cotton (julian et al., ). conversely, de keuckelaere et al. ( found cotton and polyester swabs to not be significantly different in their recovery efficiencies of hnovs gi. and gii. from nitrile gloves, polyethylene, or neoprene rubber surfaces. park et al. ( ) reported a similar result when evaluating the recovery efficiencies of four swab types (macrofoam, rayon, cotton, and polyester). the authors applied the different swabs for recovery of hnov gii. from stainless steel and toilet representative surfaces and found that rayon, cotton, and polyester were not significantly different. however, macrofoam swabs obtained significantly higher recovery efficiencies of hnov gii. in comparison to the other three swabs after h of drying on a given surface (park et al., ) . additionally, some studies found other tools and methods such as biowipes and cell scraper-aspiration methods to be potentially more efficient for enteric virus recovery from surfaces in comparison to cotton and/or polyester swabs. these studies are further examined in sections . . and . . (de keuckelaere et al., ; taku et al., ) . cloths and wipes have also been introduced as possible alternatives to swabbing methods for obtaining higher recovery efficiencies of enteric viruses from surfaces. de keuckelaere et al. ( ) evaluated two swabs (cotton and polyester) along with biowipes (biomérieux, lyon, france) composed of a mixture of fibers and microfibers (cotton, polyester, and polyamide fibers) moistened in pbs (ph . ) by recovering gi. and gii. hnovs from fcs (high-density polyethylene, nitrile gloves, and neoprene rubber). there was no significant difference among any of the three tools evaluated based on recovery efficiency from polyethylene surfaces and nitrile gloves for hnov gi. . meanwhile, the authors found significantly higher recovery efficiencies using biowipes ( . ± . %) compared to cotton swabs ( . ± . %) on the coarser rubber surface (de keuckelaere et al., ) . the authors also found that the mean recovery efficiency of biowipes for gi. from rubber surfaces was higher than using polyester swabs even though no significant difference was observed. for hnov gii. , there was no significant difference in recovery observed between all three tools tested on polyethylene surfaces and nitrile gloves even though the biowipes had significantly higher recovery efficiency ( . ± . %) on rubber surfaces compared with both polyester ( . ± . %) and cotton ( . ± . %) swabs (de keuckelaere et al., ). another study further confirmed the effectiveness of these biowipes in collecting hnov gii. at various inoculum concentrations ( to pcru) from stainless steel and ceramic fcs (tung-thompson et al., ) . the authors reported a range of mean recovery efficiencies of gii. using biowipes (biomerieux sa, grenoble, france): . - . % (stainless steel) and . - . % (ceramic). it should be noted that recovery efficiencies reported by tung-thompson et al. ( ) were generally much higher than other studies included in table . however, a few studies showed certain swabs to be more efficient for recovery of enteric viruses than cloths. for example, macrofoam swabs had a higher recovery efficiency of hnov gii. ( . ± . %) from large ( . cm ) stainless steel surfaces than antistatic cloths ( . ± . %) (park et al., ) . additionally, julian et al. ( ) determined that polyester swabs obtained higher recoveries of infectious ms than antistatic cloths as well. overall, cloths and wipes may be a valuable tool for collecting enteric viruses from fcs, and there is a need for further studies using cloths and wipes involving a greater variety of virus types, cloth types, surface types, and infectivity analyses. other surface sampling methods such as vortexing, repeated pipetting, stomaching/sonication, and cell scraping have been used for baseline information for viral persistence studies and disinfection studies (table ). the studies summarized in table use different surrogates, initial drying times, and elution buffers making it difficult to adequately compare the studies. fallahi and mattison ( ) recovered % of mnv- from stainless steel after a min drying time using a repeated pipetting method with ebss eluent. mattison et al. ( ) recovered % of fcv from stainless steel after a min drying time by vortexing for s in ebss eluent. arthur and gibson ( ) obtained recovery efficiencies of % and % for tuv from acrylic and stainless steel surfaces, respectively, after a drying time of h using a cell scraping techniques. the cell scraping technique was confirmed as possible with tuv and has also been evaluated using fcv previously (taku et al., ) . taku et al. ( ) found consistently better mean virus efficiencies for fcv using mm glycine (ph . ) from stainless steel surfaces in comparison to mm glycine (ph . ) and modified eagle's medium (ph . ) using the scraping-aspiration method. the mean fcv recovery efficiencies for the scraping-aspiration method from stainless steel were reported to be % (glycine ph . ), % (glycine ph . ), and % (modified eagle's medium). the authors suggested the modified eagle's medium complex composition may have played a role in being less efficient than the glycine buffers (taku et al., ) . taku et al. ( ) added cell scraping to the aspiration method for better recovery efficiencies speculating that cell scraping may facilitate release of virus from surface. in addition, yeargin et al. ( ) recovered a range of . % (cotton) to . % (glass) for fcv and . % (cotton) to . % (glass) for mnv- from three surface types (i.e. polyester, cotton, and glass) using a stomaching/sonication method. the authors also found the recovery efficiencies to be highest for glass and lowest for polyester and cotton for both virus types. the recovery efficiencies were also reported to be significantly different among all surface types for the same virus type while only cotton swab recoveries showed a significant difference between mnv- and fcv (yeargin et al., ) . similar to other techniques, more studies with inclusion of more virus types and standardized drying times are needed to provide information on using these alternative techniques for future persistence and environmental sampling studies. surface sampling of enteric viruses varies across studies throughout the literature. this variability in results may exist due to varying human behavior, the tool used, and/or the elution buffer type used to recover the virus from the surface as well as numerous other factors outlined in the present review. most surface sampling evaluations have focused on various swab types while there are limited studies focused on evaluation of other possible tools and techniques such as repeated pipetting and cell scraper application, historically used in a laboratory setting. as a result, food and environmental virology researchers may have difficulty in selecting the most appropriate surface sampling method for a particular study. additionally, we found that no single standard approach to recover enteric viruses from fcs exists. the following suggestions are based on our review to assist researchers in moving towards one standard methodology for optimizing the recovery of enteric viruses from fomite surfaces: • eluent buffer used to recover sample needs to be standardized. • concentrations and volumes of virus need to be more consistent and include standard low and high inoculum levels. • the impact of organic materials on enteric virus recovery from surfaces needs further investigation. • infectivity assays such as plaque assays are highly recommended for the analysis of surface sampling optimization in order to distinguish infectious particles from non-infectious viral particles. however, this is currently only possible with cultivable viruses and hnov surrogates. • results need to be reported in one standard form of measurement. • more techniques and tools need to be evaluated along with the swab protocols and these evaluations should include of a variety of human enteric viruses and their surrogates. survival of enteric viruses on environmental fomites global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis environmental persistence of tulane virus: a surrogate for human norovirus significance of fomites in the spread of respiratory and enteric viral disease year-round prevalence of norovirus in the environment of catering companies without a recently reported outbreak of gastroenteritis norovirus: specimen collection surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus methods for the recovery of a model virus from healthcare personal protective equipment possible prolonged environmental survival of small round structured viruses virus transmission via food semi-direct lysis of swabs and evaluation of their efficiencies to recover human noroviruses gi and gii from surfaces persistence and transferability of noroviruses on and between common surfaces and foods replication of human noroviruses in n stem cell-derived human enteroids evaluation of murine norovirus persistence in environments relevant to food production and processing chemical factors influencing adsorption of bacteriophage ms to membrane filters a rapid procedure for detecting noroviruses from cheese and fresh lettuce environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season applied and theoretical aspects of virus adsorption to surfaces norovirus gastroenteritis. new eng evaluation of the swab sampling method to recover viruses from fomites division of viral diseases, national center for immunization and respiratory diseases, c.d.c., . vital signs: foodborne norovirus outbreaks -united states microbiology of food and animal feed-horizontal method for determination of hepatitis a virus and norovirus in food using real-time rt-pcr-part : method for quantification test and validation of methods to sample and detect human virus from environmental surfaces using norovirus as a model virus comparison of surface sampling methods for virus recovery from fomites survival of enteric viruses in water and foodstuffs and on various surfaces a critical review of methods for detecting human noroviruses and predicting their infectivity foodborne viruses: an emerging problem influence of salts on virus adsorption to microporous filters attachment of listeria monocytogenes to stainless steel, glass, polypropylene, and rubber surfaces after short contact times characterization of physicochemical forces involved in adhesion of listeria monocytogenes to surfaces survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus problems associated with traditional hygiene swabbing: the need for in-house standardization evaluation of a new environmental sampling protocol for detection of human norovirus on inanimate surfaces noroviruses on surfaces: detection, persistence, disinfection and role in environmental transmission swabs as a tool for monitoring the presence of norovirus on environmental surfaces in the food industry survival of human enteric viruses in the environment and food improved detection of human enteric viruses in foods by rt-pcr foodborne illness acquired in the united states-major pathogens application of a swab sampling method for the detection of norovirus and rotavirus on artificially contaminated food and environmental surfaces concentration and detection of caliciviruses from food contact surfaces efficacy of commonly used disinfectants for inactivation of human noroviruses and their surrogates evaluation of a surface sampling method for recovery of human noroviruses prior to detection using reverse transcription quantitative pcr reported behavior, knowledge and awareness toward the potential for norovirus transmission by food handlers in dutch catering companies and institutional settings in relation to the prevalence of norovirus outbreaks of acute gastroenteritis transmitted by person-to-person contact, environmental contamination, and unknown modes of transmission-united states inhibition and facilitation of nucleic acid amplification recovery and disinfection of two human norovirus surrogates, feline calicivirus and murine norovirus, from hard nonporous and soft porous surfaces this review is based upon work that is supported by the arkansas biosciences institute with the grant received by author gibson key: cord- - qq xsw authors: kramer, axel; schwebke, ingeborg; kampf, günter title: how long do nosocomial pathogens persist on inanimate surfaces? a systematic review date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: qq xsw background: inanimate surfaces have often been described as the source for outbreaks of nosocomial infections. the aim of this review is to summarize data on the persistence of different nosocomial pathogens on inanimate surfaces. methods: the literature was systematically reviewed in medline without language restrictions. in addition, cited articles in a report were assessed and standard textbooks on the topic were reviewed. all reports with experimental evidence on the duration of persistence of a nosocomial pathogen on any type of surface were included. results: most gram-positive bacteria, such as enterococcus spp. (including vre), staphylococcus aureus (including mrsa), or streptococcus pyogenes, survive for months on dry surfaces. many gram-negative species, such as acinetobacter spp., escherichia coli, klebsiella spp., pseudomonas aeruginosa, serratia marcescens, or shigella spp., can also survive for months. a few others, such as bordetella pertussis, haemophilus influenzae, proteus vulgaris, or vibrio cholerae, however, persist only for days. mycobacteria, including mycobacterium tuberculosis, and spore-forming bacteria, including clostridium difficile, can also survive for months on surfaces. candida albicans as the most important nosocomial fungal pathogen can survive up to months on surfaces. persistence of other yeasts, such as torulopsis glabrata, was described to be similar ( months) or shorter (candida parapsilosis, days). most viruses from the respiratory tract, such as corona, coxsackie, influenza, sars or rhino virus, can persist on surfaces for a few days. viruses from the gastrointestinal tract, such as astrovirus, hav, polio- or rota virus, persist for approximately months. blood-borne viruses, such as hbv or hiv, can persist for more than one week. herpes viruses, such as cmv or hsv type and , have been shown to persist from only a few hours up to days. conclusion: the most common nosocomial pathogens may well survive or persist on surfaces for months and can thereby be a continuous source of transmission if no regular preventive surface disinfection is performed. within the global infection control community, there is an ongoing controversy about the appropriate treatment of inanimate surfaces in hospitals in order to prevent transmission of nosocomial pathogens within an institution. based on a lack of epidemiological data that would provide evidence of a benefit for the patient from surface disinfection (e.g., from a significant reduction of nosocomial infection rates), some scientists postulate that cleaning of surfaces with non-antimicrobial detergents is generally sufficient [ ] . others prefer cleaning of surfaces with antimicrobial agents, based on data on the risk of infection due to microbial contamination and potential transmission of nosocomial pathogens, at least in the immediate vicinity of patients [ ] [ ] [ ] . new guidelines on treatment of surfaces in hospitals take into account more parameters which are considered to be relevant for preventing the transmission of nosocomial pathogens, such as the type of ward or the expected frequency of hand contact with a surface [ , ] . irrespective of the divergent opinions regarding the appropriate treatment of surfaces, an important parameter for a fair scientific assessment remains, that is, the persistence of nosocomial pathogens on surfaces. the longer a nosocomial pathogen persists on a surface, the longer it may be a source of transmission and thus endanger a susceptible patient or healthcare worker. the aim of this review was therefore to collect and assess the data that have been published in the last decades on persistence of all types of nosocomial pathogens on surfaces, both in the context of surface disinfection and the control of nosocomial outbreaks. the literature was systematically reviewed in medline on the internet homepage of the national library of medicine without language restrictions. the search was done on december and covered all years available in medline. the following search terms were applied: persistence, survival, surface, fomite, bacteria, virus, pathogen, transmission, and nosocomial. in addition, the citations in each study found during the main search were reviewed for potential relevance. finally, standard textbooks on infection control, bacteriology and virology were examined for information. all reports with experimental evidence on the duration of persistence of a nosocomial pathogen on any type of inanimate surface were included. information from textbooks was also included, even if the chapter itself did not contain experimental evidence. at least two of the investigators decided on the relevance of each report. reports were not blinded to the investigators so that they knew the names of the authors of all studies. for a clinically relevant summary, all nosocomial pathogens were grouped according to their importance in causing hospital-acquired hand-transmitted infections [ ] and according to their mode of nosocomial transmission [ ] . the range of the reported duration of persistence was used as the principle outcome of the search for each nosocomial pathogen. in addition, parameters with potential influence on persistence were evaluated in all experimental studies. most gram-positive bacteria, such as enterococcus spp. (including vre), staphylococcus aureus (including mrsa), or streptococcus pyogenes survive for months on dry surfaces (table ). in general, there was no obvious difference in survival between multiresistant and susceptible strains of staphylococcus aureus and enterococcus spp. [ ] . only in one study was such a difference suggested, but the susceptible strains revealed a very brief survival as such [ ] . many gram-negative species, such as acinetobacter spp., escherichia coli, klebsiella spp., pseudomonas aeruginosa, serratia marcescens, or shigella spp. can survive on inanimate surfaces even for months. these species are found among the most frequent isolates from patients with nosocomial infections [ ] . a few others, such as bordetella pertussis, haemophilus influenzae, proteus vulgaris, or vibrio cholerae, however, persist only for days (table ) . mycobacteria -including mycobacterium tuberculosis and spore-forming bacteria, including clostridium difficilecan also survive for many months on surfaces (table ) . overall, gram-negative bacteria have been described to persist longer than gram-positive bacteria [ , ] . humid conditions improved persistence for most types of bacteria, such as chlamydia trachomatis [ ] , listeria monocytogenes [ ] , salmonella typhimurium [ ] , pseudomonas aeruginosa [ ] , escherichia coli [ ] , or other relevant pathogens [ , ] . only staphylococcus aureus was found to persist longer at low humidity [ ] . low temperatures, e.g., °c or °c, also improved persistence of most types of bacteria, such listeria monocytogenes [ ] , salmonella typhimurium [ ] other factors were rarely investigated and hence provide inconsistent results. longer persistence has been described with higher inocula [ ], in the presence of protein [ ] , serum [ , ] , sputum [ ], or without dust [ ] . candida albicans as the most important nosocomial fungal pathogen can survive up to months on surfaces ( table ). persistence of other yeasts was described to be similar (torulopsis glabrata months) or shorter (candida parapsilosis days). the presence of serum or albumin, a low temperature, and high humidity have been described as leading to longer persistence [ ] . most viruses from the respiratory tract such as corona-, coxsackie-, influenzavirus, sars, or rhinovirus can persist on surfaces for a few days. viruses from the gastrointestinal tract, such as astrovirus, hav, polio-and rotavirus persist for approximately months. blood-borne viruses, such as hbv or hiv, can persist for more than one week. herpes viruses such as cmv or hsv type and have been shown to persist from only a few hours up to days. [ ] or norovirus [ ] . other investigators found that per- sistence was favored on non-porous surfaces for influenzavirus [ ] , on formica and gloves for rsv [ ] , and on a telephone receiver for fcv [ ] . other parameters for a longer persistence of viruses include the presence of fecal suspension [ ] and a higher inoculum [ ] . cryptosporidium species have been reported to survive on dry surfaces for only hours [ ] . the most relevant nosocomial pathogens can persist on dry inanimate surfaces for months. in addition to the duration of persistence, some studies have also identified factors influencing persistence. a low temperature, such as °c or °c, was associated with longer persistence for most bacteria, fungi and viruses. high humidity (e.g., > %) was also associated with longer persistence for most bacteria, fungi, and viruses, although for some viruses conflicting results were reported. a few studies also suggest that a higher inoculum is associated with longer per-sistence. the type of surface material and the type of suspension medium, however, reveal inconsistent data. overall, a high inoculum of the nosocomial pathogen in a cold room with high relative humidity will have the best chance for long persistence. in most reports with experimental evidence, persistence was studied on dry surfaces using artificial contamination of a standardized type of surface in a laboratory. in most studies, bacteria were prepared in broth, water or saline. viruses were usually prepared in a cell culture medium [ ] . the main advantage is that the environmental conditions are consistent regarding temperature and air humidity. in addition, the effect of temperature or relative humidity can only be determined under controlled conditions, which are much easier to ensure in the laboratory. however, this may not always reflect the clinical situation, in which surfaces can be simultaneously contaminated with various nosocomial pathogens and different types of body fluids, secretions etc. yet the question remains: what is the clinical evidence for the role of surfaces in nosocomial infections? in hospitals, surfaces with hand contact are often contaminated with nosocomial pathogens [ ] [ ] [ ] , and may serve as vectors for cross transmission. a single hand contact with a contaminated surface results in a variable degree of pathogen transfer. transmission to hands was most successful with escherichia coli, salmonella spp., staphylococcus aureus (all %) [ ] , candida albicans ( %) [ ] , rhino virus ( %) [ ] , hav ( % - %) [ ] , and rota virus ( %) [ , ] . common modes of transmission from inanimate surfaces to susceptible patients figure common modes of transmission from inanimate surfaces to susceptible patients. susceptible patient direct transmission compliance in hand hygiene: ~ % to more surfaces [ ] or other subjects [ ] . contaminated hands can also be the source of re-contaminating the surface, as shown with hav [ , ] . compliance rates of healthcare workers in hand hygiene are known to be around % [ ] . due to the overwhelming evidence of low compliance with hand hygiene, the risk from contaminated surfaces cannot be overlooked (figure ). the main route of transmission is via the transiently contaminated hands of the healthcare worker [ ] [ ] [ ] . an outbreak of nosocomial infections due to acinetobacter baumannii in a neurosurgical intensive care unit may serve as an example. a direct correlation was found between the number of environmental isolates obtained during screening and the number of patients who were colonized or infected with the same strain during the same calender month [ ] . during outbreaks, the environment may play a significant role for transmission of nosocomial pathogens, as suggested by observational evidence. this has been described for various types of microorganisms, such as acinetobacter baumannii [ ] [ ] [ ] , clostridium difficile [ ] [ ] [ ] , mrsa [ , ] , pseudomonas aeruginosa [ , ] , vre [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , sars [ , ] , rota- [ , ] , and norovirus [ ] . however, the evidence to support a role of environmental contamination is not equally strong for all types of nosocomial pathogens. for clostridium difficile, mrsa, and vre, data are stronger than for other pathogens, such as pseudomonas aeruginosa or acinetobacter baumannii, of which multiple types were detected in the environment, and which did not always correlate with the acquired strain [ ] . the role of surface disinfection for the control of nosocomial pathogens has been a contentious issue for some time [ ] . routine treatment of clean floors with various types of surface disinfectants (some of them had rather poor bactericidal activity) has been described to have no significant impact on the incidence of nosocomial infections [ ] . disinfection of surfaces in the immediate environment of patients, however, has been described to reduce acquisition of nosocomial pathogens such as vre [ ] or acinetobacter baumannii [ ] . it is therefore advisable to control the spread of nosocomial pathogens at least in the direct inanimate environment of the patient by routine surface disinfection. most nosocomial pathogens can persist on inanimate surfaces for weeks or even months. our review supports current guidelines which recommend a disinfection of surfaces in specific patient-care areas in order to reduce the risk of transmission of nosocomial pathogens from inanimate surfaces to susceptible patients. gk is a paid employee of bode chemie gmbh & co. kg, hamburg, germany. all authors contributed to the conception, review of studies, and analysis of data. all authors were involved in drafting and revising the manuscript. all authors approved the final version of the manuscript. routine surface disinfection in health care facilities: should we do it? surface disinfection: should we do it disinfection and the prevention of infectious disease pseudomonas aeruginosa outbreak in a haematologyoncology unit associated with contaminated surface cleaning equipment anonymous: guidelines for environmental infection control in health-care facilities epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs nosocomial spread of viral disease survival of enterococci and staphylococci on hospital fabric and plastic long-term in-vitro survival of an epidemic mrsa phage-group iii- strain nosocomial and community-acquired infections in germany. summary of the results of the first national prevalence study (nidep) untersuchungen über das verhalten grampositiver und gramnegativer bakterien in trockenem und feuchtem milieu. zentralblatt für bakteriologie und hygiene, i abt orig b survival of bacteria under dry conditions from a viewpoint of nosocomial infection chlamydia trachomatis can be transmitted by a nonporous plastic surface in vitro survival and growth characteristics of listeria monocytogenes and salmonella typhimurium on stainless steel and buna-n rubber untersuchungen zur lebensdauer von bakterienstämmen im staub unter dem einfluß unterschiedlicher luftfeuchtigkeit. zentralblatt für bakteriologie und hygiene, i abt orig b persistence of escherichia coli o on farm surfaces under different environmental conditions influence of relative humidity and suspending menstrua on survival of acineto survival of enteric viruses on environmental fomites persistence of norwalk virus on environmental surfaces and its transfer to food survival of influenza viruses an environmental surfaces possible transmission by fomites of respiratory syncytial virus survival on uncommon fomites of feline calicivirus, a surrogate of noroviruses comparative efficacy of handwashing agents against cytomegalievirus the emerging nosocomial pathogens cryptosporidium, escherichia coli o :h , helicobacter pylori, and hepatitis c: epidemiology, environmental survival, efficacy of disinfection, and control measures survival of human enteric viruses in the environment and food computer keyboards and faucet handles as reservoirs of nosocomial pathogens in the intensive care unit survival of acinetobacter baumannii on bed rails during an outbreak and during sporadic cases environmental contamination due to methicillin-resistant staphylococcus aureus: possible infection control implications the survival and transfer of microbial contamination via cloths, hands and utensils an experimental model for study of candida survival and transmission in human volunteers transmission of experimental rhinovirus infection by contaminated surfaces survival of hepatitis a virus on human hands and its transfer on contact with animate an inanimate surfaces prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray rotavirus survival on human hands and transfer of infectious virus to inanimate and nonporous inanimate surfaces effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces transmission of viruses via contact in a household setting: experiments using bacteriophage strain phixi as a model virus effect of fecal contamination on diarrheal illness rates in day-care centers outbreak of shigella sonnei in a clinical microbiology laboratory outbreaks of infection with methicillin-resistant staphylococcus aureus on neonatal and burns units of a new hospital role of environmental cleaning in controlling an outbreak of acinetobacter baumannii on a neurosurgical intensive care unit an outbreak of imipenem-resistant acinetobacter baumannii in critically ill surgical patients distribution of multi-resistant gram-negative versus gram-positive bacteria in the hospital inanimate environment a nosocomial outbreak of multiresistant acinetobacter baumannii originating from an intensive care unit control of nosocomial clostridium difficile transmission in bone marrow transplant patients prospective evaluation of environmental contamination by clostridium difficile in isolation side rooms acquisition of clostridium difficile from the hospital environment a purpose built mrsa cohort study outbreak of vancomycin-resistant enterococci in a burn unit experience of vancomycin-resistant enterococci in a children's hospital control of an outbreak of vancomycinresistant enterococcus faecium in an oncology ward in south africa: effective use of limited resources management of an outbreak of vancomycin-resistant enterococci in the medical intensive care unit of a cancer center role of environmental contamination as a risk factor for acquisition of vancomycin-resistant enterococci in patients treated in a medical intensive care unit epidemiology of colonization of patients and environment with vancomycin-resistant enterococci transfer of vancomycin-resistant enterococci via health care worker hands sars in a hospital visitor and her intensivist sars in hospital emergency room. emerging infectious diseases prevalence of rotavirus on high-risk fomites in day-care facilities detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction management of hospital outbreaks of gastro-enteritis due to small roundstructured viruses hota b: contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? routine disinfection of patients' environmental surfaces. myth or reality? reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures control of an outbreak of multidrug-resistant acinetobacter baumannii-calcoaceticus colonization and infection in an intensive care unit (icu) without closing the icu or placing patients in isolation the survival of acinetobacter calcoaceticus inoculated on fingertips and on formica the inanimate environment of an intensive care unit as a potential source of nosocomial bacteria: evidence for long survival of acinetobacter calcoaceticus medizinische mikrobiologie und infektiologie microbial survival in the environment enhanced survival of campylobacter jejuni in association with wood isolation of clostridium difficile from the environment and contacts of patients with antibiotic-associated colitis epidemiologic aspects of clostridium difficile-induced diarrhea and colitis review of clostridium difficile-associated diseases transmission of chlamydia pneumoniae diphtheria bacilli in floor dust survival of verocytotoxigenic escherichia coli o in soil, water and on surfaces. symposium series bacterial adherence and viability on cutting board surfaces the microbiocidal action of a new silver-containing polymer (spi-argent ii) recovery of vancomycin-resistant enterococci on fingertips and environmental surfaces survival of vancomycin-resistant and vancomycin-susceptible enterococci on dry surfaces anthropozoonoseerreger ohne familienzugehörigkeit: listerien, brucellen, francisellen und erysipelothrix pseudomonas pyocyanea infections in hospitals environmental contamination with an epidemic strain of pseudomonas aeruginosa in a liverpool cystic fibrosis centre, and study of its survival on dry surfaces survival of s. typhimurium in floor dust: a possible reservoir of infection in institutions zur Überlebensdauer von shigellen auf plastikwerkstoffen. zeitschrift für die gesamte hygiene survival of shigella dysenteriae type on fomites better environmental survival of outbreak vs. sporadic mrsa isolates survival of cholera vibrios in food, water and fomites. public health papers a quantitative study of the survival of two species of candida on porous and non-porous environmental surfaces and hands hygiene und infektionen im krankenhaus edited by: thofern e and botzenhart k. stuttgart survival of human coronaviruses e and oc in suspension and after drying on surfaces: a possible source of hospital-acquired infections nosocomial infections due to human coronaviruses in the newborn stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation survival of cytomegalievirus on environmental surfaces maynhard je: survival of hepatitis b virus after drying and storage for one week resistance of aids virus at room temperature. the lancet handbuch der virusdesinfektion survival of hiv- : activity after disinfection, temperature and ph changes, or drying survival of herpes simplex virus in water specimens collected from hot spa facilities and on plastic surfaces survival and disinfection of parainfluenza viruses on environmental surfaces virus survival in the environment papillomavirus is resistant to desiccation survival of pseudorabies virus in the presence of selected diluents and fomites an investigation of the possible transmission of rhinovirus colds through direct contact experimentelle ergebnisse über die stabilität von pockenviren unter labor-und umweltbedingungen. zentralblatt für veterinärmedizin the authors declare that they have no acknowledgements. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /pre pub key: cord- -tncjfdtp authors: hackney, raymond w.; crawford, james j.; tulis, jerry j. title: using a biological indicator to detect potential sources of cross-contamination in the dental operatory date: - - journal: the journal of the american dental association doi: . /jada.archive. . sha: doc_id: cord_uid: tncjfdtp abstract the authors conducted a study using surveillance monitoring methodology to identify operatory contamination and to evaluate the effectiveness of infection control procedures. viridans streptococci were evaluated as biological indicators of oral contamination. viridans streptococci, abundant in human saliva, were detected on operatory surfaces after dental treatments were finished and surfaces were disinfected. the findings validate current concepts of infection control as demonstrated in barrier methods. a gallup poll indicated that two-thirds of the adult u.s. population is treated in dental offices each year. protecting a major portion of the populace from infections transmitted by saliva-and blood-contaminated operatory surfaces and equipment is an unending challenge for practicing dentists. the task of protecting patients and dental workers during the past decade has prompted dramatic change in what is required of dental practice. in addition to the bloodborne pathogens standard of the occupational safety and health administration, infection control guidelines published by the centers for disease control and prevention to protect patients have been mandated in all states, according to federal law. [ ] [ ] [ ] in recent years, instrument cleaning, disinfection and sterilization have received detailed attention and definition. [ ] [ ] [ ] [ ] [ ] patients can be protected from cross-infections only if each patient's oral tissues are not handled alternately with operatory equipment and surfaces contaminated with saliva and blood during the care of previous patients. preventing cross-contamination requires identification of the sources of contamination, as well as the careful implementation of well-designed barriers and aseptic techniques. this article addresses the difficult task of infection control assessment and monitoring for oral contamination on dental operatory surfaces handled during dental treatment. the concepts and findings we describe in this article affirm the design of current infection control methodologies. , [ ] [ ] [ ] in addition, this study also supports the importance of monitoring the potential for cross-infection in practice, research and the assessment of new dental equipment and methods. without adequate control procedures, agents of both respiratory and bloodborne diseases left on dental equipment can be transmitted to successive dental patients. intact or injured oral tissues are vulnerable to agents of hepatitis b and c, hiv, and herpes simplex and viruses. infection of oral and respiratory passages can result from transfer of pathogenic bacterial strains of streptococci, staphylococci and pneumococci; influenza, measles and mumps viruses; or varicella-zoster, cytomegalovirus, respiratory syncytial virus, rhinovirus, adenovirus, coronavirus, coxsackievirus or transmission of pathogenic yeasts and bacterial respiratory pathogens from patients' mouths to the mouths of successive patients after radiographic examinations. thus, contaminated operatory surfaces can act as fomites when infection control procedures are not followed. sampling and dye studies have shown that surfaces of operatory equipment handled during oral treatments become heavily contaminated. [ ] [ ] [ ] [ ] as saliva contamination is not visible, contaminated sites are easily overlooked. in a busy practice, time allowed between patients for thorough cleaning and disinfecting is often inadequate. these factors make rendering operatory equipment and surfaces free of contamination a difficult challenge. these observations have contributed to the development of guidelines for operatory asepsis. , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we found nothing in the literature that provided detailed documentation and evaluation of a method of assessing contamination of contact surfaces in the dental operatory, how much contamination is encountered in private operatories or an evaluation of efforts made by private office personnel in preparing operatories for safe reuse. thus, we designed this study to establish a basis for evaluating infection control procedures and equipment, and to propose an initial standard for assessing oral contamination of operatory surfaces. although pathogens can be found in bodily fluids of infected people, shedding of those pathogens is intermittent and epstein-barr virus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] hiv from human sources dried on contaminated surfaces becomes inactivated quite rapidly ( to percent reduction within several hours). however, hepatitis b can survive at percent humidity for seven days. staphylococcus aureus can survive on dried surfaces for a mean of five days. one group of investigators found that when dried on patients' paper charts, herpes viruses survived approximately three hours when mixed with saliva and more than four hours when mixed with blood. rhinovirus survived up to hours in saliva mixed with saline; streptococcus pyogenes survived more than two days, and s. aureus survived more than five days (viable salivary bacteria could be detected for up to five days). when plasticcone x-ray machines were inoculated with bacterial cultures, s. aureus was cultivated from the dry surface after hours, and streptococcus pneumoniae and s. pyogenes after hours. mycobacterium tuberculosis can survive for six to eight months in dried sputum protected from direct sunlight. lamp handles, bracket table handles, air-water syringes, suction hose handles, handpieces, switches, drawer handles, chair controls, clinicians' chairs and charts are frequently handled by oral health care workers whose hands are contaminated with blood and saliva. , these workers may then touch their own eyes, nose, mouth or skin lesions. a significant study by autio and colleagues demonstrated the unpredictable. thus, testing for such pathogens can be counterproductive. this problem was handled in the science of water sanitation by designing tests to detect coliform bacteria, indigenous to the healthy human intestine, in an effort to protect drinking water from all fecal waste contamination. like the intestinal tract, the oral cavity hosts specialized indigenous microbes, which can serve as indicators of oral contamination in the testing of dental office equipment and surfaces. for an oral microbe to be an indicator organism, it must meet the following criteria: dit must be common to the human mouth; dit must survive for a useful period of time outside the mouth on surfaces and equipment; dit must be present in low numbers in nondental environments in which there is low potential for oral contamination; dit must be relatively easy to recover and distinguish from other bacteria recovered from dental operatory surfaces; dit must be recoverable from operatory surfaces and equipment known to be contaminated. viridans streptococci are common to the human mouth, are easy to detect when cultured on blood agar, and would be logical indicators of oral contamination if they meet the aforementioned criteria. , [ ] [ ] we found no reports in which such oral streptococci were evaluated as indicators of oral contamination of dental equipment surfaces. we also found few data on the survival of oral streptococci with regard to environmental temperature and humidity. investigators have documented heavy contamina-tion of clinicians' smocks, cuffs and equipment used during treatments, but they did not differentiate oral bacteria from ordinary skin bacteria. seven common oral streptococcus species compose a group called viridans streptococci. they produce α-hemolysis, a zone of partial hemolysis-a greenish discoloration around each colony grown on blood agar. this characteristic makes these microorganisms easy to distinguish from other bacteria found in dust and on skin that might also contaminate clinical surfaces, suggesting the usefulness of α-hemolytic streptococci, or ahs, as standard indicators for detecting oral contamination and for evaluating operatory asepsis. in this study, we assessed the validity of oral ahs as an indicator of oral contamination in the following manner: dassessing the consistency and abundance of ahs in mouths of a sample of patients; ddetermining the distribution of ahs in nondental environments, both clinical and nonclinical; devaluating environmental survival of ahs on operatory materials; dusing ahs as an indicator of contamination after cleaning and disinfection in private dental offices. survey of dental patients' saliva for ahs. the number of ahs commonly found in saliva was determined from saliva samples of randomly selected general dentistry patients at dental school clinics at the university of north carolina at chapel hill. the mean age of the patients in the survey was . years; ages ranged from to years. of the patients, were male and were female. each saliva sample was diluted - and . milliliter was plated on sheep blood agar. the number of both ahs and nonhemolytic colony-forming units, or cfus, on each plate was counted. overall surface sampling methodology. we chose the swab-rinse method for all sampling because most of the surfaces encountered either were irregular in shape and unsuitable for replicate organism detection and counting sampling or were too large for the rinse method. [ ] [ ] [ ] the swab-rinse method consisted of using a sterile cotton swab moistened in a sterile recovery medium to sample equipment and other surfaces potentially touched by contaminated hands. the entire digital contact area of a surface was sampled. the sampled surface was rubbed several times with back-and-forth strokes (about to centimeters long); then the swab was rotated, and the surface was rubbed with strokes perpendicular to the original strokes. the swab was broken off in a tube containing . ml of the recovery medium and transported to the laboratory for inoculation of culture plates. to prevent growth of the bacteria in the recovery medium, the samples were kept on ice until they were processed. within one hour of their collection, the samples were taken to the laboratory and processed. spread plates were prepared for each sample. each tube was vortexed for . minute to release the bacteria from the cotton swabs. the spread plates were prepared by placing . ml of the sample in -millimeter petri dishes containing columbia colistin naladixic acid, or cna, sheep blood agar, an enriched medium selective for gram-positive organisms. the sample was spread evenly over the agar surface with a sterile glass spreader. the plates were incubated in candle jars at c for hours. after incubation, ahs colonies were counted. isolates were identified according to the framework described by facklam and facklam and carey. three recovery media were used: trypticase soy broth, or tsb; letheen broth; and dey/engley, or d/e, neutralizing broth. after disinfectants dry on a surface, the residual disinfectant can be reactivated when moistened by the recovery media from the cotton swab. letheen broth and d/e neutralizing broth contain ingredients that neutralize disinfectants. however, it is known that these neutralizing ingredients also can have bacteriostatic effects on, and some degree of toxicity for, the recovered bacteria. before the sampling in the private offices and in the general environment, we performed tests to determine which of the recovery media was most sensitive for recovery of ahs. letheen broth effectively neutralized residual phenolic disinfectants and was less toxic to the recovered ahs than was the d/e neutralizing broth. d/e broth was approximately six times less sensitive than letheen broth. iodophors, chlorine and hypochlorites are sufficiently neutralized by the organic material in letheen broth, or other nutrient media, such as tsb. tsb was even more sensitive than letheen broth when there were no residual disinfectants recovered in the sample. we chose tsb for sampling surfaces where disinfectants were not used, or where use was limited, because it is not toxic to the bacteria. recovery of ahs from surfaces in the general environment. we evaluated the occurrence of ahs in the general-nondental-environment by sampling surfaces commonly handled or touched in a general medical clinic, an ophthalmology clinic and a barber shop. we sampled surfaces and the entire digital contact area of each equipment handle using swabs moistened with tsb. samples were taken in the afternoon after the last of the patients or customers had been seen. the staff at the medical clinic used a phenolic disinfectant to clean the examination table between patients. other surfaces were cleaned daily with detergent and water. surfaces sam-pled in the general medical clinic included faucet handles, light handles, countertops and examination tables. in the ophthalmology clinic, a percent solution of household bleach ( . percent sodium hypochlorite) was sprayed and wiped on all surfaces in the examination room after patients with eye infections were seen. the frequency of visits by patients with eye infections varied from daily to weekly. otherwise, surfaces were cleaned with detergent and water. the ophthalmology clinic surfaces we sampled included the head adjustment handle, countertops, the scope adjustment handle, the lens adjustment handle, patient chair armrests and the patient chair headrest. disinfectants were not used on a routine basis in the barber shop. surfaces sampled in the barber shop included armrests, countertops, clippers, drawer handles, faucet handles, the vacuum/air blower handle and scissors. microbial sampling in the dental operatory. surfaces in the dental operatory were sampled before and after dental procedures were performed in a dental school clinic at the school of dentistry at the university of north carolina at chapel hill, where licensed general dentists treated patients. surfaces of equipment handles were sampled, including the entire digital contact area of each piece. swabs used to sample surfaces that had been disinfected were moistened with d/e neutralizing broth. surfaces sampled before the procedure were the handpiece base and holder, the air-water syringe handle and holder, the syringe water, suction handles and holder, the bracket tray handle and eyeglasses worn by the dentist. a total of samples were taken, before dental treatments began, from surfaces that had been cleaned. we observed the entire dental procedure, noting and counting the number of times each surface was touched by the potentially contaminated hands of the dentist, the dental assistant or both. we also noted handwashing and glove changes. after the dental treatment and before cleanup, the same sur- jada, vol. , november the tubes of recovery medium also were incubated and growth subcultured on columbia colistin naladixic acid sheep blood agar. † no α-hemolytic streptococci, or ahs, detected. ‡ counts of to cfus were estimated when no growth occurred on the plates and growth was detected in the recovery medium alone. faces were resampled, as were dental instruments and other surfaces that were touched with potentially contaminated hands. a total of samples were taken after dental treatments from surfaces that were touched or were potentially contaminated with saliva. after sampling, spread plates were prepared in the laboratory, as previously described. microbial survival in private dental offices after cleaning and disinfection. environmental surfaces in private dental practices were sampled for ahs in the morning before dental procedures began and at the end of the day after cleanup. both sets of samples were from operatories that were "clean" and ready for the next patient. surfaces sampled included items such as handpieces, air-water syringe handles and tips, suction handles, lamp handles, door handles, telephone receivers, bracket tray handles, patient seat buttons, dentist's seat controls, x-ray units and water from the air-water syringe. the surfaces were sampled with a sterile cotton swab moistened with letheen broth (broth containing lecithin and tween detergent (difco laboratories) to neutralize resid-ual disinfectants still remaining on the surfaces). we scrubbed each item or surface vigorously with the swab, using back-andforth and perpendicular strokes and rotating the swab several times. the swab was remoistened in the recovery medium two or three times for each sample; each time, the swab was pressed against the side of the tube to remove excess moisture. all items of a given type in an operatory (handpieces, for example) were sampled with a single swab. as we sampled each item, we wore gloves and used aseptic techniques. the samples were kept on ice and processed in the laboratory as previously described. the tubes with recovery medium were also incubated at c for hours. growth from the tubes was streaked on columbia cna blood agar plates. after incubation, the plates were examined for the growth of ahs colonies. survey of dental patients' saliva for ahs. the average number of ahs cfus counted in the survey was × per ml of saliva, ranging from × to × . there was an average of × nonhemolytic cfus per ml of saliva, ranging from × to × . recovery of ahs from surfaces in the general environment. ahs were detected in two of the three areas sampled; these results are summarized in ( ) / ( ) / ( ) / ( ) * samples were taken after cleanup/disinfection of operatory surfaces. samples yielded low counts of ahs colonies. ten cfus were detected in three positive samples, and cfus were detected in a fourth. dfrom the ophthalmology clinic, fewer than nine cfus were detected in one of samples. the streptococci were detected only in the tube of sampling broth, which was incubated to detect streptococci in the sample that did not grow on the . ml of plated sample. dfrom the general medical clinic, none of the samples yielded growth of ahs. didentification of the ahs detected in nondental environments showed five to be streptococcus mitis and one to be s. sanguis i. microbial sampling in the dental operatory. of samples taken before dental treatments from surfaces that had been cleaned (in clinic a), three ( percent) were positive for ahs. two of these samples were from bracket tray handles and one was from the air-water syringe handle and holder. a total of samples were taken after dental treatments from surfaces that were touched or were potentially contaminated with saliva. forty-nine ( percent) of these were positive for ahs. the four operatory surfaces most frequently touched during the dental procedures observed were the air-water syringe handle (touched times per treatment), the handpiece (touched nine times per treatment), the suction handles (touched eight times per treatment) and the lamp handle (touched seven times per treatment). the average time per treatment was . hours. ahs were detected on the handpiece and air-water syringe handle on percent of the samples. the suction handles were positive for ahs in percent of the samples. the lamp handle was not sampled because it had been covered with a plastic barrier that is removed and discarded after dental treatment. other items touched by dental personnel with potentially contaminated hands included dental instruments such as pliers, syringes, explorers, scalpels, tweezers, probes, mirror, amalgamator, camera, rubber cement container, spatula, drawer handles, lamp switch, refrigerant spray, floss holder, pencil, ruler, scissors, x-ray units, bur wrenches and cavity varnish containers. items that were positive for ahs were dental instruments, hand mirror, amalgamator, ultrasonic scaler and eyeglasses. microbial survival in private dental offices. ahs were detected on percent ( of ) of the surfaces sampled in the morning and percent ( of ) of the surfaces sampled in the afternoon, for a combined total of percent ( of ) of the surfaces sampled. the fisher exact test was used to investigate the significance of the morning sampling with the afternoon sampling results. the difference was significant (p = . ). the fisher exact test was also used to compare the afternoon sampling results in the private dental offices with the sampling results in the nondental areas ( , as the samples in the nondental areas were also taken in the afternoon. this difference was significant (p = . ). ahs were detected in all of the dental offices. in one of the offices, no ahs were detected in the morning samples, but four of samples were positive in the evening. the operatory with the highest number of contaminated surfaces had a combined total of positive samples of a total of ( percent). sampling results of the private offices are summarized in table , which also lists the type of disinfectant used in each office. the most frequently contaminated surface was the x-ray unit (eight of , percent), followed by the handpiece ( of , percent) and the patient chair buttons ( of , percent). these results are presented in table . six of the samples had high numbers of ahs: , to > , cfus. the numbers of cfus recovered in samples from the private dental operatories are summarized in table . a major goal of this investigation was to determine whether certain oral bacteria found in human saliva could serve as biological indicators of the contamination of operatory equipment. a bacterial indicator of oral contamination would have to be easy to cultivate and recognize, abundant in the mouth, present in low numbers in general environmental areas where there is a low potential for oral contamination, able to survive on environmental surfaces, and detectable on dental operatory surfaces where there is known contamination. in accordance with these criteria, literature data and the results obtained in this study, the best indicator of oral contamination appears to be ahs. the following observations support this conclusion. physiological appearance. ahs have an unusual physiological appearance that makes them easy to recognize on blood agar plates. their α-hemolysis is a result of the bacterial production of hemolysin, which causes a breakdown of red blood cells around a colony on blood agar. the zone of hemolysis is a mixture of lysed and incompletely lysed cells that results in a green or brownish color. , the term "viridans" comes from the latin term "viridis," meaning "green." ß-hemolysis, exhibited by other streptococci, appears as a clear zone of completely lysed red blood cells. all seven of the common species found in saliva have α-hemolytic strains, although the strains of streptococcus salivarius are predominantly nonhemolytic ( percent). α-hemolysis gives oral streptococci a distinguishing characteristic among the other flora growing on the cul-ture plate. ease of culturing and identification. ahs were relatively easy to culture and identify; they grew well on sheep blood agar at c. because growth conditions that provide increased carbon dioxide are favorable for streptococci, studies were conducted using candle jars in which colonies were visible after to hours. typical colonies were transparent to opaque, to mm in diameter, with an α-hemolytic zone of to mm after hours' incubation. positive samples, those with colonies exhibiting α-hemolysis, should be confirmed for the presence of streptococci with gram's stain and catalase test. presence in saliva. ahs were found in high numbers in saliva. the survey of saliva from patients who visited the dental school clinics showed that ahs averaged about × organisms per ml of saliva, ranging from × to × . the nonhemolytic colonies of the various species of the viridans streptococci averaged about half the number of the ahs colonies, although there were some patients with more nonhemolytic jada, vol. , november * numbers of colony-forming units, or cfus, are based on colony counts grown from . milliliters of the -ml recovery medium used to suspend each sample. the tubes of recovery medium also were incubated and growth subcultured on columbia cna sheep blood agar. † counts of to cfus were estimated when no growth occurred on the plates and growth was detected in the recovery medium alone. than ahs colonies. presence in general environment. ahs were detected in low numbers and frequency in the general environment. although the ahs were detected in samples from nondental environments, they were present in low numbers. one or two α-hemolytic colonies were observed on spread plates of four of the samples taken in the barber shop. none of the other spread plates for the nondental samples grew ahs colonies, although one sample from the ophthalmology clinic grew the indicator organisms in the recovery medium. the fisher exact test was used to compare the afternoon sampling results in the private dental offices with the sampling results in the nondental areas ( percent, five of ), since the samples in the nondental areas were also taken in the afternoon. this difference was significant (p = . ). the difference is attributed to the activity of saliva-contaminated hands' touching surfaces in a dental operatory despite the efforts of dental personnel to clean and disinfect those surfaces. although this activity does not take place in barber shops or medical clinics, ahs were detectable there nevertheless. ahs also are dispersed into the environment through sneezing, coughing and talking. detection of low numbers of ahs in the general environment is acceptable; however, a higher standard should be applied to an environment in which instruments and fingers of clinic personnel touch or penetrate the mucous membranes of patients. survival on dental operatory surfaces. ahs survived on environmental surfaces for several days. however, relative humidity has a pronounced ef-fect on the survival of ahs in saliva dried on surfaces. there is accelerated die-off at high relative humidities, or rh. at percent rh, the die, or d, value-the time for percent to die, or one logarithm reduction-was demonstrated to be only two hours, whereas at lower rh of percent and percent, the d value was hours and hours, respectively. we used the fisher exact test to compare the private dental office sampling results from the morning ( percent [ of ] of the samples were positive for ahs) with those from the afternoon ( percent [ of ] positive for ahs). the difference was significant (p = . ). this difference is attributed to the die-off of the indicator organisms during the approximately hours after the last patients were seen the day before. the rh in the private dental offices ranged from to percent when the samples were taken. presence on operatory surfaces. ahs were detectable on contaminated operatory surfaces. the indicator organisms were isolated from surfaces immediately after dental procedures and before cleanup. in private office operatories, the indicator organisms were found on surfaces that had been cleaned and were ready for the next patient. thirty-nine percent ( of ) of samples taken from "clean" operatories in private practices were positive for the indicator organisms, clearly showing the potential for cross-contamination between patients. we compared the findings of positive samples among total samples with the goal of zero positive samples among total sam-ples. the probability that a proportion that large would happen by chance is far less than one in , . although the primary criterion for interpretation of the monitoring results is whether or not ahs are detected, actual colony counts recovered might assist in evaluating the potential for cross-contamination. surfaces with higher counts of ahs would indicate a higher risk of cross-contamination between patients. ten ( percent) of the samples had high counts (> ), estimated to be > , cfus recovered in sampling. six of these had very high counts, ranging from to more than , a total considered too numerous to count but estimated to be from , to more than , cfus recovered. it should be understood, however, that the swab-rinse sampling methodology is not a precise measurement of the amount of contamination on a surface. colony counts would depend on variables such as the swabbing technique used and the condition of the surface sampled. colony counts also vary depending on the amount of saliva contamination on a dental care worker's gloves before he or she uses an item and the amount of digital contact he or she makes with the item. for these reasons, any indication of residual contamination should be considered significant in efforts to provide a safe treatment environment. implications of the findings. the environment presented to patients should be free of oral bacteria from previous patients; thus, the goal is that ahs should not be detected on any of the operatory surfaces. since each of the offices was sam-pled twice, a total of sets of samples were taken. as shown on table , only one of the sets was negative for all samples. this finding indicates that the time necessary for thorough cleaning and disinfection is not available in a busy dental practice. disinfection practices should include initial surface cleaning to physically remove debris and much of the contamination. well-cleaned surfaces then should be thoroughly wetted again with fresh disinfectant, allowing as much contact time as possible, according to the manufacturer's instructions. , all of the offices surveyed stated that operatory surfaces were disinfected between patients. the types of disinfectants used in each office are listed in table . however, a number of surfaces were left contaminated despite the use of various disinfectants. our finding indicates the difficulty of completely disinfecting all irregular operatory equipment surfaces with consistency. this observation supports the concept that cleaning and disinfection of equipment surfaces is not the most effective or reliable approach to infection control in the busy dental practice. asepsis implications and recommendations. alternatives to complete reliance on disinfection procedures can and should be implemented to control cross-contamination. , , , , - , , a more effective control method is the use of inexpensive, single-use, disposable plastic bags over surfaces that must be touched during treatments, such as the airwater syringe, the lamp handle, the suction handle, the dental control unit and even the chair. covers can be replaced rapidly between patients, eliminating the need for disinfection unless the bag comes off or its integrity is broken. , another effective approach is to prevent direct contact of contaminated gloved hands with occasionally contacted surfaces. this can be achieved in several ways. foot controls rather than chair buttons should be used to adjust seats and to operate water faucets for handwashing. dentists and dental assistants should use a paper towel or remove gloves to hold phones or to touch other surfaces that must not be contaminated during treatments. handpieces and other intraoral dental equipment should be designed to be removed and sterilized between appointments. sampling methodology. the swab-rinse method is preferred for microbial surface sampling in the dental operatory, because it is a simple method suitable for the irregular surfaces encountered in the operatory. the recovery medium should have disinfectant neutralizers if the surface has been treated with a disinfectant that leaves a residual that is reactivated when the surface is moistened. letheen broth effectively neutralizes phenolic disinfectants, quaternary ammonium compounds and iodophor disinfectants, and is more sensitive (not as toxic) for recovering the indicator organisms than d/e neutralizing broth. , incubating the recovery broth and then streaking the resultant culture on blood agar increases the sensitivity of the sampling method. sampling consistency is critical. it is recommended that the moistened swab be pressed firmly against the surface, using vigorous scrubbing, reversing directions, with perpendicular strokes, while rotating the swab frequently. all areas of a given surface should be sampled (unless it is too large to be practical), with the swab being remoistened two or three times during the sampling. during moistening and remoistening, the swab should be pressed and rotated against the side of the tube to remove excess moisture. more than one instrument can be sampled with a single swab. the goal in dental asepsis is to break the chain of transfer of blood and blood-contaminated saliva from each patient's mouth to surfaces in the dental operatory and to other patients via contaminated equipment or the hands of dental personnel. in this study (performed before the use of disposable plastic covers became widely recommended), the extensive detection of ahs on unprotected, inadequately disinfected surfaces should be interpreted as a potential for cross-contamination. our detection of ahs in the operatory on unprotected disinfected surfaces indicated the inadequacy of surface disinfection practices. these findings validate and reinforce current concepts of infection control advocated and used widely in dentistry , : duse of single-use plastic covers over surfaces handled with contaminated gloved hands during treatment, as barriers to contamination; davoidance of unnecessary touching of unprotected items and surfaces directly with contaminated gloves without using an additional clean barrier such as a paper towel or forceps; dsterilization of all other items or equipment that must be handled in the treatment field and cannot be protected in another fashion. this study indicates the usefulness-possibly for a number of applications-of an infection control surveillance monitoring methodology in dental practice environments using biological indicators. these surveillance methods can aid in evaluating equipment and techniques developed for infection control. sampling for indicator organisms also can be used epidemiologically to help determine the routes of infection transmission when investigating outbreaks in a dental clinic or practice. outside consultants or public health organizations required to evaluate asepsis in dental practices can use this technique for indicator organisms as part of an overall monitoring program. dental schools can use the technique as a teaching tool to show students the potential for crosscontamination and to teach or evaluate aseptic techniques and infection control practices. more imminently, sampling for indicator organisms can serve as a process control by dental practitioners. this can help identify hazards in dental practice before the public is harmed, and can be used to raise dental personnel's level of awareness of the potential for disease transmission. heightened awareness can encourage continued adherence to infection control procedures. such self-evaluation by the dental profession could eliminate any potential sources of crosscontamination that might have thus far escaped scrutiny by the profession or the public, ideally preventing any eventual need for greater outside controls of dental care asepsis. i d o you have comments or questions about this article? jada now offers an online resource called ask the author, which can put you in touch with the author of one featured article per issue. check out ask the author in the ada publishing co. portion of ada online at "http://www.ada.org". : . . occupational safety and health administration. standard, occupational exposure to bloodborne pathogens. federal register recommendations for preventing transmission of human immunodeficiency virus and hepatitis b virus to patients during exposureprone invasive procedures the public health and welfare act centers for disease control. recommended infection-control practices for dentistry infection control and management of hazardous materials for the dental team how to choose and use environmental surface disinfectants oral bacteria as biological indicators for dental asepsis (dissertation) university of north carolina at chapel hill the art and science of operative dentistry wilford hall usaf medical center and office of the assistant secretary of defense; . . crawford jj. new light on the transmissibility of viral hepatitis in dental practice and its control transmission of rhinovirus colds by self-inoculation low occupational risk of human immunodeficiency virus infection among dental professionals herpetic whitlow: report of a case with multiple recurrences getchell-white si, donowitz lg, groschel dhm. the inanimate environment of an intensive care unit as a potential source of nosocomial bacteria: evidence for long survival of acinetobacter calcoaceticus survival of herpes simplex virus and other selected microorganisms on patient charts: potential source of infection interpatient microbiological cross-contamination after dental radiographic examination miller ch, palenik cj. infection control and management of hazardous materials for the dental team barriers can minimize occupational exposure risks to contagions clinical asepsis in dentistry evaluation of spatter and aerosol contamination during dental procedures council on dental materials and devices and council on dental therapeutics. infection control in the dental office baltimore: williams & wilkins; . . crawford jj. sterilization, disinfection, and asepsis in dentistry cross infection control in dentistry: a practical illustrated guide oral microbiology a bacteriologic census of human saliva indications of the sanitation level in a dental clinic physiological differentiation of viridans streptococci biosafety committee. biosafety reference manual a comparative evaluation of methods for determining the bacterial contamination of surfaces comparative evaluation of the cotton swab and rodac methods for the recovery of bacillus subtilis spore contamination from stainless steel surfaces manual of clinical microbiology the use of inactivators in the evaluation of disinfectants ecologic studies of rheumatic fever and rheumatic heart disease taranta a, moody md. diagnosis of streptococcal pharyngitis and rheumatic fever oral streptococci with emphasis on streptococcus mutans council on dental materials and devices and council on dental therapeutics. current status of sterilization instruments, devices, and methods for dental office dental asepsis key: cord- -xyj mc g authors: a, jishnu; s jayan, jitha; saritha, appukuttan; a.s., sethulekshmi; venu, gopika title: superhydrophobic graphene-based materials with self-cleaning and anticorrosion performance: an appraisal of neoteric advancement and future perspectives date: - - journal: colloids surf a physicochem eng asp doi: . /j.colsurfa. . sha: doc_id: cord_uid: xyj mc g abstract lotus like materials having superhydrophobicity is attaining greater demand due to the possibility of molding them into different high end applications. the major issue related to self-cleaning superhydrophobic surfaces is their restricted mechanical properties. the development of nanotechnology has brought many advantages in the fabrication and properties of superhydrophobic surfaces and thus it enhanced the demand of superhydrophobic surfaces. many scientific groups have studied and reported about the superhydrophobicity exhibited by graphene and its analogous derivatives. the fabrication of the devices having properties ranging from anti-sticking and self-cleaning to anti-corrosion and low friction is made possible by the incorporation of this wonderful two-dimensional material. this review focuses on the preparation and properties of graphene based superhydrophobic coating materials with special mention to the wide range of applications rendered by them. composite materials with superhydrophobic traits have got extensive consideration owing to their excellent characteristic properties such as self-cleaning, hindered corrosion ( ) , and application in water resistant electronic devices ( ) ( ) ( ) . the excellent water repellent properties observed for several natural objects such as lotus leaf, butterfly wings, rice plants etc. motivated scientists towards developing materials having exceptional water repellency ( ) . superhydrophobic surfaces are those facets having a water contact angle of or more ( ) and these materials are prepared with sufficient surface abrasion and minimum surface energy ( ) . both industrial and domestic applications are possible with "superhydrophobic surfaces" which made them ranked th in journals of material science discipline between and ( ) . superhydrophobic nanocoatings extend the area of nanotechnology and superhydrophobicity into a new dimension which has an essential role in improving the properties of coatings. the technologies and materials in coating industry are advancing day by day to improvise the efficiency of coatings by the incorporation of cost effective and greener concepts. undoubtedly, coating is one of the finest methods to alter the solid interface properties by paving a new protecting layer over the substrate via chemical or physical process. so the development of superhydrophobic nanocoatings are attaining more attraction due to its possible extended high end application ( ) . the surface wetting performance can be generally classified into categories hydrophilic, hydrophobic, superhydrophilic and superhydrophobic according to their water contact angle (wca). hydrophilic and hydrophobic have their wca within the range of ° < θ < ° and ° < θ < ° respectively. superhydrophilic and superhydrophobic domains are of considerable interest owing to the utmost surface wetting traits having wca differing in the stretch of ° < θ < °and ° < θ < ° respectively. the superhydrophobic regime is important as it experiences a perfect non wetting capability with an exceptionally elevated water contact angle that result in the rolling of water droplets easily ( ) . the work of jiang et al. ( ) on nanostructure based superhydrophobicity triggered the curiosity of scientific community and it was followed by several works on superhydrophobic nano materials. wenzel model gives a fair explanation of the contact angle of a substrate which is in contact with a liquid. cassie and baxter further proposed a model which deals with j o u r n a l p r e -p r o o f heterogeneous surfaces comprising of two fractions, the former deals with solid liquid and latter with the liquid air interface. the composition of the surface together with its roughness regulate the surface dampening and the water contact angle of the surface ( , ) . the nano-micro hierarchical roughness plays an eminent role in manipulating the characteristics of superhydrophobic nanocoatings and they are classified as inorganic, organic and inorganicorganic materials ( ) . silica encompassed materials are the most common choices for superhydrophobic nanocoating area. in fact, they are hydrophilic, but through chemical treatment they obtain superhydrophobicity ( , ) . carbonaceous materials such as carbon nanotubes, carbon nanofibers and fullerenes have gained focus in superhydrophobic nanocoatings in the recent years ( ) . inorganic and organic nanomaterials are also utilized as promising materials for enhancing the flexibility and molecular framework of these coatings ( ) ( ) ( ) . yuan et al. ( ) developed superhydrophobic ldpe with lotus-leaf-like characteristics and contact angle in the range of °. a vast array of inorganic fillers could be employed for designing hybrid superhydrophobic nanocoatings. qing et al. ( ) developed zno/polystyrene nano composite coatings having a wca of °. graphene, a younger cohort material, is nothing but the allotrope of carbon isolated by basic mechanical exfoliation in the earliest years of st century ( ) . it is a mono-atomic thin sheet of carbon arranged in a hexagonal honeycomb like crystalline lattice and it has attracted worldwide interest within no time because of fascinating properties arising from the two-dimensional structure and existence of sp bonded carbon atoms ( ) . graphene is believed to have a better future for the fabrication of various carbonaceous technical devices, owing to its superior conductivities at room temperature and inertness towards corrosion ( ) . graphene was isolated in and prior to that intercalated clays and fullerenes were used as coating materials for corrosion resistance ( , ) . for the last few years, application of graphene and graphene-related materials are topics of increasing research interests due to many outstanding features which make them suitable for a passive layer formation that protects metals from oxidation and corrosion( - ). the surface wettability of graphene has been discussed a lot and many efforts have been taken to control the same by modifying the surface of graphene( ). in order to reduce surface energy and increase surface roughness, a lot of methods have been employed by means of layer-by-layer assemblies ( ) ( ) ( ) , spraying ( ) , electrospinning ( , ) , spin coating( ), electrochemical reaction and deposition ( ) ( ) ( ) ( ) , chemical vapor deposition( )etc. though application of superhydrophobic nanocoatings is the main destination of all researches, the theory, materials and moulding has also fascinated the research community( ). self-cleaning is the most promising property of graphene coated superhydrophobic surfaces as water droplets can roll on the surface and take away the dirt sticking on the surface effectively. j o u r n a l p r e -p r o o f numerous artificial superhydrophobic nanocoatings with self-cleaning traits have been synthesized by different methods and are applied in diverse domains based on their applications ( ) ( ) ( ) ( ) ( ) ( ) . graphene coated nano-composite materials can be effectively used in antiicing and de-icing methods ( ) ( ) ( ) ( ) and used in biosensors, biomedical implants and devices, food packaging, industrial and marine equipment ( ) ( ) ( ) . graphene is an oil repellent material and hence could be used for effective separation of oil and water ( ) . it also imparts anti-corrosion property to a coating system ( ) . diverse varieties of graphene, mainly single layer/bilayer graphene films, graphene oxide (go), graphene nanoplatelets and graphene nanoribbons can be synthesized by modern techniques ( , ) . now a days, nanopellets of graphene are extensively used for the synthesis of composite materials via mixing with metals, ceramics and polymers to tailor the properties for various applications( ). graphene coatings are generally synthesized by chemical vapour deposition. nilsson and his coworkers studied the drawbacks regarding the corrosion inhibition properties of graphene coatings on metal surfaces and concluded that graphene coated by cvd acts as corrosion inhibitor only at low gas pressures ( ) . choi and co-workers( ) synthesized graphene/nafion nanohybrids with hierarchical petal-like, porous structure and superhydrophobicity (wca= °) by regulating the structures with respect to the chemical composition. lee et al. ( ) presented another simple route towards the fabrication of superhydrophobic graphene surface via thermal reduction method and they obtained a transparent nano-sphere structure with excellent wca as shown in ngyuen and co-workers synthesized graphene-based sponges with enormous absorption capacities ( times their own weight) there by making them apt for large-scale application( ). the increased surface roughness in nanoscale dimension was responsible for the superhydrophobicity with a ca of ( ). table the superhydrophobicity of a surface is purely dependent on the functionalities present on the surface as well as the morphology and contact angle and hence characterization of the coating is to account the elemental change as shown in figure . in one of our studies, we have utilized the technique of edax to analyze the elements present in the self-assembled nano porous coreshell tio -go hybrid material( ). durability analysis of superhydrophobic surface is very important as it has an immense effect in determining the application. mechanical durability is one of the crucial components in meeting the real-world application. the fabrication of a durable superhydrophobic material as a coating is an important aspect as it easily gets tampered by mechanical and chemical action ( ) . hierarchical nanostructures were behind this excellent superhydrophobicity ( ) . in another study, it was observed that the graphene/polyurethane sponges made by dip coating method were capable of showing enhanced mechanical durability and thermal and chemical stability. the sponges also showed good elastic recovery and the test using lubricating oil confirmed the recyclability as well ( ) . superhydrophobic surfaces made by laser induction process for desalination application were capable of showing enhanced mechanical durability as well as thermal stability. even after water jet treatment and scotch tape test, the surface maintained the superhydrophobic character. moreover, the surfaces were capable of retaining the nature even after seven cycles of scotch test ensuring the desalination application. the surface maintained the same pattern even after the removal of the tape, but later the degradation occurred due to the failure of adhesion and it is not associated with the failure in superhydrophobicity ( ) . it is observed that the topology of graphene/polypropylene surface is highly efficient to keep the water away and it forms a perfect cassie-baxter state. interestingly, after abrasion, the new surfaces were having increased roughness and maintains the hydrophobicity ( ) . in another study, a stretchable, robust and superhydrophobic graphene based thermoplastic polyurethane (tpu) composite with excellent mechanical durability was prepared. knife scratch and hand rub test were carried out in order to understand the durability. the composites were capable of exhibiting superhydrophobicity even after these harsh tests and water easily rolled off from the surface. sand paper abrasion test revealed that the surfaces were capable of maintaining the superhydrophobic behavior even after five cycles of . kpa of abrasion. moreover, at a low load of kpa, the surface maintained the behavior even after cycles due to the tensile strength of pristine graphene, physical support of tpu to the partially embedded nanoplatelets of graphene and the elastic nature of tpu ( ) . graphene based superhydrophobic surfaces tend to show excellent mechanical durability than rgo based systems. hence modification of rgo is usually done to increase the durability. recently, superhydrophobic hexadecyltrimethoxysilane-grafted rgo based melamine sponges having excellent mechanical and chemical stabilities were made. these materials maintained the mechanical stability even after cycles .due to the interaction between rgo and the grafted material, it shows chemical inertness as well ( ) . nanofibers of thermoplastic polyurethane (tpu) prepared via electrospinning were decorated using graphene for imparting hydrophobicity. even after , and % stretching, the fibers retained the superhydrophobicity due to the surface modification using graphene and they also maintained the superhydrophobicity in harsh chemical environment( ). j o u r n a l p r e -p r o o f corrosion generally describes the damage to metal or its alloy by means of chemical or electrochemical interactive reactions that occur on the surface of the metal due to the environmental exposure. this serious problem leading to the loss of metal as scrap is a major economic issue faced throughout the world. oiling, painting, electroplating etc. are the common methods followed to avoid this damage ( ) . ( ) . reproduced with permission from springer. the presence of organic pollutants in water is a major threat to the ecosystem. hence oil -water separation is considered as a major solution for environmental problems. oil water separation is a problem that is gaining momentum in the present scenario. hence the development of oil water separating materials from superhydrophobic graphene coatings is considered very important. conventional materials used for this process include zeolites, activated carbon, natural clays, straw, wool fibers etc. these materials exhibit low efficiency in the separation process due to their hydrophilic nature. hence the fabrication of novel superhydrophobic materials is the need of the hour. superhydrophobic surfaces that are oleophilic or super oleophilic can be employed to separate oil and water by concentrating oil into a semi solid phase. tai and co-workers employed sponges for effective oil water separation ( ) . go sponges devoid of any positively charged groups exhibit the capability to adsorb anionic dyes via π-π interactions( ). jayaramalu et al. ( ) made a biomimetic composite for oil-water separation using highly fluorinated go and nanocrystalline zeolite imidazole framework zif- (figure ). this oil water separation is necessary to avoid the spilled oil in oceans and other water sources. due to j o u r n a l p r e -p r o o f the self-assembly of the micro-mesoporous structure, the material was able to show a wca of ° with an oil ca of °. the sponges incorporated with these hybrid materials were able to show high absorption capacity to oils, polar and non-polar solvents. since these sponges float over water, they can easily extract oil. oil absorption capacity of the sponge and the hybrids are shown in figure . moreover the reusability of the fabrics was confirmed by the sorption-mechanical squeezing test. the absorption capacity was determined by simply immersing the cotton/rgo fabric in inorganic solvents and oil. the soaked fabric absorbed the oil and the absorbed oil was separated by simple squeezing and are suitable for the reuse. it was also suitable for removing oil/solvents about - times of its weight. cao et al. ( ) made anticorrosive and superhydrophobic coatings using fluorinated polydopamine/chitosan/rgo composite. by following the self-polymerization, polydopamine was anchored onto the surface of rgo. d structured chitosan/rgo hybrid hydrogels had oil absorption capacity - times higher than their weight. nguyen et al. ( ) used the superhydrophobic pdms/go sponges for oil water separation. these materials were able to absorb the spilled oil to about to times higher than their weight. but the absorption capacity was found to be reduced after repeated use in the case of oil. whereas, the sponges were able to retain the absorption capacity to organic solvents even after repeated cycles. xiao et al. ( ) fabricated go/nanofiber gels for oil-water separation. higher wca ensures easy transport of oil into these aerogels. this can be monitored by directly immersing the material into the oil-water medium as shown in figure and absorption capacity can be evaluated from its weight. yang et al. ( ) synthesized silica incorporated go by sol-gel process, for separating oil from water. zhou et al. ( ) used graphene/polyurethane based superhydrophobic sponge for oil-water separation. it showed an absorption efficiency of times higher than its weight due to the superhydrophobic network structure (figure ) . table . anti-bacterial property is very effective for biosensing and biomedical applications, food packaging and for industrial as well as marine equipment manufacturing. amine functionalized in another study, shateri-khalilabad and yazdanshenas ( ) ( ) .even though graphene shows excellent optical absorption, adsorption is not that much efficient, hence the incorporation of plasmonic nanoparticles together with graphene could enhance the adsorption behavior of the mask. superhydrophobicity together with electrothermal efficiency helps the surface to remain dry and clean in the glaze ice condition ( ) . the removal of surface ice can be easily done in graphene due to its highly hierarchical structure and moreover the partially embedded structure shows excellent durability against sand paper abrasion( ). the antibacterial, and mechanical robustness of functionalized and hybrid graphene based superhydrophobic materials can be exploited for making cost effective facial masks and pp kits. apart from medical applications, graphene based superhydrophobic materials can also be utilized for anti-icing, sensing, pollution control and self-cleaning applications. switchable superhydrophobic graphene based surfaces can also be utilized in the development of smart materials. thus there lies a vast prospect for graphene based superhydrophobic surfaces in the development of value added products in future. the authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. reversible switching between superhydrophilicity and superhydrophobicity why superhydrophobic surfaces are not always icephobic tailoring of the electrical properties of carbon black-silica coatings for de-icing applications an electrically and mechanically self-healing composite with pressure-and flexion-sensitive properties for electronic skin applications creating robust superamphiphobic coatings for both hard and soft materials superhydrophobic surfaces buoyancy increase and drag-reduction through a simple superhydrophobic coating global research report superhydrophobic nanocoatings: from materials to fabrications and to applications stick and slide" ferrofluidic droplets on superhydrophobic surfaces super-"amphiphobic" aligned carbon nanotube films resistance of solid surfaces to wetting by water wettability of porous surfaces one-step synthesis of superhydrophobic coating on cotton fabric by ultrasound irradiation superhydrophobic rtv silicone rubber insulator coatings preparation of superhydrophobic, long-neck vase-like polymer surfaces preparation and anti-icing property of a lotus-leaf-like superhydrophobic low-density polyethylene coating with low sliding angle mechanically robust superhydrophobic polymer surfaces based on protective micropillars facile approach in fabricating superhydrophobic zno/polystyrene nanocomposite coating electric field effect in atomically thin carbon films. science ( -) removal of methylene blue from aqueous solution by graphene oxide impermeable atomic membranes from graphene sheets self-cleaning antireflective coatings assembled from peculiar mesoporous silica nanoparticles a facile layer-by-layer deposition process for the fabrication of highly transparent superhydrophobic coatings transparent superhydrophobic films based on silica nanoparticles mechanically durable carbon nanotube− composite hierarchical structures with superhydrophobicity, self-cleaning, and low-drag spray deposition of electrospun tio nanoparticles with self-cleaning and transparent properties onto glass superhydrophobic terpolymer nanofibers containing perfluoroethyl alkyl methacrylate by electrospinning influence of the average surface roughness on the formation of superhydrophobic polymer surfaces through spin-coating with hydrophobic fumed silica one-step electrodeposition process to fabricate corrosion-resistant superhydrophobic surface on magnesium alloy advantage of super-hydrophobic surface as a barrier against atmospheric corrosion induced by salt deliquescence corrosion resistance and long-term durability of superhydrophobic nickel film prepared by electrodeposition process simultaneous fabrication of superhydrophobic coatings on cathodic and anodic copper surfaces with micro/nano-structures one-step chemical vapor deposition and modification of silica nanoparticles at the lowest possible temperature and superhydrophobic surface fabrication self-cleaning fibers via nanotechnology: a virtual reality micro-, nano-and hierarchical structures for superhydrophobicity, self-cleaning and low adhesion wetting and superhydrophobicity a review on self-cleaning coatings facile creation of bio-inspired superhydrophobic ce-based metallic glass surfaces bio-inspired self-cleaning surfaces preparation and anti-icing of superhydrophobic pvdf coating on a wind turbine blade role of water vapor desublimation in the adhesion of an iced droplet to a superhydrophobic surface preparation of superhydrophobic surfaces on al substrates and the anti-icing behavior anti-icing superhydrophobic coatings antibacterial fluorinated silica colloid superhydrophobic surfaces antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms superhydrophobic graphene/cellulose/silica aerogel with hierarchical structure as superabsorbers for high efficiency selective oil absorption and recovery water/oil repellency and work of adhesion of liquid droplets on graphene oxide and graphene surfaces exploring graphene as a corrosion protection barrier highly crystalline graphene oxide nano-platelets produced from helical-ribbon carbon nanofibers graphene nanomaterials: fabrication, properties, and applications a review on graphene-based polymer composite coatings for the corrosion protection of metals graphene coatings: probing the limits of the one atom thick protection layer superhydrophobic graphene/nafion nanohybrid films with hierarchical roughness a route towards superhydrophobic graphene surfaces: surface-treated reduced graphene oxide spheres superhydrophobic and superoleophilic properties of graphene-based sponges fabricated using a facile dip coating method in situ preparation of graphene/polypyrrole nanocomposite via electrochemical co-deposition methodology for anti-corrosion application thiolated graphene-based superhydrophobic sponges for oil-water separation a cost-effective method for preparing mechanically stable anti-corrosive superhydrophobic coating based on electrochemically exfoliated graphene preparation of superhydrophobic electroconductive graphene-coated cotton cellulose high performance graphene-based foam fabricated by a facile approach for oil absorption multifunctional superhydrophobic polymer/carbon nanocomposites: graphene, carbon nanotubes, or carbon black? bioinspired fabrication of superhydrophobic graphene films by two-beam laser interference biomimetic graphene surfaces with superhydrophobicity and iridescence laser-induced graphene in controlled atmospheres: from superhydrophilic to superhydrophobic surfaces hydrophobic bacteria-repellant graphene coatings from recycled pencil stubs fabrication of superhydrophobic reduced-graphene oxide/nickel coating with mechanical durability, self-cleaning and anticorrosion performance graphene-based nanostructured hybrid materials for conductive and superhydrophobic functional coatings p-phenylenediamine strengthened graphene oxide for the fabrication of superhydrophobic surface superhydrophobic lotus-leaf-like surface made from reduced graphene oxide through soft-lithographic duplication characterization of superhydrophobic polymer coating synthesis, characterization, and surface wettability properties of amine functionalized graphene oxide films with varying amine chain lengths preparation and characterization of polyvinylidene fluoride/graphene superhydrophobic fibrous films superhydrophobic polyvinylidene fluoride/graphene porous materials graphene oxide as a prospective graft in polyethylene glycol for enhancing the toughness of epoxy nanocomposites nanoribbon/polyurethane sponge for efficient oil/water separation at static and dynamic states the stability of the superhydrophobic surfaces robust superhydrophobic graphene-based composite coatings with self-cleaning and corrosion barrier properties superhydrophobic hybrid membranes by grafting arc-like macromolecular bridges on graphene sheets: synthesis, characterization and properties solution-processable, durable, scalable, fluorine-grafted graphene-based superhydrophobic coating for highly efficient oil/water separation under harsh environment synthesis of self-assembled and porous nano titaniagraphene oxide hybrids for toughening the epoxy formation of superhydrophobic microspheres of poly (vinylidene fluoride-hexafluoropropylene)/graphene composite via gelation superhydrophobic functionalized graphene aerogels facile fabrication of superhydrophobic octadecylamine-functionalized graphite oxide film triblock copolymer grafted graphene oxide as nanofiller for toughening of epoxy resin constructing mechanochemical durable and self-healing superhydrophobic surfaces fabrication of superhydrophobic reduced-graphene oxide/nickel coating with mechanical durability, self-cleaning and anticorrosion performance durable superhydrophobic/superoleophilic graphene-based foam for high-efficiency oil spill cleanups and recovery engineering polydimethylsiloxane with two-dimensional graphene oxide for an extremely durable superhydrophobic fabric coating super-hydrophobic graphene coated polyurethane (gn@ pu) sponge with great oil-water separation performance robust superhydrophobic laser-induced graphene for desalination applications fabrication of superrepellent microstructured polypropylene/graphene surfaces with enhanced wear resistance a stretchable and super-robust graphene superhydrophobic composite for electromechanical sensor application reduced graphene-based superhydrophobic sponges modified by hexadecyltrimethoxysilane for oil adsorption a highly stretchable, super-hydrophobic strain sensor based on polydopamine and graphene reinforced nanofiber composite for human motion monitoring corrosion behavior of superhydrophobic surfaces: a review fabrication of self-cleaning superhydrophobic nickel/graphene hybrid film with improved corrosion resistance on mild steel carbon nanotube-reinforced siloxane-pmma hybrid coatings with high corrosion resistance high-performance polystyrene/graphenebased nanocomposites with excellent anti-corrosion properties improvement of anticorrosion ability of epoxy matrix in simulate marine environment by filled with superhydrophobic poss-go nanosheets superhydrophobic epoxy coating modified by fluorographene used for anti-corrosion and self-cleaning a novel hydroxyl epoxy phosphate monomer enhancing the anticorrosive performance of waterborne graphene/epoxy coatings one-step synthesis of superhydrophobic polyhedral oligomeric silsesquioxane-graphene oxide and its application in anti-corrosion and anti-wear fields go@ cusilicate nano-needle arrays hierarchical structure: a new route to prepare high optical transparent, excellent self-cleaning and anticorrosion superhydrophobic surface environmental performance of graphene-based d macrostructures biomimetic superhydrophobic/superoleophilic highly fluorinated graphene oxide and zif- composites for oil-water separation reduced graphene oxide-coated cotton as an efficient absorbent in oil-water separation facile synthesis of fluorinated polydopamine/chitosan/reduced graphene oxide composite aerogel for efficient oil/water separation graphene/nanofiber aerogels: performance regulation towards multiple applications in dye adsorption and oil/water separation surface roughness induced superhydrophobicity of graphene foam for oil-water separation one-pot synthesis of robust superhydrophobic, functionalized graphene/polyurethane sponge for effective continuous oilwater separation synthesis of graphene oxide-sio coated mesh film and its properties on oil-water separation and antibacterial activity the preparation of superhydrophobic graphene/melamine composite sponge applied in treatment of oil pollution graphene sponge for efficient and repeatable adsorption and desorption of water contaminations a biomimetic, multifunctional, superhydrophobic graphene film with self-sensing and fast recovery properties for microdroplet transportation d superhydrophobic reduced graphene oxide for activated no sensing with enhanced immunity to humidity fabrication of a mechanically-stable anti-icing graphene oxide-diatomaceous earth/epoxy coating reusable and recyclable graphene masks with outstanding superhydrophobic and photothermal performances a superhydrophobic/electrothermal synergistically anti-icing strategy based on graphene composite fabrication of superhydrophobic titanium surfaces with superior antibacterial properties using graphene oxide and silanized silica nanoparticles using graphene/tio nanocomposite as a new route for preparation of electroconductive, self-cleaning, antibacterial and antifungal cotton fabric without toxicity antimicrobial features of organic functionalized graphene-oxide with selected amines three-dimensional hierarchical and superhydrophobic graphene gas sensor with good immunity to humidity passive anti-icing and active deicing films key: cord- -ninzeazy authors: fiorillo, luca; cervino, gabriele; matarese, marco; d’amico, cesare; surace, giovanni; paduano, valeria; fiorillo, maria teresa; moschella, antonio; la bruna, alessia; romano, giovanni luca; laudicella, riccardo; baldari, sergio; cicciù, marco title: covid- surface persistence: a recent data summary and its importance for medical and dental settings date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: ninzeazy recently, due to the coronavirus pandemic, many guidelines and anti-contagion strategies continue to report unclear information about the persistence of coronavirus disease (covid- ) in the environment. this certainly generates insecurity and fear in people, with an important psychological component that is not to be underestimated at this stage of the pandemic. the purpose of this article is to highlight all the sources currently present in the literature concerning the persistence of the different coronaviruses in the environment as well as in medical and dental settings. as this was a current study, there are still not many sources in the literature, and scientific strategies are moving towards therapy and diagnosis, rather than knowing the characteristics of the virus. such an article could be an aid to summarize virus features and formulate new guidelines and anti-spread strategies. coronavirus disease (covid- ) is an infectious respiratory disease caused by the virus called severe acute respiratory syndrome coronavirus (sars-cov- ), belonging to the coronavirus family. an infected person may experience symptoms after an incubation period that could vary from about to days (there have rarely been cases of incubation periods of days), during which time the person could still be contagious. to limit transmission, precautions should be taken, such as adopting careful personal hygiene, washing hands frequently and wearing masks. coronavirus mainly affects the lower respiratory tract and causes a number of symptoms described as flu-like, including fever, cough, shortness of breath, muscle pain, tiredness and gastrointestinal complaints such as diarrhea. in severe cases, pneumonia, acute respiratory distress syndrome, sepsis and septic shock could occur, up to the death of the patient. among the collective preventive measures, it should be noted that in , during the sars epidemic, the major collective catering companies in china and hong kong adopted the obligation to wear surgical masks for their service personnel to protect both the workers of companies and the public. this professional category is particularly exposed to potentially infectious contacts, both active and passive. in the same way, a person in charge of equipment who is not equipped with the appropriate personal protective equipment (ppe) will find themselves exposed to contact with the dirty dishes and recent food remains of a large number of customers. further interventions in the restaurant sector may include a prohibition on the distribution of buffets for both food and dishes [ ] [ ] [ ] [ ] . in recent weeks there has been about a great deal of discussion about the contamination and decontamination of inanimate surfaces. in fact, the duration before inactivation of the covid- virus on surfaces (liquid, solid or gaseous) is still debated. in most cases, the spread between people occurs through the respiratory droplets emitted by an infected individual through coughing or sneezing, which, are subsequently inhaled by a healthy person who is nearby. this also caused an initial diffidence on the part of people in purchasing products of any kind coming (including by post) from the areas affected by the epidemic, leading to economic damage. it is possible to become infected by touching surfaces or objects where the virus is present and then bringing your hands towards your mouth, nose or eyes [ ] [ ] [ ] [ ] [ ] [ ] . in ideal conditions, the virus can in fact persist on different surfaces for hours or days. the surfaces most exposed to this type of transmission include, for example: public transport handholds [ ] [ ] [ ] [ ] . the viral titration, viral assay or viral count is the count, made in the laboratory, of the number of viral particles of a given virus under examination present in a biological sample. it is used in microbiological research, diagnostics and in the production of antiviral vaccines-all situations that require knowledge of the amount of virus being analyzed or used. we used tissue-culture infectious dose (tcid) per milliliter for covid- persistence evaluation [ ] [ ] [ ] [ ] [ ] . the aim of this article is to evaluate, through the analysis of the current literature, how long this virus can remain active on different surfaces. it is too early to be able to carry out a review with meta-analysis of the literature given the incredible relevance of this topic, but it is certainly a step to clarify this pandemic. the following questions were used to develop the study framework according to the pico (population/intervention/comparison/outcomes) guidelines: • what is the persistence of sars-cov- on surfaces? • what is the mean persistence of coronaviruses compared to sars-cov- ? an investigation methods protocol was used, according to the prisma statement; the aim of the prisma statement is to help authors improve the reporting of systematic reviews and meta-analyses. it can be used as a basis for reporting reviews of other types of research. the use of checklists like prisma is likely to improve the reporting quality of a systematic review and provides substantial transparency in the selection process of papers in a systematic review. furthermore, pico guidelines have been used to prepare summary questions [ ] [ ] [ ] . the full texts of all studies related to the main revision topics were obtained for comparing the inclusion parameters: • sars-cov- features articles; the persistence of other coronaviruses. the following were the exclusion criteria: • not enough information regarding the topic; • articles published prior to january ; • no access to the title and abstract. research was conducted in five electronic databases, including medline, pubmed, and embase. in addition, a manual search was conducted for relevant studies published. digital and manual searches were then performed. the data search was performed in order to add significant studies and to increase the sensitivity of this study. for the search we used keywords according to medical subject headings (mesh). (sars-cov- or coronavirus) and (persistence or surface) terms were investigated on information sources, as specified in section . . first, the manuscript titles list was highlighted to exclude irrelevant publications and search errors. the final selection was performed by reading the full texts of the papers in order to approve each study's eligibility based on the inclusion and exclusion criteria. data selection and revision was performed by independent reviewers of three different affiliations (luca fiorillo, messina; giovanni surace, reggio calabria; alessia la bruna, milan). they singularly analyzed the obtained papers. results obtained were compared and discussed with a fourth independent reviewer (gabriele cervino, university of messina) when a consensus could not be reached. for the stage of the full-text articles' revision, a complete independent analysis was performed. results were singularly analyzed and items about viruses' persistence on different materials were evaluated and shown. we created a table to show a summary of inherent virus features. the grade of bias risk was independently considered, as reported in [ ] [ ] [ ] . potential causes of bias were investigated: • selection bias; • performance bias and detection bias; • attrition bias; • reporting bias; • examiner blinding, examiner calibration, standardized follow-up description, standardized residual graft measurement, standardized radiographic assessment. we conducted a manual synthesis of article results. during the first search, studies were obtained. after applying inclusion and exclusion criteria, only the remaining articles were further analyzed. then, articles were manually selected, and finally articles were obtained ( figure ). • performance bias and detection bias; • attrition bias; • reporting bias; • examiner blinding, examiner calibration, standardized follow-up description, standardized residual graft measurement, standardized radiographic assessment. we conducted a manual synthesis of article results. during the first search, studies were obtained. after applying inclusion and exclusion criteria, only the remaining articles were further analyzed. then, articles were manually selected, and finally articles were obtained ( figure ). results of individual studies were shown after an accurate analysis in table . the aim of this table is to summarize coronaviruses' persistence time. van doremalen et al. [ ] evaluated the stability of sars-cov- and sars-cov- in aerosols and different surfaces. they evaluated these viruses' decay rates using bayesian linear regression. they conducted their experiment using aerosols (< µm) containing sars-cov- ( . % tissue-culture infectious dose (tcid ) per milliliter) or sars-cov- ( . - . tcid per milliliter) generated by a nebulizer. ten different experimental conditions involving sars-cov- or were evaluated. results on infectious titer reduction are shown in table . the data were expressed as % tissue-culture infectious dose (tcid ). it is important to specify that the limit of detection for this experiment was . × . tcid per liter of air for aerosols; . tcid per milliliter of medium for plastic, steel, and cardboard; and . tcid per milliliter of medium for copper. kampf et al. [ ] showed how different coronaviruses could persist on different types of inanimate surfaces. they also evaluated some environmental characteristics, such as temperature or humidity. they showed how human coronavirus could be influenced by temperature, as or • c reduced the duration of persistence of coronaviruses on inanimate surfaces. however, at the temperature of • c, the persistence could be greater than or equal to days. another important result is that the persistence was longer with higher inocula. warnes et al. [ ] evaluated coronaviruses' persistence on metal and non-metal samples. they inoculated plaque forming units (pfu) on different materials: polyfluorotetraethylene (teflon; ptfe), polyvinyl chloride (pvc), ceramic tiles, glass and stainless steel. coronaviruses' persistence was at least days (and days for silicon rubber) at • c. despite this, it could be rapidly inactivated by brass and copper nickel surfaces in less than min. copper nickel surfaces were effective but less than brass copper; in these cases, the inactivation time was up to min in the fingertip contamination model. warnes et al. demonstrated how a higher percentage of copper could lead to superior antiviral properties. another important factor reported in this study was that the release of ions from copper and the formation of reactive oxygen species (ros) take part in the deactivation of the virus. furthermore, the authors report that following an analysis carried out with a transmission electron microscope (tem), the virus was normally present on common surfaces such as stainless steel, but on copper surfaces it appeared to be damaged and intact particles were few. it was not possible to conduct a bias risk analysis according to the prisma statement as specified in the previous section. unfortunately, the limited number of articles obtained does not allow for the realization of a systematic review. the individual studies were analyzed, and the experiments leading to the results shown in table were rigorously conducted and are repeatable analyses [ ] . the literature concerning the characteristics of these viruses is still scarce, especially if one considers only covid- . the aspects related to the persistence of the virus on surfaces not only represent an environmental and public health problem concerning schools, roads, offices. it is a much bigger problem if hospitals, operating theaters, and sanitary waiting rooms are considered, especially in the new and continuously increasing "covid departments". knowing how the virus behaves in contact with surfaces and with different disinfectants could be important for the sanitization of medical environments. in particular, some authors deal with the optimization of infection control in operating rooms. some devices are used in hospital operating rooms for single use only, but other devices, surfaces, handles and cords could be transmission vehicles [ ] . ong et al. [ ] evaluated the presence of coronavirus in a hospital room of covid- patients. some surfaces, such as the toilet bowl and the sink, were positive. room air samples and samples collected after cleaning were negative. the time span varied according to the characteristics of the type of surface: the less-porous ones like plastic and steel were the worst because they absorb droplets less easily, preserving the active virus. additionally, the different environmental conditions could affect the amount of ventilation of the rooms and the humidity [ ] . according to van doremalen et al. [ ] , aerosol and surface virus transmission is plausible, since it can remain viable and infectious for hours or days. kampf et al. [ ] showed how human coronaviruses can remain infectious on surfaces for up to days at room temperature. this is an important factor about coronaviruses' spread. from this, it is easy to see that if someone tends to touch the environment often-especially if not properly disinfected-the possibility of becoming infected increases. furthermore, the droplets present in the form of an aerosol of an infected patient can not only easily spread, but also easily settle and last for several hours on a surface. kampf et al. [ ] investigated different biocidal agents on coronaviruses. they demonstrated how ethanol ( %- %), -propanol ( %- %), the combination of % -propanol with % -propanol, glutardialdehyde ( . %- . %), formaldehyde ( . %- %) and povidone iodine ( . %- . %) readily inactivated coronavirus infectivity by approximately log or more. sodium hypochlorite required a minimal concentration of at least . % to be effective. hydrogen peroxide was effective with a concentration of . % and an incubation time of min. an important finding is the ineffectiveness of chlorhexidine. within min, a concentration of . % revealed no efficacy against coronavirus. it is a result that does not support some guidelines for dentistry [ ] . kampf et al. [ ] concluded that these viruses can remain on surfaces up to days and that surface disinfection could be performed with . % sodium hypochlorite or %- % ethanol for minute. according to warnes et al. [ ] , coronavirus persists in an infectious state on surfaces for several days. warnes et al. [ ] demonstrated the survival of coronaviruses on different surfaces for up to days. concerning the persistence of the virus on different surfaces, and in particular on metals containing copper, these findings are interesting and could lead to the development of new surfaces with viricidal or bactericidal properties. in confined environments, especially if poorly ventilated, viral particles of less than . µm in size may remain in the environment as a secondary aerosol. studies on the topic indicate that a sneeze could release up to million droplets into the air, less than a million from a cough and about from speaking out loud. the droplets eliminated from the airways, if larger than µm, from a height of m settle on flat surfaces in - s and reach horizontally about . m away, then evaporate rapidly, dry and become solid material. this material reaches a size of - µm. studies on tuberculosis have shown that this material, maintaining its infectious capacity, could be inhaled and, thanks to its size, reach the most peripheral parts of the lungs, becoming a secondary biological aerosol [ ] . there has not been much discussion about the importance of ventilating environments to prevent sars-cov- infection, and although the viral particles have not been studied sufficiently for their ability to achieve dangerous concentrations from a distance in confined environments, increased ventilation in an environment is believed to reduce the cross-infection of airborne diseases. therefore, existing recommendations could be amended to include ventilating public spaces, including means of transport, with suitable means. it is essential to focus on preventing infection by using ventilation suitable to reduce the infectious capacity of the coronavirus. it has been widely demonstrated that natural ventilation causes better air exchange compared to mechanical ventilation-up to changes per hour of rooms when the windows are completely open [ ] . most international guidelines recommend about changes per hour for isolation rooms in case of infections. medical and dental staff will have to work safely, and this is complicated by the fact that there are still no official guidelines that tell them how to behave. moreover, as suggested by spagnuolo et al., dentists should avoid the scheduling of any patient: only such urgent dental diseases can be considered during the covid- outbreak. the waiting time of patients in dental offices could highly predispose patients to be infected. when dentists treat patients, they should intercept potentially infected persons before they reach the operating areas; for example, identifying those with a fever measuring > . • c and posing a few questions about the patient's general health status in the last days, and about the risk of having been in contact with other infected persons. dentists as well as medical office employees should always be covered by facial masks, and they should remind patients to maintain the -m distance with each other during their waiting time [ , ] . disinfecting surfaces is one of the aspects to which to give greater attention, being done with the detergents already in use today, along with the washing of hands and the use of suitable ppe. in regards to ppe, surgical masks must be used by those who could transmit the virus, but those who work in contact with the patient's aerosol must use ffp and ffp (filtering facepiece particles) masks. there should only be one patient in waiting rooms. this is also true in clinical areas; in the case of minors who need an escort, the escort must be at a distance from operators, and must wear a surgical mask. at the end of each session, the surfaces will then have to be cleaned, and the air exchanged. the same procedures should be adopted in the waiting room and in other areas where the patient might pass or touch objects. certainly, some tools, such as quick tests (once validated), can become useful in the hands of the doctor/dentist to understand if the patient, or some member of their team, is potentially infected. we are aware of the limitations of this article; it is not possible to make a review of the literature due to the lack of data. so, this is only a summary of the available information. further studies are needed in order to verify the persistence times of coronavirus on surfaces more precisely. however, we have considered proceeding with the creation of this summary, while respecting the guidelines of systematic reviews and also not having sufficient numbers. the reason for this choice lies in the incredible topicality and reliability of the data obtained, with the hope that this work could be widely and immediately used to contain the pandemic. the purpose of this summary is to draw a line and clarify the characteristics of sars-cov- with regard to its persistence on surfaces. this article is of great interest and could be used to write new guidelines by epidemiologists, having a clear summary of the current situation. from our analysis it is possible to deduce some aspects. the virus can reach surfaces in the form of an aerosol. therefore, following nebulization through people (sneezing or coughing) or electromedical machinery, infection via surfaces should be considered, since the latter could remain viable and infectious for hours or days. on average, the different coronaviruses persist in an infectious state on surfaces for several days, even up to nine. surface disinfection could be performed with . % sodium hypochlorite or %- % ethanol for minute. copper has shown antiviral properties-so much so that the virus appears damaged or altered on copper surfaces. other experiments are certainly needed, on different surfaces or even on biological surfaces, to better understand the persistence times of this virus and promote adequate standards. a novel coronavirus (covid- ) outbreak: a call for action suspending classes without stopping learning: china's education emergency management policy in the covid- outbreak reverse logistics network design for effective management of medical waste in epidemic outbreaks: insights from the coronavirus disease (covid- ) outbreak in wuhan (china) facing the covid- outbreak: what should we know and what could we do? open access epidemiological data from the covid- outbreak characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention immediate psychological responses and associated factors during the initial stage of the coronavirus disease (covid- ) epidemic among the general population in china risk management of covid- by universities in china covid- : fighting panic with information is covid- receiving ade from other coronaviruses? microbes infect backcalculating the incidence of infection with covid- on the diamond princess novel coronavirus disease (covid- ): paving the road for rapid detection and point-of-care diagnostics prevention is better than the cure: risk management of covid- understanding unreported cases in the covid- epidemic outbreak in wuhan, china, and the importance of major public health interventions short-term forecasts of the covid- epidemic in guangdong and zhejiang covid- ) outbreak: what the department of endoscopy should know )f-fdg pet/ct findings of covid- : a series of four highly suspected cases similarity in case fatality rates (cfr) of covid- /sars-cov- in italy and china the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? the prisma extension statement the effects of the prisma statement to improve the conduct and reporting of systematic reviews and meta-analyses of nursing interventions for patients with heart failure the prisma extension statement for reporting of systematic reviews incorporating network meta-analyses of health care interventions: checklist and explanations the cochrane collaboration's tool for assessing risk of bias in randomised trials association between risk-of-bias assessments and results of randomized trials in cochrane reviews: the robes meta-epidemiologic study robis: a new tool to assess risk of bias in systematic reviews was developed aerosol and surface stability of sars-cov- as compared with sars-cov- persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents human coronavirus e remains infectious on common touch surface materials risk of bias assessment: ( ) overview. zhonghua liu xing bing xue za zhi perioperative covid- defense: an evidence-based approach for optimization of infection control and operating room management surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus (sars-cov- ) from a symptomatic patient covid- transmission in dental practice: brief review of preventive measures in italy assessing the dynamics and control of droplet-and aerosol-transmitted influenza using an indoor positioning system covid- outbreak: an overview on dentistry d printing beyond dentistry during covid epidemic: a technical note for producing connectors to breathing devices acknowledgments: in this covid- emergency period, thanks go to all clinicians and researchers who every day risk their lives for research. the authors declare no conflict of interest. key: cord- -tjrz ulk authors: harris, debra; taylor, keyanna p.; napierkowski, katie; zechmann, bernd title: indoor finish material influence on contamination, transmission, and eradication of methicillin-resistant staphylococcus aureus (mrsa) date: - - journal: herd doi: . / sha: doc_id: cord_uid: tjrz ulk objective: the purpose of this study was to evaluate environmental surface materials used in healthcare environments for material composition, methicillin-resistant staphylococcus aureus (mrsa) viability, and a comparison of two disinfectants, a bleach germicidal cleaner and decon , a novel disinfectant. background: contaminated environmental surfaces have been associated with outbreaks of healthcare-associated illness (hais). one in every patients in u.s. acute care hospitals acquire a healthcare-associated illness, leading to consequences such as elevated morbidity, mortality, and a decrease in quality of life. in the patient environment, mrsa can remain viable from hours to up to days. methods: environmental surface materials were evaluated as new and worn. material composition and properties were assessed to evaluate surface integrity and the influence on the disinfection of mrsa. inoculated materials were used to assess mrsa viability over time and the efficacy of a manufacturer’s recommended cleaning and disinfection product compared to a novel disinfectant. results: environmental surface materials respond differently in appearance and roughness, when mechanically worn. when measuring mrsa survival, at hr, mrsa colony forming unit (cfu) counts were reduced on the copper sheet surface and solid surface with cupric oxide. by hr, all mrsa counts were zero. bleach and the novel disinfectant were equally effective at disinfecting mrsa from all surface types. conclusions: this study highlights a gap in knowledge about the impact of type and wear of environmental surface materials used in healthcare environments on contamination with epidemiologically important organisms. in conclusion, environmental surface material wear, properties, and cleaning and disinfection efficacy are important factors to consider when addressing hais. healthcare-associated infections (hais) are a leading cause of illness and death in the united states and worldwide (han et al., ) . annually, an estimated . million patients suffer from hais in the united states, leading to about , deaths (klevens et al., ) . a recent study found that one of every patients in u.s. acute care hospitals acquires an hai, with the most prevalent pathogens being clostridium difficile infection (cdi) and methicillin-resistant staphylococcus aureus (mrsa; klevens et al., ; magill et al., ) . most hais are thought to be preventable; however, published mitigation guidelines are incongruent, obstructing a clear path for reduction success (stone, ) . unresolved is the role of environmental surface contamination across the continuum of healthcare spaces as a contributing factor for hais. during the last decade, substantial scientific evidence has increased, demonstrating that contamination of environmental surfaces in hospital rooms plays an important role in the transmission of several key healthcare-associated pathogens including mrsa, vancomycin-resistant enterococcus, cdi, and norovirus (boyce, ; otter et al., ; weber et al., ; weber et al., ; weinstein & hota, ) . one pathogen often linked in the literature to contaminated surfaces in hospitals is the gram-positive pathogen, mrsa. mrsa is a bacteria that is resistant to many antibiotics and may cause severe health problems including skin infections, fever, chest pain, fatigue, and muscle aches. transmission of mrsa can occur directly from environment to patient or through healthcare workers, with one study showing that % of nurses contaminated their gloves by touching objects in the room of patients with mrsa without ever having touched the patient (boyce et al., ) . furthermore, mrsa can remain viable for up to days on surfaces and up to weeks on cotton blanket material (beard-pegler et al., ; duckworth & jordens, ) . the ability to survive on surfaces for an extended time is a clear justification that environmental surface materials play a significant role. this previous literature shows a clear justification for the claim that environmental surfaces in the patient environment should be given major consideration in preventing the transmission of mrsa. furthermore, mrsa can remain viable for up to days on surfaces and up to weeks on cotton blanket material. the purpose of this study was to evaluate environmental surfaces used in healthcare environments for material influence in disinfection of mrsa, material composition, and compare a common disinfectant and decon (decon seven systems, scottsdale, az), a biological disinfectant and chemical contaminant. the purpose of this study was to evaluate environmental surfaces used in healthcare environments for material influence in disinfection of mrsa, material composition, and compare a common disinfectant and decon . this experimental study sought to simulate contamination of new and worn environmental surface materials by patients or healthcare workers infected with mrsa through contact with surfaces and producing aerosols through coughing or sneezing. this study did not include human subjects or animals. five types of environmental surface samples were evaluated including stainless steel (ss), copper sheet (cs), high-pressure laminate (hpl), acrylic polymer solid surface (aps), and scu with a total number of environmental surface samples (table ) . samples used as supplied were considered "new" representing environmental surface materials for initial use. the worn samples were abraded using the taber rotary platform abrasion tester (taber industries, north tonawanda, ny) to represent worn samples with a specific end point to simulate years of normal wear for surface materials utilizing astm g - , modified for accelerated wear. material documentation was assessed to determine the manufacturer's intended useful life. failure tests were conducted on high-pressure plastic laminate to set a baseline. visual assessment for wear was reached, indicated by wear through of printed pattern/color in all four quadrants of the sample was used to determine failure (american national standards institute, ) . the first test utilized the cs- wheel and the material failed at , cycles, the second test utilized the cs- wheel and the material failed at , cycles, and the final test utilized the s- wheel and the material failed at cycles. all tests used a wheel loading of , g and a vacuum suction level of ( %) with a height of the vacuum pick up nozzle set to . mm ( . in.) above the sample surface. based on these tests, it was determined that the s- wheel was to be used on all materials at . cycles ( ) to simulate years of accelerated use across all material types (ansi, ) . based on these tests, it was determined that the s- wheel was to be used on all materials at . cycles ( ) to simulate years of accelerated use across all material types (ansi, ) . with the accelerated wear test established, samples were abraded in ambient room conditions (temperature at f [ . c]; humidity at %). each sample was weighed before and after the abrasion test and analyzed for mass loss. the data were analyzed for percent mass loss by type of material and within the type of material comparing new and worn. the data were transformed to satisfy assumptions of normality and homogeneity of variances. analysis of variance was conducted on the transformed data to determine whether there was a difference in the five environmental surface materials. since there was a difference in the means of the transformed variables (p < . ), post hoc multiple comparisons were conducted. tukey's honestly significant difference test for pairwise comparisons was used to compare new and worn within material type and compare for mass loss by type of material. scanning electron microscopy (sem) and atomic force microscopy (afm) were used for surface characterization of new and worn samples. for sem, small samples of the different surfaces ( cm ; . in.) were mounted on aluminum mounts with carbon tape. samples were then imaged with a hitachi tm plus tabletop sem (hitachi high technologies america, irving, tx) in low vacuum mode, with kv, and mm ( . in.) working distance. for afm studies, the same samples were mounted on metal discs with super glue and imaged with a dimension icon afm (bruker corporation, billerica, ma) in peak force tapping mode. surface roughness was analyzed with the software nanoscope version . . the initial bacterium received was a freeze-dried pellet of staphylococcus aureus subspecies aureus rosenbach. to prepare the initial bacterium into a suspension for inoculation, serial dilutions were completed using a smear plate method and optical densities were recorded using a thermo electron spectronic dþ single beam spectrophotometer (thermo fisher, waltham, ma). the serial dilutions and recorded optical densities were used to determine the bacteria's growth rate and calculate suspension concentration. utilizing the same method for serial dilutions, the suspension used to inoculate the surface material samples had a concentration of . Â cfu/ml. coded and sterilized samples ( ), triplicates of new materials, were inoculated with a prepared mrsa suspension equivalent to . Â cfu/ml. samples were inoculated with, thirty ml (or . ml) droplets of the prepared suspension were placed into each sample. this size droplet was used to better simulate small droplets of contaminated body fluids from colonized patients and to ensure that the suspension would dry on each surface. this drop size and number of drops vary in comparison to lankford et al. ( ) who used three hanging drops of approximately ml (. ml) to inoculate surfaces. to determine the length of time mrsa survives on environmental surface materials in a controlled environment, samples were inoculated and stored covered with sterile petri dishes in an incubator at c ( f) and cultured by tryptic soy agar contact plates at the designated time points. the time points measured were min, hr, hr, and hr. evaluating the effectiveness of manufacturer's recommended cleaning and disinfecting processes for environmental surface materials in healthcare facilities and laboratories required inoculation of samples (triplicates) that were then stored uncovered in a sterigard iii advance model, class ii type a/b hood (the baker company, sanford, me) for approximately hr. two hours were chosen to ensure that surfaces would dry before being contact plated. once surfaces were dry, they were contact plated and then cleaned and disinfected by spraying and air drying in the fume hood with a minimum contact time of min. the chemical disinfectant used to assess recommended cleaning and disinfection was clorox healthcare bleach germicidal cleaner (the clorox company, oakland, ca). these procedures were replicated with another set of samples, triplicates of new and worn samples for the materials, to assess the effectiveness of decon , a disinfectant and decontaminant developed for bio and chemical warfare, using recommended cleaning and disinfecting processes for environmental surface materials in healthcare facilities and laboratories. the conditions in the fume hood were recorded as at . c ( f) and % relative humidity and . c ( f) and % relative humidity for the healthcare germicidal bleach and decon , respectively. for each test, after incubation, plates were counted and recorded. for statistical analysis related to the length of time mrsa survives on the environmental surface materials, the cfu measurements were treated as ordinal categories, including too few to count (tftc) and too many to count (tmtc). the ordinal categories were , tftc, a numerical count for - , and tmtc. these categories were collapsed into treatments of three groups (cs and scu, aps and hpl, and ss) to determine whether the category was dependent on material type at each time ( min, hr, hr, and hr) using fisher's exact test. percent mass loss by type of environmental surface material. there was an interaction between material type and wear, but only for cs (p < . ). there were statistically significant differences between cs and the aps (p ¼ . ), between cs and the scu (p ¼ . ), and between cs and ss sheet (p ¼ . ). significant differences were also found between hpl and the aps (p ¼ . ) and ss (p ¼ . ). multiple comparisons showed no difference in percentage mass loss between ss, aps, or the scu. no difference was shown between the scu and hpl nor between hpl and cs. cs had the most percent mass loss ( %) and the ss had the least percent mass loss ( %; figure ). percent mass loss of new and aged samples by environmental surface material type. when comparing new and worn surfaces of the same material type, all materials except ss indicated a significant difference: ( ) cs (p ¼ . ), ( ) aps (p ¼ . ), ( ) scu (p ¼ . ), and ( ) hpl (p ¼ . ). microscopy. sem and afm of new and worn samples revealed differences in surface topography among the different samples (figures and ) . while new ss showed the least surface roughness of all analyzed surfaces ( nm), new aps and hpl showed the highest surface roughness ( and nm). while the worn surface of ss and scu appeared smooth in the sem and afm (figures and -a of cs samples ( . %) but decreased surface roughness of scu ( . %) and aps ( . %; table ). when assessing the length of time mrsa survives on the environmental surface samples, gmrsa survived between and hr, while for the aps and ss, mrsa expired between and hr. at min, all surfaces besides scu showed too many cfus to count (> ). at hr, every surface type showed too many cfus to count. at hr, cs, scu, and hpl showed no cfus, while the aps and ss surface samples showed low numbers (ranging from to ) of mrsa cfus still present. lastly, at hr, all surfaces showed no cfus remaining. environmental surface materials were evaluated for dependence of type of material. at min and two hr, all material types measured tmtc, indicating that cfu counts were independent of material type. at hr, there was an association between material type and cfu counts (p ¼ . ). the combined copper category had all cfu measurements of at this time, while one of the plastics and ss had cfu measurements in the countable range ( - ). at hr, the cfu count was independent of the material type. when comparing cs and scu at each time point, at min, cs was more likely to have tmtc cfus than the scu. at hr, both materials had tmtc cfus. at hr, both types of materials had no cfus with no significant differences. the effectiveness of a manufacturer's recommended cleaning and disinfecting process was tested using healthcare bleach germicidal cleaner on all inoculated surface material types, with results showing that mrsa was eradicated. similarly, to the healthcare bleach germicidal cleaner, the effectiveness of decon was tested, with results showing that all surface types inoculated with mrsa were eradicated. however, after inoculation and before cleaning and disinfection, cs and scu showed lower cfus of mrsa compared to all other surfaces. the purpose of this study was to evaluate a selection of environmental surface materials used in healthcare environments to determine differences in material composition and influence on the viability of mrsa; and compare a common disinfectant, healthcare bleach germicidal cleaner to decon , an epa registered biological disinfectant for norovirus. in this study, all surfaces of the materials performed differently when worn except for ss. ss is a hard metal and did not lose significant mass, nor change surface characteristics after wearing. all other materials lost significant mass after the wear study. when comparing materials, cs and hpl significantly lost mass compared to ss and the two solid surfaces. in this study, all surfaces of the materials performed differently when worn except for ss. microscopy of the new and worn environmental surface materials revealed differences in surface topography among the different materials. of the new materials, ss (brushed) and hpl appeared to have the smoothest surface, while the aps and hpl appeared to have the roughest surface. of the worn materials, the surfaces of the ss and the scu were smooth, compared to the aps, hpl, and cs, which appeared flaky. these results suggest that the surface of the ss did not significantly change; however, after wear, the scu became smoother, while the remaining surface materials increased in roughness. future research should investigate if topographical changes increase or decrease the risk of forming biofilms or providing reservoirs for pathogens. cleaning efficacy is influenced by the surface contamination, properties of each surface material, the cleaning and disinfection technology used, and the efficiency of environmental services staff. environmental surfaces with differing material properties may be a factor that should be considered in specification with concern for cleaning and disinfection of patient environments where hais are common. mrsa survival time from undisturbed surfaces in a controlled environment was between and hr. however, a reduction in mrsa cfus was found on the cs and solid surface infused with cupric oxide between and hr after inoculation. previous studies echo similar results that focus on the use of copper in surface materials as a natural antimicrobial agent that degrades cell structure and reduces both gram-negative and gram-positive bacteria (eser et al., ; esolen et al., ; jing et al., ; schmidt et al., ) . sharpe and schmidt ( ) found that antimicrobial copper alloy products reduce the bioburden over time and that the bioburden was reduced on adjacent materials, suggesting a potential microbiological halo effect, which should be studied further. additional studies with similar results indicated the efficacy of copper as an antimicrobial surface for hais such as mrsa (humphreys, ; schmidt et al., schmidt et al., , . to be registered with the environmental protection agency (epa, ) as an antimicrobial product and to use the claim for "continuous reduction" of bacterial, antimicrobial copper alloy products must achieve a log reduction in viable bacteria within a -hr contact time. . . . a reduction in mrsa cfus was found on the cs and solid surface infused with cupric oxide between and hr after inoculation. healthcare bleach germicidal cleaner and decon were effective in disinfecting mrsa on all surface types. there was no interaction between material type and disinfection. the centers for disease control and prevention have established guidelines for cleaning and disinfection in healthcare settings, which recommend cleaning and disinfecting noncritical environmental surfaces with epa registered low-or intermediate-level disinfectants (rutala et al., ) . however, our study utilized decon , a biological and chemical decontaminant developed for biowarfare to eradicate biological contaminants such as antibiotic-resistant escherichia coli, antibiotic-resistant klebsiella pneumoniae, clostridium difficile, human coronavirus, and sars-cov- . future studies should test for efficacy and material compatibility. decon were effective in disinfecting mrsa on all surface types. this study had several limitations to consider. when conducting the wear study, a modified standard was used to develop a protocol for accelerated wear testing. this study only tested for mechanical wear, but future studies should utilize methods for accelerated life testing that include wear, heat, ultraviolet (uv) light, humidity, and chemical erosion. mrsa time points for survival should be selected for higher sensitivity. the crucial time frame for tracking the growth of mrsa in a controlled environment was between min and hr. reducing the inoculum to decrease mrsa cfus would be beneficial as well as counting all cfus so that the data would be numerical and not categorical. future research should aim to develop a validated methodology to conduct accelerated life testing of environmental surfaces. a methodology for testing accelerated life of environmental surfaces should be comprehensive including more factors that may elucidate limits of the materials and potential incompatibilities such as chemical exposure, temperature, humidity, and uv light. future research should continue to evaluate how long pathogens are viable on surface materials to determine significant differences that may inform specifications for the built environment. observing the prevalence of mrsa on surfaces between hr and hr postinoculation is a key time frame to discover the specific lifetime of mrsa on various environmental surfaces. future studies should examine the role of environmental surface materials on pathogen survival, and cleaning and disinfection efficacy in an applied healthcare environment. this study was conducted in a controlled laboratory environment without soil loads. other research has shown that mrsa can live on surfaces up to days and cotton blankets for up to weeks. future studies should consider expanding the experiments to use soil and to conduct field experiments where mrsa survival and cleaning protocols may vary. finally, future studies should utilize an expanded selection of environmental surface materials, pathogens, and cleaning methods. the results of this study suggest that environmental surfaces behave differently when worn; therefore, a careful selection of materials should be considered in high use environments like healthcare. antimicrobial materials, and cleaning and disinfection methods and technologies should be studied further in the context of pathogen contamination and transmission in healthcare environments. while chemical disinfection and cleaning procedures in the healthcare environment have been studied extensively, this study highlights a gap in knowledge regarding the impact of type and wear of environmental surface materials used in healthcare environments on pathogens with epidemiologically important organisms. in conclusion, environmental surface material wear, properties, and cleaning and disinfection efficacy are important factors to consider when addressing hais. environmental surface material wear, properties, and cleaning and disinfection efficacy are important factors to consider when addressing the specification of materials for healthcare environments. the impact of wear on environmental surface material properties may provide a reservoir for pathogens. copper and copper-infused surfaces reduced the bioburden, suggesting a continuous reduction that may contribute to a productive reduction in overall contamination. cleaning and disinfection methods such as novel disinfectants should be considered in the context of pathogen contamination and transmission in healthcare environments. nema standards publication ld - -high-pressure decorative laminates observations on the resistance to drying of staphylococcal strains environmental contamination makes an important contribution to hospital infection environmental contamination due to methicillin-resistant staphylococcus aureus: possible infection control implications adherence and survival properties of an epidemic methicillinresistant strain of staphylococcus aureus compared with those of methicillin-sensitive strains protocol for the evaluation of bactericidal activity of hard, non-porous copper containing surface products antimicrobial activity of copper alloys against invasive multidrug-resistant nosocomial pathogens the efficacy of self-disinfecting bedrail covers in an intensive care unit cleaning hospital room surfaces to prevent health care-associated infections: a technical brief self-disinfecting and microbiocide-impregnated surfaces and fabrics: what potential in interrupting the spread of healthcare-associated infection inhibition of biofilm growth on polymer-mwcnts composites and metal surfaces estimating health careassociated infections and deaths in u.s. hospitals assessment of materials commonly utilized in health care: implications for bacterial survival and transmission & emerging infections program healthcare-associated infections and antimicrobial use prevalence survey team evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings guideline for disinfection and sterilization in healthcare facilities sustained reduction of microbial burden on common hospital surfaces through introduction of copper antimicrobial copper alloys decreased bacteria on stethoscope surfaces copper surfaces are associated with significantly lower concentrations of bacteria on selected surfaces within a pediatric intensive care unit control and mitigation of healthcare-acquired infections: designing clinical trials to evaluate new materials and technologies economic burden of healthcareassociated infections: an american perspective the role of the surface environment in healthcareassociated infections role of hospital surfaces in the transmission of emerging health care-associated pathogens: norovirus, clostridium difficile, and acinetobacter species contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? clinical infectious diseases the authors would like to thank dr. erica bruce for the support provided by her laboratory. we would also like to acknowledge statisticians, dr. jeanne hill and dr. amy maddox, for their contribution to analysis for this study. finally, the authors would like to thank dr. diane hartman for her generosity for sharing her knowledge and expertise. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the authors disclose receipt of the following financial support for the research, authorship, and publication of this article: this work was supported by the university research committee (urc) at baylor university, grant number . debra harris, phd, cid, aahid https://orcid. org/ - - - keyanna p. taylor https://orcid.org/ - - - key: cord- -fnn wshy authors: moccia, giuseppina; motta, oriana; pironti, concetta; proto, antonio; capunzo, mario; de caro, francesco title: an alternative approach for the decontamination of hospital settings date: - - journal: j infect public health doi: . /j.jiph. . . sha: doc_id: cord_uid: fnn wshy background: the increasing emergence and spread of multiresistant microorganisms in hospital wards is a serious concern. traditional protocols are often not sufficient to protect patients susceptible to serious and life-threatening infections, therefore new strategies for decontaminating hospital environments are crucial to reducing microbial transmission and the spread the nosocomial infections. the adoption of modern technologies is indicated to supplement traditional methods and to improve desired levels of surface disinfection. aim: this work aims to report the development, implementation, and validation of cleansing and sanitizing procedure for critical clinical settings through the innovative use of disposable cloths pre-impregnated with solutions containing different active formulations and biocidal agents, relating to the areas to be treated (low, moderate, high-risk). methods: the implementation and validation of the sanitizing system were conducted in different wards of two healthcare structures. the protocol for the study involved a structured selection of representative surfaces, such as the floor, bathroom, desk, and beds. microbiological analyses were performed according to iso - : . findings: the efficiency of the proposed system was measured through the estimation of total microbial count values on the different surfaces before and after the sanitization operations by traditional methods and by the system described here. the results demonstrated a significant reduction in the microbial count that always fell below the threshold value. for the analyzed surfaces such as shower tray, bathroom floor, toilet edge, the traditional system had an effectiveness of less than %, whereas pre-impregnated cloths succeed to eliminate about % of the bacteria present. as an example, on the floor we observed a microbial count reduction from > to cfu/ cm( ) with the new method ( % of colonies were destroyed), while with the traditional one we have a reduction from > to cfu/ cm( ) ( % of microbial colonies). moreover, the advantages of using this sanitization system are not limited to disinfecting surfaces and limiting cross-contamination but involve all activities related to the cleaning and disinfection operations, including the training and education of the operators and traceability of the operations. conclusions: the innovative disinfection and cleaning protocol used in the present study proved to be a highly valuable alternative to the traditional cleaning procedures in healthcare settings for the sanitizing process of all kinds of surfaces. all tools were specifically designed to improve disinfection efficiency and to reduce the problems associated with traditional methods, such as preventing cross-contamination events, limiting the physical efforts of operators, and avoiding incorrect practices. our findings add support to the knowledge that an effective sanitization procedure is critical in minimizing microorganisms' transmission and cross-contamination. the recent public health emergency of coronavirus disease declared by the world health organization (who) introduced the need for new strategies to face the pandemic. in particular, hospitals have to use appropriate protocols to manage their space, staff, suppliers, patients to reduce infection, and in-hospital transmission. the virus aerosol deposition on protective apparel or floor surface and their subsequent resuspension is a potential transmission pathway and effective sanitization is critical in minimizing aerosol transmission of pathogens. cleaning practices are of utmost importance to improve the healthiness of indoor environments and hospitals to prevent further spread and nosocomial infections diffusion [ ] [ ] [ ] [ ] . hospitals and some critical clinical departments, such as intensive care units, oncological units, departments with dialyzed patients, are considered potential points for opportunistic pathogenic microbes [ , ] . pathogens can spread from patient to patient through contact with inanimate surfaces, including medical equipment and the environment close to the patient [ ] [ ] [ ] . there is clinical evidence of a close relationship between environmental hygiene and transmission of microorganisms which produces nosocomial infections such as environmental contamination from methicillin-resistant staphylococcus aureus, the transmission of norovirus, clostridium difficile, pseudomonas aeruginosa, and acinetobacter spp. [ ] [ ] [ ] [ ] [ ] [ ] ] . various approaches have been used to disinfect critical environments but there are still many difficulties related to traditional equipment used and to implementation of procedures. moreover, in many cases, due to lack of formal surveillance, the rate of healthcareassociated infections is high and compliance with hygiene is low. strategies for the decontamination of hospital environments are described in guidelines proposed by various international committees [ ] , in particular, the advisory committee for the practice of controlling sanitary infection underlines the importance of correct cleaning operations to promote decontamination and the necessary use of disinfectants to reduce the microbial contamination in hospitals [ ] [ ] [ ] . although traditional cleaning protocols are applied and appropriate disinfectants are used in the right concentrations, statistical analyses revealed increasing infections and data suggest that traditional protocols are not sufficient to protect patients susceptible to serious and life-threatening infections [ ] . we call for extra care and attention on the proper design, use, and disinfection of the surfaces in hospitals to minimize the potential spread of microorganisms and the risk of cross-contamination thus reducing the infections. traditional cleaning methods in medical and healthcare facilities include dry cleaning and dusting. these methods can disperse dust particles throughout the area surrounding the patient, negatively influencing the quality of the air and promoting contamination. moreover, they cannot be able to contrast the dry deposition of the airborne dispersed microorganisms in a patient room, where it has been hypotized that contamination may come from the resuspension of dust particles from the floors or other hard surfaces [ ] [ ] [ ] . multiple drug resistance (mdr) microorganisms and healthcare-associated infections (hais) remain significant challenges, both in hospitals and in long-term care facilities [ , , , ] . they may cause patients or residents to suffer from additional ailments, and sometimes result in death (in italy it is estimated in . cases per year). the existing problem of mdr microorganisms and hais show us that, in order to properly follow the primum non nocere principle in healthcare, we should take into account all of the epidemiological, biological, organizational, and social aspects of healthcare. in this study, we propose a multidisciplinary approach based on the use of disposable microfibre or spunlace cloths impregnated with different disinfectant solutions to be used in the cleaning operations of healthcare settings. this approach also considers of primary importance the use of a specifically designed trolley and mandatory training and education of the operators. this procedure leads to an improvement in air quality, disinfection of surfaces close to the patient, and a significant reduction in cross-contamination. in addition to the antimicrobial efficacy, the disinfectant products proposed have low toxicity, low flammability index, low skin irritation and no residue are even detectable. the cleaning activity is part of a complex holistic approach that combines the need to ensure the disinfection of the area close to the patient with the safety of the cleaning operator, considering also the right training and education. some studies have highlighted the importance of decontamination processes after the use of reusable detergents and/or cloths to avoid cross-contaminations [ ] . in common cleaning protocols, the same mop is used to clean a large area, without any difference between contaminated and uncontaminated spaces. there is a mandatory practice required for the washing procedure with frequent replacement of disinfectant solutions (e.g. after every three or four chambers, with intervals of no more than min) and high temperature treatment of the mop. however, cloths and mop, subjected to continuous high temperature decontamination treatments, progressively lose their cleaning capability crumbling their texture. on the other hand, disinfection methods used in many intensive care units and other healthcare facilities include the innovative use of antimicrobial wipes. these products seem to be more efficient to remove the microbial load on surfaces [ ] . the use of alcohol wipes has also been shown to reduce the microbial load usually present on toilets [ ] . however, very elaborate procedures are required to ensure maximum effectiveness of the antibacterial wipes, otherwise even these, if not properly used, can favor the spread of microorganisms (e.g. tables, headboards) [ ] . operators should not use the same wipe repeatedly to clean and disinfect different surfaces and have to be correctly trained. disposable wipes pre-impregnated with a disinfectant can also be used for low-level disinfection when spot cleaning of non-critical surfaces is required [ ] . an important question raised by the use of disinfectants for non-critical surfaces, such as wardrobe and bedside table, and in healthcare facilities is the contact time specified on the product label, which is often too long to be practiced. the labels of most products registered by epa (environmental protection agency) to be used against hbv (hepatitis b virus), hiv (human immunodeficiency virus), or mycobacterium tubercolosis specify a contact time of at least min. such a long contact time is not practical for surface disinfection in a healthcare environment, since most healthcare facilities apply a disinfectant leading it to dry for about min [ ] . the method proposed here uses disposable microfiber or spunlace cloths, pre-impregnated with a mixture specifically formulated to ensure a detergent and disinfectant synergic action. according to who guidelines the detergent and disinfectant solutions must meet the requirements for disinfectant/antimicrobial products for contaminated surfaces and may contain different active ingredients or mixtures of these, for example, alcohol, chlorine [ , , ] , phenols, polyphenols [ ] , quaternary ammonium salts [ ] , tertiary amines, chlorhexidine gluconate. the formulations used for the pre-impregnation contain increasing concentrations of the selected disinfectant as required for the sanification of environments in the different areas of a hospital (e.g. low, moderate, and high-risk areas). the staff dedicated to cleaning operations represents the most critical point in the cleaning and sanitizing processes of hospitals. the hospital staff or the company that carries out the cleaning oper- ations must ensure that its manager and all the operators involved in the service, do their work or duties safely, guaranteeing a result capable of satisfying the requirements, the expected quality levels, and the objectives of the activity carried out. the training plans should include, in addition to a general basic course for health service workers, targeted courses on the order to be performed. it is necessary that the staff employed is well trained on the protocols drawn up according to the environment to be cleaned. in particular, it has to be employed personnel who have been properly trained to carry out cleaning activities e.g. in the infectious diseases and nuclear medicine departments, operating rooms, intensive or sub-intensive therapy services, which require greater attention to the cleaning process, avoiding turn-over. employees must perform only the functions for which they were trained; they must receive specific training for the areas of intervention; they also need to be trained on the biological and chemical hazards to which they could be exposed on the intervention site. the environmental intervention program and its actuation mechanism may vary according to the size of the structure and to the services provided. the staff training course programs include mainly the following themes: cleaning (products and procedures) and environmental disinfection; use of work equipment; correct use of the trolley, that is specifically designed to contain separately pre-impregnated cloths for the different surfaces and allows to avoid contact between dirty and clean cloths; definition of internal paths (clean/dirty); personal hygiene; hand washing; adoption of measures to prevent the transmission of infections; use of the supplied devices; staff clothing; disposal of medical waste; risk management; quality plan; significant impact. other training topics in compliance with the safety of the workers themselves may be the prevention of risks from the hospital environment; method of execution of the rooms service; correct use of protective devices; collection, transport, and disposal of dangerous substances and/or preparations, written protocols for daily disinfection activities, traceability cards, checklists. documentation of work and registration of all phases can avoid legal problems and could increase professional credibility according to presidential decree / and ec directive / [ ] . tracing processes helps the operator to identify errors and possibly to remedy. therefore, the processing and use of traceability cards, (see fig. ), represent a medical-legal protection tool for operators and healthcare companies. the traceability system must document the process steps: operator data and signature, date and time, process steps, hospital structure reference, any notes or observations, etc. in order to monitor and make traceable all the phases of the cleaning process and simplify the organization of the operator's work, each activity must be recorded on special cards/checklists and signed by the operator who performed the interventions. the introduction of traceability cards has proven to be a very useful tool integrated with the training and educational programs adopted by the hospitals for the implementation and validation of this new cleaning strategy for the selected hospital settings, leading to an overall improvement in the cleaning and sanitization activities of hospital wards. in order to use measurable outcomes to investigate the hospital cleaning as a scientific process, we performed microbiological analyses in three different environments according to international standard uni en iso - : , which specifies a horizontal method for enumeration of microorganisms able to grow and form colonies in a solid medium after aerobic incubation at • c. a specific quantity of the test samples, or a specified quantity of an initial suspension, was surface plated on a solid agar culture medium contained in petri dishes. other plates were prepared under the same conditions using decimal dilutions either of the test sample or of the initial suspension. the plates were incubated under aerobic conditions at • c for h. the number of microorganisms per gram of sample or the number of microorganisms per milliliter of samples was calculated from the number of colonies obtained on the plates containing less than colonies/plate. all samples were collected before and after disinfection processes, both by the traditional system and the one proposed by us, to evaluate the efficiency of the different cleaning protocols in the same hospital wards. the standard cleaning protocol of the hospitals included cleaning with a reusable mop dipped in a solution of water and a disinfectant, the same sanitization solution used with the innovative system. once dipped the mop was used to clean approximately square meters room, following which the mop was double dipped and reused in adjacent rooms. for the cleaning protocol purpose in this study, microfiber wipes were used that, thanks to their positive charge (containing polyester and nylon), attract negatively charged particles such as dust, liquid, and pathogens. microfiber wipes used had the following characteristics: high density with × surface area than a conventional cotton-loop mop; were split to create gripping "hooks" that make them highly absorbent; weigh of g and specifically g/m ; various dimensions such as × cm for surface cleaning and × cm for floors and larger surface; more than % of disinfectant solution impregnated could be absorbed ( , ml/cm of disinfectant solution). one single cloth was used for each medium surface/room to avoid contamination between different rooms: operator removed the wipe from the packet, seal the packet to avoid other wipes drying out, start wiping surfaces and at the end, it was thrown in the trash. the cleaned surface was allowed to air dry for min and then we collected samples for bacterial culture. for each room - high-touch surfaces, such as sink, desk, shower, bed were chosen for the assessment of the cleanliness, and the cleaning staff was not informed about the sampling. about - samples were collected in triplicate for each room, in different locations, as reported in tables a, b, . statistical analysis was performed with graphpad prism . (graphpad software, inc., san diego, ca). the results were considered significant at p-value < , . disposable pre-impregnated wipes with many different disinfectants were obtained from cle.pr.in. srl and a specially designed cleaning trolley was delivered by femir srl. wipes used in this work were impregnated with a sanitization solution of % alcohol, % chlorine, phenols, % quaternary ammonium salts, % chlorhexidine gluconate, all solutions were tested in the same conditions. all trolleys were designed to prevent cross-contamination events, limit the operators' physical efforts, avoid incorrect postures, and equipped with advanced accessories: • n. closed l bag holders with divider and pedal set; • green base with wheels Ø mm, for outdoors; • n. drawers of l (green, white, gray, cream) for arranging the different types of cloths to be used for cleaning; • gray bar for handle hook; • hooks for storing cleaning tools; • plastic container to hold waste bags; the tray must be inserted interlocking on the handlebar handle of the trolley. • rolled paper holder, completely in plastic, which can be inserted into the edge of the container. it is complete with a transparent plastic cover, to avoid contact of the paper with the work environment. the trolley was designed to separate the storage of cloths from the waste section, to avoid contamination of the material and clean instruments. the surfaces were also made without corners and edges, and caps and covers were affixed to close any holes that are difficult to reach during cleaning operations. furthermore, the basic idea of the project was that for each ward a specific trolley was used that had not to be moved in different wards to avoid cross-contamination events. the advantages of using this innovative cleaning system compared to traditional methods are shown by the results of tables a, b, , which summarize the results of the swabs carried out in different areas of two healthcare facilities, one private and one public. the results showed in these tables were obtained with microfiber wipes pre-impregnated with ethanol %, however all disinfectant solutions were tested and gave the same performance. the significant reduction in the microbial count, that always falls below the threshold value, reported thereafter, demonstrates the effectiveness of the system proposed here. the following tables show that for the analyzed surfaces such as shower tray, bathroom floor, toilet edge, etc. the traditional system has little efficacy, with the effectiveness of less than %, whereas pre-impregnated cloths succeed to eliminate about % of the bacteria present. cross-contaminations, disinfection, and cleaning strategies play an important role in the everyday organization of hospitals and many scientific studies are reporting precise protocols for sanitizing healthcare environments [ ] [ ] [ ] . in the framework of the present international outbreak related to coronavirus disease covid- , rigorous measures are necessary to optimize the qual- ity of care provided to infected patients and to reduce the risk of pathogen transmission to other patients or healthcare operators. regardless of the type of surface (hands, environmental surfaces, fabrics), the objective of a cleaning and sanitizing procedure is to reduce contamination to an acceptable level of safety by applying operating methods designed to remove pathogens from surfaces. the procedure described in this work is based on a holistic approach for the decontamination and sanitization of surfaces in healthcare facilities with different risk levels (low, moderate, high). a careful analysis of the characteristics of the antimicrobial agent chosen to disinfect the critical surface was necessary and it was crucial to define the right composition of disinfectant solutions so that these were not corrosive to the treated surfaces and did not release strong odors. regarding the use of disinfectants, guidelines exist to define the minimum concentration of biocide in an optimal disinfectant solution to control the spread of harmful bacteria and other microorganisms. the disinfectant formulation must contain enough antimicrobial agents to be effective for a given application. the presence of a greater quantity of antimicrobial agent does not provide any advantage to the resulting composition, essentially it could only produce a disadvantage on the treated surface and, more generally, on environmental pollution. on the other hand, having a lower concentration of antimicrobial agent will make the composition less effective than necessary for the required use. results, reported in table a , were obtained using microfiber wipes pre-impregnated with ethanol % to clean different surfaces in a patient room of public healthcare structure and showed a better performance than traditional cleaning protocols: for exam-ple on the floor we observed a microbial count reduction from > to cfu/ cm with the new method ( % of colonies were destroyed), while with traditional one we have a reduction from > to cfu/ cm ( % of microbial colonies). moreover, in literature it was demonstrated that the microfiber system has superior microbial removal efficiency compared to cotton string mops [ , ] , however, there is no experimental data on pre-impregnated wipes with various disinfectant solutions. previous experimental data record efficacy principally for cleaning and disinfection process with microfiber/steam technology or wipes dampened with water, where authors demonstrated that the introduction of microfiber into the hospital sanitization process, in particular into operating room environment, has been environmentally and fiscally beneficial [ ] [ ] [ ] [ ] . the antimicrobial efficacy of a pre-impregnated cloth is also related to the interaction of the disinfectant solution with the support. the interaction of the support with the disinfectant solution could lead to a decreasing amount of biocide in the solution. therefore, in the realization of this work, we used pre-saturated cloths which means cloths that are saturated by the manufacturer with the desired disinfectant solution and delivered to the user in a wet format. saturated pre-impregnated cloths solve, or at least reduce, the problem of decreasing the availability of the active ingredient because they offer the possibility of changing the concentration of disinfectant in the solution during the manufacturing process which is consistent with the desired percentage of biocide agent (solution concentration). the characteristics of the surface to be cleaned have also to be considered when choosing the cleaning support (spunlace or microfiber), in fact, in some cases, the presence of non-uniform surfaces seems to favor the use of microfiber wipes that are more adaptable, even in most hard-toreach spaces. in table results showed that the total microbial load estimated on sampling performed on different surfaces in a staff room of public healthcare structure was reduced more than % cfu/ cm , only for floor attending room we have a reduction of %. a possible weakness related to the use of pre-impregnated spunlace or microfiber could be the quick evaporation of the solvent if the package is not properly closed and there is no good adhesion between wipes and surfaces, with less efficiency of the system. the use of a disinfectant solution pre-impregnated microfiber or spunlace cloth for different kinds of surfaces was supported by an appropriated trolley designed to help the operator during the cleaning procedure. all tools were specifically designed to improve disinfection efficiency and to eliminate the problems associated with traditional methods, such as preventing cross-contamination events, limiting the physical efforts of operators, avoiding incorrect postures and errors. moreover, this innovative method for cleaning and disinfecting floors avoid the risks of falling or tripping because the presence of wet floors is significantly reduced thanks to the use of pre-impregnated microfiber cloths. in addition, this procedure involves the use of ergonomic cleaning tools capable of reaching even difficult-to-reach surfaces, significantly reducing the use of stairs and stools. however all tools must be cleaned and disinfected to improve cleaning procedures, otherwise they could be one of the possible sources for contamination. this system shows several advantages for the operator, for occupation quality and work satisfaction, and helps in protecting the environment [ , ] according to the new parameters provided for by current regulations and international guidelines. the use of pre-impregnated cloths reduces the amount of water to be used and spills into the pipes, also eliminating the disinfectant solutions that are thrown into the sewers daily. this item was also deeply investigated through an lca (life cycle assessment) study that allowed to confirm that the system can be suitably included in an epd (environmental product declaration) system [ ] . the procedure implemented in this work provides evidence on the sanitization of hospital settings and examination of different products in terms of commodity, variety, composition, and chemical nature, demonstrating that pre-impregnated microfiber cloths could be a useful alternative for hospital infection-control program. quantitative assessment of the level of cleanliness by swab cultures revealed the better performance of the proposed method highlighting the vital role that infection prevention and control play in improving healthcare systems. this approach also considers of primary importance the use of a specifically designed trolley and mandatory training and education of the operators. this procedure leads to an improvement in air quality, disinfection of surfaces close to the patient, and a significant reduction in cross-contamination. an additional advantage of this system is the simultaneous dusting, cleaning, and disinfection action which avoids passing three times on the same surface. overall this study showed that the use of disposable preimpregnated cloths has many advantages: • the cleaning operator will be able to achieve higher cleaning and decontamination standards than traditional methods, thanks to the synergistic action of microfiber cloths (which already show high microbicide capacity on their own) and of detergent and disinfectant mixtures. • the use of pre-impregnated cloths for dusting and wet cleaning will prevent phenomena of air dispersion of contaminated particles • the patient who occupies sanitized rooms with disposable cloths will be better protected from accidental cross-contamination diffusion. • the cleaning operator is less exposed to chemical, biological, physical, and postural risks. • the hospital will be less exposed to the risk of contamination and the spread of nosocomial infections and will be able to more easily manage the personnel involved in the cleaning procedures because these will be faster, less tiring, and require fewer extraordinary interventions (weekly, monthly). deaths: leading causes for estimating health care-associate disinfections and deaths in us hospitals bacterial contamination of inanimate surfaces and equipment in the intensive care unit microorganisms in confined habitats: microbial monitoring and control of intensive care units, operating rooms, cleanrooms and the international space station airborne hydrogen peroxide for disinfection of the hospital environment and infection control: a systematic review a renaissance for the pioneering s rrna gene ific basic concepts of infection control. portadown: international federation of infection control study on the effectiveness of disinfection with wipes against methicillin-resistant staphylococcus aureus and implications for hospital hygiene mopping up hospital infection health-care-associated infections: risk factors and epidemiology from an intensive care unit in northern india uses of inorganic hypochlorite (bleach) in health-care facilities new approach for evaluating the public health risk of living near a polluted river determination of c/ c carbon isotope ratio assessment of perchloratereducing bacteria in a highly polluted river healthcare infection control practices advisory committee. guideline for disinfection and sterilization in healthcare facilities rethinking sterile: the hospital microbiome the survival and transfer of microbial contamination via cloths, hand and utensils cleaning for health report how clean is clean? proposed methods for hospital cleaning assessment who wash in health care facilities: global baseline report . world health organization surveillance of nosocomial infections: a preliminary study on hand hygiene compliance of healthcare workers two-years surveillance of fungal contamination in three hospital departments in campania region efficacy of 'sporicidal' wipes against clostridium difficile are toilet seats a vector of transmission of methicillin-resistant staphylococcus aureus the development of a new three-step protocol to determine the efficacy of disinfectant wipes on surfaces contaminated with staphylococcus aureus bacterial contamination of keyboards: efficacy and functional impact of disinfectants treatment of acute hypoxemic respiratory failure caused by chlorine exposure accidental systemic exposure to sodium hypochlorite (clorox) during hemodialysis report of a fatal case, supplemented with an experimental and clinico-epidemiological study potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance choosing disinfectants a standardized test to assess the impact of different organic challenges on the antimicrobial activity of antiseptics guidance for the decontamination of intracavity medical devices: the report of a working group of the healthcare infection society surface contamination in the operating room: use of adenosine triphosphate monitoring modern technologies for improving cleaning and disinfection of environmental surfaces in hospitals efficacy of disinfectant-impregnated wipes used for surface disinfection in hospitals: a review a laboratory evaluation of the decontamination properties of microfibre cloths improving operating room cleaning results with microfiber and steam technology microfiber and steam for environmental cleaning during an outbreak spread of bacteria on surfaces when cleaning with microfibre cloths new ftir methodology for the evaluation of ( )c/( )c isotope ratio in helicobacter pylori infection diagnosis an improved method for btex extraction from charcoal detection of diagenetic alterations by spectroscopic analysis on archaeological bones from the necropolis of poseidonia (paestum): a case study this work was financially supported by fondi di ateneo per la ricerca di base (farb ), university of salerno. we are grateful to femir srl, cleprin srl, and sanita service srl for technical support and for providing the materials. none declared. not required. key: cord- - fit q s authors: wan, bin; zhang, xinlian; luo, dongxia; zhang, tong; chen, xi; yao, yuhan; zhao, xia; lei, limei; liu, chunmei; zhao, wang; zhou, lin; ge, yuqing; mao, hongju; liu, sixiu; chen, jianmin; cheng, xunjia; zhao, jianlong; sui, guodong title: on-site analysis of covid- on the surfaces in wards date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: fit q s abstract sars-cov- has erupted across the globe, and confirmed cases of covid- pose a high infection risk. infected patients typically receive their treatment in specific isolation wards, where they are confined for at least days. the virus may contaminate any surface of the room, especially frequently touched surfaces. therefore, surface contamination in wards should be monitored for disease control and hygiene purposes. herein, surface contamination in the ward was detected on-site using an rna extraction-free rapid method. the whole detection process, from surface sample collection to readout of the detection results, was finished within min. the nucleic acid extraction-free method requires minimal labor. more importantly, the tests were performed on-site and the results were obtained almost in real-time. the test confirmed that patients contaminated seven individual sites. among the sampled surfaces, the electrocardiogram fingertip presented a . % positive rate, indicating that this surface is an important hygiene site. meanwhile, the bedrails showed the highest correlation with other surfaces, so should be detected daily. another surface with high contamination risk was the door handle in the bathroom. to our knowledge, we present the first on-site analysis of covid- surface contamination in wards. the results and applied technique provide a potential further reference for disease control and hygiene suggestions. in early , severe acute respiratory syndrome coronavirus (sars-cov- ) spread across the globe, causing more than million infections and , deaths as of aug , (dong et al., ) coronavirus can spread through the air and survives on various surfaces for considerable periods. on june , beijing reported new incidents related to the xinfadi market cluster (owen, ) covid- was discovered on the surface of chopping boards used for imported salmon at the xinfadi food market, providing that covid- could survive on material surfaces. previous researches reported that the covid- virus can inhabit the surface of materials in wards. (guo et al., ; these researchers confirmed the virus by real time reverse transcription polymerase chain reaction (rt-pcr), which typically performs deactivation, nucleic acid extraction, and rt-pcr amplification of the collected samples. however, nucleic acid extraction risks nucleic acid losses and places high demands on the detection limit. furthermore, the whole nucleic-acid extraction and amplification process requires approximately . - hours for one batch of detection. therefore, a rapid detection method should be applied j o u r n a l p r e -p r o o f journal pre-proof for on-site covid- identification in the environment. loop-mediated isothermal amplification (lamp) has achieved brilliant performance in pathogenic virus detection, accomplishing amplification within minutes (liu et al., ) . lamp assay also performs nucleic acid amplification without requiring nucleic acid extraction (lalli et al., ) , thus preventing rna damage through a tedious process. for these reasons, we applied lamp in our present report on surface-contamination detection. patients with confirmed covid- are retained for over two weeks in rooms with many living and medical apparatuses. nosocomial transmission plays a major role in viral spread and infection, especially in wards. confirmed patients living in the ward can spread viruses through coughing or even shortness of breath (ghinai et al., ) . this experiment aimed to determine the concentration of surface contaminants in wards of the chengdu center of disease control (chengdu cdc), which has been designated for the treatment of covid- patients during the disease outbreak. samples were collected from seven sites: ) bedrail; ) bedside cupboard; ) chairs; ) door handles of the bathroom; ) light switches; ) remote controller or beeper; ) fingertip of electrocardiograph (ecg) monitoring. the samples were collected on th, th and th mar, th and th apr of . the sampling site was illustrated in figure (a). the correspondences between patient clinical information and collected samples were listed in table s the surface contamination samples were collected as described index a of the hygienic standard for disinfection in hospital (chinese national standard, gb - ). briefly, a cm × cm standard scale board was placed on the surface of sampling material, and the surface was evenly rubbed within the -cm area with a cotton swab wetted with . % sodium chloride. after swiping, the cotton swab was immersed in ml . % sodium chloride solution prepared for nucleic-acid amplification detection. the collected surface-contamination samples were immediately transferred to the bsl- laboratory next to the chengdu cdc wards for analysis. viral contamination was detected by a nucleic acid extraction-free isothermal detection kit, specifically, a novel coronavirus real-time isothermal amplification kit (cat.no. pcsyhe), acquired from shanghai fosun long march medical science co., ltd and certificated by the detection process was performed following by manufacture's instructions. for each batch, we prepared sufficient reaction reagent for n tested samples plus two control samples. that is, n × μl of bst enzyme, n × μl of rt ii enzyme, and n × μl of covid- gene reaction reagent were added to a centrifuge tube, mixed by shaking, and centrifuged at low speed for a few seconds. after separation, μl aliquots were pipetted into pcr reaction tubes. the reaction tubes could be placed at ~ °c for hours at most after separation. next, μl of the samples were added to different pcr reaction tubes. a -μl negative control and μl-positive control were added to the control wells. the five positive samples from the handles were % associated with positive results from the ecg fingertips positive results. four positive cases from the handles were also associated with positive results from bedrails, and three cases were associated with positive results from cupboards and light switches. therefore, a surface contamination sample from the door handle can be interpreted as an ultra-high risk label. in daily monitoring, the bathroom door handles should be swabbed and analyzed. when the samples from a door handle report positive results, the corresponding ward poses an enhanced hygienic challenge. three of the collected samples (samples # - , # - , and # -l ) presented on six contaminated surfaces. all of these confirmed cases came from j o u r n a l p r e -p r o o f outside mainland china and presented positive symptoms. on a - scale of clinical severity, where type denotes mild cases and type represents the severest cases, sample # - was classified as type , and samples # - and # -l were classified as type . the clinical symptoms might influence the contamination degree and infection risk. further research on these relationships is ongoing. we successfully applied an extraction-free sars-cov- isothermal amplification detection method to on-site analysis of surface contamination by covid- patients in wards. for each confirmed case, seven sites in the ward were collected and analyzed. the detection process is efficient and labor-saving, as desired for on-site covid- contamination detection. among cases collected from march to april of , . % reported positive amplifications on the ecg fingertip, indicating that this surface is an important hygiene site. the correlation results also confirmed that bedrails should be regularly monitored, as contamination on bedrail surfaces is relevant to many other contaminated surfaces. although the analysis can be performed by standard real-time pcr instruments, simpler isothermal amplification fluorescent instruments are suggested for on-site analysis, as they are less expensive, smaller in size, and more easily transported than standard pcr instruments. in future works, we will analyze the relationship between different clinical symptoms and surface contamination, which may reveal the transmission mode of covid- in wards. validated the method. yuqing ge, hongju mao, sixiu liu, jianmin chen, xunjia cheng, jianlong zhao, and guodong sui: conceived the idea, coordinated the project, and wrote the manuscript. -first report about on-site detection of sars-cov- in wards. -the whole detection process could accomplish within minutes without nucleic acid extraction. -the presence of sars-cov- in wards was confirmed by nucleic acid isothermal amplification. j o u r n a l p r e -p r o o f prevention and control of major infectious diseases such as aids and viral hepatitis" ( zx - - ); the science and technology commission of shanghai municipality (nos. jc , and dz ) ，the national natural science foundation of china an interactive web-based dashboard to track covid- in real time first known person-to-person transmission of severe acute respiratory syndrome coronavirus (sars-cov- ) in the usa rapid and extraction-free detection of sars-cov- from saliva with colorimetric a sample-to-answer labdisc platform integrated novel membrane-resistance valves for detection of highly pathogenic avian influenza viruses covid- : who raises concerns about new cases in beijing environmental virus surveillance in the isolation ward of covid- sars-cov- rna detection of hospital isolation wards hygiene monitoring during the coronavirus disease outbreak in a chinese hospital the authors declare no competing interests. key: cord- - einbent authors: theodore coroneo, minas title: the eye as the discrete but defensible portal of coronavirus infection date: - - journal: ocul surf doi: . /j.jtos. . . sha: doc_id: cord_uid: einbent oculo-centric factors may provide a key to understanding invasion success by sars-cov- , a highly contagious, potentially lethal, virus with ocular tropism. respiratory infection transmission via the eye and lacrimal-nasal pathway elucidated during the influenza pandemic, remains to be explored in this crisis. the eye and its adnexae represent a large surface area directly exposed to airborne viral particles and hand contact. the virus may bind to corneal and conjunctival angiotensin converting enzyme (ace ) receptors and potentially to the lipophilic periocular skin and superficial tear film with downstream carriage into the nasopharynx and subsequent access to the lungs and gut. adenoviruses and influenza viruses share this ocular tropism and despite differing ocular and systemic manifestations and disease patterns, common lessons, particularly in management, emerge. slit lamp usage places ophthalmologists at particular risk of exposure to high viral loads (and poor prognosis) and as for adenoviral epidemics, this may be a setting for disease transmission. local, rather than systemic treatments blocking virus binding in this pathway (advocated for adenovirus) are worth considering. this pathway is accessible with eye drops or aerosols containing drugs which appear efficacious via systemic administration. a combination such as hydroxychloroquine, azithromycin and zinc, all of which have previously been used topically in the eye and which work at least in part by blocking ace receptors, may offer a safe, cost-effective and resource-sparing intervention. unexpectedly, ophthalmology may be playing a central role in the current coronavirus disease (covid- ) epidemic. the harbinger of the third zoonotic coronavirus epidemic in as many decades ( ) , was dr li wenliang, an ophthalmologist, who died following infection with covid- ( , ) . it was thought he was infected during examination of a patient with angle closure glaucoma in the second week of january . he suspected an outbreak after seeing patients with sars-like symptoms and the system failure to heed his warning may well have changed the course of world history. the second instructive case is that of the respiratory physician guangfa wang ( ) . days before pneumonia onset, his earliest symptoms related to left conjunctivitis, then catarrhal symptoms and fever which developed after - hours, slower than might be expected from older studies tracing the passage of bacteria through the lacrimal drainage system ( ) . while an n respirator was worn, offering protection from infection via oro-nasal pathways, eye protection was not. evidence that ocular and periocular tissue may be uniquely placed as an entry point for viral invasion will be reviewed. at the height of the world influenza epidemic, a landmark paper appeared, proposing transmission of acute respiratory infections via the eye and lacrimal-nasal pathway ( ) (figure ) . it was noted that this pathway had been "disregarded in planning measures for the prevention of the spread of contagious diseases" and it would appear that little has changed. figure . the lacrimatory-nasal mechanism for the mechanical disposition of organisms entering the upper respiratory tract ( ) . a third factor in this perfect storm is the well-established but not well recognized coronavirus ocular tropism ( ) . the study of oculotropic influenza and adenoviruses ( - ) sets precedents for disease patterns, bought into focus by coronaviruses. whereas influenza viruses generally represent a respiratory pathogen and only occasionally cause ocular complications, adenoviruses mirror image this disease pattern, causing severe ocular surface disease, and are often highly contagious (known as "eye hospital eye"( )) with fewer, seldom lethal, systemic manifestations. thus, all three virus types share a common ocular entrée but vary in their degree of contagion, ocular versus respiratory/system impact and lethality. taken as a whole, these disease patterns have implications for more effective management strategies. dr li wenliang's death and the apparent high infection rate in ophthalmologists (and ent surgeons) ( ), attributed to the high viral shedding from the nasal cavity, should not be unexpected. a high initial viral load is associated with poor prognosis ( ) and ophthalmologists are particularly at risk, since ophthalmic practice involves very close ophthalmologist-patient physical proximity. this is necessitated by optical imperatives to optimise image quality by close alignment of imaging devices to the eye. slit lamp biomicroscopy is the cornerstone of ophthalmic practice and the slit lamp is also used to carry out surgical procedures. the back focal distance (the distance of the subject from the front lens surface of the microscope), is set to give the surgeon sufficient space for manipulation ~ cm (figure a ). with the instrument body length added, the distance from patient to the surgeon's eye is at a convenient working distance of ~ - cm. this proximity and the possibility of cross infection between examiner and examinee has long been recognized and a protective, transparent, typically perspex ® "breath shield" was added to slit lamps. it is evident in slit lamps from as early as ( ) and may well have gained popularity during the era of the influenza pandemic. the necessity for such shields is readily apparent from studies in which exposure between two face-to-face breathing manikins is measured this study concluded that air exhaled from human respiration contains contaminants and is able to penetrate the breathing zone of other nearby persons. of interest is that exhalation flow may stratify in a horizontal layer at breathing zone height under certain conditions. thus, with slit lamp examination, the layer of air between patient and ophthalmologist may remain stable, exposing both individuals for the entire time of the examination. breath shields are often missing from slit lamps either because they are not fitted or because, as they get in the way, are removed. nares: (a), average total eye surface exposed; shaded area represents proportion of time not exposed, due to winking; (b), average total mouth area exposed in talking; shaded area represents proportion of time not exposed, due to closure; (c), average total area of crosssection of nares exposed; shaded area represents proportion of time not exposed, owing to protected position and expiration ( ) . that slit lamps and their accessory lenses are a source of infection transmission has long been known ( ) . in epidemic adenoviral keratoconjunctivitis ((akc), shipyard/eye hospital eye) ( , ) , ophthalmologists are not infrequently infected although this is not well documented ( , ) . while akc can cause considerable incapacity and has resulted in some riskminimization measures, unlike coronavirus it does not usually result in death. the earlier severe acute respiratory syndrome (sars) epidemic resulted in recommendations for how to manage eye facilities and include advice in relation to slit lamp cleaning and eye protection for staff ( , ) . slit lamp biomicroscopy is an almost optimal method of transferring material in breaths between two individuals, short of actual facial contact. a very recent study provides evidence for human-to-human transmission ( ) . pathways include direct transmission, such as cough, sneeze, droplet inhalation transmission as well as contact transmission including contact with oral, nasal, and eye mucous membranes ( ) . sars-cov- aerosol and fomite transmission is highly likely, since the virus can remain viable and infectious in aerosols for hours and on different surfaces up to days ( ) . while transmission of coronaviruses occurs via an airborne route ( ), because infected, symptomatic patients tend to develop severe lower, as opposed to upper respiratory tract infections, it has been supposed that airborne agent virus has to be small enough to penetrate directly into the lower respiratory tract to preferentially replicate there before causing disease. that transmission could occur via the ocular-nasolacrimal pathway was not considered. human eyes are located at a coign of vantage in the body, simultaneously providing information from our highest band width sense but also being exposed to risk of exposure including to airborne virus. the surface area of the eye(s) is large compared to that of the mouth and nares -( figure d ) and was recognized early as a target for "promiscuous spraying" whereby coughing could project material at least feet away ( ) . since this study ( ), a number of other studies have investigated ocular surface area and reported as a total (for two eyes) as - ( ) and - mm ( ) , indicating that the figure from maxcy's early study ( ) was a good estimate (table ) of the "visible" ocular surface. the later study ( ) also explains variability in measurements, since palpebral aperture is dependent on eye gaze direction. however, the total ocular surface area has been estimated at ~ - sq. mm/eye ( , ) , including the cornea, this representing a maximal absorptive area of ~ sq.mm, accounting for the tarsal conjunctiva and including the palpebral fornices. even this consideration underestimates the potential ocular/periocular landing zone for a viral particle. it is not an uncommon observation that makeup, applied around the eye, can "migrate" onto the ocular surface ( ) , similar to the phenomenon by which noxious agents in minute quantities are easily transferred from fingertip to periocular skin and then the eye. this may be due to subtle actions of riolan's muscle ( ) . in fact, this mechanism of transport of agents from the periocular eye lid skin to the eye (obviating the need for eyedrops) has been termed supracutaneous and has been developed as an efficient delivery system for management of dry eye syndrome ( ) . this supracutaneous mechanism, might provide a substantial periocular area in which viral particles could land and be "funnelled" onto the ocular surface and beyond. table provides available information on the size of the orbital opening ( and reviewed in ), which likely underestimates the area of eyelid skin. we have estimated eyelid skin to be ~ mm and that of the brow to be ~ mm , so that in total with the ocular surface area, a landing site of ~ , mm would be available, - orders of magnitude greater than for the nares and mouth. this does not take into account the surface area that could be attributed to the hair of the eyebrows or the eyelashes, for which estimates have not been made. eyelash aerodynamics may also play a role ( ) . eyelashes have been shown to divert airflows, acting as a passive ocular dust controlling system. they reduce evaporation and particle deposition up to %. in a comparative study ( ) , asian eyelashes had lower lift-up and curl-up angles, fewer numbers and a thicker transverse diameter as compared to caucasian eyelashes. whether these differences play any role in influencing the rate at which particles land on the ocular surface is unknown. however, it is possible that a deficiency in this mechanism could increase the risk of infection. in the study ( ) , bacillus prodigiosus (serratia marcescens) was instilled into the lacrimal sac of volunteers and subsequently was recoverable from the nose, throat and stool after mins, mins and hours respectively. it was concluded that via this ocular lacrimatory-nasal mechanism (figures and ) , the upper respiratory tract of a person wearing a properly constructed mask may be infected by exposing the eye briefly to direct droplet spray. identification of ocular surface cellular receptors utilized by respiratory viruses has provided information as to the permissiveness of ocular tissue to infection with these agents ( , ) . central to covid- pathogenicity is that cellular entry is via the cell surface angiotensin converting enzyme (ace ) receptor ( , ) . it is the only mammalian group i coronavirus known to use ace as its receptor ( ) . ace was previously identified as the receptor for sars-cov and nl ( ) . virus infectivity studies have shown that ace is essential for sars-cov- to enter hela cells ( ) . while ace mrna is known to be present in virtually all organs, surface expression of ace protein was described in lung alveolar epithelial cells and small intestinal enterocytes ( ) and it was postulated that ace might provide the coronavirus entry route. the eye and its adnexae were not investigated in this study. it was subsequently shown that ace protein is more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia ( ) and thus accessible by topical agents. an immunohistochemical study revealed both extra-and intraocular localisation of ace in human eyes ( ) . of particular interest was the localisation of ace to the epithelial cells of both the cornea and conjunctiva. apical epithelia location and whether receptors are ace remains to be confirmed. recently, more widespread distribution of sars-cov- entry factors, ace as well as tmprss protease and cathepsin b/l activity (for post-binding spike protein priming) have been documented using single-cell rna-sequencing data ( ) . while it was recognized that binding affinity of the spike protein and ace is the major determinant of sars-cov replication rate and disease severity and that viral entry also depends on protease activity, ace , rather than protease activity, may be a limiting factor for initial viral entry. this study confirmed evidence of ace in the limbus, corneal epithelium (basal/suprabasal, superficial and wing cells) and conjunctiva (basal and superficial cells) and co-location with tmprss in superficial conjunctival cells. of interest is that this study demonstrated evidence of ace in nasal goblet cells which also express genes associated with immune functions including innate and antiviral immune functions. there should be some priority for re-evaluation of these elements in the ocular surface ( ) . the density of sars-cov- entry factors and their presence or absence in the lacrimal drainage system would be of interest, since viral particles that land in the tear film need not necessarily bind to an epithelial cell immediately but could be presented with a second opportunity (a "wash through" effect) in the lacrimal drainage system. tear film resilience may well protect the underlying corneal and conjunctival epithelium so that viral adherence to the tear film could prevent access to the apical epithelial surface, which may account for the relatively low incidence of keratitis/conjunctivitis reported to date ( , ) . the tear film lipid layer plays an important role in retarding tear film evaporation and tear spillage ( ) . however, this lipophilicity and that of the periocular skin ( ) ( ) ( ) ( ) . while it was found that no viral rna was detected in the tear fluid and conjunctival secretions of infected patients without conjunctivitis in one study, it was felt that this did not eliminate the risk of transmission via this pathway ( ) . the finding that human conjunctival explant cultures were more extensively infected by sars-cov- than by sars-cov is also significant ( ) . taken together, these findings are also consistent with the concept that the tear film may protect the ocular surface from epithelial infection and convey virus downstream. the nasolacrimal system, via the nasopharynx (considered crucial for viral replication ( )) thus provides a bridge between ocular and respiratory tissues, serving as a conduit for viruscontaining fluid exchange between these sites ( ) . furthermore, beyond the anatomical linkage, it was recognized that the structure and distribution of cellular receptors in these systems is likely contributing to the tissue tropism of respiratory viruses ( ) . furthermore, there is respiratory-ocular mucosal immune interdependence with linkage via the nasolacrimal lymphoid tissue ( , ) . despite early suggestions of ocular involvement ( ) in this process, it appears that this was not taken into consideration in framing early guidelines for protection against infection. more recently, american academy of ophthalmology recommendations include protection for the mouth, nose and eyes when caring for patients potentially infected with this virus ( ). thus, the pathophysiology of ocular surface-sars-cov- interactions requires reevaluation, not only to determine the dynamics of ocular surface viral exposure and binding but also transmission via the lacrimal drainage system. less pathogenic coronaviruses as well as other respiratory viruses could be used in animal models to better elucidate this relatively unexplored pathway. thus, the ocular surface, representing a large surface area, exposed to and likely receptive to coronavirus, bearing the appropriate sars-cov- entry factors, may be an ideal point of intervention. efforts in directly targeting the ace receptor in this way are limited ( ) . potentially, blocking the ace receptor would deprive coronaviruses from their main point of tissue binding. p and p peptides and naae, a small molecule targeting ace have been developed but there have been concerns about a narrow spectrum of activity and effects on blood pressure regulation ( ) . this strategy has previously been employed, using antibodies ( ) -anti-ace but not anti-ace antibody blocked viral replication in a model system. blocking of tmprss protease and cathepsin b/l activity could also be considered, however it appears that ace , rather than protease activity, may be the viral entry rate limiting factor ( ) . a search of fda-approved drug libraries identified a number of drugs including chloroquine as potentially having anti-coronavirus actions ( ) and therefore potentially able to be repurposed. table summarises the drugs that have been identified as potential treatments for coronavirus infection, their efficacy (in vitro and in vivo) and for which there is data (for that agent or a related compound) for previous topical ocular surface usage. it is apparent that while for each of these agents, there is evidence of in vitro anti-coronavirus activity, there is a paucity of in vivo studies. this has been raised as a matter for concern in that there are precedents for paradoxical untoward drug systemic effects that result in increased disease severity when in vitro testing alone might suggest efficacy ( ) . chloroquine has long been used in the treatment of malaria and subsequently, autoimmune disorders (such as rheumatoid arthritis) as well as in oncology and for pediatric inflammatory disease. however, chloroquine has direct antiviral effects, inhibiting ph-dependent steps of the replication of flaviviruses, retroviruses, and coronaviruses ( ), exhibiting strong antiviral effects on coronavirus infection of primate cells ( , ) . the drug can be effective either before or after exposure to the virus, so could act both therapeutically and prophylactically. several mechanisms have been proposed but of particular interest is that the drug appears to interfere with terminal glycosylation of ace ( ) ( ) ( ) . chloroquine also has immunomodulatory effects, ( , ) suppressing the production/release of tumour necrosis factor and interleukin , which may mitigate the severe inflammatory cascade associated with severe covid- disease ( ) . recently, hydroxychloroquine was found to be more potent than chloroquine at inhibiting sars-cov- in vitro ( ), fortuitous, since it appears that lower toxicity has been attributed to this derivative ( ) . a recent systematic review of clinical trials utilising chloroquine or hydroxychloroquine ( ) concluded that / trials had shown favorable outcomes for patients using these drugs and / demonstrated no significant change compared to control. it was noted that all trials carried varying degrees of poor study design and bias. one such study, a pilot observational study ( ) showed that use of hydroxychloroquine and azithromycin demonstrated apparent improved clinical outcomes in / patients, expanding earlier work suggesting that combined therapy resulted in rapid reduction in viral load. clearly, more robust clinical studies are needed and have been called for ( ) . while systemic chloroquine and its derivatives appear to be associated with relatively minor side effects in the shorter term, electrocardiogram qt interval prolongation has been reported ( ) and this may be exacerbated by combination treatemtn with azithromycin ( ) . the well-known association with irreversible visual loss due to retinal toxicity is considered a late manifestation ( ) , however early-onset (~ months) has been reported in a case where the ideal dosage was exceeded for one month ( ) . a number of predisposing factors to retinal toxicity include genetic ( ) -polymorphisms in the cytochrome p gene, drug interactions ( ) and racial factors ( )), may play a role. chloroquine's anticancer activity in concert with zinc have been explained by the fact that chloroquine acts as a zinc ionophore ( ), significantly, inhibiting cellular autophagy and enhancing apoptotic cell death via inhibition of lysosome function. it transpires that zinc inhibits coronavirus polymerase activity ( ), blocking viral replication. for this reason, combination treatment for covid- with chloroquine and zinc has been suggested ( ) , although this intervention has yet to be formally reported. it is perhaps unsurprising, given the passage of time, that an understanding of viral oculotropism from the influenza epidemic has faded. yet if sars-cov- oculotropism is confirmed, this invasion entry point also represents an opportunity for intervention. given the potential toxicity of apparently efficacious drugs and issues relating to drug bioavailability, initially in the upper airways and respiratory system, local, topical therapy offers potential significant advantages. many of the proposed systemic therapies such as chloroquine, zinc and ace inhibitors have all been used topically in the eye (table ) . chloroquine as . % chloroquine phosphate eye drops have apparently been efficacious in the management of dry eye syndrome in humans ( , ) . significant side effects were not reported, however there is a report of chloroquine keratopathy in workers chronically exposed to chloroquine dust ( ) . chronic usage is unlikely in the setting here proposed. ace inhibitors have been used topically in animal models of glaucoma ( ) . in this class of drugs, agents such as telmisartan are long acting, with a mean half-life of hours ( ). zinc has traditionally been used in astringent eye drops or as an excipient, zinc sulphate ( . %) ( ) . since chloroquine and ace inhibitors seem to act at different parts of the receptor, in combination, synergy is possible or at the very least an additive effect, permitting a reduction in dosage and risk of potential side effects. however, another issue is that ace inhibitors may upregulate this receptor ( ) , perhaps increasing the risk of coronavirus infection. early reports have suggested that hypertension may be a risk factor for infection but these are unconfirmed ( ) . azithromycin has also previously been safely used topically in the human eye as a - . % solution to treat ocular infections ( , ) . in the case of hydroxychloroquine, preliminary calculations suggest that with topical application, a dosage one to two orders of magnitude higher than plasma levels (reached with systemic treatment ( )) could be achieved. at this point, clinical studies are needed expeditiously to investigate the safety and efficacy of local prophylactic regimens following exposure to sars-cov- . for instance, investigations can include initial local delivery of a combination of chloroquine, zinc and azithromycin to the eyelids and ocular surface with eye drops or a spray/aerosol. this would potentially inhibit binding of sars-cov- to the likely major entry point into the human body, would reduce the risk of systemic side effects and conserve drugs that may become scarce, since the dosage required would be much less than for systemic treatment. a spray or aerosol preparation would also allow treatment of the nasal passages ( ) and mouth ( ), given high ace receptor populations in these locations. however, if the virus is truly oculotropic, this may be superfluous. if a patient has active infection of the respiratory tree, then this combined medication could also be given in an inhaled form. the advantage of this ace targeting approach is that ace receptors from the outer cornea, through the lacrimal drainage system to the respiratory and gastrointestinal systems as well as both nasal and oral cavities, are directly accessible to a high topical drug dose. thus, the portals that make us susceptible to coronavirus attack and invasion can be used to provide protection that is likely to be safe, extensive, convenient and at low cost. the ocular surface, offering all of the benefits of local treatment familiar to ophthalmologists, may thus provide a convenient testing ground for rapid, safe and likely cost-effective trials of newer drugs that may be developed to combat this modern plague. table . another decade, another coronavirus jia zhi-fang. -ncov transmission through the ocular surface must not be ignored. the lancet the transmission of infection through the eye ocular tropism of respiratory viruses the eyes have it: influenza virus infection beyond the respiratory tract pathogenesis, transmissibility, and ocular tropism of a highly pathogenic avian influenza a (h n ) virus associated with human conjunctivitis sialic acid-containing glycans as cellular receptors for ocular human adenoviruses: implications for tropism and treatment eye hospital eye initial viral load and the outcomes of sars the history of optical instruments for the examination of the eye dispersal of exhaled air and personal exposure in displacement ventilated rooms slit lamps and lenses: a potential source of nosocomial infections? eye outbreak of epidemic keratoconjunctivitis caused by human adenovirus type d in an eye care clinic -los angeles county frequency and assortment of self-report occupational complaints among iranian ophthalmologists: a preliminary survey the prevalence of adenoviral conjunctivitis in tikrit district and the role of traditional medicine "om al kaash" in transmission of the disease and in the severity of complications precautions in ophthalmic practice in a hospital with a major acute sars outbreak: an experience from hong kong novel coronavirus disease (covid- ): the importance of recognising possible early ocular manifestation and using protective eyewear early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia transmission routes of -ncov and controls in dental practice who | update -one month into the global sars outbreak: status of the outbreak and lessons for the immediate future accessed recognition of aerosol transmission of infectious agents: a commentary the exposed ocular surface and its relationship to spontaneous eyeblink rate in elderly caucasians ocular surface area as an informative index of visual ergonomics on the size of the conjunctival sac comparison of conjunctival and corneal surface areas in rabbit and human migration of cosmetic products into the tear film supracutaneous treatment of dry eye patients with calcium carbonate new treatment of dry eye: the effect of calcium ointment through eyelid skin delivery evaluation of eyeball and orbit in relation to gender and age morphometric analysis of the orbital aperture in north indian population: a retrospective digital forensic study eyelashes divert airflow to protect the eye ethnic characteristics of eyelashes: a comparative analysis in asian and caucasian females identification of nucleolin as a cellular receptor for human respiratory syncytial virus evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission single-cell rna expression profiling of ace , the putative receptor of wuhan crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor the s proteins of human coronavirus nl and severe acute respiratory syndrome coronavirus bind overlapping regions of ace a pneumonia outbreak associated with a new coronavirus of probable bat origin tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis ace receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia immunohistochemical localization of angiotensin-converting enzyme, angiotensin ii and at receptor in human ocular tissues sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes tropism, replication competence, and innate immune responses of the coronavirus sars-cov- in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures identification of a new human coronavirus human coronavirus nl , france dry eye epidermal surface lipids lipids, proteins and corneocyte adhesion tear film lipids common features of enveloped viruses and implications for immunogen design for next-generation vaccines murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release importance of cholesterol-rich membrane microdomains in the interaction of the s protein of sars-coronavirus with the cellular receptor angiotensin-converting enzyme complexity of the tear film: importance in homeostasis and dysfunction during disease evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov- infection sars-cov- isolation from ocular secretions of a patient with covid- in italy with prolonged viral rna detection ocular manifestations of a hospitalised patient with confirmed novel coronavirus disease functional anatomy and immunological interactions of ocular surface and adnexa nasolacrimal duct closure modulates ocular mucosal and systemic cd (+) t-cell responses induced following topical ocular or intranasal immunization tears and conjunctival scrapings for coronavirus in patients with sars angiotensin receptor blockers as tentative sars-cov- therapeutics coronaviruses -drug discovery and therapeutic options angiotensin-converting enzyme is a functional receptor for the sars coronavirus the comparative efficacy and safety of the angiotensin receptor blockers in the management of hypertension and other cardiovascular diseases crystal structure of the human angiotensin-converting enzyme-lisinopril complex a virus-binding hot spot on human angiotensin-converting enzyme is critical for binding of two different coronaviruses ace x-ray structures reveal a large hingebending motion important for inhibitor binding and catalysis screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice a rapid systematic review of clinical trials utilizing chloroquine and hydroxychloroquine as a treatment for covid- evaluation of the effects of chloroquine phosphate eye drops in patients with dry eye syndrome efficacy and safety of topical chloroquine in mild to moderate dry eye disease clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid- review of azithromycin ophthalmic % solution (azasite(®)) for the treatment of ocular infections elimination of active trachoma after two topical mass treatments with azithromycin . % eye drops corneal pharmacokinetics of topically applied azithromycin and clarithromycin effects of dehydration on corneal tissue absorption of topical azithromycin in rabbits zn( +) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture influence of topical and oral zinc upon corneal wound healing influence of local antiseptics on regeneration of corneal epithelium of rabbits chloroquine paradox may cause more damage than help fight covid- effects of chloroquine on viral infections: an old drug against today's diseases? chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine new insights into the antiviral effects of chloroquine the anti-viral facet of anti-rheumatic drugs: lessons from covid- in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study the effect of chloroquine hydroxychloroquine retinopathy macular toxicity after short-term hydroxychloroquine therapy analysis of the abcr (abca ) gene in -aminoquinoline retinopathy: is retinal toxicity by chloroquine and hydroxychloroquine related to stargardt disease? therapeutic use and safety profile in the management of osteoarthritis and rheumatoid arthritis audo i; dhu vision handicaps task force rétine. différences ethniques parmi les patients souffrant de toxicité maculaire aux antipaludéens de synthèse chloroquine is a zinc ionophore zinc salts block hepatitis e virus replication by inhibiting the activity of viral rna-dependent rna polymerase improving the efficacy of chloroquine and hydroxychloroquine against sars-cov- may require zinc additives -a better synergy for future covid- clinical trials chloroquine keratopathy in chloroquine workers after topical dust exponation. a case report topical ocular hypotensive effects of the novel angiotensin converting enzyme inhibitor sch in conscious rabbits the comparative pharmacology of angiotensin ii receptor antagonists upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors clinical features of deaths in the novel coronavirus epidemic in epub ahead of print bioavailability of hydroxychloroquine tablets in healthy volunteers high expression of ace receptor of -ncov on the epithelial cells of oral mucosa key: cord- -s wjg ar authors: cobrado, l.; silva-dias, a.; azevedo, m. m.; rodrigues, a. g. title: high-touch surfaces: microbial neighbours at hand date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: s wjg ar despite considerable efforts, healthcare-associated infections (hais) continue to be globally responsible for serious morbidity, increased costs and prolonged length of stay. among potentially preventable sources of microbial pathogens causing hais, patient care items and environmental surfaces frequently touched play an important role in the chain of transmission. microorganisms contaminating such high-touch surfaces include gram-positive and gram-negative bacteria, viruses, yeasts and parasites, with improved cleaning and disinfection effectively decreasing the rate of hais. manual and automated surface cleaning strategies used in the control of infectious outbreaks are discussed and current trends concerning the prevention of contamination by the use of antimicrobial surfaces are taken into consideration in this manuscript. in spite of the growing global commitment towards an effective reduction of healthcare-associated infections (hais), it is unfortunately certain that such infections will continue to be responsible for very high morbidity, increased costs and length of stay (los) for the coming decades [ , ] . among potential sources of pathogens causing hais, the most frequent are the patient's microbiota and the hands of healthcare personnel [ ] . additionally, evidence that hightouch surfaces (hts) will work as an extra source of microbial pathogens accumulated over the years, e.g., several microorganisms can survive on medical equipment for hours to months, improved cleaning and disinfection of surfaces decrease the rate of hai, and hospital environmental screening results and the study of clonal outbreaks, all have given support to the role of contaminated hts in the transmission of pathogens between patients and healthcare personnel [ ] . from surfaces, microbial transmission may occur either through direct patient contact or, indirectly, through healthcare personnel hands or gloves [ ] . therefore, upon potentially preventable sources of microorganisms, contaminated hts deserve strong consideration. microbial pathogens most frequently involved in the contamination of hospital environmental surfaces are (methicillinresistant) staphylococcus aureus (mrsa), vancomycinresistant enterococci (vre), clostridium difficile, multidrug resistant gramme-negative bacilli (such as pseudomonas, acinetobacter and enterobacteriaceae), norovirus, coronavirus and candida species [ ] [ ] [ ] [ ] [ ] . strategies for cleaning contaminated hts may include manual and automated techniques. wipes and cloths with application of detergents or disinfectants are examples of manual techniques, while automated methods may involve uv light, hydrogen peroxide, steam vapour, ozone and hins (high-intensity narrow-spectrum light). on the other side, in order to prevent contamination of hts, antimicrobial surfaces are being developed. the inhibition of microbial adhesion with repellent films is a possible strategy, as it is the surface treatment with antimicrobial coatings of silver, copper, polycations, triclosan, bacteriophages or, even, light-activated biotoxic radicals. the aim of this manuscript is to review the role of hightouch surfaces in healthcare-associated infections, from the aetiology to strategies for surface cleaning and addressing preventive trends. as early as , spaulding proposed a classification of inanimate surfaces into three general categories based on the risk of infection if the surfaces were contaminated at the time of use [ ] . these categories can be applied to devices or instruments as follows: critical (exposed to normally sterile areas of the body; require sterilization), semi-critical (touch mucous membranes; may be sterilized or disinfected), and noncritical (touch skin or come into contact with people only indirectly; can be either cleaned and then disinfected with an intermediate-level disinfectant, sanitized with a low-level disinfectant or, simply, cleaned with water and soap). in , the cdc proposed environmental surfaces (floors, walls and other bhousekeeping surfaces^that do not make direct contact with a person's skin) as an additional category [ ] . more recently, the cdc's and healthcare infection control practices advisory committee's guidelines for environmental infection control in healthcare facilities [ ] divided surfaces into patient care items and environmental surfaces. environmental surfaces were further divided into medical equipment and patient room surfaces (table ) . over the years, research has been done in order to better target room disinfection practices. following recommendations made by the cdc to clean and disinfect hts more frequently than minimal-touch surfaces, data published in by huslage et al., based on the real frequency of contact, defined the top five most touched surfaces in hospitals: bed rails, bed surface, supply cart, over-bed table and intravenous pump [ ] . hts may be classified as non-critical items (the contact occurs with intact skin that effectively acts as a barrier to most pathogens, but not with mucous membranes) and must be subject to cleaning and disinfection procedures as recommended, but with no absolute need for sterilization [ ] . many pathogens may thrive on healthcare-associated equipment and environmental surfaces. among such organisms, mrsa, vre, c. difficile, p. aeruginosa, a. baumannii, enterobacteriaceae, stenotrophomonas maltophilia, burkholderia cepacia, norovirus, coronavirus and candida spp. may persist and contribute to the infection risk to which patients are systematically exposed. several studies have demonstrated that basic cleaning leads to mrsa elimination from environmental surfaces and enhanced cleaning may terminate outbreaks in intensive care units, with cost savings of $ , up to $ , per year [ ] [ ] [ ] [ ] . recently, two studies reported positively about pulsed xenon uv and hydrogen peroxide methods to boost the decontamination of patient rooms, contributing towards a reduction of mrsa bioburden [ , ] . its inherent ability to resist certain antimicrobial agents (such as cephalosporins and aminoglycosides) allied to a great capacity to acquire determinants of antibiotic resistance (like gene clusters of vancomycin resistance) turn enterococci into a versatile nosocomial multidrug-resistant pathogen. the number of vre infections has been increasing worldwide, most frequently afflicting patients with serious comorbidities or undergoing prolonged hospitalization [ ] . vre are known to survive for a long time in the hospital environment. viability on surfaces may range from days to months [ ] . moreover, enterococci are tolerant to heat, chlorine and some alcohol preparations, making them very resilient to conventional cleaning practices, thus becoming easily disseminated among healthcare facilities [ ] . therefore, besides thorough environmental cleaning several times a day with disinfectants, vre management protocols should include strict adoption of contact precautions and implementation of comprehensive educational programs for staff [ , ] . spores of c. difficile can hold on to a healthcare environment for more than months [ , ] . fortunately, the use of chlorine-releasing disinfectants reduces the amount of spores in the environment, with some evidence suggesting that it may reduce the risk of recurrence and transmission of c. difficileassociated infections [ ] . transmission of p. aeruginosa may easily occur from contaminated sinks to hands of healthcare personnel during washing, since this organism may thrive in biofilms that are adherent to sink traps, pipes, water lines and hospital drains [ ] , turning these fashion-organized bacteria more prone to resist to disinfectants [ ] . additionally, p. aeruginosa can resist hours to months on dry inanimate surfaces [ ] . programs to control transmission should include, therefore, repeated cleaning with chlorine-based disinfectants, physical removal of persistent biofilm, replacement of components whenever feasible and regular inspection [ , ] . the increase in the number of hais caused by a. baumannii might be explained not only by its ability to persist from days to more than months in undisturbed surfaces of healthcare equipment [ ] , but also by its high resilience to cleaning with conventional detergent and alcohol disinfectants [ ] . hence, outbreaks in hospital or other healthcare settings are difficult to contain because of the easy environmental contamination by this pathogen [ ] [ ] [ ] . targeted infection control measures may be needed, including intensive cleaning with sodium hypochlorite and subsequent measurement of cleanliness, hand hygiene training, adoption of barrier precautions and contact isolation, as well as patient surveillance [ , ] . there has been a growing concern about klebsiella pneumoniae infections, mainly because of its extensive β-lactamase resistance. k. pneumoniae are usual colonizers of the human gastrointestinal tract, pharynx and skin that may cause wound infections, pneumonia and sepsis, particularly in immunocompromised patients [ ] . more recently, given its wide dissemination and selective advantage to resist to carbapenem antibiotics, k. pneumoniae have been showing a propensity to cause outbreaks in healthcare institutions [ ] . it is known that k. pneumoniae may survive for more than months in the healthcare environment [ ] and that the origin of some outbreaks has been related to sinks and related pipes [ , ] . another member of the enterobacteriaceae family, serratia marcescens, are known to cause pneumonia, meningitis, urinary tract and bloodstream infections. mdr isolates, including colistin resistant [ ] , have been responsible for serious outbreaks among intensive care units and critically ill neonates [ ] [ ] [ ] . s. marcescens are known to survive up to months on dry inanimate surfaces [ ] and have frequently been recovered from water pipes and hospital disinfectants [ ] . because of the easy transmission and environmental persistence of enterobacteriaceae in healthcare facilities, adequate solutions aiming its eradication should ensure comprehensive educational interventions, hand hygiene training, chlorine-based cleaning and even the replacement of sinks and pipes [ , , ] . similarly to other bacteria, it can persist in biofilms that may turn cleaning products and disinfectants more ineffective [ ] . long-term control of s. maltophilia will be dependent upon the integration of an efficient cleaning strategy into a targeted healthcare facilities maintenance program [ ] . it is widely distributed in soil and water habitats and recent healthcare-associated outbreaks have been linked to b. cepacia persistence in disinfectants, drugs, medical devices (e.g., respiratory nebulizers), sinks and contiguous aerator filters [ ] [ ] [ ] . strict and repeated cleaning and replacement of aerators with flow straighteners may be required to stop outbreaks [ , ] . the origin of norovirus outbreaks in healthcare facilities has been traced not only to sites near bathroom showers and toilets but also to sites near patients, including clinical equipment (e.g., blood pressure and pulse oximeter monitors), thermometers, trolleys and soap and alcohol gel containers [ ] . after suspected or confirmed case contact, use of soap and running water is recommended [ ] , probably with a superior efficacy than ethanol-based sanitizers [ ] . however, detergent-based cleaning may be insufficient to eliminate norovirus from the environment and, therefore, hypochlorite solutions of at least ppm for an appropriate contact time represent a better strategy for cleaning [ , ] . human coronavirus, usually responsible for acute respiratory syndromes, have been causing increased concern due to contact transmission during healthcare-associated outbreaks. viral persistence on doorknobs and surgical boom shelves has already been identified, with a presumed viability of h; scrupulous environmental cleaning is certainly highly advisable in reducing the spread [ , ] . moreover, biocidal surfaces based on copper alloys are very effective in inactivating coronavirus and could be employed in high touch surfaces in order to prevent the transmission of this respiratory virus [ ] . although candida spp. are more resistant to germicidal chemicals than most vegetative bacteria, there are no specific recommendations other than general healthcare surface decontamination with disinfectants. nevertheless, in order to control a recent outbreak by a mdr c. auris, measures implemented included isolation of cases and contacts, protective clothing, screening of all other ward patients, skin decontamination with chlorhexidine, environmental cleaning with chlorine-based disinfectants and hydrogen peroxide vapour [ ] . a clinical alert issued in june by the cdc on the global emergence of invasive infections caused by the mdr c. auris recommended thorough daily and terminal cleaning and disinfection of patient rooms using an epa-registered hospital grade disinfectant with a fungal claim. preventing the environmental surface transmission of healthcare-associated pathogens general strategies based on patterns of microbial resistance to physical and chemical germicidal agents and on the instrument/surface classification, spaulding has proposed three levels of disinfection [ ] : high-level disinfection, that inactivates all vegetative bacteria, mycobacteria, viruses, fungi and some bacterial spores by the action of chemicals such as glutaraldehyde, peracetic acid and hydrogen peroxide; intermediate-level disinfection, which is effective against vegetative bacteria, some spores, mycobacteria, fungi, lipid and medium size viruses, but not against all nonlipid and small size viruses (e.g., sodium hypochlorite, alcohols, some phenolics and some iodophors); and low-level disinfection, that inactivates vegetative bacteria, fungi, enveloped viruses and some non-enveloped viruses (e.g., adenoviruses) by the action of quaternary ammonium compounds, some phenolics and some iodophors [ ] . in order to prevent the persistence of microbial pathogens on medical equipment and environmental surfaces, education of healthcare staff, checklists and assessment of the adequacy of cleaning (by direct observation, use of fluorescent markers, of atp bioluminescence systems, swab cultures or agar slide cultures) with feedback to the staff are general interventions that need to be implemented to improve the frequency of adequate cleaning [ ] [ ] [ ] . as general principles, all patient care items should be cleaned and/or decontaminated before and after use, for all patients [ , ] ; whenever these items come into contact with blood or other body fluids, stringent cleaning and disinfection is warranted before and after use [ ] . manufacturers of medical equipment usually provide care and maintenance instructions regarding servicing decontamination, compatibility with germicidal agents and water-resistance. in the absence of such instructions, the cdc and the healthcare infection control practices advisory committee (hicpac) recommend non-critical medical equipment (e.g., stethoscopes, blood pressure cuffs, equipment knobs and controls) to be subject to low or intermediate-level disinfection after cleansing, depending on the nature and degree of contamination. for instance, ethyl or isopropyl alcohol ( - % v/v) may be used to disinfect small surfaces (e.g., rubber stoppers of multiple-dose medication vials and thermometers) and surfaces of healthcare equipment (e.g., stethoscopes and ventilators) [ ] , while for large surfaces it may be impractical due to the rapid evaporation of alcohol and absence of the adequate contact time [ ] . as a whole, frequently touched environmental surfaces benefit from enhanced cleaning. routine decontamination and disinfection are practices normally included within institutional cleaning policies. nevertheless, evidence has been built in order to favour the use of less toxic detergents over disinfectants in non-outbreak situations, without losing cleaning efficacy or adding costs [ ] . detergents are less likely to contribute to the accumulation or dispersal of tolerance or resistance genes among healthcare-associated microbial isolates [ , ] . according to the cdc, for medical equipment (particularly in the case of monitor touch screens, controls and cables), a disposable plastic barrier protection can be useful whenever these surfaces, touched frequently by gloved hands, may become contaminated with body fluids or present difficulties to cleaning. manual cleaning the physical removal of soil is a very important step in the cleaning process since its presence will impede the microbicidal activity of disinfectants, if needed. in order to control the bioburden on regular wards, daily cleaning with neutral detergent wipes is usually sufficient. however, more attention is essential on high-risk intensive care units because of the easiness of microbial recontamination [ ] . moreover, patients colonized or infected with specific pathogens may demand cleaning regimens with disinfectants with registered label claims [ ] . after patient discharge, terminal or deep cleaning is usually performed by removal of all detachable objects from the room and systematically wiping all surfaces downward to the floor level, with detergent cloths or disinfectant wipes. new liquid disinfectants are under development and include: improved hydrogen peroxide disinfectants, effective in reducing bacterial levels on surfaces [ , ] , related to fewer hais [ ] and able to reduce contamination by mdr pathogens on soft surfaces such as bedside curtains [ ] ; peracetic acid and hydrogen peroxide disinfectants, a sporicidal combination that was shown to lower bacterial levels on surfaces and to reduce the contamination by mrsa, vre and c. difficile as effectively as sodium hypochlorite [ ] ; electrolyzed water (hypochlorous acid) disinfectant, which may reduce bacterial levels on surfaces near patients in a higher degree than quaternary ammonium disinfectants [ ] ; further promising, electrolyzed water has been sprayed onto medical equipment (with a short contact time and without the need for wiping because no toxic residue remains on surfaces) with a reduction of aerobic bacteria and c. difficile spores [ ] ; coldair atmospheric pressure plasma systems, which generate reactive oxygen species (ros) with bactericidal activity and have potential use as surface disinfectants [ , ] ; nebulized polymeric guanidine, under investigation for its antimicrobial activity against several healthcare-associated pathogens [ ] . together with disinfectants, novel materials for liquid application such as microfiber cloths or mops and ultramicrofiber cloths are under development. when used according to manufacturers' instructions, an increased cleaning efficacy is to be expected as compared to standard cotton cloths or mops [ ] . automated cleaning on the pathway to improve quality and ease of cleaning environmental surfaces, considerable efforts have been dedicated towards the development of automated devices. however, because of yet unsolved safety risks, mainly for patients, automated solutions are invariably targeting terminal cleaning. in most instances, these solutions do not preclude preliminary manual cleaning of surfaces to remove residual debris and reduce the bioburden. the microbicidal effect of uv light has been in use for disinfection of environmental surfaces, instruments and air. by damaging the molecular bonds in dna, a reduction in contamination by mrsa, vre and c. difficile on high-touch surfaces has been achieved [ ] . automated mobile uv light devices are easy to use, with minimal need for special staff training. nonetheless, several issues have been raised that may hinder its efficacy, namely, the time and intensity of light exposure and potential barriers that may exist between the lamp and its target surface. as such, uv light is regarded as an effective adjunct, but not a stand-alone strategy for disinfection [ ] . by producing free radicals that lead to oxidation of dna, proteins and membrane lipids [ ] , vapour and aerosol hydrogen peroxide systems have already been shown to be effective against mrsa, vre, mdr gramme-negative bacilli, c. difficile, viruses and fungi [ ] [ ] [ ] [ ] [ ] [ ] . this excellent wide spectrum antimicrobial activity is not without drawbacks, such as toxicity after accidental exposure, minor erosion of environmental polymers and damage of electronic equipment. in addition, there is the need for trained operators, long cycle times for disinfection and the cost is high [ ] . experiments suggest that vapour-phase hydrogen peroxide is a more potent oxidizer of protein than liquid-phase hydrogen peroxide [ ] and, when supplementing other strategies, microcondensation hydrogen peroxide vapour systems may have contributed to control outbreaks by mrsa, mdr grammenegative bacteria and c. difficile in intensive care units, surgical wards and long-term care facilities [ , [ ] [ ] [ ] [ ] [ ] . a novel silver-stabilized hydrogen peroxide is under investigation for its enhanced biocidal activity towards gramme positive and negative bacteria capable of producing catalase, both in planktonic and biofilm cultures. silver probably helps to stabilize and target hydrogen peroxide to the bacterial cell surface acting, therefore, synergistically [ ] . in fact, a previous report on the effect of a dry-mist system using a mixture of hydrogen peroxide ( %) and silver cations (< ppm) was effective in decontaminating burn patient rooms, as well as a fungal research laboratory: a reduction in growth of at least two log was observed for tested bacteria, mycobacteria and fungi [ ] . steam cleaning is a non-toxic and rapid method that may reduce the total bioburden from environmental surfaces by more than % [ ] , with effectiveness against mrsa, vre and gramme-negative bacilli [ ] . concerns about security when steam is applied to electrical items such as switches and buttons and risk of burns and scalds when cleaning a crowded ward are the reasons precluding its widespread use in healthcare facilities [ ] . the oxidizing capacity of ozone justifies its previous evaluation as a gaseous decontaminant for controlling c. difficile on environmental surfaces and e.coli in hospital laundries [ , ] . while it seems highly effective against vegetative bacterial cells, a smaller impact has been found in case of bacterial spores and fungi [ ] . moreover, corrosiveness and toxicity issues may restrain further the use of ozone in healthcare settings [ ] . by targeting intracellular porphyrins that absorb the light and produce ros with bactericidal activity [ ] , highintensity narrow-spectrum (hins) light stands as another light-based method with possible application for decontamination of high-touch surfaces, although its efficacy is lower than uv light. as clear advantages, hins light is safe for patients, allowing continuous decontamination of the clinical environment [ ] and it exhibits a wide-range microbicidal activity that includes mrsa, p. aeruginosa and a. baumannii [ , ] . however, hins light has yet to prove its effectiveness in clinical settings and benefits upon hai rates, given the small range of published studies [ , , ] . antimicrobial surfaces instead of focusing on the reduction of the bioburden on surfaces solely by cleaning, there are solutions designed to prevent surfaces from working as a microbial reservoir and that may be used as an adjunct to other strategies in reducing hais. antiadhesive surfaces target microbial adhesion usually by the interaction of antagonist physicochemical properties. easy-clean surfaces that are hydrophobic repel bacteria better than glass-coated controls [ ] , while hydrophilic surfaces favour water sheeting and subsequent cleaning. similarly, polyethylene glycol coated surfaces promote a hydrophilic interaction against bacteria, preventing attachment [ ] . the use of diamond-like carbon films has already been tried for medical implanted devices such as joint prostheses and stents in order to repel microbial adhesion [ ] . despite being non-toxic and appealing, the lack of biocidal properties may turn discouraging a more generalized implementation of such easy-clean technologies. currently, there are available antimicrobial coatings that can produce a microbicidal effect and could lead to an effective reduction of high-touch surface bioburden. for instance, inorganic metals have been investigated for a long time and it is known that silver binds with disulphide and sulfhydryl groups present in proteins of microbial cell wall leading to death [ ] , inhibiting not only environmental contamination but also colonization of medical implanted devices [ , ] ; copper and copper alloys may form reactive oxygen radicals that damage nucleic acid and proteins [ ] and have already demonstrated a potent antimicrobial effect when applied to surfaces, reducing the rate of healthcare-associated infections [ , ] . polycationic surfaces, such as those coated with polyethyleneimines, hydrophobically attract and kill bacteria by physically damaging the cell wall [ ] . triclosan has been in use for more than years in detergents, soaps and cosmetics. at lower concentrations, it is bacteriostatic by inhibiting an enzyme involved in fatty acid synthesis and, at higher concentrations, it is bacteric i d a l b y d e s t a b i l i z i n g m i c r o b i a l m e m b r a n e s . compatibilization of triclosan with polymers may extend the duration of its wide-spectrum antimicrobial activity [ ] and could prove effective in reducing environmental surface load of pathogens. bacteriophages applied to surfaces and targeting specific microorganisms have been attempted and mixtures of phages have been further suggested in order to effectively reduce the environmental bioburden. particularly interesting in healthcare settings is the fact that mdr pathogens keep vulnerable to the lytic action of phages [ , ] . light-activated antimicrobial surfaces, such as those coated with titanium dioxide and activated by uv light [ ] , generate reactive oxygen radicals with nonselective toxicity towards both bacteria and yeasts [ ] . similarly, photosensitized surfaces could reduce the healthcare bioburden without promoting microbial drug resistance mechanisms. although antimicrobial coatings may seem very promising, especially as an adjunct measure to more traditional and proven cleaning strategies, some concerns keep hindering its wider use in healthcare settings. robust cost-effectiveness studies are still lacking since reliable information about antimicrobial coatings durability, resistance and possible toxicity is yet somewhat insufficient [ , ] . given the high morbidity and costs associated with hais, improved strategies are urgently needed to reduce effectively the rate of infection. certainly, one good step forward would be the blockade of transmission from environmental hightouch surfaces. at the moment, manual and automated techniques for cleaning surfaces exhibit variable success. concerns over durability, resistance and toxicity may be precluding a much wider application of the novel antimicrobial coatings. admitting an albeit limited performance of the traditional cleaning methods, the supplementation with newer technology should be indicated. hence, more randomized controlled trials and cost-effectiveness studies are needed and further investigation on antimicrobial surfaces is welcomed in order to face the challenge imposed by the global advance of antimicrobial drug resistance and the pressure to reduce bed turnover times with shortages in nursing personnel, housekeeping staff and budgets. funding none to declare. ethical approval this type of study does not involve human participants and/or animals. informed consent for this type of study, informed consent is not required. multistate point-prevalence survey of health care-associated infections point prevalence survey of healthcare-associated infections and antimicrobial use in european acute care hospitals epidemiology and control of nosocomial infections in adult intensive-care units the role of the surface environment in healthcare-associated infections cleaning hospital room surfaces to prevent health care-associated infections: a technical brief importance of the environment in meticillinresistant staphylococcus aureus acquisition: the case for hospital cleaning role of environmental contamination as a risk factor for acquisition of vancomycin-resistant enterococci in patients treated in a medical intensive care unit characterization of a hospital outbreak of imipenem-resistant acinetobacter baumannii by phenotypic and genotypic typing methods the role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay acquisition of clostridium difficile from the hospital environment chemical disinfection and antisepsis in the hospital chemical disinfection of medical and surgical material, in disinfection, sterilization and preservation guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) a quantitative approach to defining bhigh-touch^surfaces in hospitals guideline for disinfection and sterilization in healthcare facilities an outbreak of mupirocin-resistant staphylococcus aureus on a dermatology ward associated with an environmental reservoir control and outcome of a large outbreak of colonization and infection with glycopeptideintermediate staphylococcus aureus in an intensive care unit evidence that hospital hygiene is important in the control of methicillin-resistant staphylococcus aureus measuring the effect of enhanced cleaning in a uk hospital: a prospective cross-over study evaluation of a pulsed-xenon ultraviolet room disinfection device for impact on contamination levels of methicillin-resistant staphylococcus aureus controlling methicillin-resistant staphylococcus aureus (mrsa) in a hospital and the role of hydrogen peroxide decontamination: an interrupted time series analysis the rise of the enterococcus: beyond vancomycin resistance how long do nosocomial pathogens persist on inanimate surfaces? a systematic review emergence and spread of vancomycin resistance among enterococci in europe successful prevention of the transmission of vancomycin-resistant enterococci in a brazilian public teaching hospital epidemiology and control of an outbreak of vancomycin-resistant enterococci in the intensive care units use of purified clostridium difficile spores to facilitate evaluation of health care disinfection regimens efficacy of cleaning products for c. difficile: environmental strategies to reduce the spread of clostridium difficile-associated diarrhea in geriatric rehabilitation distribution and transmission of pseudomonas aeruginosa and burkholderia cepacia in a hospital ward bacterial biofilms in nature and disease outbreak of multidrug-resistant pseudomonas aeruginosa colonization and infection secondary to imperfect intensive care unit room design pseudomonas aeruginosa: a formidable and ever-present adversary the effect of terminal cleaning on environmental contamination rates of multidrug-resistant acinetobacter baumannii the epidemiology and control of acinetobacter baumannii in health care facilities acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options acinetobacter outbreaks a multifaceted intervention to reduce pandrug-resistant acinetobacter baumannii colonization and infection in intensive care units in a thai tertiary care center: a -year study management of a multidrug-resistant acinetobacter baumannii outbreak in an intensive care unit using novel environmental disinfection: a -month report epidemiology of klebsiella and hospitalassociated infections outbreak of a multiresistant klebsiella pneumoniae strain in an intensive care unit: antibiotic use as risk factor for colonization and infection an outbreak of multiply-resistant klebsiella pneumoniae in the grampian region of scotland minor outbreak of extendedspectrum beta-lactamase-producing klebsiella pneumoniae in an intensive care unit due to a contaminated sink outbreak of a cluster with epidemic behavior due to serratia marcescens after colistin administration in a hospital setting evaluation and comparison of random amplification of polymorphic dna, pulsed-field gel electrophoresis and adsrrs-fingerprinting for typing serratia marcescens outbreaks molecular epidemiology of an outbreak of serratia marcescens in a neonatal intensive care unit serratia marcescens: an outbreak experience serratia marcescens klebsiella pneumoniae producing kpc carbapenemase in a district general hospital in the uk decreased transmission of enterobacteriaceae with extended-spectrum beta-lactamases in an intensive-care unit by nursing reorganization hospital cleaning in the st century outbreak of burkholderia cepacia complex among ventilated pediatric patients linked to hospital sinks multi-institutional outbreak of burkholderia cepacia complex associated with contaminated mannitol solution prepared in compounding pharmacy an outbreak of burkholderia cepacia complex pseudobacteremia associated with intrinsically contaminated commercial . % chlorhexidine solution uses of inorganic hypochlorite (bleach) in health-care facilities norovirus in the hospital setting: virus introduction and spread within the hospital environment guideline for the prevention and control of norovirus gastroenteritis outbreaks in healthcare settings effectiveness of liquid soap and hand sanitizer against norwalk virus on contaminated hands effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces middle east respiratory syndrome coronavirus on inanimate surfaces: a risk for health care transmission stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions human coronavirus e remains infectious on common touch surface materials first hospital outbreak of the globally emerging candida auris in a european hospital identifying opportunities to enhance environmental cleaning in acute care hospitals impact of an environmental cleaning intervention on the presence of methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci on surfaces in intensive care unit rooms modern technologies for improving cleaning and disinfection of environmental surfaces in hospitals cleanliness audit of clinical surfaces and equipment: who cleans what? healthcare equipment as a source of nosocomial infection: a systematic review controlling hospital-acquired infection: focus on the role of the environment and new technologies for decontamination apic guideline for selection and use of disinfectants the crucial role of wiping in decontamination of high-touch environmental surfaces: review of current status and directions for the future promises and pitfalls of recent advances in chemical means of preventing the spread of nosocomial infections by environmental surfaces bacterial adaptation and resistance to antiseptics, disinfectants and preservatives is not a new phenomenon evaluation of the efficacy of a conventional cleaning regimen in removing methicillin-resistant staphylococcus aureus from contaminated surfaces in an intensive care unit efficacy of improved hydrogen peroxide against important healthcare-associated pathogens evaluation of a new hydrogen peroxide wipe disinfectant use of a daily disinfectant cleaner instead of a daily cleaner reduced hospital-acquired infection rates effectiveness of improved hydrogen peroxide in decontaminating privacy curtains contaminated with multidrug-resistant pathogens evaluating a new paradigm for comparing surface disinfection in clinical practice comparison of cleaning efficacy between in-use disinfectant and electrolysed water in an english residential care home effectiveness of an electrochemically activated saline solution for disinfection of hospital equipment cold air plasma to decontaminate inanimate surfaces of the hospital environment cold atmospheric pressure plasma and decontamination. can it contribute to preventing hospital-acquired infections? evaluation of the efficacy of akacid plus (r) fogging in eradicating causative microorganism in nosocomial infections microbiologic evaluation of microfiber mops for surface disinfection evaluation of an automated ultraviolet radiation device for decontamination of clostridium difficile and other healthcare-associated pathogens in hospital rooms applications of ultraviolet germicidal irradiation disinfection in health care facilities: effective adjunct, but not stand-alone technology use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action controlling methicillin-resistant staphylococcus aureus (mrsa) in a hospital and the role of hydrogen peroxide decontamination: an interrupted time series analysis control of an outbreak of acinetobacter baumannii infections using vaporized hydrogen peroxide airborne hydrogen peroxide for disinfection of the hospital environment and infection control: a systematic review hydrogen peroxide vapour decontamination of surfaces artificially contaminated with norovirus surrogate feline calicivirus impact of hydrogen peroxide vapor room decontamination on clostridium difficile environmental contamination and transmission in a healthcare setting deactivation of the dimorphic fungi histoplasma capsulatum, blastomyces dermatitidis and coccidioides immitis using hydrogen peroxide vapor floor wars: the battle for 'clean' surfaces eradication of persistent environmental mrsa hydrogen peroxide vapour decontamination in the control of a polyclonal meticillin-resistant staphylococcus aureus outbreak on a surgical ward hydrogen peroxide vapor decontamination of an intensive care unit to remove environmental reservoirs of multidrug-resistant gram-negative rods during an outbreak impact of environmental decontamination using hydrogen peroxide vapour on the incidence of clostridium difficile infection in one hospital trust use of vaporized hydrogen peroxide decontamination during an outbreak of multidrug-resistant acinetobacter baumannii infection at a long-term acute care hospital antibacterial properties and mechanism of activity of a novel silver-stabilized hydrogen peroxide efficacy of hydrogen peroxide dry-mist disinfection system for hospital environment disinfection reduction in the microbial load on hightouch surfaces in hospital rooms by treatment with a portable saturated steam vapor disinfection system reduction in infection risk through treatment of microbially contaminated surfaces with a novel, portable, saturated steam vapor disinfection system hospital cleaning: problems with steam cleaning and microfibre ozone gas is an effective and practical antibacterial agent disinfection of hospital laundry using ozone: microbiological evaluation use of gaseous ozone for eradication of methicillin-resistant staphylococcus aureus from the home environment of a colonized hospital employee gaseous and air decontamination technologies for clostridium difficile in the healthcare environment helicobacter pylori accumulates photoactive porphyrins and is killed by visible light environmental decontamination of a hospital isolation room using high-intensity narrow-spectrum light high-intensity narrow-spectrum light inactivation and wavelength sensitivity of staphylococcus aureus inactivation of bacterial pathogens following exposure to light from a -nanometer light-emitting diode array clinical studies of the high-intensity narrow-spectrum light environmental decontamination system (hins-light eds), for continuous disinfection in the burn unit inpatient and outpatient settings self-cleaning coatings bacterial adhesion on peg modified polyurethane surfaces a review of modified dlc coatings for biological applications self-disinfecting surfaces: review of current methodologies and future prospects silver in health care: antimicrobial effects and safety in use dual-action hygienic coatings: benefits of hydrophobicity and silver ion release for protection of environmental and clinical surfaces copper surfaces reduce the rate of healthcare-acquired infections in the intensive care unit role of copper in reducing hospital environment contamination permanently microbicidal materials coatings triclosan antimicrobial polymers potential of bacteriophage phi ab as an environmental biocontrol agent for the control of multidrug-resistant acinetobacter baumannii antimicrobial surfaces and their potential in reducing the role of the inanimate environment in the incidence of hospital-acquired infections comparison of infectious agents susceptibility to photocatalytic effects of nanosized titanium and zinc oxides: a practical approach light-activated antimicrobial coating for the continuous disinfection of surfaces key: cord- -wdjlr z authors: szpiro, l.; pizzorno, a.; durimel, l.; julien, t.; traversier, a.; bouchami, d.; marie, y.; rosa-calatrava, m.; terrier, o.; moules, v. title: role of interfering substances in the survival of coronaviruses on surfaces and their impact on the efficiency of hand and surface disinfection date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: wdjlr z contaminated environmental surfaces are considered to represent a significant vector for hospital-acquired viral infections. in this study, we have evaluated the impact of interfering substances on sars-cov- surface stability and virucidal efficiency of hand sanitizers and surface disinfectant. to this end, surface stability of sars-cov- was measured on stainless steel in different experimental conditions, with or without an artificial mucus/saliva mixture and compared against that of human coronavirus hcov- e and feline coronavirus fcov. the impact of the mucus/saliva mixture on the virucidal efficiency of commercial alcohol hand sanitizers and surface chemical disinfectant against sars-cov- , hcov- e and fcov was then measured. our results indicate that mucus/saliva mixture did not demonstrate a beneficial effect on the surface survival of tested viruses, with temperature being an important parameter. in addition, we demonstrated that interfering substances may play an important role in the virucidal efficacy of hand sanitizers and disinfectants, highlighting the need for adapted testing protocols that better reflect current - real life -conditions of use. respiratory diseases caused by human coronavirus infection are of both medical and socio-economic importance. severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and currently sars-cov- have crossed the species barrier and entered the human population to cause severe disease. as observed for a large number of respiratory viruses, airborne transmission and fomite transmission are thought to play important roles in the epidemiology of these viruses ( ) . several reviews and models have suggested that indirect contact transmission involving contaminated surfaces could be the predominant transmission route for certain respiratory viruses, including influenza, in some settings ( ) ( ) ( ) . indeed, contaminated environmental surfaces are considered to represent a significant vector for hospital-acquired viral infections ( , ) . influenza a virus has also been shown to be transferred from stainless steel countertops to hands up to h after surface contamination ( ) . the transfer of human parainfluenza virus and rhinovirus from contaminated surface to clean fingers supports a role for fomites in the contamination of hands with both viruses ( ) . for any environmental contamination to be relevant, a virus should not only remain infectious on the recipient surface but also persist at a sufficient concentration to enable it to reach the respiratory tract via finger contamination. in general terms, the potential of a fomite to spread a given infectious agent is directly related to the capacity of the agent to survive on that surface. the surface stability of viruses is generally influenced by the type of surface, environmental factors such as relative humidity (rh) and temperature, and the presence of body fluid secretions. coronaviruses have the capacity to survive on a wide range of porous and non-porous materials ( ). a recent study has shown that sars-cov and sars-cov- , at % rh and - °c, were most all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint stable on smooth surfaces and that viable virus could be detected up to and days respectively post application ( ). chin et al showed that sars-cov- could be detected on contaminated stainless steel supports, at % rh and °c, seven days after experimental inoculation ( ) . sars-cov has been reported to stay viable for up to five days at to °c and to % rh, with an increase in temperature and humidity resulting in a rapid loss of viability ( ) . casanova et al demonstrated a higher stability in sars-cov compared to the human coronavirus hcov- e, which in a dried state retained residual infectivity even after days compared to the loss of infectivity within days observed in hcov- e ( ) . mers-cov virus could still be recovered on stainless steel after days in the °c and % rh condition, whereas it remained viable for a much shorter h at °c and % rh, and hours at °c and % rh ( ) . all studies that have tested varying temperature and rh agree that lower temperatures and higher rhs systematically favor the survival of coronaviruses ( , ) . during and after illness, viruses are shed in large numbers in body secretions, including blood, feces, urine, saliva, and nasal fluid, all of which comprise high levels of protein and other biological organic matter. while several studies appear to have reached a consensus agreement on the role of both fecal material and blood in the increased stability of viruses on environmental surface ( ) ( ) ( ) , the potential beneficial effect of respiratory secretions on surface viral survival remains unclear. mixing of highly concentrated inocula with respiratory mucus increased the infectiousness of influenza viruses, allowing its transmission for up to days ( ) and thus confirming the protective role of human mucus for the survival of respiratory viruses previously shown by parker et al ( ) . however, human rhinovirus type suspended in tryptose phosphate broth could survive for more than h incubation, though for less time when suspended in bovine mucin or in nasal secretions ( ) . no data are available concerning the transmissibility of coronaviruses from contaminated surfaces to hands. data are however available concerning other respiratory viruses including influenza a virus, parainfluenza virus and rhinovirus which were shown to be transferred to the hands ( . to . % of the viral load) after a contact of s with an inoculated surface ( , ) . several studies have found that frequent hand-surface contact can lead to rapid diffusion of surface contamination ( , ) . in view of the indirect transmissibility of viruses, compliance with both hand hygiene and surface disinfection recommendations is critical to reducing colonization and infection of the hands of entire populations but in particular the hands of health-care workers. an important consideration in the strict compliance of these recommendations is the finding in several studies that interfering substances (including body secretions) could have a strong impact on the efficiency of a disinfectant ( , ) . to simulate clean/dirty environments, standardized interfering substances are used to evaluate disinfection product efficiency according to phase en standards. while the determination of surface virucidal efficiency of a disinfectant is managed according to en standards through the use of inoculated stainless steel carriers, the virucidal efficiency of hand rub products is currently tested using the en suspension test ( , ). the efficiency of hand rub products is based upon a titer reduction of ≥ log in a maximum contact time of s to s in compliance with suspension test standards. in an attempt to better understand and thus better control the transmission of sars-cov- behind the recent and ongoing pandemic, the impact of body fluid secretions, from coughing or sneezing corresponding to an artificial mixture containing nasal mucus and saliva, on the surface stability of sars-cov- and the virucidal efficiency of disinfectant were tested. to this end, surface stability was measured on stainless steel at and °c ( % rh) with or without the artificial mucus/saliva mixture and compared against that of human coronavirus hcov- e and the standardized feline coronavirus fcov strain used in en standard tests of surface virucidal activity ( ) . the impact of the mucus/saliva mixture on the virucidal efficiency of commercial alcohol hand sanitizers (according to the en standard suspension test) and surface chemical disinfectant (according to the virucidal surface quantitative en test) against sars-cov- , hcov- e and fcov was then measured. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . table ) and epithelium mucus was put in the center of a stainless-steel disk. the deposit was dried under psm for a maximum of h. disks were then placed in a refrigerator at °c or in an oven stabilized at °c and % relative humidity for different lengths of time (from to h). the recovery of each virus at different time points was performed using the same protocol: ml of infection medium was first placed on the disk and incubated for min at room temperature. all the liquid was collected into a tube and quantified by tcid /ml (spearman and karber method). the quantification limit was determined taking into account the cytotoxicity. the limit of detection (lod) on every cell, was . log tcid /support. virucidal activity according to suspension test. the virucidal activity of three commercial hand rub products against sars-cov- , hcov- e and fcov was determined using the quantitative suspension test according to en , comparing standardized interfering substance (clean condition, . g/l bsa) and our artificial mucus/saliva mixture. a . part by volume of virus suspension and . parts by volume of interfering substance were mixed with . parts by volume of the hand rub product corresponding to the modified protocol to evaluate ready-to-use products at % according to the en standard. at the tested contact time, a tenfold dilution was made to stop the activity of the disinfectant before quantifying the remaining infectious virus using the spearman and karber method. contact times of and s were assessed at °c. titer reduction is presented as the difference between the control virus titer and that after contact with the test product. this difference is given as a reduction factor including its % confidence interval. a reduction in virus all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint to investigate the stability of sars-cov- , hcov- e and fcov on stainless steel at both and °c, a -μl droplet of solution containing virus culture diluted in either culture media or artificial mixture ( . , . and . log tcid /ml respectively for sars-cov- , hcov- e and fcov) was pipetted onto stainless steel discs and left in an incubator at or °c (relative humidity: %). figures b and c) . as observed for sars-cov- , the viral stability decreased at °c with only a few (hcov- e) or no (fcov) viable virus being detected h post application. we then evaluated the effect of an artificial mucus/saliva mixture on the stability of the three viruses under the same temperature conditions described above. the interfering mixture induced a moderate negative impact on the stability of sars-cov- at °c, with . , . and . log tcid reductions at , and h post application, respectively, compared to the condition without the artificial mixture ( figure a ). this negative impact was even stronger at °c, with no viable virus measured at h post application. artificial mucus/saliva mixture had no significant impact on the stability of hcov- e and fcov at °c (figures b and c) , however, as observed for sars-all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint cov- , it had a strong negative impact in both at °c, with no viable virus measured at h post application (figures b and c) . in order to measure the virucidal activity of alcohol hand sanitizers, the nf en +a standard protocol was followed. briefly, a volume of . ml of interfering substance (bsa . g/l or artificial saliva/epithelium mucus) was added to . ml of purified virus and to . ml of disinfectant (ready-touse product). at the tested contact times ( and s), a tenfold dilution was made to stop the activity of the disinfectant before quantifying the remaining infectious virus. of three commercial hand rub products tested, only product showed a virucidal activity at s (figure a) . to further investigate a potential impact of the mucus/saliva mixture on virucidal efficiency, we evaluated the virucidal activity of a commercial chemical surface disinfectant using both clean/dirty medical standardized interfering substances ( . g/l bsa and g/l bsa + ml/l sheep erythrocytes) and the artificial mucus/saliva mixture following en quantitative non-porous surface tests. stainless steel discs were inoculated with µl of a test suspension containing the standard modified vaccine ankara virus strain (mva) in a solution of interfering substance and let dry for min. the the role of contaminated surfaces as a potential significant vector for hospital-acquired viral infections has gained a whole new community-based dimension in the current covid- pandemic context. in that regard, the results presented in this study demonstrate the stability of sars-cov- on smooth surfaces, with infectious virus being detected up to h and h post application at °c and °c respectively. these observations confirm recent data that sars-cov- remains infectious for several days on stainless steel surface ( , ) . our data suggest a higher stability of both hcov- e and fcov at both temperatures compared to sars-cov- , with infectious particles being detected up to h post application as described previously ( ) . as previously described for coronavirus and other respiratory viruses, sars-cov- showed greatest surface-stability at low temperature ( , , ) . numerous studies have described the role of body secretions in the observed increased stability of a large number of viral strains on environmental surfaces ( ) ( ) ( ) , as demonstrated for influenza virus in contact with respiratory mucus ( ) . interestingly, the mucus/saliva mixture did not demonstrate a beneficial effect on surface survival for all tested viruses and a negative effect in sars-cov- seemed to correlate with the incubation temperature. at moderate temperatures, the mucus/saliva mixture had a strong impact on the viability of coronaviruses and quantities of virus close to the limit of detection (lod) were detected up to h. we hypothesize that the increase in temperature from to °c may activate the catalytic site of antimicrobial proteins present in nasal mucus. in this sense, the most abundant airway antimicrobial factors are lysozyme ( ), lactoferrin ( ) , and secretory leukoproteinase inhibitor ( ) . lactoferrin is a multifunctional glycoprotein with a broad spectrum of antiviral activity against a wide range of viruses in vitro including enveloped viruses ( ) , while the antiviral spectrum of lysozyme is considerably more modest ( ) . several other known antimicrobial proteins and peptides including statherin and secretory phospholipase a have been identified in nasal secretions and likely contribute to their antimicrobial properties ( ) . as observed for rhinoviruses, nasal secretions and saliva seem to reduce the ability of coronaviruses to survive on environmental surfaces at moderate temperatures ( ) . however, we show that a large quantity of infectious sars-cov- were still detectable on surfaces after h post application at °c in the presence of artificial all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint body fluid secretions from coughing or sneezing. laboratory simulations have demonstrated that hand-to-hand and fomite-to-hand contact are viable modes of transmission for influenza and other respiratory viruses depending how long the viral particles remain viable on hands and fomites ( , ) . as compared to influenza virus stability, we can hypothesize that sars-cov- remains stable on fomite as long as sufficient to contaminate hands and the risk of contaminating surfaces with sars-cov- should be emphasized in guidelines for hand and surface disinfection practices. with regards to disinfection, antiseptic hand hygiene including use of alcohol-based hand sanitizer is one of the most important measures in preventing outbreak-associated viral infections. we have shown that among the three commercially available alcohol hand sanitizers tested in our study, only one, corresponding to the ethanol-based who formulation, was associated with the mucus/saliva mixture used in this study showed a strong impact on virucidal efficiency when disinfectant was applied onto a dried inoculum-contaminated surface; in contrast however, it had no impact on virucidal efficiency tested using the suspension protocol with no significant difference shown between standardized clean medical condition and mucus/saliva mixture. we hypothesize that the dilution factor of the mucus/saliva mixture used in the modified suspension protocol at %, all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint regularly used to evaluate ready-to-use hand rub products, may explain this difference. however, mucus/saliva interference had a similar impact on virucidal activity on surfaces as observed with the standardized medical dirty condition. in summary, the democratization of the use of alcohol-based hand sanitizers from healthcare facilities to the general public as a barrier measure to prevent outbreak-associated respiratory viral infections emphasizes the importance of assessing the potential impact of interfering substances rich in protein or other biological organic matter, which are usually present in soiled hands and might significantly impact virucidal efficiency. our results advocate for the development of standardized phase /step testing protocols according to simulated practical conditions reflecting new "real life" needs, notably considering the impact of omnipresent secretions from coughing or sneezing on the evaluation of the virucidal activity of hand sanitizers. hcov- e (b) and fcov (c) on stainless steel. viral suspensions diluted in either culture media or artificial mucus/saliva mixture ( . , . and . log tcid /ml respectively for sars-cov- , hcov- e and fcov) were deposited onto stainless steel discs and left in an incubator at or °c (relative humidity: %). the inoculated supports retrieved at desired time points were immediately soaked with ml of viral culture media. the limit of detection (lod) for all experiments was . log tcid /support. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . virucidal activity of three commercial hand rub sanitizer products against sars-cov- , hcov- e and fcov. virucidal activity was determined using the quantitative suspension test according to en , comparing the standardized interfering substance ("clean" condition, . g/l bsa (a) with the artificial mucus/saliva mixture (b). the remaining infectious viruses were quantified by the spearman and karber method. the quantification limit was determined taking into account the cytotoxicity of the products ( . log tcid ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . virucidal activity of a commercial surface disinfectant. the purified virus was mixed with the interfering substance ("clean" condition: bsa . g/l, artificial saliva + epithelium mucus, "dirty" condition: bsa g/l + ml/l of sheep erythrocytes). a volume of μl of the mix was deposited on a previously functionalized stainless-steel disk (see previously). the inoculum was left to dry before . ml of the disinfectant ( , and % concentrations) was deposited on the disk. the remaining infectious viruses were quantified by the spearman and karber method. the quantification limit was determined taking into account the cytotoxicity of the product ( . log tcid ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination significance of fomites in the spread of respiratory and enteric viral disease transmission of influenza a in human beings informing optimal environmental influenza interventions: how the host, agent, and environment alter dominant routes of transmission survival of human coronaviruses e and oc in suspension and after drying on surfaces: a possible source of hospital-acquired infections microbicides and the environmental control of nosocomial viral infections role of fomites in sars transmission during the largest hospital outbreak in hong kong the occurrence of influenza virus on household and day care center fomites potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus and rhinovirus aerosol and surface stability of hcov- (sars-cov- ) compared to sars-cov- stability of sars-cov- in different environmental conditions. the lancet microbe effects of air temperature and relative humidity on coronavirus survival on surfaces stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions the effects of temperature and relative humidity on the viability of the sars coronavirus survival of hepatitis a virus in feces after drying and storage for month the effects of relative humidity and temperature on the survival of human rotavirus in faeces survival of human immunodeficiency virus in suspension and dried onto surfaces survival of influenza virus on banknotes resistance of the melbourne strain of influenza virus to desiccation survival of human rhinovirus type dried onto nonporous inanimate surfaces: effect of relative humidity and suspending medium characterization of cellular transcriptomic signatures induced by different respiratory viruses in human reconstituted airway epithelia. sci rep standard practice for evaluation of effectiveness of decontamination procedures for surfaces when challenged with droplets containing human pathogenic viruses chemical disinfectants and antiseptics -quantitative non-porous surface test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area -test method and requirements -phase , step all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity characterizing dynamic transmission of contaminants on a surface touch network the influence of interfering substances on the antimicrobial activity of selected quaternary ammonium compounds chemical disinfectants and antiseptics -quantitative non-porous surface test without mechanical action for the evaluation of virucidal activity of chemical disinfectants used in the medical area -test method and requirements chemical disinfectants and antiseptics -quantitative suspension test for the evaluation of virucidal activity in the medical area -test method and requirements lysozyme and lactoferrin in sputum from patients with chronic obstructive lung disease bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms antibacterial activity of antileukoprotease antivirals against animal viruses virucidal activity of world health organization-recommended formulations against enveloped viruses, including zika, ebola, and emerging coronaviruses efficacity of ethanol against viruses in hand disinfection key: cord- -affuyn z authors: poggio, claudio; colombo, marco; arciola, carla renata; greggi, tiziana; scribante, andrea; dagna, alberto title: copper-alloy surfaces and cleaning regimens against the spread of sars-cov- in dentistry and orthopedics. from fomites to anti-infective nanocoatings date: - - journal: materials (basel) doi: . /ma sha: doc_id: cord_uid: affuyn z the latest diffusion of severe acute respiratory syndrome coronavirus (sars-cov- ), responsible for the coronavirus disease (covid- ), has involved the whole world population. even if huge efforts to control the pandemic have been done, the viral spread is still continuing. covid- is reported as a zoonosis jumped from bats and pangolins to humans. after infection in humans, sars-cov- is found in the nasopharyngeal and salivary secretions. the virus has also been detected in the blood plasma of infected patients. the viral spread occurs through droplets exhaled from the nose and mouth of the infected people when they breath or talk, or through droplets propelled as a dense cloud by chough or sneeze. the virus can also be delivered as an aerosol from blood plasma, through surgical procedures. following these ways, the virus can disperse in the air, then reaching and settling on the exposed surfaces. how long the virus will survive on a surface depends on the material the surface is made from. infection via high-touch surfaces should be prevented. copper alloy coatings, combined with efficient hygienic/disinfectant procedures and careful surgical practice, could be helpful to health protection in dental practice and can also be adopted in orthopedic traumatology. viruses can live on many surfaces outside the human body and persist on inanimate surfaces like metal, glass or plastic for days [ ] . sars-cov- (acronym for severe acute respiratory syndrome coronavirus ), responsible for the current outbreak that causes covid- (acronym for "corona virus disease "), is reported to be able of surviving on inanimate surfaces for days. although person-to-person transmission (through the droplets emitted by infected people when they breath, talk, cough or sneeze) has been referred as the primary way to spread the virus, however an indirect traumatology procedures that involve incising tissues, truncating vessels, cleaning and irrigating wounds, electro cauterizing bleeding tissues, treating broken bones and injured areas around broken bones, realigning limb, reaming bone, drilling bone, aspirating hemorrhagic synovial fluid after traumatic hemorrhage [ ] . materials , , x of bones, realigning limb, reaming bone, drilling bone, aspirating hemorrhagic synovial fluid after traumatic hemorrhage [ ] . routes of transmission in outpatient practice (adapted from ge et al. [ ] ). genetic material of sars-cov- has recently been demonstrated in the plasma of patients with covid- , thus feeding concerns for virus shedding during surgical procedures [ ] . in a recent study, the prevalence of orthopedic surgeons who had become infected with sars-cov- in wuhan (china) hospitals was found high. exposure to viral infection was traced. orthopedic surgeons operating in the wards were exposed to the highest risk of infection (close to %). interestingly, the public places of the hospital turned out to be areas at high risk of infection (over %), even higher than that associated with operating rooms ( . %) [ ] . it should be emphasized that during the viral epidemic, orthopedic trauma services remain necessarily open and active, contributing to the chances of the virus to spread from asymptomatic infected patients who come to the observation of orthopedic surgeons [ ] . the risk of covid- infection in orthopedic practice and the personal protective equipment have been recently considered [ , ] . recent studies have shown that sars-cov- can survive from hours to days on a multiplicity of artificial surfaces like metal, glass or plastic. kampf et al. [ ] states that human coronaviruses can persist for up to days at room temperature, and that veterinary coronaviruses can even persist for up to days. it is noteworthy that increasing temperatures to °c or more can shorten coronavirus survival, even if there is no established correlation between the variations in temperature and the spread of coronavirus [ ] . the life span of the virus on a surface depends on many factors, including the temperature, humidity and ventilation of the room and the type of surface [ ] . this denotes that the people may contract the virus and become infected by touching an infected object [ ] . the load of sars-cov- on non-living surfaces (fomites) is still unclear and probably variable, but the disinfection/cleaning of the surrounding surfaces appears to reduce the viral load, especially that of the objects touched by the infected person and the nearby area, where the maximum viral load is present [ ] . a person can be infected with the coronavirus by touching a surface or an object that has viral particles on it and then by touching mouth, nose, or eyes [ ] . face-touching is a common habit of people: it is considered a vector in self-inoculation. an interesting article that dealt with environmental hygiene focused on the importance of the transmission of respiratory tract infections genetic material of sars-cov- has recently been demonstrated in the plasma of patients with covid- , thus feeding concerns for virus shedding during surgical procedures [ ] . in a recent study, the prevalence of orthopedic surgeons who had become infected with sars-cov- in wuhan (china) hospitals was found high. exposure to viral infection was traced. orthopedic surgeons operating in the wards were exposed to the highest risk of infection (close to %). interestingly, the public places of the hospital turned out to be areas at high risk of infection (over %), even higher than that associated with operating rooms ( . %) [ ] . it should be emphasized that during the viral epidemic, orthopedic trauma services remain necessarily open and active, contributing to the chances of the virus to spread from asymptomatic infected patients who come to the observation of orthopedic surgeons [ ] . the risk of covid- infection in orthopedic practice and the personal protective equipment have been recently considered [ , ] . recent studies have shown that sars-cov- can survive from hours to days on a multiplicity of artificial surfaces like metal, glass or plastic (see table ). kampf et al. [ ] states that human coronaviruses can persist for up to days at room temperature, and that veterinary coronaviruses can even persist for up to days. it is noteworthy that increasing temperatures to • c or more can shorten coronavirus survival, even if there is no established correlation between the variations in temperature and the spread of coronavirus [ ] . the life span of the virus on a surface depends on many factors, including the temperature, humidity and ventilation of the room and the type of surface [ ] . this denotes that people may contract the virus and become infected by touching an infected object [ ] . the load of sars-cov- on non-living surfaces (fomites) is still unclear and probably variable, but the disinfection/cleaning of the surrounding surfaces appears to reduce the viral load, especially that of the objects touched by the infected person and the nearby area, where the maximum viral load is present [ ] . a person can be infected with the coronavirus by touching a surface or an object that has viral particles on it and then by touching mouth, nose, or eyes [ ] . face-touching is a common habit of people: it is considered a vector in self-inoculation. an interesting article that dealt with environmental hygiene focused on the importance of the transmission of respiratory tract infections through the contact of contaminated hands with face. the article pointed out that a not small portion of human respiratory tract infections is transmitted through this path. infection is likely to occur at a rate proportional to the number of contacts the hands have with the facial areas, in particular with the facial mucous membranes [ ] . frequent hand hygiene, both before and after patient contact, is an effective way to reduce colonization and limit transmission of infection for themselves and for patients: washing hands and not touching face are the best ways to minimize the chance of picking up the coronavirus from surfaces [ ] . van doremalen et al. [ ] investigated the survival of sars-cov- in the air and on surfaces: they simulated how virus could be spread by infected people onto common surfaces in domestic or hospital sceneries, by coughing, sneezing or touching/handling objects. they also explored how long the virus remained infectious on these surfaces. they tested viral vitality on plastic, stainless steel, copper, and cardboard. they also simulated an aerosol suspension with the virus, creating a mist of tiny droplets: in that way, it was possible to determine if the virus could remain in the air and how long. under these experimental conditions, sars-cov- showed to be active and infectious on plastic and stainless-steel surfaces from to days, on cardboard for up to h, and on copper for h. sars-cov- virus was detectable in aerosols for up to h [ ] . these times should vary in real conditions, due to the influence of factors such as temperature, humidity, ventilation, dust, fingerprints, organic debris, and the amount of virus deposited. additionally, the characteristics of the surface must be taken into careful consideration: generally, smooth surfaces are more easily sanitized. on the other hand, the difficulty in cleaning increases with the roughness of the surface, as debris could penetrate the cracks and resist traditional alcohol-based cleaning treatments. these results obviously imply that people can catch the sars-cov- through the air and after touching contaminated objects. for these reasons, frequent disinfection of touched objects and surfaces play a fundamental role in reducing viral cross contamination [ ] . in a recent article all the sources currently present in the literature concerning the persistence of the different coronaviruses in the environment as well as in medical and dental settings have been described and critically analyzed [ ] . world health organization (who) advises frequent, correct and consistent disinfection/cleaning procedures of environments and surfaces, which must be thoroughly cleaned with water and simple disinfectants [ ] . hospital disinfectants such as alcohol or bleach-based products are considered effective. these products must be used following scrupulously the manufacturer's instructions. particular attention must be paid to touched surfaces such as doors, handles, toilets, desks, switches and sinks: these surfaces can be frequently disinfected and cleaned with household disinfectants. there is a not negligible probability of environmental contamination. since the potential for viral transmission through inanimate objects is strong, every effort should be made to consistently and effectively clean and disinfect surfaces and objects. however, these findings do not change the cleaning practices for coronaviruses, including recommendations issued by the us centers for disease control and prevention (cdc) [ ] . it is essential that appropriate protocols for daily cleaning and disinfection are part of the measures to prevent infection. cleaning procedures remove large numbers of microorganisms from surfaces and should always precede disinfection (a less lethal anti-microbial process than sterilization). routine cleaning and disinfection can achieve the removal and elimination of the main pathogenic microorganisms, although not necessarily of all microbial forms (e.g., bacterial spores) [ , ] . the role of cleaning and disinfection is more critical for surfaces of medical offices that are more exposed to pathogens, including clinical contact surfaces (e.g., frequently touched surfaces such as light handles, bracket trays, switches on electrical equipment, dental units, computer equipment) in the patient-care area. when these surfaces are touched, even if the operators pay their attention, microorganisms can be transferred to other surfaces, instruments or to the nose, mouth, or eyes of healthcare personnel or patients. although hand hygiene is the key to minimizing the spread of microorganisms, clinical contact surfaces should be protected by single-use barriers or cleaned and disinfected between patients. for the disinfection procedures, the american environmental protection agency (epa)-registered hospital disinfectants or the detergents/disinfectants labeled for use in health care settings should be used. it should also be noted that a disinfectant product can be used as a detergent only if the label explicitly indicates that the product is suitable for that use. the environmental infection control measures for sars-cov- are not unexpected. the management of sars-cov- in health care settings (especially in those patient-care areas in which aerosol-generating procedures are conducted) involves a strict adhesion to the manufacturer's recommendations for routine cleaning and disinfection, for example the use of water and cleaners to pre-clean surfaces before applying disinfectants. additionally, the use of dedicated medical equipment, as ffp or n face masks, are needed [ ] . different types of biocidal agents, such as alcohols, hydrogen peroxide, benzalkonium or sodium hypochlorite chloride, are applied worldwide for disinfection. it has been proven that disinfectants with - % ethanol or . % sodium hypochlorite can reduce coronavirus contamination on surfaces within one minute of exposure. cleaning should be done after disinfection for contaminated surfaces. kampf et al. [ ] reported that human coronaviruses could be inactivated on surfaces with ethanol (between - %), -propanol (between - %), the combination of % -propanol with % -propanol, glutardialdehyde (between . - . %), formaldehyde (between . - %), and povidone iodine (between . - . %). sodium hypochlorite requires a minimal concentration of at least . % to be effective. hydrogen peroxide is effective with a minimal concentration of . % and an application time of min. kampf et al. [ ] have discussed the evidence on the disinfectant efficacy of benzalkonium chloride, which is still being tested and is not always effective. at contact times within min, a concentration of . % revealed no efficacy against enveloped human coronavirus, whereas a concentration of . % proved quite effective against a non-enveloped coxsackie virus. finally, using ozone is another potential effective measure to counteract viruses. ozone can be conveyed as a gas or a gel to sanitize the environment. it has been extensively studied and used for many years and its effectiveness has been demonstrated against viruses, bacteria and fungi [ , [ ] [ ] [ ] [ ] [ ] , even if no tests have been conducted on sars-cov- yet. the important thing when disinfecting a surface is to bring the potential infectious dose of the virus below a level that would cause disease. a correlation between sars-cov- burden and the severity of clinical course of the covid- infection in five patients has been recently presented [ ] . the authors described three different clinical types of evolution with detailed viral sampling: ( ) two paucisymptomatic women, ( ) a two-step disease progression in two young men and ( ) an -year-old man with a rapid evolution towards multiple organ failure. the viral burden both in plasma and in the respiratory tract during infection was found correlating with the severity of infection. [ ] . this interpretation has been discussed by joynt and wu, who have highlighted that the impossibility to differentiate between infective and noninfective (dead or antibody-neutralized) viruses remains a major limitation in establishing a correlation between the viral rna burden and the severity of the infection [ ] . the potential use of biocidal surfaces to provide constant antiviral activity against continual surface recontamination could help to limit the spread of respiratory viruses. each material able to inactivate sars-cov- should be investigated and applied on critical surfaces in medical and dental offices, with the aim to reduce the number of viruses potentially deposed on devices, tools and furniture. copper has shown antiviral properties: the efficacy of the copper against bacteria and fungi is known since antiquity. for centuries, long before the knowledge about germs or viruses, people were aware of the disinfecting power of copper. in ancient times, egyptian and babylonian soldiers put the metal shavings of their bronze swords (made of copper and tin) into open wounds to reduce infection. greeks, romans and aztecs used copper to treat headaches and ear infections. and in india, copper vessels have been used for millennia in the transportation of water [ ] . the metal nanoparticles of copper, magnesium, titanium, gold, and zinc have been proved to be bactericidal at nano-levels [ ] . new antiviral effective metal materials may be provided by the advancement of nanotechnology. the activity of the metal nanoparticles against viruses as an advanced -although still unexplored-field has been discussed in a noteworthy review [ ] . bacterial adhesion to the surface is the first step in the pathogenesis of implant infection, as the implant provides a surface to which bacteria can adhere. modifying the implant surface with a repellent coating to reduce bacterial adherence can decrease the risk of infection. as observed since long ago heparin surface treatment of a material is effective in hindering bacterial adhesion [ ] . in case of nanoparticles also it has been proposed that the differential antiviral effectiveness of silver or gold nanoparticles against different viruses depends on their surface functionalization with different ligands [ ] . the antibacterial properties of copper chelates and copper nanoparticles have been tested against e. coli, s. aureus and e. faecalis [ , ] . cu-amino acids chelate proved ten times more effective than cu nanoparticles and cu-edta chelate. currently, copper has been recognized as the best antimicrobial metal by the american environmental protection agency (epa). epa has even approved the registrations of copper alloys as "antimicrobial materials with public health benefits" allowing manufacturers to make legal claims to the public health benefits of products made of registered alloys. the agency has also approved a long list of antimicrobial copper products made from alloys, like bedrails, handrails, over-bed tables, sinks, faucets, doorknobs, toilet hardware, computer keyboards, health club equipment, and shopping cart handles [ ] . in addition to the copper alloys, other hard surfaces embedded with copper oxide microparticles have been approved by the epa [ ] with demonstrated potent antimicrobial efficacy [ ] . they are indeed in use in several hospitals in the usa [ ] . these polymeric-based copper-oxide-impregnated surfaces are easier to incorporate and more adapt than copper alloys and thus their use in the context of dental clinics is very relevant. many functional nanoparticles with remarkable antiviral ability, such as quantum dots, gold and silver nanoparticles, nanoclusters, carbon dots, graphene oxide, silicon materials, polymers and dendrimers have been studied and the possible antiviral mechanism of action described. however, biocompatibility and biosafety issues in the use of nanoparticles are far from being solved [ ] . warnes et al. [ , ] investigated the efficacy of a range of copper alloys to inactivate human coronavirus e (hucov- e). a rapid inactivation of human coronavirus occurs on brass and copper nickel surfaces at room temperature ( • c): copper ion release and generation of reactive oxygen species (ros) are involved in the inactivation of hucov- e on copper and copper alloy surfaces. the final effect is the fragmentation of the viral genome, ensuring that inactivation is irreversible. figure gives a representation of the virus inactivation drawn from the knowledge of the effects of copper nanoparticles [ ] and copper alloys [ ] on the integrity of the viral capsid and viral rna. exposure to copper surfaces results in morphological changes to human coronavirus particles visible in transmission electron microscopy (tem) [ ] . colin et al. [ ] investigated the efficacy of copper alloy touch surfaces in healthcare facilities to prevent bacterial spreading and environmental bacterial contamination in healthcare facilities from inanimate surfaces to patients or healthcare workers. dentists and other health workers, although working with gloves, can have a high risk of contamination following a direct contact with mouth of infected patient. hygiene is for sure the first strategy to prevent healthcare-associated infections, but the professionals can touch many devices and surfaces in the operative room and viruses too like sars-cov- can spread through touch of surfaces. indeed, by multiple surface-skin transfer, a contamination on a primary surface can somehow very quickly spread across an entire dental unit, or dental furniture, or medical clinic, or surgical service. a specific strategy to protect these surfaces may represent a way to limit the contamination for users and for patients, also considering that most of the microorganisms can survive on these surfaces for an extremely long time. copper shields or sheaths or coatings applied on critical frequently touched surfaces could be a solution. in fact, rapid inactivation, irreversible destruction of viral rna, and massive structural damage were observed in coronavirus exposed to copper and copper alloy surfaces. incorporation of copper alloy surfaces in conjunction with effective cleaning regimens and good clinical practice could help to control transmission of respiratory coronaviruses, including mers and sars [ , ] . copper alloy is well-known for its antimicrobial properties and several studies have highlighted that copper alloy can break down the microbial load on the surfaces it coats and of counteracting infection in intensive care units. recently, an effective antiviral activity by copper alloy against rna viruses has been demonstrated. copper acts against the virus with a dual harmful mechanism, on one side by degrading the viral rna and on the other destructing the viral capsid, which would hinder the entry of copper [ ] . the suggestion that emerges from these studies, which deserves to be emphasized, is that copper alloy surfaces could express antiviral activity against other rna viruses that are believed to be able to spread from touch surfaces, including coronavirus (although not there are still published data for them) and the ebola virus [ ] . colin et al. [ ] investigated the efficacy of copper alloy touch surfaces in healthcare facilities to prevent bacterial spreading and environmental bacterial contamination in healthcare facilities from inanimate surfaces to patients or healthcare workers. dentists and other health workers, although working with gloves, can have a high risk of contamination following a direct contact with mouth of infected patient. hygiene is for sure the first strategy to prevent healthcare-associated infections, but the professionals can touch many devices and surfaces in the operative room and viruses too like sars-cov- can spread through touch of surfaces. indeed, by multiple surface-skin transfer, a contamination on a primary surface can somehow very quickly spread across an entire dental unit, or dental furniture, or medical clinic, or surgical service. a specific strategy to protect these surfaces may represent a way to limit the contamination for users and for patients, also considering that most of the microorganisms can survive on these surfaces for an extremely long time. copper shields or sheaths or coatings applied on critical frequently touched surfaces could be a solution. in fact, rapid inactivation, irreversible destruction of viral rna, and massive structural damage were observed in coronavirus exposed to copper and copper alloy surfaces. incorporation of copper alloy surfaces in conjunction with effective cleaning regimens and good clinical practice could help to control transmission of respiratory coronaviruses, including mers and sars [ , ] . copper alloy is well-known for its antimicrobial properties and several studies have highlighted that copper alloy can break down the microbial load on the surfaces it coats and counteract infection in intensive care units. recently, an effective antiviral activity by copper alloy against rna viruses has been demonstrated. copper acts against the virus with a dual harmful mechanism, on one side by degrading the viral rna and on the other destructing the viral capsid, which would hinder the entry of copper [ ] . the suggestion that emerges from these studies, which deserves to be emphasized, is that copper alloy surfaces could express antiviral activity against other rna viruses that are believed to be able to spread from touch surfaces, including coronavirus (although not there are still published data for them) and the ebola virus [ ] . colin et al. [ ] also suggest the use of copper for door handles. and indeed, even the usual gesture of closing or opening a door by touching its handle causes microbial contamination, as well as a sharing of microbes with those who will touch the handle after us, and thus ultimately an indirect transfer of microbes from one individual to another. copper handles can reduce the average bacterial burden up to % in respect to the traditional stainless-steel handles commonly utilized in hospitals, dental settings, schools, and other public places. inactivation of bacteria and viruses by copper relies on cu + and cu ++ hydroxyl radicals generated by an electrochemical process during aqueous corrosion of metallic copper [ , ] . in the case of copper alloys, copper ions in solution are produced from copper in the sequestered oxide layer formed over the surface of the alloy or released but chelated with some molecular species in solution [ ] . details of metallic corrosion processes producing biocide copper and the antimicrobial and antivirus effects of copper alloys are discussed by john r. scully [ ] . another interesting aspect of copper application is through "nanoparticles" (nps): they have been defined by the encyclopedia of pharmaceutical technology as solid colloidal particles ranging in size from one to nm (one micron) [ ] . nps are endowed with multifaceted chemical properties and biological activities and promise many possible uses in the biomedical field. the evolution of metallic nps has conducted to the development of a new family of antimicrobial materials. highly ionic metallic nps are of interest due to their extremely high surface areas and numerous reactive surface sites with unusual crystal morphologies. cu-based nps can be produced using different techniques (chemical treatment, thermal treatment, electrochemical synthesis, photochemical methods, sonochemical techniques). a new plastic antimicrobial agent including polypropylene with embedded cu metal or copper oxide nps was examined by delgado et al. [ ] . so, hopefully soon, cu-nps based materials could probably be used as effective antimicrobial systems in pharmaceutical, biomedical, and medical fields to combat pathogenic microorganisms. in dentistry, the use of copper has been proposed as implant surface treatment [ ] , as endodontic irrigating solution [ ] , and as overlay for handles in dental offices [ ] . moreover, nps lend themselves to being used in sterilizing medical devices, as well as in preparing chemical disinfectants or coatings. however, more in-depth studies will be necessary to succeed in minimizing the toxicity of the metal and metal oxide nanoparticles and to apply them as safe antimicrobials in the medical field. although they are effective against virulent microbes and have resistance to heat, their toxicity at higher concentrations is a limit. toxicity-minimized nps might be promising for controlling and treating different infectious diseases in the future [ , ] . the high care in cleaning and disinfecting all the devices, the furniture and the environment in medical, surgical and dental field, for daily practice, represents the key for guaranteeing the protection of professionals and patients. the new human coronavirus sars-cov- emerged in wuhan, china, in late is now causing a pandemic. so, its diffusion may affect medical practice, due to its aerosol production and surface stability. operators must protect themselves and must prevent cross-infections, minimizing the risk of new infections and paying attention to decontamination of dental offices and surgical services [ , ] . dental professionals are exposed to high risk of contagion through patient's saliva, blood, and aerosol/droplet during dental practice [ ] [ ] [ ] [ ] . the use of handpieces under irrigation generate aerosol particles of saliva, blood, and secretions and facilitates the contamination of the environment and instruments, dental apparatuses, and surfaces [ ] [ ] [ ] [ ] [ ] [ ] . sars-cov- can occur through inhalation of aerosol/droplets from infected individuals or by direct contact with mucous membranes, oral fluids, and contaminated instruments and surfaces [ , ] . orthopedic surgeons too that operate in traumatology services are to be included among the professionals at high risk of generating aerosols. and indeed, orthopedists use surgical procedures that cause the spread of fluids, tissues and debris of biological materials, similarly to what happens for dental professionals in their practice and recommendations to protect the orthopedic and trauma surgeons and the personnel of the musculoskeletal health facilities have been recently published [ , ] . the available literature and actual clinical experience are still not able to suggest which protections to use when treating patients during covid- pandemic in dental practice and in medical clinic, so each kind of device or shield or surface available has to be considered and evaluated [ ] [ ] [ ] [ ] . given this context, the use of copper surfaces brings a new perspective for constant and inherent disinfection. these copper surfaces already play an effective role in thwarting the viral spread, even in non-optimal use. according to the reports, metal and metal oxide nanoparticles represent a group of materials which were investigated in respect to their antimicrobial effects. in the present review, we focused on the recent research works concerning antimicrobial activity of metal and metal oxide nanoparticles together with their mechanism of action. reviewed literature indicated that the particle size was the essential parameter which determined the antimicrobial effectiveness of the metal nanoparticles. it was therefore proposed that in addition to the existing infection control armamentarium, the incorporation of copper into surfaces could effectively reduce environmental contamination, thus rendering healthcare facility surfaces safer in dentistry as well as in other surgical settings: anyhow, the use of antimicrobial copper is not a substitute for good hygienic practices. other experimental evaluations on different surfaces or even on biological surfaces are certainly needed, to better understand the times of persistence of this virus and promote adequate standards. indirect virus transmission in cluster sars-cov- rna detection of hospital isolation wards hygiene monitoring during the coronavirus disease outbreak in a chinese hospital de contagione et contagiosis morbis et eorum curatione ueber die nachsten aufgaben zur erforschung der verbreiturigs weise der phtisie photopolarimetrical properties of coronavirus model particles: spike proteins number influence aerosol and surface stability of sars-cov- as compared with sars-cov- turbulent gas clouds and respiratory pathogen emissions potential implications for reducing transmission of covid- saliva: potential diagnostic value and transmission of -ncov rapid detection of sars-cov- in saliva: can an endodontist take the lead in point-of-care covid- testing? transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination covid- ): implications for clinical dental care covid- and dentistry: prevention in dental practice, a literature review overview of transnational recommendations for covid- transmission control in dental care settings characterization of aerosols produced during surgical procedures in hospitals possible aerosol transmission of covid- and special precautions in dentistry. version coronavirus disease : coronaviruses and blood safety covid- and the risk to health care workers: a case report disease among orthopaedic surgeons in wuhan, people's republic of china surgical considerations in patients with covid- : what orthopaedic surgeons should know personal protective equipment: current best practices for orthopedic teams persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation the effects of temperature and relative humidity on the viability of the sars coronavirus face touching: a frequent habit that has implications for hand hygiene a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions covid- surface persistence: a recent data summary and its importance for medical and dental settings world health organization. decontamination and reprocessing of medical devices for health-care facilities centers for disease control and prevention (cdc). cleaning and disinfection for households interim recommendations for u.s. households with suspected or confirmed coronavirus disease protection and disinfection policies against sars-cov- (covid- ) survival of human coronaviruses e and oc in suspension and after drying on surfaces: a possible source of hospital-acquired infections severe acute respiratory syndrome coronavirus- (sars-cov- ): an update ozone disinfection of chlorine-resistant bacteria in drinking water proxies to monitor the inactivation of viruses by ozone in surface water and wastewater effluent ozonized gel against four candida species: a pilot study and clinical perspectives clinical and virological data of the first cases of covid- in europe: a case series understanding covid- : what does viral rna load really mean? the sword and the knife: a comparison of ancient egyptian treatment of sword injuries and present day knife trauma silver nanoparticles as a new generation of antimicrobials metal nanoparticles: the protective nanoshield against virus infection in vitro adhesion of staphylococcus epidermidis on heparin-surface-modified intraocular lenses antimicrobial activity of metals: mechanisms, molecular targets and applications antimicrobial properties of copper nanoparticles and amino acid chelated copper nanoparticles produced by using a soya extract metallic copper as an antimicrobial surface office of pesticide programs potent bactericidal efficacy of copper oxide impregnated non-porous solid surfaces reduced health care-associated infections in an acute care community hospital using a combination of self-disinfecting copper-impregnated composite hard surfaces and linens an overview of functional nanoparticles as novel emerging antiviral therapeutic agents human coronavirus e remains infectious on common touch surface materials inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity copper alloy touch surfaces in healthcare facilities: an effective solution to virus inactivation by copper or iron ions alone and in the presence of peroxide polypropylene with embedded copper metal or copper oxide nanoparticles as a novel plastic antimicrobial agent novel antiviral characteristics of nanosized copper(i) iodide particles showing inactivation activity against pandemic h n influenza virus the covid- pandemic, part : can antimicrobial copper-based alloys help suppress infectious transmission of viruses originating from human contact with high-touch surfaces? corrosion evaluation of antibacterial properties of copper nanoparticles surface coating on titanium dental implant copper nanoparticles as potential antimicrobial agent in disinfecting root canals. a systematic review metal-based nanoparticles as antimicrobial agents: an overview antimicrobial activity of the metals and metal oxide nanoparticles prevention and control of novel coronavirus infection in department of stomatology covid- ): emerging and future challenges for dental and oral medicine transmission routes of -ncov and controls in dental practice pathogenicity and transmissibility of -ncov-a quick overview and comparison with other emerging viruses covid- transmission in dental practice: brief review of preventive measures in italy covid- : its impact on dental schools in italy, clinical problems in endodontic therapy and general considerations covid- outbreak: an overview on dentistry covid- coronavirus: recommended personal protective equipment for the orthopaedic and trauma surgeon working through the covid- outbreak: rapid review and recommendations for msk and allied health personnel this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors express their greatest thanks to lucio montanaro (irccs rizzoli orthopaedic institute) for his masterly suggestions and for critical reading the article. the contributions by the " per mille" research grants to the rizzoli orthopaedic institute of bologna and by the rfo and pallotti legacy research grants to dimes of the university of bologna are gratefully acknowledged. the authors of this study have no conflict of interest to disclose. key: cord- - oe nj j authors: wang, hua; shen, guoli; yu, ruqin title: aspects of recent development of immunosensors date: - - journal: electrochemical sensors, biosensors and their biomedical applications doi: . /b - - . - sha: doc_id: cord_uid: oe nj j this chapter focuses on the recent developments in the field of immunosensors. immunosensors incorporate the specific immunochemical reaction with the modern transducers including electrochemical (potentiometric, conductometric, capacitative, impedance, amperometric), optical (fluorescence, luminescence, refractive index), and microgravimetric transducers. these immunosensor devices with dramatic improvements in the sensitivity and selectivity possess the abilities to investigate the reaction dynamics of antibody–antigen binding and the potential to revolutionize conventional immunoassay techniques. with the rapid development of immunological reagents and detection equipments, immunosensors have allowed an increasing range of analytes to be identified and quantified and in particular, simple-to-use, inexpensive, and reliable immunosensing systems have been developed for areas such as outpatient monitoring, large screening programs, and remote environmental surveillance. immunosensors with lowered detection limits and increased sensitivities have been developed in various fields, particularly in clinical analysis. a noticeable development trend is also observed in the development of immunosensors combining with other techniques such as flow injection analysis (fia) or capillary electrophoretic (ce) analysis, which complement and improve the present immunoassay methods. belov et al. have proposed a novel immunophenotyping method for leukemias which uses a cluster of differentiation antibody microarray, and a microarray of enzyme-linked immunosorbent assay has been developed for autoimmune diagnosis of systematic rheumatic disease. development of microfluidic immunosensor systems for proteomics and drug discovery have also been reported in recent years where the microfluidic system integrates multiple processes in a single device to improve analytical performance by reducing the reagent consumption and the analysis time, and increasing reliability and sensitivity through automation. immunosensors are affi nity ligand-based biosensing devices that involve the coupling of immunochemical reactions to appropriate transducers. in recent decades, immunosensors have received rapid development and wide applications with various detection formats [ ] [ ] . the general working principle of the immunosensors is based on the fact that the specifi c immunochemical recognition of antibodies (antigens) immobilized on a transducer to antigens (antibodies) in the sample media can produce analytical signals dynamically varying with the concentrations of analytes of interest. here, the highly specifi c reaction between the variable regions of an antibody and the epitopes of an antigen involves different types of bonding, basically hydrophobic and electrostatic interactions, van der waals force, and hydrogen bonding. the antigenantibody reaction is reversible and, owing to the relative weakness of the forces holding the antibody and antigen together, the complex formed would dissociate in dependence upon the reaction environment (e.g. ph and ion strength). the strength of the binding of an antibody to an antigen could be characterized by its affi nity constant (k), which is of the order between ϫ and ϫ l mol Ϫ . the high affi nity and specifi city of this antigen-antibody binding reaction defi nes the unique immunosensor characteristics. the general immunosensor design consists of three individual parts in close contact: a biological recognition element, a physicochemical transducer, and an electronic part. antibodies or antibody derivatives (antigens or haptens) usually serve as the biological recognition elements, which are either integrated within or intimately associated with a physicochemical transducer. this recognition reaction defi nes the high selectivity and sensitivity of the transducer device. the electronic part is used to amplify and digitalize the physicochemical output signal from the transducer devices such as electrochemical (potentiometric, conductometric, capacitative, impedance, amperometric), optical (fl uorescence, luminescence, refractive index), and microgravimetric devices. gizeli and lowe [ ] suggested that an ideal immunosensor design should possess the following specifi cations: the ability to detect and quantify the antigens (antibodies), the capacity to transform the binding event without externally added reagents, the ability to repeat the measurement on the same device, and the capacity to detect the specifi c binding of the antigens (antibodies) in real samples. all of these specifi cations have been the main issues to pursue in developing immunosensors applied in various fi elds. as an important branch of immunoassay techniques, immunosensors possess all essential performance characteristics of immunoassays. they show high selectivity, sensitivity, reversibility and effi cient reagent usage. at the same time, the immunosensors are generally simple to operate, and easy to realize automation, digitization, and miniaturization. they may bypass some inherent problems of traditional analytical methods. therefore, immunosensors have been the subject of expanding interest in the immunochemical studies with enormous potential in clinical diagnosis [ ] [ ] ] , environmental analysis [ ] [ ] , and biological process monitoring [ ] . as for the medical diagnosis of some diseases, herein considerable efforts have been devoted to the development of precise, rapid, sensitive, and selective immunosensors by measurement of the markers or pathogenic microorganisms responsible for the diseases, such as proteins, enzymes, viruses, bacteria, and hormones [ , [ ] [ ] . chagas' disease, an american trypanosomiasis caused by the hemofl agellate trypanosoma cruzi, is an example. an amperometric immunosensor has been recently proposed to probe the presence of antibodies against t. cruzi in blood donors, and to follow the antibody decay during treatment of chagasic patients with the available drugs [ ] . yuan et al. reported a novel potentiometric immunosensor for detection of hepatitis b surface antigen by immobilizing hepatitis b surface antibody on a platinum electrode [ ] . a piezoelectric immunosensor was developed for the on-line detection of severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) in sputum in the gas phase. compared to other sars detection techniques, this method can rapidly test sars-cov at low cost [ ] . moreover, the determination of some tumor markers plays an important role in diagnosing, screening, and determining the prognosis of a cancer disease. such tumor markers to be detected are often found in abnormally high amounts in the blood, urine, or tissue of patients with certain types of cancers. the examples include carcinoembryonic antigen (cea), carbohydrate antigen - (ca - ), carcinoma antigen (ca ), alpha-fetoprotein (afp), prostate specifi c antigen (psa), ca - and human chorionic gonadotropin (hcg) [ ] [ ] [ ] . wilson proposed an electrochemical immunosensor for the simultaneous detection of two tumor markers of cea and afp [ ] . an increasing number of immunosensors have been utilized to analyze a series of biochemical targets for diagnosing infectious diseases, although there are still problems concerning the assay of analytes in real sample matrixes [ ] . since immunosensors usually measure the signals resulting from the specifi c immunoreactions between the analytes and the antibodies or antigens immobilized, it is clear that the immobilization procedures of the antibodies (antigens) on the surfaces of base transducers should play an important role in the construction of immunosensors. numerous immobilization procedures have been employed for diverse immunosensors, such as electrostatic adsorption, entrapment, cross-linking, and covalent bonding procedures. they may be appropriately divided into two kinds of non-covalent interactionbased and covalent interaction-based immobilization procedures. this type of immobilization of immunoactive entities is based on the non-covalent interactions between the antibody or antigen molecules and the transducer substrates, and usually refers to hydrophobic interaction, electrostatic interaction, van der waals force, and hydrogen bonding. one notices that besides pure physical adsorption, some weak chemical interactions are also involved here. the non-covalent interactions may vary from the different substrates of transducers. for a non-polarity sensing substrate, the antibody or antigen molecules can be adsorbed through the hydrophobic interaction and van der waals force. wenmeyer et al. attached anti-digoxin antibodies at the surfaces of polystyrene microtubes by direct adsorption interaction, achieving the determination of digoxin with a detection limit of pg ml Ϫ [ ] . while for the charged substrates, the non-covalent interactions are mainly associated with the electrostatic interactions. the most typical layer-by-layer technique of self-assembly has attracted considerable attention in biomolecular immobilizations [ ] [ ] [ ] [ ] [ ] . caruso and coworkers assembled polyallylamine hydrochloride/polystyrene sulfonate layers on the self-assembled monolayer of mercaptopropionic acid, providing a charged polyelectrolyte layer on the transducer surface [ ] . the biomolecules of avidin and anti-immunoglobulin (igg) antibodies were then well immobilized through electrostatic interaction. a novel biosensing interfacial design strategy has been developed for immobilizing the antibodies onto the positively charged surfaces of plasma-polymerized fi lm (ppf) via electrostatic interaction through a polyelectrolyte-mediated layer [ ] . the immunosensors so prepared exhibited excellent response sensitivity due to the low disturbance of the electrostatic adsorption immobilization to the activity of antibody. the ppf surfaces can be regenerated repetitively by changing the ph of the buffer solutions to remove the polyelectrolyte-mediated layer. moreover, antibodies or antigens may be physically entrapped into the fi lms of organic high polymers or inorganic materials (e.g. sol-gel, graphite powder) with stereo meshy structures. of these entrapment immobilizations, the sol-gel-based immobilizations have recently attracted much attention due to their ability to encapsulate biomolecules at low temperature, as well as the physical tenability, optical transparency, mechanical rigidity, and low chemical reactivity [ ] [ ] . most applications of the sol-gel-based immobilizations have been primarily directed to the optical immunosensors [ , , ] and the electrochemical immunosensors [ , [ ] [ ] [ ] [ ] . martínez-fàbregas et al. proposed a polishable entrapment immobilization based on rigid biocomposite materials consisting of graphite powder, rabbit igg, and methacrylate (or epoxy resins) [ ] . the surface of the immunosensor can be regenerated by simply polishing to obtain a fresh layer of immunocomposite ready for next immunoassay. the aforementioned physical interaction-based immobilization procedures are demonstrated to be operated simply and rapidly. however, their immobilization stability might be infl uenced by the bulk metal surfaces and environmental factors such as temperature, ph, and ion strength of solution, resulting in a loss of bioactivity or denaturation of the proteins. moreover, the gradual elution of proteins physically adsorbed may occur during the analytical performances, which may in turn bring about some problems associated with loss of detection sensitivity and low reproducibility of the sensors. in recent years, nanomaterials (e.g. noble metals, magnetic oxides, and carbon nanoparticles or nanotubes) with unique physical and chemical properties have been successfully applied to modify immunosensing interfaces to achieve greatly improved immobilization of antibodies or antigens [ ] [ ] [ ] . some pioneering works have shown that the assembly of the gold nanoparticle layer on an electrode would lead to substantially increased electrode surface areas available for direct adsorption of biological entities, thus offering the possibility of the great enhancement of analytical sensitivity [ , [ ] [ ] [ ] [ ] [ ] . for example, a new immobilization procedure of antibodies for capacitive immunosensor has been recently proposed using thiol compound and gold nanoparticles [ ] . it was here demonstrated that the proposed immobilization procedure could retain the high biological activity of immobilized entities and provide favorable sensing performances. moreover, magnetic nanoparticles as special carriers for immobilizing biomolecules have also been the current hot subject of a series of investigations for the construction of different immunosensors [ ] [ ] [ ] . the easy localization of magnetic beads was used to generate a sensing layer at the surface of a piezoelectric sensor, where the magnetic beads bearing antibodies were immobilized with the help of a permanent magnet at the surface of the crystal [ ] . more recently, an amperometric immunosensor has been developed by employing a kind of core-shell magnetic nanoparticle of (cdfe o øsio ) to immobilize antibody onto the electrode surface with a magnet fi eld [ ] . additionally, magnetic beads may be applied to label or attach antibodies (antigens) for the magneto-detection of the immune complex based on the perturbation of a magnetic fi eld, which could be quantifi ed using a suitable electronic device [ ] . compared with the conventional immobilization methods, these magnetism-driven immobilization procedures may have some merits such as simple manipulation, easy biomolecule modifi cation, low cost, and repeatable regeneration. the covalent interaction procedures, typically the cross-linking methods, are the most popular immobilization manipulation for fabricating various immunosensors. due to the lack of an amount of active covalently binding sites at some transducer substrates (e.g. metals, semiconductor, or optical fi bers), the precoatings of the base transducers with thin fi lms are generally necessary for covalently binding the antibodies or antigens by using the functional reagents such as glutaraldehyde, carbodiimide succinimide ester, maleinimide, and periodate. many traditional coating materials, such as polyethyleneimine [ ] [ ] , (γ-aminopropyl) trimethoxysilane [ ] [ ] , and copolymer of hydroxyethyl-and methyl-methacrylate [ ] , are often used as the mediate layers for immunoactive molecule immobilization. in recent decades, however, some new coating or functionalized fi lm techniques (materials) have been introduced into this fi eld. self-assembled monolayers (sams) offer promising functionalized fi lms for the immobilization of antibodies or antigens [ ] [ ] . since sulfur donor atoms strongly coordinate on noble metal substrates (e.g. au, ag, and pt), various sulfur-containing molecules such as disulfi des (r-ss-r), sulfi des (r-s-r), and thiols can form various functionalized sams of highly organized and compact construction. the applications of the sam technique in the immobilization of biomolecules have been widely documented [ ] [ ] [ ] . knoll and coworkers presented a versatile biotin-functionalized sam, on which the biotinylated antibodies can be readily immobilized through an avidin mediator [ ] . mixed sams composed of long-chain thiols with carboxylic and hydroxyl groups are also used to attain a specifi c and stable affi nity interface of immunosensors [ ] [ ] . langmuir-blodgett (lb) fi lms are other useful alternatives to traditional mediate layers [ ] [ ] [ ] . lb fi lms, which are usually prepared by transferring a monolayer on a solid substrate, have great potential in helping to control the orientation and surface density of the antibodies. hirata et al. [ ] successfully prepared the lipid-tagged antibody/phospholipid monolayers with high immobilization properties using the lb technique. vikholm et al. demonstrated the incorporation of lipid-tagged single-chain antibodies into lipid monolayers obtaining desirable retention of antibody activities [ ] [ ] . moreover, recent years witness a newly emerged ultra-thin polymer fi lm, plasma-polymerized fi lm (ppf), which is reported with successful applications in various immunosensor designs [ , [ ] [ ] [ ] . ppfs, which are generally prepared by using glow discharge or plasma of organic vapors, are extremely thin, homogeneous, mechanically and chemically stable, with strong adhesion to the substrates. karube's group fi rst reported the application of ppf to qcm immunosensors [ ] . they verifi ed that the resultant sensors were more reproducible from batch to batch, and might have lower noise and higher sensitivity than sensors using some conventional organic coatings (e.g. polyethylenimine). this kind of functionalized fi lm may offer promising alternatives in interfacial design of immunosensors of various transducers. in recent years, various nanomaterials are found to be skillfully applied in combination with the covalent interaction-based immobilization procedures for immunosensors. carbon nanotubes (cnts), for example, have been recognized as the quintessential nano-sized materials since their discovery in [ ] . these nanotubes are now chemically functionalized for the immobilization of biological entities for different biosensors, i.e. electrochemical devices [ ] [ ] [ ] . pantarotto et al. successfully bound a model pentapeptide and a virus epitope of foot-and-mouth disease onto single-walled cnts [ ] . they found that the cnts-loaded peptide might retain the structural integrity to be well recognized by monoclonal or polyclonal antibodies, indicating the potential applications for diagnostic purposes and vaccine delivery. a silica nanoparticles-based immobilization strategy was also proposed by wang et al. for direct immunosensing determination of toxoplasma gondii-specifi c igg [ ] . herein, the preparation strategy could allow for antigens covalently bound with higher loading amount and better retained immunoactivity compared to the commonly applied crosslinking methods. the aforementioned covalent interaction-based procedures may usually allow for the immunoactive proteins immobilized with high stability and repeatability, and the robust covalent bonds may favor the low noise of detection. nevertheless, problems associated with these covalent bond immobilizations are the decrease of binding capacity of antibodies (antigens) in the immobilization process. such a phenomenon may be presumably contributed to the partial loss of the immunoactive sites and the random orientation of antibody molecules bound on the transducer surfaces. in addition, cross-linking can produce a three-dimensional multilayer matrix that creates diffusion barriers and transport limitations, resulting in long immunoreaction time and low sensitivity [ ] . it is established that the oriented immobilization of antibodies has low infl uence on their immunological activities to a certain degree [ ] [ ] [ ] [ ] [ ] , which antigen binding capacity was demonstrated with a factor of - higher than that of antibodies randomly immobilized [ ] . therefore, special interest has been given to the development of the orientation-controlled immobilization techniques for antibodies, i.e. mostly through proteins a or g to specifi cally bind the antibody fc fragment, or by directly binding the chemical groups at antibody fc region [ , [ ] [ ] [ ] . lee et al. utilized the self-assembled layer of thiol group-modifi ed protein a for the oriented immobilization of antibodies [ ] . an increased binding capacity was further observed. as another illustrative instance, a protein a-based orientationcontrolled immobilization strategy for antibodies was proposed for the fabrication of a qcm immunosensor using nanometer-sized gold particles and amine-terminated ppf [ ] . moreover, in recent years, there has emerged another oriented immobilization methodology for antibodies through their native thiol (-sh) groups, which were liberated after the splitting of the intact igg into two antibody fragments [ ] [ ] . karyakin et al. reported a site-oriented immobilization strategy of antibodies on the gold electrode surfaces by use of native sulfi de groups of igg fragments obtained by reduction of intact igg [ ] . they found that antibodies immobilized by this procedure showed an antigen binding capacity - times higher than that of non-specifi cally adsorbed intact ones traditionally used. in general, an ideal immobilization should have the following characteristics: (i) a suffi cient loading amount of active antigens or antibodies at the transducer surface; (ii) the immobilized antigens or antibodies staying stable during the measurement process; (iii) the immobilization process having no infl uence to the sensing behavior of the transducer; and (v) the ability of sensor regeneration. an effective dissociation of the antigen and regeneration of antibody, i.e. by using gly-hcl buffer (ph . ), for cost effectiveness is of practical interest in real immunosensor applications [ ] . there are mainly three types of transducers used in immunosensors: electrochemical, optical, and microgravimetric transducers. the immunosensors may operate either as direct immunosensors or as indirect ones. for direct immunosensors, the transducers directly detect the physical or chemical effects resulting from the immunocomplex formation at the interfaces, with no additional labels used. the direct immunosensors detect the analytes in real time. for indirect immunosensors, one or multiple labeled bio-reagents are commonly used during the detection processes, and the transducers should detect the signals from the labels. these indirect detections used to need several washing and separation steps and are sometimes called immunoassays. compared with the direct immunosensors, the indirect immunosensors may have higher sensitivity and better ability to defend interference from non-specifi c adsorption. the majority of known immunosensor devices belong to the group of electrochemical immunosensors. electrochemical immunosensors may possess several advantages, for example high sensitivity, low cost, and portable design. the principle of their operation is based on the electrochemical detection of the labeled immunoagents or markers such as enzymes, metal ions, or other electroactive compounds, thus providing an opportunity to analyze complex multicomponent mixtures for diagnosing diseases or monitoring the status of patients [ ] . the kinds of detection transducer for electrochemical immunosensors can be mainly subdivided into potentiometric, conductometric, capacitive, impeditive, and amperometric (metal and graphite electrodes) devices. potentiometric transducers now belong to the most mature transducers with numerous commercial products. for potentiometric transducers, a local equilibrium is established at the transducer interface at near-zero current fl ow, where the change in electrode or membrane potential is logarithmically proportional to the specifi c ion activity. the relationship of logarithmical proportionality constitutes the fundamental principle of all potentiometric transducers such as the ion-selective electrodes (ise). the groups of biosensors are characterized as simple in preparation, robust in operation, and moderately selective in analytical performance [ ] [ ] [ ] [ ] [ ] [ ] . janata fi rst proposed a potentiometric transducer for immunosensing and named it "immuno-electrode" [ ] , using the immuno-electrode to detect concanavalin a through covalent attachment to the surface of a pvc membrane deposited on a platinum electrode. the incorporation of ises, ph electrodes or gas-sensing electrodes into potentiometric immunosensors to improve their assay sensitivity has been extensively investigated by rechnitz and coworkers, i.e. for immunochemical measurements of digoxin and human igg [ ] [ ] . d'orazlo et al. reported the indirect measurements of immunoagents using ion-selective electrodes [ ] . a potentiometric immunosensor based on a molecularly imprinted polymer was prepared as a detecting element in micro total analysis systems with the intent of providing easy clinical analysis [ ] . moreover, the ion-selective fi eld-effect transistor (isfet) as a semiconductor device is generally constructed by substituting an ion-sensing membrane for the metal gate of a fi eld-effect transistor (fet) [ ] . the isfet is able to respond to the surface potential change resulting from the specifi c immunochemical reaction between the immobilized antibodies and the free antigens. the ph-sensitive isfets, as the most widely used sensor of this type, are fabricated with a large range of possible insulators (i.e. sio , si n , and al o ) and enzyme labels (i.e. urease, peroxidase, and glucose oxidase) [ ] [ ] . nevertheless, only a few examples of isfet-based immunosensors could be found in the literature [ ] [ ] [ ] . for example, zayats et al. report the impedance measurements on an isfet device that can be used to detect antigen-antibody interactions on the gate surface [ ] . in the meantime, they performed complementary surface plasmon resonance (spr; see above) experiments to illustrate that the isfet impedance measurements and the spr reveal comparable sensitivities. conductometric transducers, as the oldest electrochemical devices, seem not to enjoy wide applications due to their poor selectivity. for example, yagiuda et al. proposed a conductometric immunosensor for the determination of methamphetamine (ma) in urine [ ] . the decrease in the conductivity between a pair of platinum electrodes might result from the direct attachment of ma onto the anti-ma antibodies immobilized on the electrode surface. the system was claimed to be a useful detection technique of ma in comparison with a gas chromatography-mass spectrometry method. capacitance and impedance transducers with high sensitivity are widely employed for various immunosensing assays [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the capacitance sensors are essentially based on the principle that the electrolyte capacitance of an electrode depends on the thickness and dielectric behavior of the dielectric layer on the electrode surface and the solid/solution interface. dijksma et al. designed an immunosensor for the direct detection of interferon-γ at the attomolar level by using the ac impedance approach [ ] . the immobilization processes of antibodies (antigens) play an important role in these immunosensors, and the sensitivity of a capacitive immunosensor increases with the decreasing thickness of the insulating layer. shen and coworkers fabricated a heterostructure of au/o-aminobenzenethiol layer/covalent-coupling antibody/electrode for the direct detection of the antibody-antigen interaction by capacitance measurements [ ] . a capacitive immunoassay based on antibody-embedded ultra-thin alumina sol-gel fi lms (∼ to nm) was reported and used for direct determination of antigens with a detection limit as low as ϳ ng ml Ϫ [ ] . fernandez-sanchez et al. reported a successful integration of the lateral fl ow immunoassay format and impedance detection for prostate-specifi c antigen of tumor marker, where the electrochemical transducer was coated with a ph-sensitive polymer layer [ ] . although capacitance and impedance immunosensors can directly be utilized to investigate the antibody-antigen interaction without the need of other reagents and a separation step, their analytical sensitivity is limited in clinical applications [ ] . in order to amplify the capacitance or impedance response to immunoreaction for the sensitive detection of various clinical markers, different labels have been used including enzymes, fl uorophores, and metal chelates [ ] [ ] . ruan et al. developed an immunosensor based on enzyme-stimulated precipitation for the detection of escherichia coli o :h using an electrochemical impedance spectroscopy [ ] . another illustrative example was the sensitized immunosensor proposed by chen et al. [ ] . in their study, a receptor protein was directly adsorbed on a porous nanostructure gold fi lm to perform a sandwich immunoreaction with the precipitation of insoluble product on the electrode. the impedance signals so amplifi ed showed good linearity with the content of igg in the range . - ng ml Ϫ with a detection limit of . ng ml Ϫ . a new strategy of signal amplifi cation was also introduced for highly sensitive impedance measurements using biotin-labeled protein-streptavidin network complex [ ] . amperometric immunosensors, as the most popular immunosensing formats, are based on the measurement of the currents resulting from the electrochemical oxidation or reduction of electroactive species at a certain constant voltage. this kind of immunosensor usually uses a complex three-electrode measuring system consisting of a working electrode (e.g. gold, glassy carbon, or carbon paste), a reference electrode (e.g. ag/agcl), and a conducting auxiliary electrode (e.g. platinum). since most antibodies and antigens are not electrochemically active, there are only a few applications available for direct amperometric sensing. therefore, most amperometric immunosensors are indirect ones which can detect mainly the redox currents associated with electroactive or catalytic labels [ - , , - ] . aizawa et al. fi rst developed an amperometric immunosensor for the determination of human chorionic gonadotropin using an amperometric oxygen electrode [ ] . among the labels used, enzymes are the most popular ones in different types of immunoassays, such as horseradish peroxidase (hrp) or glucose oxidase. an immunosensor was designed for determining isopentenyl adenosine based on the electro-polymerization of polypyrrole and poly(m-phenylenediamine) entrapped with hrp on the glassy carbon electrode [ ] . a design strategy of reagentless immunosensor was reported for the detection of carcinoma antigen- antibodies by direct hrp-labeled electrochemistry [ ] . due to the high sensitivity inherent in these transducers by enzymatic catalysis, amperometric immunosensors can obtain a much higher sensitivity than the classical elisa. for the immunosensors used in clinical applications, their surfaces should be capable of renewal. yu et al. developed a renewable amperometric immunosensor for the determination of schistosoma japonium (sj) antibody by using the paraffi n graphite-sj antigen biocomposite paste electrodes which might be regenerated by polishing the surface [ ] . ionescu and his collaborators have developed two similar amperometric immunosensors for cholera antitoxin immunoglobulins, where the cholera toxin biorecognition entities were bound to a biotinylated polypyrrole fi lm or pyrrole-biotin and lactitobionamide electropolymerized copolymer [ ] [ ] . moreover, nano-sized particles or sol-gel matrixes have also been increasingly employed in the design of amperometric immunosensors with enhanced analytical performance [ , [ ] [ ] . for example, an electrochemical immunosensor has been developed for probing complement iii (c ) by use of nanogold particle monolayer as the sensing interface [ ] . with the coupling of sol-gel and screen-printing technologies, a sensitive thick fi lm immunosensor was fabricated by dispersion of rabbit immunoglobulin g, graphite powder, and a binder in the solgel solution [ ] . a new hrp-labeled amperometric immunosensor for determination of chorionic gonadotrophin in human serum was constructed by immobilizing hcg within titania sol-gel on a glassy carbon electrode [ ] . anodic stripping voltammetry as an electrochemical assay technique has been well adopted for sensitive measurements of heavy metals such as copper and silver, which may also offer an attractive way of sensitive immunosensor development [ ] [ ] . an immunosensor was designed by coupling immunoassay with the square wave anodic stripping voltammetry technique involving copper ion-labeled antigen in the competitive immunoreaction [ ] . this immunosensor might allow rapid, accurate, and inexpensive detection of gibberellin acid with a concentration as low as µg ml Ϫ . chu et al. designed a silverenhanced colloidal gold metalloimmunoassay for the determination of schistosoma japonicum antibody (sjab) in rabbit serum [ ] . in their study, after the immunoreaction of sjab target with immobilized sj antigens, colloidal gold-labeled secondary antibody was introduced to favor the silver enhancement process. an acidic solution was further used to dissolute silver metal atoms, followed by the sensitive determination of dissolved silver ions using anodic stripping voltammetry. in addition, many immunoreaction signal-amplifi ed methods or processes have also been adopted for the development of sensitive amperometric immunosensors. willner's group reported an amplifi ed immunosensing scheme of chronopotentiometry and faradaic impedance spectroscopy by way of a bio-catalyzed precipitation of the insoluble product onto the gold electrode [ ] . they also designed a variation of this scheme with signal amplifi cation by employing liposomes labeled with biotin and hrp as a probe to amplify the sensing of antigen-antibody interactions [ ] . in this case, the electrode with the antigen-antibody complex was exposed to the biotinylated anti-igg antibody, and further the biotin-labeled hrp-liposomes through an avidin bridge to achieve the biocatalyzed precipitation of an insoluble product on the conductive support. since almost all optical phenomena at sensing surfaces (e.g. adsorption, fl uorescence, luminescence, scatter or refractive index, etc.) can be used for biochemical sensing designs, optical immunosensors are considered as one of the most promising alternatives to the traditional immunoassays in clinic diagnosis and environmental analysis. in recent years, there has been an increased trend in the use of optical transduction techniques in immunosensor technologies due to the advantages of applying visible radiation, non-destructive operation mode, and the rapid signal generation and reading [ , [ ] [ ] . the optical immunosensors may be divided into two types of approaches: direct optical immunosensors and indirect immunosensors depending upon the use of labeled signaling molecules. surface plasmon resonance (spr) as a direct and reliable optical transducer is commonly based on the evanescent wave, in which a thin gold layer is generally deposited on a prism serving as an optically rarer medium [ ] [ ] . not requiring additional labels and separation steps, the direct spr immunosensors have been proven to be powerful analytical tools for rapid real-time monitoring the immunological targets. schofi eld and dimmock developed a spr system in combination with the fl ow system for detection of infl uenza virus by use of carboxylated dextran polymer matrix to couple monoclonal antibody of hc [ ] . in order to validate the feasibility of spr immunosensor as a tool for diagnosing type i diabetes, choi et al. modifi ed mixed sams onto the optical substrate achieving the immuno-response detection for monoclonal antibodies of anti-glutamic acid decarboxylase [ ] . moreover, the fatty acidbinding protein assay has an application potential in clinical analysis for diagnosis of myocardial infarction. a direct optical immunosensor based on spr was developed for detecting the human heart-type fatty acid binding protein with a detection limit of ng ml Ϫ [ ] . highly sensitive spr-based immunosensors using self-assembled protein g have also been successfully applied for the detection of microbes such as salmonella typhimurium and legionella pneumophila [ ] [ ] . more importantly, several instrument systems using spr technology have been commercially available, such as the biacore ™ system from pharmacia biosensor, the iasys ™ system from affi nity sensors, and so on. nevertheless, at present, there are still some unsolved problems for these spr devices, such as non-specifi c adsorption and poor analytical sensitivity to analytes of low molecular weight. fluorescence immunosensors, as the total internal refl ection fl uorescence devices, continue to prove themselves as another promising type of sensitive and selective optical immunoassay technique, in which labels are sometimes used [ ] . when the fl uorescence-labeled antibodies or antigens are attached to the transducer surface and enter the evanescent fi eld, the incident light will excite fl uorescent molecules producing a fl uorescent evanescent wave signal to be detected. the optic-fi ber immunosensor system by fl uorescence enhancement or quenching is separation-free, reagentless and applicable to the determination of various proteins by antigen-antibody reactions [ ] [ ] [ ] [ ] [ ] . maragos et al. described the development of a fl uorescence polarizationbased competition immunoassay for fumonisins in maize using fumonisin-specifi c monoclonal antibodies [ ] . a fl uorescence-based immunosensor array for simultaneous determination of multiple clinical analytes was developed by rowe et al. [ ] . in their study, the patterned array of recognition elements was immobilized onto the planar waveguide to "capture" the analytes from the samples to be quantifi ed by means of fl uorescent detector molecules. moreover, in recent years, quantum dots as the most suitable fl uorescence labels have received increasing applications for developing fl uorescence immunosensors due to their high fl uorescence quantum yield and sensitivity to environmental changes upon binding proteins. aoyagi et al. proposed a reagentless, regenerable, and portable optic immunosensor for the ultra-sensitive detection of a model sample of igg based on changes in fl uorescent intensity of fl uorescent quantum dot-labeled protein a [ ] . an antibody for leukemia cell recognition was attached to the luminophore-doped nanoparticle through silica chemistry, yielding an optical microscopy imaging technique for the identifi cation of leukemia cells [ ] . experimental results in this report showed that the new technique using the antibodycoated luminophore nanoparticles could allow leukemia cells to be easily and clearly identifi ed with high effi ciency. chemiluminescence sensors have also been extensively applied in routine clinical analysis as well as biomedical research due to the advantages of no radioactive wastes, simple instrumentation, low detection limit, and wide dynamic range [ , [ ] [ ] [ ] [ ] [ ] . a chemiluminescent immunosensor for carbohydrate antigen - (ca - ) was described by lin et al., with ca - immobilized on the cross-linked chitosan membrane [ ] . the decrease of the immunosensor chemiluminescent signal was proportional to the ca - concentration in the range . - u ml Ϫ , with the detection limit of . u ml Ϫ . pandian et al. developed an automated chemiluminometric immunoassay for the measurement of hcg [ ] . it was demonstrated that the immunoassay might facilitate exploration of hcg utility for down syndrome screening, early pregnancy detection, and differentiation of invasive from non-invasive trophoblastic disease. an optical microbiosensor has been newly designed for the diagnosis of hepatitis c virus (hcv) by using a novel photo-immobilization methodology based on a photo-activable electro-generated polymer fi lm [ ] . herein, the immunosensor using optical fi ber photochemically modifi ed was tested for the determination of anti-e protein antibodies through chemiluminescence reaction. another published study presented the use of electrogenerated luminol chemiluminescence in a homogeneous immunosensor, where digoxin was labeled with luminol through a luminol-bsa-digoxin conjugate [ ] . the prepared chemiluminescence immunosensor in a competitive format was shown allowing for the detection of free digoxin with the concentration as low as . µg l Ϫ . microgravimetric immunosensors may incorporate high sensitivity of piezoelectric response and high specifi city of antibody-antigen immunoreaction. the detection principle of these devices is generally based on adsorbate recognition where the selective binding may cause the changes in mass loading and interfacial properties (i.e. viscoelasticity and surface roughness), which can be recognized by a corresponding shift in the oscillation frequency [ ] [ ] [ ] [ ] . outstanding features of these sensors include low cost, simple usage, high sensitivity, and real-time output. microgravimetric immunosensors have two kinds of sensing formats, gas phase and solution phase sensing. the sensitivity to the mass change in air on the transducer surface is about hz ng Ϫ for a bulk acoustic wave device with mhz of fundamental frequency, which can be described by the sauerbrey equation [ ] . the microgravimetric transducer is thus mainly known as the quartz crystal microbalance (qcm). microgravimetric immunosensors in solution phase sensing were used for the quantifi cation of a number of biological targets [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nüsslein's group reported a qcm assay for bacteria using a cell-selective polymer fi lm, with desirably low detection limit and no need for prior sample treatment [ ] . wang and coworkers initially developed an integrated qcm immunosensor array composed of four kinds of leukemic lineageassociated probes to explore the differentiated leukocyte antigens for immunophenotyping of acute leukemia [ ] . in their study, the probes (crystals) of the array were immobilized separately with fab fragments of leukemic lineage-associated monoclonal antibodies (markers). the developed immunosensor array was demonstrated to be able to rapidly identify normal cells from leukemic blasts and defi ne the leukemic blasts within certain phenotypic groups (lineages). recently, a qcm immunosensor using protein a for antibody immobilization has been described for the detection of salmonella typhimurium in chicken meat sample by simultaneous measurements of the resonant frequency and motional resistance [ ] . based on the modifi cation of mixed sams on gold electrodes for covalently binding antigens, another piezoelectric immunosensor has been recently developed to detect antisperm antibody [ ] . the analytical results for evaluating several clinical specimens by the developed method were found to be in satisfactory agreement with those given by the classical elisa. despite many salient successes, the use of qcm-based immunosensors for trace biological target detection is still challenged by its relatively low intrinsic sensitivity. kim et al. incorporated the immunomagnetic separation with the qcm-based impedance technique achieving a new immunoassay for quantifying salmonella typhimurium with very high sensitivity of cell detection [ ] . herein, antibodies immobilized on magnetic particles were delivered into the sample medium to capture the targets. the resultant immunocomplex was further magnetically collected onto the piezoelectric crystal to be quantifi ed with impedance spectroscopy. through the enzyme-catalyzed formation of a precipitate on the qcm surface, a mass-amplifi ed microgravimetric immunosensor was proposed in combination with a sandwich enzyme-linked immunoassay [ ] . su and his coworkers successfully used qcm for detection of dengue virus [ ] . the authors immobilized two monoclonal antibodies on the crystal that act specifi cally against the dengue virus envelope protein and non-structural protein. the sensitivity reported for the fabricated piezoelectric immuno-chip was -fold greater than the conventional sandwich elisa method. a highly sensitive microgravimetric biosensor has been developed incorporating noble metal particle-amplifi ed sandwiched immunoassay and silver enhancement reaction [ ] . upon the formation of the sandwiched immunocomplex, the sensor surfaces were coated with gold nanoparticles serving as the nucleation sites to catalyze silver ion reduction. the silver metal deposition would result in a large change in frequency responses, achieving approximately two orders of magnitude improvement in human igg quantifi cation. moreover, there is another important type of microgravimetric immunosensor which is based on the immunological agglutination events. the agglutination immunoreaction of antibody-bearing suspensoids such as polymers, microbeads, and naoparticles may induce a corresponding change in the solution parameters (i.e. density and viscosity) and the interfacial properties of the crystal monitored by the qcm device [ ] [ ] [ ] [ ] . in contrast to the common conventional piezoelectric assays, the qcm sensing format offers a unique advantage in that the immobilization of antibodies or antigens on the crystal is not necessary. the kind of qcm-sensing methods are widely recognized to be simple, sensitive, and feasible for detecting relevant targets responsible for many clinical diseases [ ] [ ] [ ] [ ] [ ] [ ] . kurosawa et al. fi rst developed an agglutination-based piezoelectric immunoassay using antibody-bearing latex, termed as the latex piezoelectric immunoassay (lpeia), for detecting c-reactive protein [ ] . recently, it has been demonstrated that the lpeia could be greatly improved by using gold nanoparticles as replacements for latex particles, resulting in a novel agglutination-based piezoelectric immunoassay for directly detecting anti-t. gondii immunoglobulins in infected rabbit sera and bloods [ ] . in recent years, considerable efforts have been devoted to the development of cantilever-based immunosensors with unique enantio-selective antibodies [ ] [ ] . these devices are mainly used for quality and process control, and diagnostic biosensing for medical analysis. they may have fast responses and high sensitivity and are suitable for mass production. lee et al. fabricated a piezoelectric nanomechanical cantilever by a novel electrical measurement. they found that this technique might allow for the labelfree detection of a prostate-specifi c antigen (psa) with a detection sensitivity as low as pg ml Ϫ [ ] . a microfabricated cantilever was utilized to perform the direct (label-free) stereo-selective detection of trace amounts of an important class of chiral analytes, the r-amino acids, based on immunomechanical responses involving nanoscale bending of the cantilever. the major advantages of the microcantilever sensors over more traditional scale transducers such as the qcm reside in the superior sensitivity to minute quantities of analytes and the ability to micro-fabricate compact arrays of cantilevers to facilitate simultaneous and high throughput measurements [ ] . moreover, mass-sensitive magnetoelastic immunosensors are exploited to design extraordinarily versatile and useful sensor platforms [ ] . magnetoelastic sensors are well established and benefi t from mass sensitivity compared to that of a surface acoustic wave (saw) sensor. however, they may cost much less and are much smaller in size than saw devices. ruan et al. proposed a mass-sensitive magnetoelastic immunosensor based on the immobilization of affi nity-purifi ed antibodies on the surface of a micrometer-scale magnetoelastic cantilever achieving the highly sensitive detection of escherichia coli o :h [ ] . in addition, imaging ellipsometry (ie) has also been developed as a new kind of immunosensor, i.e. for the detection of pathogens of yersinia enterocolitica [ ] . as another example, a label-free multi-sensing immunosensor based on the combination of ie and the protein chip was reported to be able to detect multiple analytes simultaneously, and even to monitor multiple biological interaction processes in situ and in real-time conditions [ ] . immunosensors incorporate the specifi c immunochemical reaction with the modern transducers including electrochemical (potentiometric, conductometric, capacitative, impedance, amperometric), optical (fl uorescence, luminescence, refractive index), and microgravimetric transducers, etc. [ ] . these immunosensor devices with dramatic improvements in the sensitivity and selectivity possess the abilities to investigate the reaction dynamics of antibody-antigen binding and the potential to revolutionize conventional immunoassay techniques. with the rapid development of immunological reagents and detection equipments, immunosensors have allowed an increasing range of analytes to be identifi ed and quantifi ed. in particular, simple-to-use, inexpensive and reliable immunosensing systems have been developed to bring immunoassay technology to much more diverse areas, such as outpatient monitoring, large screening programs, and remote environmental surveillance [ ] . however, there are still some unsolved problems associated with the immobilization of immunoactive entities, nonspecifi c adsorption from sample backgrounds (e.g. blood, serum, plasma, urine, and saliva) and practical applications of various transducer devices. the current development of new immunosensors should aim at solving the problems of clinical analysis in medicine and of chemical analysis in the food industry and biotechnology. the development trends of immunosensors are likely to be primarily driven by the requirements of analytical practice on the improvement in sensitivity, selectivity, rapidity, and especially effi ciency of assays (i.e. immunosensing array or microfl uidic system). immunosensors with lowered detection limits and increased sensitivities have been developed in various fi elds, particularly in clinical analysis. for example, the sandwich immunoassay using enzyme-functionalized liposomes as the catalytic label is proposed to obtain the substantially improved assay sensitivity, as validated in the immunoassay of cholera toxin [ ] . meanwhile, as the latest paradigm of development topic, nanomaterials with unique chemical and physical properties should continue to be exploited to offer important possibilities for new immunosensor designs [ ] . a noticeable development trend is also observed in the development of immunosensors combining with other techniques such as fl ow injection analysis (fia) or capillary electrophoretic (ce) analysis, to complement and improve the present immunoassay methods [ ] [ ] . moreover, the miniaturization and automation of immunosensing devices should be another important intention of development to facilitate the signifi cantly shortened analysis time and simplifi ed analytical procedure (i.e. one-step analysis). of note, protein and antibody array technologies are envisaged to have potential for biomedical and diagnostic applications in recent years [ ] [ ] [ ] [ ] [ ] . belov et al. have proposed a novel immunophenotyping method for leukemias using a cluster of differentiation antibody microarray [ ] . a microarray of enzyme-linked immunosorbent assay has been developed for autoimmune diagnosis of systematic rheumatic disease, where the high titers of antinuclear antibodies against various nuclear proteins and nucleoprotein complexes might be detected with high throughput [ ] . at the same time, the screen-printing techniques may also appear to be the most promising technology for immunosensor array to be commercialized on a large scale and widely applied in clinical diagnosis. moreover, there have been increasing reports focusing on the development of microfl uidic immunosensor systems for proteomics and drug discovery in recent years [ ] . microfl uidic system integrating multiple processes in a single device generally seeks to improve analytical performance by reducing the reagent consumption and the analysis time, and increasing reliability and sensitivity through automation. the micro total analysis systems (µtas) are already under development and should represent the future of high throughput immuno-tests [ ] . in addition, with the development of protein engineering technology and molecular biology techniques, more fl exible antibodies suitable for immunosensing applications may be expected. for example, the recombinant or fusion approach is powerful in the production of antibodies and antibody derivates. use of various new generations of antibodies should lead to the enhancement of activity and stability of the immobilized bio-species and even the improvement of the regeneration and sensitivity of the immunosensors. as an inspiringly illustrative instance, aptamers are beginning to emerge as a class of synthetic oligonucleotides or molecules that rival antibodies in both therapeutic and diagnostic applications [ ] [ ] [ ] . baldrich and coworkers fi rst demonstrated the exploitation of an aptamer in an extremely rapid and highly sensitive displacement assay, the displacement enzyme-linked aptamer assay, using enzymelabeled target as a suboptimal displaceable molecule [ ] . to sum up, immunosensors are now becoming one of the most widely used analytical techniques, embracing a vast repertoire of analytes that are detected by a diverse range of transducer devices. the enormous potential of immunosensors in clinical diagnosis, environmental analysis, and biological process monitoring has been widely accepted and increasing efforts have been devoted to these fi elds. in particular, with the continual development of transducer technology, laser technology, nano-sized material technology, and antibody engineering technology, immunosensors based on the application of these technologies should be inevitably powerful tools in increasingly wide analytical areas [ ] . aboul-enein, immunosensors in clinical analysis immunosensor principles and applications to clinical chemistry immunosensors for pesticide determination in natural waters immunochemical techniques for environmental analysis: i. immunosensors single-and dual-fractal analysis of hybridization binding kinetics: biosensor application biosensors: fundamentals and applications immunosensors: technology and opportunities in laboratory medicine immunosensor for the diagnosis of chagas' disease ultrasensitive potentiometric immunosensor based on sa and oca techniques for immobilization of hbsab with colloidal au and polyvinyl butyral as matrixes piezoelectric immunosensor for sars-associated coronavirus in sputum some methodological issues associated with tumour marker development: biostatistical aspects electrochemical and chemiluminescent immunosensors for tumor markers electrochemical immunosensors for the simultaneous detection of two tumor markers competitive heterogeneous enzyme immunoassay for digoxin with electrochemical detection effect of anionic surfactant on interactions between lysozyme layers adsorbed on mica interaction between adsorbed layers of lysozyme studied with the surface force technique quartz crystal microbalance study of dna immobilization and hybridization for nucleic acid sensor development a novel approach of antibody immobilization based on n-butyl amine plasma-polymerized fi lms for immunosensors a novel biosensing interfacial design based on the assembled multilayers of the oppositely charged polyelectrolytes affi nity of antifl uorescein antibodies encapsulated within a transparent sol-gel glass sol-gel-derived thick-fi lm amperometric immunosensors schistosoma japonicum antibody assay by immunosensing with fl uorescence detection using , Ј, , Ј-tetramethylbenzidine as substrate renewable amperometric immunosensor for schistosoma japonium antibody assay an amperometric immunosensor based on an electrochemically pretreated carbon-paraffi n electrode for complement iii (c ) assay a renewable amperometric immunosensor for phytohormone β-indole acetic acid assay amperometric immunosensors based on rigid conducting immunocomposites nanomaterials in analytical chemistry development of an aggregation-based immunoassay for anti-protein a using gold nanoparticles determination of atrazine and alachlor in natural waters by a rapid-magnetic particle-based elisa infl uence of common cross-reactants: deethylatrazine, deisopropylatrazine, simazine and metolachlor colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of the enzyme electrode by electrodeposition morphology-dependent electrochemistry of cytochrome c at au colloid-modifi ed sno electrodes a novel biosensing interfacial design produced by assembling nano-au particles on amine-terminated plasma-polymerized fi lms application of impedance spectroscopy for monitoring colloid au-enhanced antibody immobilization and antibodyantigen reactions a reusable capacitive immunosensor with a novel immobilization procedure based on , -hexanedithiol and nano-au self-assembled layers piezoelectric immunosensor based on magnetic nanoparticles with simple immobilization procedures core-shell magnetic nanoparticles applied for immobilization of antibody on carbon paste electrode and amperometric immunosensing the use of coated paramagnetic particles as a physical label in a magneto-immunoassay development of a piezoelectric immunosensor for the detection of human erythrocytes detection of viruses and bacteria with piezoimmunosensors piezoelectric immunosensor for the detection of candida albicans microbes piezoelectric crystal biosensor system for detection of escherichia coli piezoelectric immunosensor for the detection of immunoglobulin m formation and structure of self-assembled monolayers self-assembled monolayers for biosensors: a tutorial review quartz-crystal microbalance immunosensor for schistsoma-japonicum-infected rabbit serum biotin-functionalized self-assembled monolayers on gold-surface-plasmon optical studies of specifi c recognition reactions a piezoelectric immunoassay based on selfassembled monolayers of cystamine and polystyrene sulfonate for determination of schistosoma japonicum antibodies molecular recognition between genetically engineered streptavidin and surface-bound biotin enhanced performance of an affi nity biosensor interface based on mixed self-assembled monolayers of thiols on gold microscopic characterization of langmuir-blodgett fi lms incorporating biosynthetically lipid-tagged antibody layer formation of a lipid-tagged single-chain antibody and the interaction with antigen incorporation of lipid-tagged single-chain antibodies into lipid monolayers and the interaction with antigen a novel method of immobilizing antibodies on a quartz crystal microbalance using plasma-polymerized fi lms for immunosensors a plasmapolymerized fi lm for surface plasmon resonance immunosensing a reusable piezo-immunosensor with amplifi ed sensitivity for ceruloplasmin based on plasma-polymerized fi lm helical microtubules of graphitic carbon carbon nanotube/tefl on composite electrochemical sensors and biosensors electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides novel immunoassay for toxoplasma gondii-specifi c immunoglobulin g using a silica nanoparticle-based biomolecular immobilization method self-assembled monolayers as the coating in a quartz piezoelectric crystal immunosensor to detect salmonella in aqueous solution fabrication of self-assembled protein a monolayer and its application as an immunosensor a protein a-based orientation-controlled immobilization strategy for antibodies using nanometer-sized gold particles and plasma-polymerized fi lm atomic force spectroscopybased study of antibody pesticide interactions for characterization of immunosensor surface in situ quartz crystal microbalance monitoring of fabЈ-fragment binding to linker lipids in a phosphatidylcholine monolayer matrix: application to immunosensors oriented immobilization of antibodies and its applications in immunoassays and immunosensors use of protein a as an immunological reagent and its application using fl ow injection: a review comparative study of igg binding to proteins g and a: nonequilibrium kinetic and binding constant determination with the acoustic waveguide device site-directed immobilization of glycoproteins on hydrazidecontaining solid supports oriented immobilization of antibodies onto the gold surfaces via their native thiol groups nanogold particle-enhanced oriented adsorption of antibody fragments for immunosensing platforms regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies immunosensors in biology and medicine: analytical capabilities, problems, and prospects potentiometric sensors: aspects of the recent development potentiometric microbiological assay of gentamicin, streptomycin, and neomycin with a carbon dioxide gas-sensing electrode potentiometric enzyme immunoassay for digoxin using polystyrene beads homogeneous potentiometric enzyme immunoassay for human immunoglobulin g ion electrode measurements of complement and antibody levels using marker-loaded sheep red blood cell ghosts potentiometric immunosensor using artifi cial antibody based on molecularly imprinted polymers protein detection with a novel isfet-based zeta potential analyzer ion sensitive fi eld effect transducer-based biosensors improvement of urease based biosensor characteristics using additional layers of charged polymers an isfetbased immunosensor for the detection of β-bungarotoxin a new assay format for electrochemical immunosensors: polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by ph-sensitive fi eld-effect transistor probing antigen-antibody binding processes by impedance measurements on ion-sensitive fi eld-effect transistor devices and complementary surface plasmon resonance analyses: development of cholera toxin sensors development of a conductivity-based immunosensor for sensitive detection of methamphetamine (stimulant drug) in human urine development of an electrochemical immunosensor for direct detection of interferon-γ at the attomolar level capacitive immunosensor for transferrin based on an o-aminobenzenthiol oligomer layer ultrathin alumina sol-gel-derived fi lms: allowing direct detection of the liver fi brosis markers by capacitance measurement disposable noncompetitive immunosensor for free and total prostate-specifi c antigen based on capacitance measurement direct detection of immunospecies by capacitance measurements capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol fi lms on gold electrodes electrochemical sensors based on impedance measurement of enzyme-catalyzed polymer dissolution: theory and applications real-time monitoring of immunochemical interactions with a tantalum capacitance fl ow-through cell direct detection of biomolecules by electrochemical impedance measurements study of immunoglobulin g thin layers obtained by the langmuir-blodgett method: application to immunosensors on-line monitoring of monoclonal antibody production with regenerable fl ow-injection immuno systems a capacitative immunosensor measurement system with a lock-in amplifi er and potentiostatic control by software a label-free electrochemical immunosensor based on gold nanoparticles for detection of paraoxon immunobiosensor chips for detection of escherichia colio :h using electrochemical impedance spectroscopy impedance immunosensor based on receptor protein adsorbed directly on porous gold fi lm amplifi cation of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy enzyme immunosenser: iii. amperometric determination of human cherienic gonadotropin by membrane-bound antibody application of redox enzymes for probing the antigen-antibody association at monolayer interfaces: development of amperometric immunosensor electrodes amperometric immunosensor based on polypyrrole/poly(m-phenylenediamine) multilayer on glassy carbon electrode for cytokinin n -(d -isopentenyl) adenosine assay reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen- amperometric immunosensors based on protein a coupled polyaniline-perfl uorosulfonated ionomer composite electrodes application of photoisomerizable antigenic monolayer electrodes as reversible amperometric immunosensors amperometric immunosensor for direct detection based upon functional lipid vesicles immobilized on nanowell array electrode electronic transduction of photostimulated binding interactions at photoisomerizable monolayer electrodes: novel approaches for optobioelectronic systems and reversible immunosensor devices an indirect perfl uorosulfonated ionomer-coated electrochemical immunosensor for the detection of the protein human chorionic gonadotrophin an amperometric immunosensor based on nafi onmodifi ed electrode for the determination of schistosoma japonicum antibody development of amperometric and microgravimetric immunosensors and reversible immunosensors using antigen and photoisomerizable antigen monolayer electrodes comparison between the performances of amperometric immunosensors for cholera antitoxin based on three enzyme markers construction of amperometric immunosensors based on the electrogeneration of a permeable biotinylated polypyrrole fi lm amperometric immunosensor for probing complement iii (c ) based on immobilizing c antibody to a nano-au monolayer supported by sol-gelderived carbon ceramic electrode reagentless amperometric immunosensor for human chorionic gonadotrophin based on direct electrochemistry of horseradish peroxidase immunosensor for rapid detection of gibberellin acid in the rice grain silver-enhanced colloidal gold metalloimmunoassay for schistosoma japonicum antibody detection chronopotentiometry and faradaic impedance spectroscopy as methods for signal transduction in immunosensors liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplifi cation of antigen-antibody or oligonucleotide-dna sensing processes by the precipitation of an insoluble product on electrodes fiber-optic chemical sensors and biosensors fiber-optic chemical sensors and biosensors surface plasmon resonance sensors: a review surface plasmon resonance-based immunoassays determination of affi nities of a panel of iggs and fabs for whole enveloped (infl uenza a) virions using surface plasmon resonance enhanced performance of a surface plasmon resonance immunosensor for detecting ab-gad antibody based on the modifi ed self-assembled monolayers sensing fatty acid binding protein with planar and fi ber-optical surface plasmon resonance spectroscopy devices surface plasmon resonance immunosensor for the detection of salmonella typhimurium immunosensor for detection of legionella pneumophila using surface plasmon resonance applications of fluorescence in immunoassay fluorescence polarization as a means for determination of fumonisins in maize reagentless and regenerable immunosensor for monitoring of immunoglobulin g based on non-separation immunoassay an array immunosensor for simultaneous detection of clinical analytes development of fl uorescence change-based, reagent-less optic immunosensor conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers clinical applications of chemiluminescence chemiluminescent immunosensor for ca - based on antigen immobilization on a cross-linked chitosan membrane fully automated chemiluminometric assay for hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen) optical fi ber immunosensor based on a poly(pyrrole-benzophenone) fi lm for the detection of antibodies to viral antigen homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin use of a quartz vibrator for weighing thin layers on a microbalance piezoelectric immunosensors -theory and applications piezoelectric quartz crystal biosensors characterization of a quartz crystal microbalance with simultaneous mass and liquid loading specifi c recognition of bacteria by surface-templated polymer fi lms immunophenotyping of acute leukemia using an integrated piezoelectric immunosensor array improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection detection of antisperm antibody in human serum using a piezoelectric immunosensor based on mixed self-assembled monolayers impedance characterization of a piezoelectric immunosensor. part ii: salmonella typhimurium detection using magnetic enhancement amplifi ed mass immunosorbent assay with a quartz crystal microbalance development of immunochips for the detection of dengue viral antigens au nanoparticle-and silver-enhancement reaction-amplifi ed microgravimetric biosensor latex piezoelectric immunoassay detection of agglutination of antibody-bearing latex using a piezoelectric quartz crystal a piezoelectric immunoagglutination assay for toxoplasma gondii antibodies using gold nanoparticles detection of antistreptolysin o antibody: application of an initial rate method of latex piezoelectric immunoassay improvement of latex piezoelectric immunoassay: detection of rheumatoid factor latex piezoelectric immunoassay: effect of interfacial properties polymer agglutination-based piezoelectric immunoassay for the determination of human serum albumin immunoassay of prostatespecifi c antigen (psa) using resonant frequency shift of piezoelectric nanomechanical microcantilever enantioselective sensors based on antibody-mediated nanomechanics invited paper: wireless magnetoelastic resonance sensors: a critical review magnetoelastic immunosensors: amplifi ed mass immunosorbent assay for detection of escherichia coli o :h immunosensor for detection of yersinia enterocolitica based on imaging ellipsometry a label-free multisensing immunosensor based on imaging ellipsometry electrochemical and quartz crystal microbalance detection of the cholera toxin employing horseradish peroxidase and gm -functionalized liposomes prussian blue modifi ed amperometric fia biosensor: one-step immunoassay for α-fetoprotein capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format protein and antibody arrays and their medical applications immunophenotyping of leukemias using a cluster of differentiation antibody microarray interdigitated array microelectrode-based electrochemical impedance immunosensor for detection of escherichia coli o :h antibody arrays for high-throughput screening of antibody-antigen interactions a microarray enzyme-linked immunosorbent assay for autoimmune diagnostics microfl uidic immunosensor systems micro total analysis system (µ-tas) in biotechnology analytical applications of aptamers aptamers: an emerging class of molecules that rival antibodies in diagnostics displacement enzyme linked aptamer assay