key: cord-0905717-ylxqh3sj authors: Jayamohan, Harikrishnan; Sant, Himanshu J.; Gale, Bruce K. title: Applications of Microfluidics for Molecular Diagnostics date: 2012-08-09 journal: Microfluidic Diagnostics DOI: 10.1007/978-1-62703-134-9_20 sha: b472f58728eedf75411b7f5c6941663d792312cc doc_id: 905717 cord_uid: ylxqh3sj Diagnostic assays implemented in microfluidic devices have developed rapidly over the past decade and are expected to become commonplace in the next few years. Hundreds of microfluidics-based approaches towards clinical diagnostics and pathogen detection have been reported with a general theme of rapid and customizable assays that are potentially cost-effective. This chapter reviews microfluidics in molecular diagnostics based on application areas with a concise review of microfluidics in general. Basic principles of microfabrication are briefly reviewed and the transition to polymer fabricated devices is discussed. Most current microfluidic diagnostic devices are designed to target a single disease, such as a given cancer or a variety of pathogens, and there will likely be a large market for these focused devices; however, the future of molecular diagnostics lies in highly multiplexed microfluidic devices that can screen for potentially hundreds of diseases simultaneously. mobility has aided the rapid spread of infectious diseases from region of origin to other parts of the world as seen during the 2009 H1N1 pandemic. This mobility has highlighted the need for rapid, portable diagnostic (point-of-care [POC] ) devices at ports of entry to prevent global spread of infections. Current laboratory culture methods for pathogens take a day or more to provide results ( 2 ) . Clearly, methods need to be developed to aid rapid and site-relevant diagnosis of disease. For certain other types of infections, in both the developed and developing worlds, the diagnostic tests need to be repeated periodically to measure response to therapy and monitor the disease condition. One such case is monitoring the viral load (number of viral particles per milliliter of blood) for infections like HIV (Human immunode fi ciency virus) and hepatitis C. Sub-Saharan Africa is a region heavily affected by the AIDS pandemic. The lack of standard laboratory facilities and trained laboratory technicians in these regions is a serious bottleneck ( 3 ) . Similar problems exist in medically underserved areas of the USA. A simple POC platform could enable increased access to treatment for patients in such low-resource settings. In the developed world, the strategy to deal with major disease burdens such as cancer is shifting from a therapeutic to diagnostic mode ( 4 ) , as the cost of treating disease falls dramatically if it is found early. Ischemic heart diseases and cerebrovascular diseases, which are the major causes of mortality in the developed world, can be targeted by effective diagnostics ( 1 ) . With projected US healthcare costs of $4.4 trillion by 2018, expanding conventional expensive diagnostic solutions is not a viable option ( 5 ) . Rapid, low-cost diagnostic tools that can be dispersed throughout a community for easy access, possibly even in the home, would provide substantial bene fi t by allowing more rapid diagnosis and monitoring of disease and infection. Homeland security is another key sector where portable molecular biology tools are needed to detect a variety of biological agents ( 6 ) . The US Departments of Health and Human Services (HHS) and Agriculture (USDA) maintains a list of biological agents and toxins de fi ned as select agents "that have the potential to pose a severe threat to public, animal or plant health, or to animal or plant products" ( 7 ) . Again, there is a need for rapid, inexpensive detection, identi fi cation, and quanti fi cation of pathogens to help reduce this threat. Hence, there is an unmet need for simple, low-cost/cost-effective, accurate, portable/point-of-care diagnostic tools for rapid identi fi cation of disease markers and pathogens in a variety of settings. The FDA (Food & Drug Administration), de fi nition of a "simple test" provides a benchmark for features for an ideal diagnostic tool (Table 1 , ( 1, 8 ) ). There is consensus that for such an ideal diagnostic tool, micro fl uidics will certainly be required and will likely make up the critical components of the device ( 9 ) . Micro fl uidics can be de fi ned as "science and technology of systems that process or manipulate small (10 -9 to 10 -18 liters) amounts of fl uids, using channels with dimensions of tens to hundreds of micrometers" ( 10 ) . Lab-on-achip (LOC) refers to the application of micro fl uidics in chemical, biological analysis and diagnostics. The ultimate objective of LOC devices is to integrate the entire gamut of laboratory capabilities on a micro fl uidic chip (11) (12) (13) . Some of the features of micro fl uidics that make the technology attractive for lab-on-a-chip point-of-care applications are: The availability of fabrication methods to manufacture small Smaller length scales result in faster analyses and higher sepa-• ration ef fi ciencies, reducing response times. The high speed analysis also makes micro fl uidics a suitable candidate for highthroughput applications. Straightforward integration of multiple components/func-• tionalities (sample preparation, detection, data processing) on a single device. Potentially fully automated and simple to use, enabling use by • laypeople. Portability and a small footprint should allow fi eld and clinic • use, as well as possibly allowing more widespread diagnostics. Pervasive diagnostics should greatly increase the likelihood of personalized medicine having a signi fi cant impact on society. Highly parallel analyses will allow multiple tests to be run • simultaneously, either on the same sample or multiple samples. Micro fl uidic devices can in principle be used to obtain parameters like proteomic, metabolomic, and genetic data of each individual for personalized care ( 15 ) . Figure 1 provides a basic generalized schematic of a micro fl uidic LOC device with sample-in/readout capabilities. The fi gure shows some of the various technologies that might be involved in sample preparation, analysis or separation, and detection. Figure 2 is an example of a real nucleotide analysis system developed at the State of Utah Center of Excellence for Biomedical Micro fl uidics. 1 . A schematic diagram of a conceptual lab-on-a-chip device designed to perform a variety of unit operations and unit processing steps including: sample preparation (e.g., fl uid handling, , derivatization, lysis of cells, concentration, extraction, and ampli fi cation), sample separation (e.g., electrophoresis, liquid chromatography, molecular exclusion, fi eld-fl ow fractionation), and detection (e.g., fl uorescence, UV/Vis absorption, amperometric, conductivity, Raman, electrochemical). Micro fl uidic devices have been steadily developing over the past 30 years, but most of the progress related to diagnostic applications has been made in about the past 15 years ( 10, 16 ) . A major driver for micro fl uidic development was the focus on genomics and The micro fl uidic system consists of fi ve different components: (i) a disposable micro fl uidic cartridge containing a glass fi ber fi lter (inset fi gure); (ii) a PDMS-micro fl uidic chip for fl ow control; (iii) micro fl uidic chambers for mixing, metering, pumping, and reactions; (iv) a pneumatic micropump to deliver the eluted sample to downstream assays; and (v) a vacuum pump to control the on-chip valves. The extraction chip also has provision for thermal lysis and reverse transcription (not shown). ( b ) Prototype of a test socket for characterization of a carbon nanotube-based electrochemical nanosensor array. The test socket provides both fl uidic and electrical interface to the nanosensor chip (inset fi gure) that detects nucleotide hybridization. ( c ) Prototype of a shuttle PCR chip with three temperature zones and which is fabricated using polycarbonate lamination. The heaters and thermocouples are shown with a manifold for on-chip fl uidic control. The fl uidic interface for the extraction system is designed so that it can be readily connected to the downstream assays such as hybridization and PCR. molecular biology in the 1980s especially on microanalysis techniques like high-throughput DNA sequencing. The initial micro fl uidic devices were inspired by the microelectronics industry and relied on photolithography and MEMS fabrication techniques. Hence, most of the earliest micro fl uidic devices were fabricated in silicon and glass. The origins of micro fl uidics as used in diagnostic and molecular biology applications can be traced to microanalytical tools like gas-phase chromatography (GPC), high-pressure liquid chromatography (HPLC), and capillary electrophoresis (CE) developed in the mid-1990s ( 16 ) . Rapid progress was made on these tools at this time and many of the developed concepts are still in use today. presented an automated enzyme assay in which nanoliter volumes of substrate, enzyme, and inhibitor were mixed using electrokinetic fl ow ( 23 ) . Microchip-based capillary electrophoresis (CE) for separation and relative quantitation of human serum proteins was achieved ( 24 ) . Some of the other separation methods like free-fl ow electrophoresis (FFE) ( 25 ) , capillary gel electrophoresis ( 26 ) and capillary array electrophoresis (CAE) ( 27 ) were reported. These devices were primarily fabricated in silicon and glass and lead to the work on related components like micropumps, microvalves and sensors. There are a few examples of plastic devices before 2000. Delamarche et al . used elastomeric micro fl uidic networks to pattern immunoglobulin with high resolution on a variety of substrates (gold, glass, polystyrene) ( 28 ) . Freaney et al . developed a prototype miniaturized chemical analysis system comprising biosensors and a microdialysis interface for on-line monitoring of glucose and lactate in blood ( 29 ) . In the 1990s, to counter the threat of biological and chemical weapons, the US Defense Advanced Research Projects Agency (DARPA) supported development of " fi eld-deployable micro fl uidic" devices and was a driver for academic research in micro fl uidics ( 10 ) . The fi rst lab-on-a-chip emerged with the concept of a "miniaturized total analysis system" or μ TAS, involving a silicon chip analyzer with sampling, sample pretreatment, separation, and detection functionalities embedded on an integrated system ( 30 ) . Electroosmotic pumping was the primary actuation mode used in these early μ TAS systems especially since separation was one of the objectives and pumping could be controlled using simple electronics and no moving parts ( 31 ) . Seiler et al . reported amino-acid separation on chip and their detection using laser-induced fl uorescence ( 32 ) . Other applications involving biomolecules and cells emerged during the period. These include fl ow cytometry ( 33 ) , DNA ampli fi cation (PCR) ( 34 ) and cellular metabolism studies ( 35 ) on a microfabricated chip. A host of innovations in micro fl uidic devices came forth in the period from 1994 to 1997. These include, "reactor chambers for continuous precolumn and postcolumn labeling reactions" ( 36, 37 ) , high speed ef fi cient separations ( 38 ) , on-chip static mixing ( 39 ) , separation of oligonucleotides ( 40 ) , DNA ( 41 ) , and amino acids ( 42 ) , and cell manipulation by electrical fi elds ( 43 ) . There was also work on separation modes like synchronized cyclic capillary electrophoresis ( 44 ) up with a miniaturized mass spectrometer incorporating an integrated plasma chamber for electron generation, an ionization chamber, and an array of electrodes acting as the mass separator ( 49 ) . All of these systems would fi nd their way into later diagnostic micro fl uidic devices. The introduction of polymer-based soft lithography offered a cheaper alternative to silicon and glass in micro fl uidic device fabrication ( 50 ) . Most of the exploratory research in micro fl uidics is currently performed on polymer-based devices primarily made of poly(dimethylsiloxane) (PDMS), a soft elastomer ( 10 ) . Soft lithography techniques for micro fl uidic devices have been reviewed multiple times along with many of the structures and devices than can be produced ( 51, 52 ) . Related polymer-based methods like microcontact printing and microtransfer molding enabled rapid fabrication of micrometer scale structures ( 53 ) . Three dimensional structures were reported using a layer-by-layer structuring using microtransfer molding ( 54 ) . Other plastics, hybrid materials, and packaging techniques were soon developed, including a variety of low cost plastic prototyping and manufacturing methods for micro fl uidics ( 55, 56 ) . Martynova et al . reported micro fl uidic devices fabricated in Poly-(methyl methacrylate) (PMMA) by imprinting them with an inverse three-dimensional image of the device micromachined on silicon ( 57 ) . Wang et al . developed a low temperature bonding process using a sodium silicate layer as an adhesive for glass micro fl uidic devices. Micro fl uidic interconnects for connecting vertically stacked micromachined channels and to external tubing on the same plane was demonstrated by Gonzalez and co-workers ( 58 ) . Additional landmark work included Johnson et al . fabricating nanometer wide channels on silicon, SiO 2 , and gold substrates by exposing them to a metastable argon atom beam in the presence of dilute vapors of trimethylpentaphenyltrisiloxane ( 59 ) . Lorenz et al . reported the characterization of SU-8 negative photoresist for the fabrication of high aspect-ratio structures ( 60 ) . Larsson et al . fabricated 3D microstructures by conventional CD-injection molding against a silicon master produced by wet and deep reactive ion etching (DRIE) ( 61 ) . Silicon micromachining methods based on DRIE, silicon fusion bonding (SFB) ( 62 ) , and electron cyclotron resonance (ECR) source were reported ( 63 ) . Dozens of other techniques have also been reported, but cannot all be reviewed here. Micro fl uidics was inspired by the microelectronics industry and hence most of the initial devices were fabricated in silicon using photolithography and related technologies. The success of the microelectronics and MEMS industries in manufacturing thousands of miniaturized components in parallel at very low costs was thought to be applicable to micro fl uidics. While this may eventually prove to be true, low cost micro fl uidic devices made using photolithographic techniques have proven to be the exception rather than the rule, since the numbers of identical micro fl uidic chips manufactured for current and foreseeable markets tend to be more in the 10,000 s −100,000 s, where batch processing does not provide suf fi cient cost savings. Packaging and other post processing steps like reagent introduction have also proven challenging and expensive, and consequently other manufacturing methods currently appear to be more in favor. Thus, while most of the earliest work in micro fl uidics was in silicon, the majority of current devices are now made in glass or a variety of plastics. Nonetheless, silicon and glass manufacturing technique are important in micro fl uidics, because molds for rapid and inexpensive manufacturing of plastic devices are still often made of silicon or glass. Standard silicon and glass manufacturing techniques are based on microlithography, subtractive techniques (etching), and additive techniques ( 64 ) . Microlithography involves the use of an energy beam to transfer a geometric pattern to a substrate. Depending on the type of energy beam used, these can be divided into: photolithography, electron beam lithography, X-ray lithography, and ion lithography. Photolithography involves using light to transfer a geometric pattern from a photo mask to a light sensitive chemical called photoresist. This is followed by a development process using a developer solution to create a positive or negative image of pattern onto the photoresist. Other techniques like X-ray lithography, extreme ultraviolet (UV) lithography, ion particle lithography, scanning probe lithography, and nanoimprint lithography are being increasingly used due to their capability in producing sub-100 nm structures. Of these, nanoimprint lithography, a type of embossing, is a low cost, high throughput and high resolution method that has the potential to be used for low-cost mass manufacture of micro and nano fl uidic devices in a variety of materials, but especially for direct embossing of plastics ( 65 ) . Subtractive techniques involve dry and wet etching, which are primarily used with glass and silicon devices. Wet etching involves chemical removal of layers from a material and is typically used to etch silicon, silicon dioxide, silicon nitride, metals, and glass. Dry etching refers to the removal of material by bombarding it with ions. Sputtering, ion beam milling and plasma etching (reactive ion etching and deep reactive ion etching) are some of other methods used in silicon etching. Additive technologies involve techniques to deposit fi lms. Methods to deposit thin fi lms include: thermal oxidation of silicon, chemical vapor deposition (CVD), and physical vapor deposition (PVD). Methods to deposit thick fi lms usually involve a spinning or electroplating technique. These thick fi lms are often patterned using photolithography and then used as molds for micro fl uidic devices. The reader can refer to the text by Madou ( 64 ) for an in-depth description of the techniques described above, as well as a description of other micromanufacturing techniques. In the past decade, silicon and glass have been largely displaced by plastics as the ideal substrate for micro fl uidic devices ( 10 ) . Six primary considerations have been behind this transition. First, silicon is relatively expensive compared to plastics because micro fl uidics tends to take up larger areas than microelectronic chips (and silicon costs are measured by area). Second, the electronic advantages of silicon are not typically required in micro fl uidic devices. Third, silicon is not transparent, so troubleshooting micro fl uidic devices during development can be dif fi cult and optical detection techniques cannot be employed. Fourth, silicon processing typically requires processes found in expensive cleanrooms that are also relatively slow. The development of rapid and inexpensive polymer processing methods has proven compelling. Fifth, silicon is relatively brittle and is not ideal for devices that experience signi fi cant "handling." Sixth, silicon is incompatible with the strong potentials used in electrokinetic pumping and capillary electrophoresis (CE). Silicon does have some advantages, such as well controlled surface properties, but these have not proven suf fi cient to drive micro fl uidic development. Polymers, due to their lower cost, ease of fabrication and physical properties, are now the primary materials used in micro fl uidic research. Many micro fl uidic components, such as pumps and valves, work better when fabricated in the less rigid polymer medium as compared with silicon. The permeability of polymer to gases make it suitable for work with living mammalian cells. PDMS, an optically transparent, soft elastomer has been used for various micro fl uidic devices since its introduction ( 10 ) . Most polymer devices are made using a molding, embossing, or casting techniques, although direct processing means, such as laser-based or knife-based manufacturing is increasing ( 56 ) . Soft lithography is the technique of replicating structures from a master mold or stamp onto an elastomeric (PDMS) substrate. Fabrication using PDMS is simple and does not require expensive facilities, and prototyping can often be done in less than a day. The reader can refer to in-depth reviews of soft lithography for detailed insight into the method ( 51, 52 ) . Interestingly, not many commercial products use devices fabricated in PDMS due to a gap between academic and industrial practices, although this is starting to change ( 66 ) . In addition, PDMS has limited application due to its hydrophobic surface and tendency to swell in organic solvents ( 67 ) . Although polymers are the preferred material for most micro fl uidic applications today, silicon and glass are still relied upon for building specialized devices that need chemical and thermal stability ( 68 ) . In the nascent fi eld of nano fl uidics, silicon and glass are used due to their mechanical stability ( 10 ) . Some of other methods that are used in micro fl uidic fabrication are xerography ( 69 ) , laser micromachining ( 70 ) and polymer stereolithography ( 71 ) . An innovative method for creating low cost disposable micro fl uidic diagnostic devices (paper-based analytical devices [ μ PADs]) was introduced by Martinez et al . ( 72, 73 ) . The fl uid movement is controlled primarily by evaporation and capillary forces. Although the technology is very promising, more work needs to be done to bring forth real world μ PAD applications. Recently, micro fl uidic devices fabricated on engineered plastics, such as cyclo-ole fi n copolymers (COC) ( 74 ) , and photocurable per fl uoropolyether (PFPE) ( 65 ) have been reported. A major boom in micro fl uidics research has occurred in the last 10-12 years as is re fl ected by the number of published journal papers using the term: 26 papers were published before 1990, 341 in the 1990s, 15773 in the 2000s and 3,322 in the fi rst 2 years of this decade. While the number of papers each year appears to be leveling off, the impact of micro fl uidics is likely to continue to grow. Another consequence of this large body of literature is that it becomes infeasible to cover all the important papers and developments in a chapter such as this. As most reviews on micro fl uidics for diagnostic applications have focused on the physical methods behind the device operation and not as much on the applications, this work will focus on some speci fi c diagnostic developments and application areas. We examine the micro fl uidic applications in diagnostics for diabetes, cardiac related conditions, and infections related to bacteria, virus, and HIV. We also review the applications in pharmacogenomics and devices for low resource settings. We discuss some of the methods used in fabricating these micro fl uidic devices and the challenges in mass production. Included is a section on some of the commercial diagnostic products using micro fl uidic technology. The reader may refer to supplemental reviews for the theory behind micro fl uidics ( 10, (75) (76) (77) (78) and methods used in micro fl uidic LOC detection ( 14, 67, 79 ) . There is an increasing need in the developing and developed world for new cost-effective diagnostic technologies, albeit for different reasons. In developed countries, health care costs are rising rapidly, and containment is an issue. In developing countries, delivery of medical services to remote and resource poor areas is dif fi cult and the needs are enormous, as infectious disease is a critical barrier to economic and social development. Interestingly, the two problems tend to converge towards one solution: micro fl uidic diagnostic devices. The Grand Challenges in Global Health (GCGH) initiative, a major effort to achieve scienti fi c breakthroughs against infectious diseases that cause signi fi cant problems in the developing world, has identi fi ed seven long-term goals in global health ( 80 ) , most of which revolve around eliminating infectious disease. Infectious diseases constitute a huge burden in developing countries (32.1%) using disability-adjusted life year (DALY) metrics compared to developed countries (3.7%) and account for 50% of infant deaths ( 1, 81 ) . The major concerns in terms of DALY are infections due to viruses (HIV/AIDS, measles, hepatitis B, hepatitis C, and viral gastroenteritis [rotavirus]); bacteria (cholera, tuberculosis, pertussis, tetanus, and meningitis); and parasites (malaria, Lymphatic fi lariasis, leishmaniasis, and trypanosomiasis). The three most devastating diseases are malaria, tuberculosis, and HIV. In 2009, there was an estimated 169-294 million cases of malaria worldwide, resulting in about 781,000 deaths. Of these 85% of deaths were in children under 5 years of age [ 82 ) . There was an estimated 14 million people infected with TB and about 1.7 million related deaths in 2009. TB is a major cause of deaths in HIV infected patients with about 380,000 of the 1.7 million deaths being reported in people with HIV ( 83 ) . An estimated 33.3 million people are living with HIV worldwide of which about 67.5% live in sub-Saharan Africa ( 84 ) . There has been an estimated 1.8 million AIDS related deaths, 73% of those being in sub-Saharan Africa. Thus, early infectious disease detection and management is a high priority in low-resource settings and a major driver of micro fl uidic diagnostic devices. Infectious diseases are not limited to developing countries. Recent outbreaks like H1N1 in fl uenza A demonstrate the rapid spread of infectious diseases from a country of origin to the rest of the world. In April 2009, USA and Mexico reported 38 cases of H1N1 in fl uenza. By June 2009 when World Health Organization declared a pandemic, there were a reported 28,774 cases and 144 deaths in 74 countries. The H1N1 in fl uenza pandemic had a total of 43,677 reported cases in the USA as of July 2010 ( 85 ) . Estimates of unreported cases are a much higher fi gure at 1.8-5.7 million cases ( 86 ) . In contrast, chronic diseases that require consistent monitoring are the major disease burden for high-income countries. These diseases include: ischemic heart disease, cerebrovascular disease, cancers, and diabetes mellitus. Global mortality and disease burden projections suggest that these chronic conditions common to highincome countries will also become a priority for low-income countries by the year 2030 ( 1 ) . Thus, the driver for micro fl uidic diagnostics in developed countries is the need for consistent, accurate, and affordable diagnostics for chronic disease. Bacterial detection is a key need in areas including: clinical diagnostics, monitoring of food-borne pathogens, and detection of biological threat agents. Harmful bacteria are the source of diseases like gastroenteritis and cholera. From a bioterrorism perspective, pathogenic bacteria pose serious risk. Under favorable temperature and in the presence of moisture and nutrition, bacteria spread rapidly. For a list of bacterial diseases and corresponding causative agents the reader can refer to a review by Ivnitski et al . ( 87 ) . Conventional methods to detect and identify bacteria require growing a small number of bacteria into colonies of higher numbers. Hence conventional methods take 18-24 h at a minimum ( 87 ) . Also, conventional methods require complex equipment, highly trained technicians, and cannot be fi eld deployable or used in point-of-care settings. There are primarily two modes of pathogen detection: immunosensing and nucleic acid-based detection. In immunosensing, a binding interaction between probing antibodies and antigens of target cells (analyte) is detected. A variety of mechanisms can be used to detect this interaction, such as: optical, electrical or electrochemical impedance, cantilever, quartz crystalline microbalance, surface plasmon resonance (SPR), and magnetoresistivity. Nucleic acid-based sensors detect DNA or RNA targets from the analyte organisms ( 88, 89 ) . The polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) is used to amplify the nucleic acids to enhance the detection signal ( 90 ) . Optical detection methods are often preferred due to their high selectivity and sensitivity ( 91, 92 ) . A variety of micro fl uidic devices have been developed for bacteria using optical means. A micro fl uidic system for detection of Escherichia coli using laser-optical fi ber fl uorescence detection was reported by Xiang et al . They reported detection limits an order of magnitude higher than that achieved for conventional fl uorescence microscope ( 93 ) . Gao Electrical and electrochemical modes of bacterial detection have also been widely reported. The primary advantage of the method is the ease of fabricating microelectrodes in the microchannel by lithography and the absence of labeling steps ( 96 ) . A micro fl uidic sensor based on impedance measurement of E. coli was constructed by Boehm et al . ( 97 ) . The selectivity of the sensor to different bacterial strains was demonstrated by positive identi fi cation of E. coli in a suspension of E. coli and M. catarrhalis . A microfabricated electrochemical sensor array for detection of bacterial pathogens in human clinical fl uid samples was demonstrated. The device consisting of a set of 16 sensors was able to detect relevant bacterial urinary pathogens ( E. coli , Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and Klebsiella-Enterobacter group) and could in principle be used as a point-of-care device for rapid diagnosis of urinary tract infections ( 98 ) . Table 2 lists a number of detection methods for bacterial diagnostics and, for a comprehensive list of electrical and electrochemical bacterial detection, the reader can refer to a review by Jinseok et al . ( 96 ) . Micro fl uidic devices have also been applied to the detection of parasites. A "micro fl uidic fl ow-through membrane immunoassay with on-card dry reagent storage" was developed by Stevens et al . for the detection of Plasmodium falciparum ( 99 ) . As noted earlier in this chapter, effective virus detection and disease management is critical in public health, the biotechnology industry, and biodefense. Some of the most deadly historical epidemics like smallpox, yellow fever, and Spanish fl u were due to viral agents. In the twenty-fi rst century, HIV, rotavirus, and measles are found to be among the leading contributors to global disease burden ( 100 ) . Many deadly viruses such as Variola virus (small pox), Rift Valley fever virus, and Venezuelan Equine Encephalomyelitis virus have been known to be developed as potential biological agents ( 101 ) . POC devices to detect these bio-agents are extremely critical for global biosecurity. The small size, simple biology, and lack of reproductive ability outside the host cell add to the complexity in detecting viruses. The primary methods for virus detection are serology, viral antigen detection, and nucleic acid detection. Serologic tests detect the presence of antibodies that the immune system produces in response to viral infection. Viral antigen detection typically relies on immunoassays as described previously. Nucleic acid detection involves ampli fi cation of the viral genome using PCR and the subsequent detection of the ampli fi ed genome. HIV is one of the primary targets of micro fl uidic diagnostic research efforts. Conventional HIV diagnostic assays are based on an enzyme assay (EIA/ELISA) followed by western blot and requires trained laboratory personnel. Universal access to HIV diagnostics is stymied by the lack of trained technicians, patient motivation, and laboratory access especially in rural areas and the developing world. For instance, about 83% of HIV patients remain undiagnosed in Kenya ( 102 ) . Thus, a simple, inexpensive diagnostic tool for HIV would be readily welcomed. The number of CD4 + T-lymphocytes per microliter of HIV-infected blood is a critical monitor of disease state and this measurement is needed to make informed antiretroviral therapy (ART) treatment decisions. Therefore, the primary mechanisms for HIV detection in POC micro fl uidic devices are enumeration of CD4 + T-lymphocytes and HIV viral load quanti fi cation. To be successful, the POC device needs to detect around 200 CD4 + cells/ μ L and 400 copies/mL of HIV from whole blood ( 103 ) . Towards this goal, Sia et al . reported a micro fl uidic immunoassay, "POCKET (portable and cost-effective)" for quantifying anti-HIV-1 antibodies in the sera of HIV-1 infected patients. The device consisted of a PDMS slab with microchannels placed orthogonally to a polystyrene stripe patterned with HIV-enveloped antigen. The HIV-1 infected patient serum sample is fl owed through the microchannels to quantify anti-HIV-1 antibodies. Although the device was able to identify the sera of HIV-1-infected patients from those of non-infected patients, it could not make a correlation of the output data with HIV disease states ( 103 ) . Lee et al . developed a RT-PCR-based POC diagnostic chip for HIV. The chip relies on HIV markers p24 (a major core protein encoded by the HIV gag gene) and gp120 (an external envelope protein encoded by envelope gene) for diagnostic purposes ( 104 ) . Cheng et al . reported a POC micro fl uidic CD4 + T-cell counting device. The device works in two stages, initial depletion of monocytes from whole blood and subsequent CD4 + T cell capture. The strategy of contaminant (monocytes) depletion prior to CD4 + T cell isolation enhances the performance in low CD4 count (200 cells/ μ L) scenarios ( 105 ) . Other label-free CD4 + T-lymphocyte capture techniques have been reported. Although the micro fl uidic devices themselves are disposable and usually cheap, they still require expensive optical microscopes to count the captured CD4 + T-cells ( 106, 107 ) . A lensless portable CCD-based micro fl uidic platform developed by Demirci et al . overcomes this limitation. The captured label-free CD4 + T-lymphocytes are detected by a charge coupled device (CCD) sensor using lensless shadow imaging techniques and counted using automatic cell counting software in a few seconds ( 108 ) . Micro fl uidic diagnostics have been designed for other viral agent infections like in fl uenza, severe acute respiratory syndrome (SARS) and dengue fever. These diseases have been of serious concern to global public health organizations especially in the last few years. The in fl uenza virus causes respiratory tract infection and is found to be severely morbid in children and the elderly ( 112 ) . The challenge with diseases like in fl uenza is that there is a large variety of the viruses and they are constantly changing. For example, the in fl uenza A virus can subdivided into H1N1 and H1N3 based on the glycoproteins (hemagglutinin and neuraminidase) present in the viral envelope. The 2009 in fl uenza pandemic was caused by a novel H1N1 strain with genes from fi ve different fl u viruses ( 113 ) . Thus, the diagnosis of in fl uenza alone is not suf fi cient; discovery of the type of in fl uenza is also critical. Some of the conventional diagnostic methods for in fl uenza virus are enzyme-linked immunosorbent assays (ELISA), immuno fl uorescence assays, serological hemagglutination inhibition assays, real-time polymerase chain reaction (PCR) assays, and complement fi xation tests. Most of these methods are complicated, relatively costly and require a lengthy process and expensive apparatus ( 114 ) . Several micro fl uidic systems have been shown for in fl uenza detection. An immunomagnetic bead-based micro fl uidic system for detection of in fl uenza A virus has been demonstrated recently. In fl uenza A viral particles are initially bound to monoclonal antibody (mAb)conjugated immunomagnetic beads using a suction type micro-mixer. Subsequently the virus-bound magnetic complexes are fl uorescently labeled by developing mAb with R-phycoerythrin. An external optical detection module is used to analyze the optical intensity of the magnetic complex. The system displayed better performance than conventional fl ow cytometry systems in terms of limit of detection ( 114 ) . However, the expensive external optical detection module could restrict its use in POC low-resource settings. Yamanaka et al . reported a micro fl uidic RT-PCR chip for rapid detection of in fl uenza (AH1pdm) virus of swine-origin. A disposable electrical printed chip was used for electrochemical detection of the PCR amplicon ( 112 ) . The electrochemical method is better for use in low-resource settings compared to the optical methods reported above due to the absence of expensive external detection units. A Magnetic Integrated Micro fl uidic Electrochemical Detector (MIMED) for detection of H1N1 in fl uenza virus from throat swab samples has recently been developed ( 115 ) . ( 118 ) . The fl uid is actuated using electrokinetic methods and the limit of detection was reported to be 30 pM. Weng et al . developed a suction-type, pneumatically driven micro fl uidic device for the detection of dengue infection ( 119 ) . A detection limit of 10 PFU/ ml was reported for the device. A "lab-on-a-disc" centrifugal micro fl uidics-based portable ELISA system was developed for detection of the antigen and the antibody of Hepatitis B virus ( 120 ) . The limit of detection of antigen and antibody were reported as 0.51 ng/mL and 8.6 mIU/mL, respectively. Heinze et al . developed a micro fl uidic immunosensor for detection of bovine viral diarrhea virus ( 121 ) . An integrated micro fl uidic assay for targeted ribonucleic acid (RNA) extraction and a one-step reverse transcription loop-mediated-isothermal-ampli fi cation (RT-LAMP) process for the detection of nervous necrosis viruses was reported by Wang et al . ( 122 ) . In 2010, there were an estimated 1,500 cancer related deaths per day in the USA and about 1.4 million new cases of cancer were reported. By 2020, cancer related deaths are estimated to be 10.3 million globally. The cancer mortality rate per 100,000 Americans has dropped from 194 to 190 since 1950, an insigni fi cant drop compared to drop in mortality rates for other diseases. Most of the improvements in cancer survival rates are due to improvements in early diagnosis rather than treatment. For instance, for cancers of the breast, colon, rectum, and cervix, early detection has proved to reduce mortality signi fi cantly. Hence, the National Cancer Institute has emphasized a shift from therapeutic to preventive mode in its 2010 vision document. Existing methods of cancer diagnostics rely on invasive techniques like taking a biopsy and then examining the cell morphology. Further, conventional methods could be inconclusive in disease detection in its early stages ( 123 ) . Other techniques like immunoassays (ELISA) have been used to detect cancer biomarkers. Although ELISAs are very sensitive, they can be time consuming, expensive and are mostly carried out in a laboratory requiring skilled personnel. In most cases, immunoassays look for only one biomarker and are not sensitive enough to detect very low biomarker levels especially at early stages of the disease. POC devices which are accurate, fast and economic are needed. This would enable improved diagnosis, monitoring of the progress of the disease, and response to therapy. Advances in oncology have led to identi fi cation of biomarkers associated with different kinds of cancers. For a comprehensive list of cancer biomarkers, the reader can refer to reviews in literature (124) (125) (126) (127) (128) (129) . There are multiple factors responsible for carcinogenesis. This along with the "heterogeneity in oncogenic pathways" makes it imperative that a range of biomarkers need to be analyzed for cancer diagnostics ( 123, 130 ) . Hence POC devices with multiplexed capability to detect multiple biomarkers are needed. Although research into cancer diagnostic devices is moving forward, commercialization of the technology still remains a challenge ( 123 ) . Here, we review some of the recent research in micro fl uidics POC devices for cancer diagnostics. Legendre et al . reported work into the design and development of a micro fl uidic device for diagnosis of T-cell lymphoma. The system accepts a whole blood sample as the input, extracts the DNA, ampli fi es target sequences of the T-cell receptor-gene, and eletrophoretically resolves the products for detection of a signature consistent with monoclonality ( 131 ) . Diercks et al . demonstrated a micro fl uidic device that measured multiple proteins (tumor necrosis factor, CXC chemokine ligand 2, interleukin 6 and interleukin 1b) at pg/mL concentrations in nanoliter volumes. Antibody-coupled polystyrene microspheres labeled with embedded fl uorophores were used to detect the analyte (proteins). Optical detection of captured analyte was performed off-chip using a confocal microscope, which proved to be a disadvantage in terms of lack of device portability ( 132 ) . A similar fl uorescence approach has been used to detect vascular endothelial growth factors in human plasma ( 133 ) . An on-chip nuclear magnetic resonance (NMR)-based biosensor was developed for the multiplexed identi fi cation of cancer markers (epidermal growth factor receptors EGFR and Her2/neu). The design consists of a microcoil array for NMR measurements, micro fl uidic channels for sample handling and a permanent magnet to generate a polarizing magnetic fi eld, all integrated into a handheld device ( 134 ) . Mass spectroscopy-based micro fl uidic detection of cancer-speci fi c biomarkers (proliferating cell nuclear antigen, cathepsin D, and keratins K8, K18, and K19) was demonstrated by Lazar ( 135 ) . Other mass-based methods like quartz crystal microbalance (QCM) have been used in cancer biomarker detection. For instance, Zhang et al . demonstrated detection of human lung carcinoma cells using a micro fl uidic surface modi fi ed piezoelectric sensor ( 136 ) . Recently, Von Muhlen et al . have reported a microcantilever-based "suspended microchannel resonator" sensing device for detection of activated leukocyte cell adhesion molecules ( 137 ) . Zani et al . demonstrated an electrochemical method for detection of prostate speci fi c antigen (PSA) cancer markers. The method works based on the differential pulse voltammetry-based electrochemical detection of protein coated paramagnetic microparticles that selectively capture the analyte (PSA) ( 138 ) . Similar electrochemical detection methods for breast cancer markers have been reported ( 139 ) . A micro fl uidic-based amperometric electrochemical detection system for carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) was developed by Kellner et al . The on-chip fl uid function is handled by computer controlled syringe pumps and reports enhanced performance due to fully automated fl uidic operations ( 140 ) . But the external computer control system and syringe pumps prove to be a bottleneck in their use for POC applications. Hence miniaturization and integration of the fl uid handling functions within the micro fl uidic chip is necessary for POC use. Cardiovascular diseases (CVD) are responsible for nearly half of the deaths in the western world. Studies suggest the acute and long term fi nancial burden of cardiac disease to be substantial ( 141 ) . It is reported that 5% of myocardial infarction (MI) patients are incorrectly discharged from emergency departments (ED). Hence for timely and effective intervention against cardiovascular diseases, there is a need for rapid and accurate diagnostic tools ( 142 ) . For the accurate "diagnosis, prognosis, monitoring and risk strati fi cation of patients with acute coronary syndromes" (ACS), biochemical markers play a fundamental role ( 142 ) . In clinical settings, in 50-70% of patients with ACS related cases, ECGs give ambiguous results. In such cases, cardiac marker levels could provide critical information for informed decision on the suitable treatment. As a de fi nite indicator of disease condition a combination of cardiac markers need to be explored ( 143 ) . For a review of cardiac biomarkers, the reader can refer to McDonnell et al . ( 142 ) . There is a difference of opinion with regard to the use of POC technologies for cardiac biomarker diagnostic, with some suggesting it to be an alternative to conventional lab analyzers ( 144, 145 ) and others questioning the accuracy of the technologies ( 146, 147 ) . The following section provides a review of micro fl uidic devices used in cardiac biomarker detection. Most of the diagnostic mechanisms for biomarkers involve two steps, an initial immunoassay to capture the analyte (biomarker) and subsequent detection of the captured analyte. chemiluminescence-based detection sensor for alpha-fetoprotein (AFP). Super-paramagnetic microbeads were used to capture the biomarker ( 153 ) . Use of magnetic microbeads results in higher surface to volume ratio for ef fi cient analyte capture and enables on-chip actuation using an integrated electromagnet. A digital micro fl uidic platform detection device for cTnI was developed by Sista et al . ( 154 ) . The fl uidic actuation is performed by electrowetting, obviating the need for any off-chip fl uid handling apparatus. SPR-based micro fl uidic detection of cardiac marker B-type natriuretic peptide (BNP) was reported by Kurita et al . ( 155 ) . Electrochemical methods have been applied to detection of cardiac markers. Unlike optical methods, these do not need an often expensive, off-chip optical detection device and could be suitable for POC applications. Tweedie et al . presented a micro fl uidic-based impedimetric sensing device for cardiac enzymes ( 156 ) . The i-STAT system (Abbott Point of Care Inc., USA) is a commercial test cartridge for electrochemical detection of cTnI ( 157 ) . The device can detect cTnI in the range of 0-50 ng/ml and has gained good acceptance as a diagnostic tool for MI ( 143 ) . Other electrochemical-based detection methods for detection of myoglobin ( 158 ) , cTnI ( 159 ) and CRP ( 160 ) ( 157 ) . Table 3 lists the set of published work and commercial devices for micro fl uidic cardiac marker detection. Micro fl uidic-based technology is ideal for developing highly parallel diagnostic assays that would allow high-throughput screening, but there has been limited success in this area. The lack of success is not due to problems with micro fl uidic devices; for example, drug screening requires high-throughput methods to fi nd and test different drug candidates. Micro fl uidic high-throughput screening (HTS) techniques have been applied to drug discovery to perform thousands of tests in parallel with some success (162) (163) (164) . As of now these methods haven't been applied in micro fl uidic diagnostics for several reasons. Current diagnostics are typically performed in large hospitals or reference labs. In these labs, most tests are Cardiac STATus™ Not reported ( 142 ) batched and performed using robots in a highly parallel, high throughput approach. Replacing these robots by using micro fl uidics is unlikely in the short term due to the large infrastructure already developed. Essentially, a solution to this problem already exists, so adoption of micro fl uidics for these assays will only occur if there are compelling assay improvements. In addition, if an assay can be performed in a batch mode using micro fl uidics, it is likely to be able to be performed in the clinic or POC setting, and for nearly the same price. Thus, micro fl uidics is likely to be driven to the POC rather than to large reference laboratories. The reverse of high-throughput screening (multiple samples with one target) is multiplexed screening, where one sample is tested for multiple agents or biomarkers. A few examples of multiplexed screening have already been provided, especially for cardiac biomarkers, but highly multiplexed diagnostics are still being developed. Multiplexed screening is likely to have a more signi fi cant impact on diagnostics than high throughput screening, especially with the move towards personalized medicine. Micro fl uidics has been combined with microarray technology, which is used regularly in genomics and proteomics, and which will likely have diagnostic applications in the future; however, this is beyond the scope of the chapter. More relevant are micro fl uidic devices that can diagnose multiple diseases simultaneously. A recently released product that uses "mesoscale" fl uidics can simultaneously diagnose 15 respiratory diseases associated with viruses ( 165 ) . A challenge with getting the device to commercialization is that regulatory agencies such as the FDA require individual validation of each assay, meaning that multiplexing must clear very challenging regulatory requirements, which will likely limit substantial multiplexing in the near future. Nevertheless, micro fl uidics will probably lead to highly multiplexed assays that can perform 100s or 1,000s of diagnostic assays on one sample. About 1,200 patents related to micro fl uidics have been issued in the USA through 2010. In spite of immense academic interest in micro fl uidics and signi fi cant research investment directed towards both academic and industrial organizations, relatively few commercial products based on micro fl uidics have been introduced into the market ( 166, 167 ) ; however, the rate of introduction is increasing and many barriers are coming down. One of the reasons cited for lack of commercial success is the lack of a potential "blockbuster" end-user product that could generate billions of dollars in revenue. Until the industry can fi nd a product with high volume demand, the fabrication costs due to lack of "economies of scale" are going to remain high. Existing materials like PDMS, which are hugely popular in research, have not succeeded in the industry due to issues with manufacturability and scaling ( 168 ) . Most of the LOC products are still focused on the business-to-business segment and not the business-to-consumer ( 167 ) . There needs to be more focused research on micro fl uidic product development including issues like manufacturability and cost dynamics and a simultaneous search for new application areas where micro fl uidics could be applied. Table 4 provides a sample of micro fl uidic companies and products in the market. More comprehensive lists are available ( 169 ) . Micro fl uidic diagnostic devices have been developing at a rapid rate over the past few years. While the potential for these devices was fi rst recognized more than 20 years ago, the realization of that potential has been slow, even though thousands of devices and methods have been published. The continuing development of applications and micro fl uidic manufacturing methods, including platform technologies that can be customized easily for each diagnostic test, will be the drivers of success. Very recent progress and an emphasis on global health has helped move the fi eld towards POC devices that will likely become ubiquitous in the years ahead. While most micro fl uidic devices have one diagnostic target, devices capable of diagnosing 100s or 1,000s of diseases will likely be developed and commercialized in the next decade, making micro fl uidics a major driver of disease diagnostics. Pointof-care diagnostics for global health Point-of-Care Testing and Molecular Diagnostics: Miniaturization Required Nanotechnology: Emerging Developments and Early Detection of Cancer, A Two-Day Workshop sponsored by the National Cancer Institute and the National Institute of Standards and Technology Updated and Extended National Health Expenditure Projections Multiplex diagnostic platforms for detection of biothreat agents National select agent registry: overview. Department of Health & Human Service, Washington DC 8. Food and Drug Administration (2008) Recommendations: Clinical Laboratory Improvement Amendments of 1988 (CLIA Waiver Applications for Manufacturers of In Vitro Diagnostic Devices Micro fl uidics and point-of-care testing The origins and the future of micro fl uidics Latest developments in micro total analysis systems Latest developments in micro fl uidic cell biology and analysis systems Micro total analysis systems: latest achievements Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis systems Continuous sample pretreatment using a freefl ow electrophoresis device integrated onto a silicon chip Threedimensional micro fl ow manifolds for miniaturized chemical analysis systems Electroosmotic pumping and valveless control of fl uid fl ow within a manifold of capillaries on a glass chip Open channel electrochromatography on a microchip A microsystem mass spectrometer Polymer microstructures formed by moulding in capillaries Soft lithography Soft lithography in biology and biochemistry Patterning self-assembled monolayers using microcontact printing: a new technology for biosensors? Fabrication of three dimensional micro structures: Microtransfer molding Xurography: Micro fl uidic Prototyping with a Cutting Plotter Fabrication and packaging: Low-cost MEMS technologies Fabrication of plastic micro fl uid channels by imprinting methods Fluidic interconnects for modular assembly of chemical microsystems Using neutral metastable argon atoms and contamination lithography to form nanostructures in silicon, silicon dioxide, and gold SU-8: a low-cost negative resist for MEMS Silicon based replication technology of 3D-microstructures by conventional CD-injection molding techniques Silicon fusion bonding and deep reactive ion etching; a new technology for microstructures A novel etchdiffusion process for fabricating high aspect ratio Si microstructures Fundamentals of microfabrication: the science of miniaturization High Resolution Soft Lithography: Enabling Materials for Nanotechnologies Wasatch Micro fl uidics, LLC, www.micro fl .com Micro fl uidics for medical diagnostics and biosensors Continuous-fl ow thermal gradient PCR Xurography: rapid prototyping of microstructures using a cutting plotter CO2-laser micromachining and back-end processing for rapid production of PMMA-based micro fl uidic systems Polymer microfabrication technologies for micro fl uidic systems Three-dimensional micro fl uidic devices fabricated in layered paper and tape Diagnostics for the developing world: micro fl uidic paper-based analytical devices Thermoplastic micro fl uidic device for on-chip puri fi cation of nucleic acids for disposable diagnostics Micro fl uidic diagnostic technologies for global public health Micro fl uidics: Fluid physics at the nanoliter scale Developing opto fl uidic technology through the fusion of micro fl uidics and optics Control and detection of chemical reactions in micro fl uidic systems Innovations in optical micro fl uidic technologies for point-ofcare diagnostics Grand challenges in global health: the ethical, social and cultural program Lab-on-achip devices for global health: past studies and future opportunities World malaria report: 2010. World Health Organization United Nations Millennium Declaration Situation updates -Pandemic (H1N1) 2009, World Health Organization Estimates of the prevalence of pandemic (H1N1) Biosensors for detection of pathogenic bacteria Sample to answer: a fully integrated nucleic acid identi fi cation system for bacteria monitoring Micro fl uidic sample preparation: cell lysis and nucleic acidpuri fi cation Integrated Micro fl uidics for Serotype Identi fi cation of Foot and Mouth Disease Virus An overview of foodborne pathogen detection: In the perspective of biosensors Pathogen detection: A perspective of traditional methods and biosensors Miniaturized immunoassay micro fl uidic system with electrokinetic control Multiplexed high-throughput electrokinetically-controlled immunoassay for the detection of speci fi c bacterial antibodies in human serum An integrated micro fl uidic platform for sensitive and rapid detection of biological toxins An overview of recent strategies in pathogen sensing On-chip micro fl uidic biosensor for bacterial detection and identi fi cation Use of electrochemical DNA biosensors for rapid molecular identi fi cation of uropathogens in clinical urine specimens Enabling a micro fl uidic immunoassay for the developing world by integration of on-card dry reagent storage Micro-and nanotechnology for viral detection Potential biological weapons threats Tackling HIV through robust diagnostics in the developing world: current status and future opportunities Nano/Micro fl uidics for diagnosis of infectious diseases in developing countries A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics Enhancing the performance of a point-of-care CD4+ T-cell counting microchip through monocyte depletion for HIV/AIDS diagnostics A micro fl uidic device for practical label-free CD4+ T cell counting of HIV-infected subjects A microchip approach for practical label-free CD4+ T-cell counting of HIV-infected subjects in resource-poor settings Integrating micro fl uidics and lensless imaging for point-of-care testing Cell detection and counting through cell lysate impedance spectroscopy in micro fl uidic devices Label Free Detection of CD4+ and CD8+ T Cells Using the Opto fl uidic Ring Resonator An integrated micro fl uidic system for counting of CD4+/ CD8+ T lymphocytes. Micro fl uid Rapid detection for primary screening of in fl uenza A virus: micro fl uidic RT-PCR chip and electrochemical DNA sensor Emergence and pandemic potential of swineorigin H1N1 in fl uenza virus Rapid detection of in fl uenza A virus infection utilizing an immunomagnetic bead-based micro fl uidic system Genetic Analysis of H1N1 In fl uenza Virus from Throat Swab Samples in a Micro fl uidic System for Pointof-Care Diagnostics Virus analysis by electrophoresis on a micro fl uidic chip Opto-fl uidic micro-ring resonator for sensitive label-free viral detection Enhanced on-chip SERS based biomolecular detection using electrokinetically active microwells A suction-type micro fl uidic immunosensing chip for rapid detection of the dengue virus A fully automated immunoassay from whole blood on a disc Micro fl uidic immunosensor for rapid and sensitive detection of bovine viral diarrhea virus An integrated micro fl uidic loop-mediated-isothermal-ampli fi cation system for rapid sample pre-treatment and detection of viruses Biosensors for cancer markers diagnosis Nanotechnology for early cancer detection Biomarkers in cancer screening, research and detection: present and future: a review Mining the plasma proteome for cancer biomarkers Antibody fragments as probe in biosensor development How does DNA sequence motif discovery work? Biomarkers for prostate cancer Electrochemical biosensors: towards point-of-care cancer diagnostics Toward a Simpli fi ed Micro fl uidic Device for Ultra-fast Genetic Analysis with Sample-In/Answer-Out Capability: Application to T-Cell Lymphoma Diagnosis A micro fl uidic device for multiplexed protein detection in nano-liter volumes Internally calibrated quanti fi cation of VEGF in human plasma by fl uorescence immunoassays in disposable elastomeric micro fl uidic devices Chip-NMR biosensor for detection and molecular analysis of cells Micro fl uidic bioanalytical platforms with mass spectrometry detection for biomarker discovery and screening A micro fl uidic system with surface modi fi ed piezoelectric sensor for trapping and detection of cancer cells Label-free biomarker sensing in undiluted serum with suspended microchannel resonators A New Electrochemical Multiplexed Assay for PSA Cancer Marker Detection Integrated micro fl uidic platform for the electrochemical detection of breast cancer markers in patient serum samples Automated microsystem for electrochemical detection of cancer markers Cost Estimation of Cardiovascular Disease Events in the US Cardiac biomarkers and the case for point-of-care testing Lab-on-a-chip based immunosensor principles and technologies for the detection of cardiac biomarkers: a review Evaluation of a pointof-care assay for cardiac markers for patients suspected of acute myocardial infarction Risk strati fi cation of chest pain patients by pointof-care cardiac troponin T and myoglobin measured in the emergency department A rapid troponin I assay is not optimal for determination of troponin status and prediction of subsequent cardiac events at suspicion of unstable coronary syndromes Lack of concordance between a rapid bedside and conventional laboratory method of cardiac troponin testing: impact on risk strati fi cation of patients suspected of acute coronary syndrome Silane-dextran chemistry on lateral fl ow polymer chips for immunoassays Toward one-step point-of-care immunodiagnostics using capillary-driven micro fl uidics and PDMS substrates Highly sensitive rapid, reliable, and automatic cardiovascular disease diagnosis with nanoparticle fl uorescence enhancer and MEMS Design and testing of a disposable micro fl uidic chemiluminescent immunoassay for disease biomarkers in human serum samples Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on crossfl ow chromatography Rapid analysis of alphafetoprotein by chemiluminescence micro fl uidic immunoassay system based on super-paramagnetic microbeads Development of a digital micro fl uidic platform for point of care testing On-chip enzyme immunoassay of a cardiac marker using a micro fl uidic device combined with a portable surface plasmon resonance system Fabrication of impedimetric sensors for label-free Point-of-Care immunoassay cardiac marker systems, with passive micro fl uidic delivery Cardiac point of care testing: a focused review of current National Academy of Clinical Biochemistry guidelines and measurement platforms Development of a myoglobin impedimetric immunosensor based on mixed self-assembled monolayer onto gold Electrochemical Immunosensor for Simultaneous Detection of Dual Cardiac Markers Based on a Poly (Dimethylsiloxane)-Gold Nanoparticles Composite Micro fl uidic Chip: A Proof of Principle Electrochemical impedance immunosensor based on three-dimensionally ordered macroporous gold fi lm Detection of multiple cardiac markers with an integrated acoustic platform for cardiovascular risk assessment Microand nano fl uidic systems for high-throughput biological screening Situ Microarray Fabrication and Analysis Using a Micro fl uidic Flow Cell Array Integrated with Surface Plasmon Resonance Microscopy Caliper LifeSciences-Target ID/Validation Applications ) search issued patents for "micro fl uidic" in title or abstract, United States Patent and Trademark of fi ce Micro fl uidic lab-on-a-chip platforms: requirements, characteristics and applications It's the economy FluidicMEMS.com's list of micro fl uidics/lab-on-a-chip companies The authors thank Keng-Min Lin for the schematic diagram in Fig. 1 . The authors would like to thank the Nano Institute of Utah for funding this work through a nanotechnology training fellowship.