key: cord-354137-6oe8nj1j
authors: Wang, Hua; Shen, Guoli; Yu, Ruqin
title: Aspects of recent development of immunosensors
date: 2008-05-20
journal: Electrochemical Sensors, Biosensors and their Biomedical Applications
DOI: 10.1016/b978-012373738-0.50011-8
sha: 
doc_id: 354137
cord_uid: 6oe8nj1j

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 [1] [2] . 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 5 ϫ 10 4 and 1 ϫ 10 12 L mol Ϫ1 . 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 [3] 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 [1] [2] 4] , environmental analysis [5] [6] , and biological process monitoring [7] . 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 [1, [8] [9] . 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 [10] . 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 [11] . 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 [12] . 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 19-9 (CA19-9), carcinoma antigen 125 (CA125), alpha-fetoprotein (AFP), prostate specifi c antigen (PSA), CA15-3 and human chorionic gonadotropin (HCG) [13] [14] [15] . Wilson proposed an electrochemical immunosensor for the simultaneous detection of two tumor markers of CEA and AFP [15] . 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 [1] .

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 50 pg mL Ϫ1 [16] . 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 [17] [18] [19] [20] [21] . 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 [19] . 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 [20] . 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 [22] [23] . Most applications of the sol-gel-based immobilizations have been primarily directed to the optical immunosensors [14, 22, 24] and the electrochemical immunosensors [23, [25] [26] [27] [28] . 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) [28] . 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 [29] [30] [31] . 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 [11, [32] [33] [34] [35] [36] . For example, a new immobilization procedure of antibodies for capacitive immunosensor has been recently proposed using thiol compound and gold nanoparticles [36] . 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 [37] [38] [39] . 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 [37] . More recently, an amperometric immunosensor has been developed by employing a kind of core-shell magnetic nanoparticle of (CdFe 2 O 4 øSiO 2 ) to immobilize antibody onto the electrode surface with a magnet fi eld [38] . 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 [39] . 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 [40] [41] , (γ-aminopropyl) trimethoxysilane [42] [43] , and copolymer of hydroxyethyl-and methyl-methacrylate [44] , 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 [45] [46] . 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 [47] [48] [49] . Knoll and coworkers presented a versatile biotin-functionalized SAM, on which the biotinylated antibodies can be readily immobilized through an avidin mediator [48] . 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 [50] [51] . Langmuir-Blodgett (LB) fi lms are other useful alternatives to traditional mediate layers [52] [53] [54] . 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. [52] 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 [53] [54] . 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 [19, [55] [56] [57] . 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 [55] . 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 1991 [58] . These nanotubes are now chemically functionalized for the immobilization of biological entities for different biosensors, i.e. electrochemical devices [59] [60] [61] . Pantarotto et al. successfully bound a model pentapeptide and a virus epitope of foot-and-mouth disease onto single-walled CNTs [61] . 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 [62] . 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 [63] . It is established that the oriented immobilization of antibodies has low infl uence on their immunological activities to a certain degree [64] [65] [66] [67] [68] , which antigen binding capacity was demonstrated with a factor of 2-8 higher than that of antibodies randomly immobilized [68] . 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 [64, [69] [70] [71] .

Lee et al. utilized the self-assembled layer of thiol group-modifi ed protein A for the oriented immobilization of antibodies [64] . 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 [65] . 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 [72] [73] . 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 [72] . They found that antibodies immobilized by this procedure showed an antigen binding capacity 20-30 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 2.3), for cost effectiveness is of practical interest in real immunosensor applications [74] .

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 [75] . 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 [76] [77] [78] [79] [80] [81] . Janata fi rst proposed a potentiometric transducer for immunosensing and named it "immuno-electrode" [78] , 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 [79] [80] . D'Orazlo et al. reported the indirect measurements of immunoagents using ion-selective electrodes [81] . 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 [82] . 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) [83] . 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 2 , Si 3 N 4 , and Al 2 O 3 ) and enzyme labels (i.e. urease, peroxidase, and glucose oxidase) [84] [85] . Nevertheless, only a few examples of ISFET-based immunosensors could be found in the literature [86] [87] [88] . 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 [88] . 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 [89] . 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 [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] . 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 [90] . 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 [91] . A capacitive immunoassay based on antibody-embedded ultra-thin alumina sol-gel fi lms (∼20 to 40 nm) was reported and used for direct determination of antigens with a detection limit as low as ϳ1 ng mL Ϫ1 [92] . 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 [93] .

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 [14] . 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 [103] [104] . Ruan et al. developed an immunosensor based on enzyme-stimulated precipitation for the detection of Escherichia coli O157:H7 using an electrochemical impedance spectroscopy [103] . Another illustrative example was the sensitized immunosensor proposed by Chen et al. [104] . 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 0.011-11 ng mL Ϫ1 with a detection limit of 0.009 ng mL Ϫ1 . A new strategy of signal amplifi cation was also introduced for highly sensitive impedance measurements using biotin-labeled protein-streptavidin network complex [105] .

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 [25-26, 28, 106-116] . Aizawa et al. fi rst developed an amperometric immunosensor for the determination of human chorionic gonadotropin using an amperometric oxygen electrode [106] . 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 [108] . A design strategy of reagentless immunosensor was reported for the detection of carcinoma antigen-125 antibodies by direct HRP-labeled electrochemistry [109] . 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 [25] . 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 [117] [118] . Moreover, nano-sized particles or sol-gel matrixes have also been increasingly employed in the design of amperometric immunosensors with enhanced analytical performance [23, [119] [120] . For example, an electrochemical immunosensor has been developed for probing complement III (C 3 ) by use of nanogold particle monolayer as the sensing interface [119] . 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 [23] . 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 [120] . 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 [121] [122] . An immunosensor was designed by coupling immunoassay with the square wave anodic stripping voltammetry technique involving copper ion-labeled antigen in the competitive immunoreaction [121] . This immunosensor might allow rapid, accurate, and inexpensive detection of gibberellin acid with a concentration as low as 1 µg mL Ϫ1 . Chu et al. designed a silverenhanced colloidal gold metalloimmunoassay for the determination of Schistosoma japonicum antibody (SjAb) in rabbit serum [122] . 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 [123] . 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 [124] . 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 [1, [125] [126] . 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 [127] [128] . 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 HC10 [129] . 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 [130] . 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 200 ng mL Ϫ1 [131] . 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 [132] [133] . 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 [134] . 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 [134] [135] [136] [137] [138] . Maragos et al. described the development of a fl uorescence polarizationbased competition immunoassay for fumonisins in maize using fumonisin-specifi c monoclonal antibodies [135] . A fl uorescence-based immunosensor array for simultaneous determination of multiple clinical analytes was developed by Rowe et al. [137] . 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 [138] . 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 [139] . 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 [14, [140] [141] [142] [143] [144] . A chemiluminescent immunosensor for carbohydrate antigen 19-9 (CA19-9) was described by Lin et al., with CA19-9 immobilized on the cross-linked chitosan membrane [141] . The decrease of the immunosensor chemiluminescent signal was proportional to the CA19-9 concentration in the range 2.0-25 U mL Ϫ1 , with the detection limit of 1.0 U mL Ϫ1 . Pandian et al. developed an automated chemiluminometric immunoassay for the measurement of HCG [142] . 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 [143] . Herein, the immunosensor using optical fi ber photochemically modifi ed was tested for the determination of anti-E 2 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 [144] . The prepared chemiluminescence immunosensor in a competitive format was shown allowing for the detection of free digoxin with the concentration as low as 0.3 µg L Ϫ1 .

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 [145] [146] [147] [148] . 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 1 Hz ng Ϫ1 for a bulk acoustic wave device with 9 MHz of fundamental frequency, which can be described by the Sauerbrey equation [145] . 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 [42, 65, [146] [147] [148] [149] [150] [151] [152] [153] . 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 [149] . 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 [150] . 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 [152] . 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 [153] . 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 [154] . 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 [155] . Su and his coworkers successfully used QCM for detection of dengue virus [156] . 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 100-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 [157] . 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 [158] [159] [160] [161] . 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 [158] [159] [160] [161] [162] [163] . 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 [158] . 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 [159] .

In recent years, considerable efforts have been devoted to the development of cantilever-based immunosensors with unique enantio-selective antibodies [164] [165] . 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 10 pg mL Ϫ1 [164] . 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 [165] .

Moreover, mass-sensitive magnetoelastic immunosensors are exploited to design extraordinarily versatile and useful sensor platforms [166] . 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 O157:H7 [167] . 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 [168] . 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 [169] .

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. [1] . 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 [9] . 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 [170] . 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 [29] . 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 [171] [172] . 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 [173] [174] [175] [176] [177] . Belov et al. have proposed a novel immunophenotyping method for leukemias using a cluster of differentiation antibody microarray [174] . 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 [177] . 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 [178] . 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 [179] . 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 [180] [181] [182] . 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 [182] .

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 [9] .

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 3,3Ј,5,5Ј-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 3 ) 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 SnO2 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 1,6-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 coliO157:H7 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 6 -(D 2 -isopentenyl) adenosine assay

Reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen-125

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 3 ) based on immobilizing C 3 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 CA19-9 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 O157:H7

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 GM1-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 O157:H7

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