Antibodies as target for affinity biosensors

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Trends in Analytical Chemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

Antibodies as target for affinity biosensors Stéphanie Patris, Marie Vandeput, Jean-Michel Kauffmann * Faculty of Pharmacy, Université libre de Bruxelles (ULB), Campus Plaine, CP 205/6, 1050 Brussels, Belgium

A R T I C L E

I N F O

Keywords: Biosensor Immunoassay Immunosensor Label Label-free Optical Piezoelectric Electrochemical

A B S T R A C T

Antibodies or immunoglobulins (Ig) are proteins produced by the immune system to protect the body by identifying and neutralizing pathogens. The determination of antibodies is an important area in bioanalysis because their presence provides information about pathologies such as infections, allergies, auto-immune diseases and cancers. Antibodies can be readily detected and quantified by using immunosensors. This review provides an up-to-date overview of immunosensors for the determination of antibodies which can be implemented in the clinical area, for point-of care applications or in routine laboratories. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

3. 4.

Introduction ............................................................................................................................................................................................................................................................. Applications ............................................................................................................................................................................................................................................................. 2.1. Immunoglobulin E (IgE) ......................................................................................................................................................................................................................... 2.2. Autoimmune diseases ............................................................................................................................................................................................................................. 2.2.1. Anti-gliadin ................................................................................................................................................................................................................................ 2.2.2. Anti-transglutaminase antibodies ..................................................................................................................................................................................... 2.3. Bacterial infections .................................................................................................................................................................................................................................. 2.3.1. Helicobacter pylori .................................................................................................................................................................................................................... 2.3.2. Salmonella .................................................................................................................................................................................................................................. 2.3.3. Bacillus mycobacterium tuberculosis .................................................................................................................................................................................. 2.4. Viral infections .......................................................................................................................................................................................................................................... 2.4.1. Measles ....................................................................................................................................................................................................................................... 2.4.2. Human immunodeficiency virus (HIV) ........................................................................................................................................................................... 2.4.3. Pseudorabies virus .................................................................................................................................................................................................................... 2.4.4. Fever virus .................................................................................................................................................................................................................................. 2.4.5. Epstein-Barr virus ..................................................................................................................................................................................................................... 2.4.6. Bovine herpesvirus-1 ............................................................................................................................................................................................................... 2.5. Parasites ....................................................................................................................................................................................................................................................... 2.5.1. Echinococcus granulosus ......................................................................................................................................................................................................... 2.5.2. Babesia bovis ............................................................................................................................................................................................................................. 2.5.3. Schistosoma japonicum ........................................................................................................................................................................................................... 2.5.4. Toxoplasma gondii .................................................................................................................................................................................................................... 2.6. Vaccination ................................................................................................................................................................................................................................................. 2.6.1. Influenza virus .......................................................................................................................................................................................................................... 2.6.2. Hepatitis B virus ...................................................................................................................................................................................................................... 2.6.3. Tetanus toxin ............................................................................................................................................................................................................................ 2.6.4. Tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B and hepatitis B ................................................................................................ 2.7. Cancer .......................................................................................................................................................................................................................................................... Analytical figures of merit .................................................................................................................................................................................................................................. Conclusion ................................................................................................................................................................................................................................................................ References ................................................................................................................................................................................................................................................................

In memory of Prof. M. Mascini. * Corresponding author. Tel.: 00 32 2 6505215; Fax: 00 32 2 6505225. E-mail address: [email protected] (J.-M. Kauffmann). http://dx.doi.org/10.1016/j.trac.2015.12.005 0165-9936/© 2015 Elsevier B.V. All rights reserved.

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1. Introduction Marco Mascini highlighted in 1992 the interest of applying biosensors in medical fields: “Electrochemical biosensors have found wide interest in clinical chemistry and medicine. Physiologists, cardiologists, diabetologists dreamt for years about the possibility of continuously monitoring chemical parameters to feedback appropriate action to restore the values to normal levels [1].” This dream is still relevant today and allergologists, infectiologists and gastroenterologists can be added to the potential users of biosensors and particularly biosensors for antibodies determination. Biosensors can be classified either as a function of the biological recognition element or as a function of the transducer. A distinction can also be made between (i) biocatalytic biosensors (i.e. comprising an immobilized enzyme, whole cell, organelle) for which the recognition and binding of the analyte are inducing chemical change(s) and (ii) affinity biosensors (i.e. comprising an immobilized antibody, antigen, DNA, aptamer, membranous receptor) for which the binding event does not involve a chemical reaction [2]. Immunosensors faced the most important research trends in affinity biosensor development. They correspond, generally, to a combination of an immobilized antibody used for the detection of a target antigen and a transducer to convert the immunological reaction into a measurable signal. The antibody is selected as a function of the target. Immunosensors exploit the same transducers as biosensors. They can be differentiated into electrochemical, optical, piezoelectric, magnetic, calorimetric, mechanical, i.e. atomic force miscrocopy (AFM) cantilever immunosensors [3]. A vast number of immunosensors have been developed for the determination of drug compunds [4], pesticides [5], biomarkers [6,7], pathogens [8] or toxins [9] with the antibody being used as immobilized probe [10]. It is interesting, however, to consider the antibody as the target of an immunosensor. Indeed, an antibody is a biological protein giving informations about the immunological status, infection, allergy, auto-immune diseases etc. . . An antibody is traditionally detected by immunoprecipitation, agglutination or neutralization assays (use of an animal) and, more frequently, by applying an enzyme immunoassay such as an enzyme-linked immunosorbent assay (ELISA). ELISAs are time consuming, and need a qualified technician and a relatively sophisticated instrumentation. In contrast, immunosensors are miniaturized integrated devices which allow rapid, easy-to-use and on-site detection (point of care) [11]. Antibodies or immunoglobulins (Ig) are subdivided into classes or isotypes according to the structure of the constant domains of the heavy chains: IgG (70-75%), IgA (15%), IgM (10%), IgE (less than 1%), IgD (less than 1%) [12]. IgGs are the main class of antibodies circulating in blood (12 mg/mL in serum). They are synthesized by plasma cells as humoral immune response to a contact of the immune system with antigens. IgGs are glycoproteins of 150 kDa containing two identical heavy chain polypeptides and two identical light chains. IgMs are the first antibody synthesized by the plasma in primary reaction after initial exposure to an antigen. The IgAs are produced under secretory form in mucosal tissues (i.a. in the intestinal lumen, the respiratory tract and the saliva) where they neutralize toxins. IgEs are utilized during immune defense against parasitic infestation. IgEs also play a role in various allergic diseases [12]. Generally, an antigen has several epitopes which cause several antibodies formation, these are polyclonal antibodies. In clinical biology, the search of an antibody is always a search of polyclonal antibodies. The strategy applied with immunosensors is based, most of the time, on a sandwich-type format. The antigen corresponding to the target antibody is immobilized onto the transducer. After sample incubation, a labeled antibody against IgG or IgA (depending on the isotype of the target antibody) is added in order to allow the de-

Fig. 1. Some strategies at immunosensors. A. Sandwich-type immunosensor measurement in multi steps, the reaction between the label and reagent causes the signal. B: Label free immunosensor measurement in one step, the antigen-antibody binding induces the signal.

tection (Fig. 1A). Label-free biosensors allow a direct detection of the affinity event such as in surface plasmon resonance (SPR) biosensors, piezoelectric sensors or impedimetric based sensors. Such configurations allow real time detection with a reduced number of steps and with reduced time of analysis (Fig. 1B). SPR biosensors measure a refractive index change due to the binding of an analyte to its biospecific partner immobilized onto a gold surface [13]. Piezo and impedimetric sensors are built with the antigen immobilization onto a quartz crystal microbalance or an electrode substrate, respectively. After antigen-antibody binding, the former measures a mass change and the latter a modification of the impedance at the electrode-solution interface [10]. To the best of our knowledge, there are no immunosensors for antibody assay on the market yet. Several commercially available transducers using a screen printed electrode (SPE) or the SPR technology can be customized for antibody assays. Semi quantitative immunochromatographic assays are available, however, such as the immunochromatography stick for the detection of tetanus antibodies (Quick TetanCheck®, Tétanos Quick Stick® [14]). In this review, we have chosen to present different immunosensors classified by their application and not by the transducer. Immunosensor applications will be dedicted (i) to an IgE boost (allergy) (ii) to antibodies raised in autoimmune diseases, during bacterial, viral and parasitic infections, (iii) to monitor levels of antibodies in the context of a vaccination and (iv) to antibodies developed in response to some cancers. 2. Applications 2.1. Immunoglobulin E (IgE) Some electrochemical immunosensors are dedicated to the determination of IgEs. Because the IgEs play an important role in type I hypersensitivity, their presence in blood is considered as a marker of allergy. Kreuzer et al. have developed an immunosensor for measurement of allergy related IgEs in human blood samples. The immunosensor is based on a competitive immunoassay using an anti-IgE modified screen-printed carbon electrode (SPE) and amperometric detection of p-aminophenol produced by alkaline phosphatase (AP). The immunosensor measures the IgE level within a 30 min time interval [15].

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The determination of IgEs produced in the case of peanuts allergy can be realized with immunosensors. Liu et al. have compared several immunosensor-like methodologies for sensitive detection of antibodies to a peptide sequence from the major peanut allergen, Arachis hypogaea 2. Faradaic and nonfaradaic impedance strategies were compared to amperometric detection. Measurements employ a sandwich-type immunoassay format with a secondary antibody labeled with horseradish peroxidase (HRP) to bind the target antibody on the sensor and provide amplified signals. The best impedimetric sensor configuration featured HPR-catalyzed precipitation of the enzyme product onto the sensor measured by nonfaradaic impedance. This sensor configuration provides the best limit of detection with respect to label-free impedimetric immunosensors and amperometric immunosensors [16]. 2.2. Autoimmune diseases Autoimmune diseases are caused by an overactivity of the immune system against compounds and tissues normally present in the body. The result is the production of antibodies against the body itself [17]. It is in this context that several groups have focused on the development of immunosensors for the diagnosis of autoimmune diseases. The celiac disease is an autoimmune disease characterized by a permanent intolerance to the different protein fractions of gluten found in various types of cereals. The pathology is diagnosed by serological tests for anti-gliadin antibodies (IgA and IgG), antiendomysial antibodies, anti-tissue transglutaminase antibodies and endoscopic biopsy [18]. The patients should follow a strict glutenfree diet. 2.2.1. Anti-gliadin A SPE immunosensor with impedance detection was developed by Balkenhohl and Lisdat for the determination of antigliadin antibodies. Gliadin is the protein part of the cereals forming the gluten with glutenin. To develop this immunosensor, the surface of a screen-printed gold working electrode is modified with gliadin. A first incubation of the modified electrode with the serum allows interaction between anti-gliadin antibodies and gliadin. A second incubation with a solution containing anti-IgG antibodies labeled with HRP allows the detection of the immunocomplex by electrochemical impedance spectroscopy (EIS). The substrate selected to be oxidized by the peroxidase is 3-amino-9-ethylcarbazol. The oxidation product, 3-azo-9-ethylcarbazole, precipitates on the surface of the electrode which induces a modification of the impedance at the working electrode surface-solution interface which can be assessed by EIS. An impedance spectrum is recorded using the redox couple ferri/ferrocyanide [19]. Another sandwich type immunosensor to determine anti-gliadin antibodies was elaborated with amperometric detection of 3,3′,5,5′-tetramethylbenzidine (TMB) oxidized by HRP [20]. A fiber-optic biosensor for anti-gliadin was developed with gliadin, the antigen, electrostatically self-assembled onto the surface of a tapered optical fiber [21]. 2.2.2. Anti-transglutaminase antibodies Several immunosensors for the diagnosis of celiac disease based on the search for anti-transglutaminase antibodies (IgA and IgG) have been described. These antibodies may be responsible for damage to the small intestine and they are directed against transglutaminase. The gliadine, the substrate of this enzyme, generates a situation of cellular aggression causing the release of transglutaminase and inflammation leading to the recruitment of immune cells. The principle of such an immunosensor is based on the modification of an SPE modified by single-walled carbon nanotubes with gold particles and transglutaminase. The binding with anti-transglutaminase autoantibodies is then evidenced by an anti-human IgA or IgG labeled with

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AP. The enzymatic substrate is 3-indoxyl phosphate (IP). The enzymatic hydrolysis of IP generates an indoxyl intermediate which reduces silver ions present in solution. The detection is performed by voltammetric oxidation of deposited metallic silver [22]. An amperometric immunosensor based on the covalent immobilization of tissue transglutaminase has been described by Giannetto et al. This immunosensor has the particularity to detect the enzyme in its open conformation. Indeed, such conformation is considered as most relevant for this biomarker [23]. The antibody linked to the immobilized antigen is revealed by anti-IgG or anti-IgA labeled by HRP and an amperometric detection via the redox species thionin [23]. A magneto-immunosensor for anti-transglutaminase antibodies has been developed. The antigens are immobilized onto magnetic beads to select the target antibodies and the detection in the presence of H2O2 by square wave voltammetry is possible through a HRP labeled anti-IgG and with o-phenylendiamine as substrate [18]. An electrochemical immunosensor array with detection of quantum dots (QDs) was developed for the detection of anti-transglutaminase IgG in human sera. The sensor uses an 8-channel screen-printed carbon electrochemical array. The antigen is immobilized by adsorption onto the array. After the immunological reaction with human serum containing the antibodies of interest, an anti-human IgG labeled with CdSe/ZnS QDs is incubated and electrochemical detection of Cd2+ released from QDs is performed [24]. Dulay et al. have developed a sandwich-type immunosensor for anti-transaminase antibodies with an amperometric detection of 3,3’,5,5’-tetramethylbenzidine diimine generated from 3,3’,5,5’-tetramethylbenzidine (TMB) by HRP in the presence of H2O2. The characterization of the modified electrode is carried out by impedance measurements [25]. A simple methodology for electrochemical immunosensors without blocking step was developed for anti-transglutaminase determination in one step. All the reagents for a sandwich-type immunoassay are incubated during the same time in order to reduce the analyze time [26]. The anti-IgG used is labeled with a CdSe/ZnS QD and the electrochemical measurements are performed as previously described [24]. 2.3. Bacterial infections 2.3.1. Helicobacter pylori Helicobacter pylori (H. pylori) is a bacterium which can be found in the stomach of healthy people but which can be involved in some peptic ulcers. In the case of the presence of the bacterium, the ulcer is treated with a proton pump inhibitor drug in combination with two antibiotics (clarithromycin and metronidazole or amoxicillin). It is essential to identify H. pylori before initiating such medications. An alternative to the invasive endoscopy to demonstrate the presence of H. pylori is the search for IgG anti-H. pylori in serum. Messina et al. have reported an amperometric immunoreactor for rapid and sensitive quantification of human serum IgG antibodies to H. pylori. The serum sample is introduced in a bioreactor flow cell and the antibodies of interest react immunologically with the purified H. pylori antigens that are immobilized on a rotating disk. The bound antibodies are detected by HRP labeled anti-IgG. The enzyme HRP, in the presence of H2O2, catalyzes the oxidation of hydroquinone (HQ) to p-benzoquinone (BQ). The electrochemical reduction back of BQ to HQ is detected on a glassy carbon electrode surface located close to the rotating disk [27]. A microfluidic magnetic immunosensor coupled to a gold electrode has also been developed. The anti-IgG used in this work is labeled by AP and the substrate is p-aminophenyl phosphate. The latter is converted to p-aminophenol and the electroactive product detected at the gold layer electrode by amperometry [28]. The determination of antiH. pylori has been previously performed at the surface of a SPE by applying the same experimental scheme [29]. A portable immunosensor coupled to a laser-induced fluorescence (LIF)

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detection system has been developed to determine anti-H. pylori. The principle is similar to above described system (anti-IgG labeled with AP) but the used enzymatic substrate is 4-methylumbelliferyl phosphate which is converted to soluble fluorescent methylumbelliferone by AP. This fluorescent product is finally quantified by LIF detection [30]. 2.3.2. Salmonella The Salmonella typhi bacterium is responsible of typhoid disease. It is possible to diagnose this disease either by culture isolation from clinical specimens or by the detection of antibodies in the patient’s serum. An amperometric sensor for detection of antibodies to S. typhi has been developed in this context. The immunoassay is performed onto a SPE onto which a S. typhi antigen has been immobilized. After following the incubation of a serum sample containing anti- S. typhi, AP labeled anti-IgG is spiked onto the SPE. The product 1-naphthol, which is formed due to the hydrolysis of the substrate 1-naphthyl phosphate by AP, is detected by amperometry [31]. A SPR immunosensor for the detection of S. typhi antibodies has been also developed [32]. It consisted of a 4-mercaptobenzoic acid modified gold surface with immobilized recombinant flagellin protein (an antigen of S. typhi). In order to optimize the parameters affecting the response (i.e. pH, temperature), the modified gold chip has been studied by SPR and EIS. The developed assay allows a detection of S. typhi antibodies in less than 10 min. Salmonella enterica serovar enteritidis is one of the major causes of bacterial gastroenteritis in humans. The spread of this disease can occur e.g. through contaminated egg consumption. It is important to detect infected flocks as soon as possible by an adequate monitoring program and avoiding eating the contaminated eggs. As an alternative to current chicken serology, testing of eggs for antibodies against Salmonella could be performed. Thomas et al. have evaluated the sensitivity, specificity and discriminatory capacity of a SPR biosensor (Biacore 3000) for detecting antibodies in egg yolk against the lipopolysaccharide (LPS) fraction of S. enterica serovar enteritidis. The SPR biosensor performances appear to be better than that of the commercial ELISA [33]. 2.3.3. Bacillus mycobacterium tuberculosis Bacillus mycobacterium tuberculosis is the bacterium responsible of tuberculosis. In order to diagnose tuberculosis, the search for the antigen lipoarabinomannan (LAM) can be conducted. Antibodies anti-LAM can be also identified. A SPE immunosensor was developed by using gold nanoparticles (AuNPs) labeled protein A (Au-SPA) as the electrochemical tag for the detection of anti-LAM. Protein A is a protein produced by the bacterium Staphylococcus aureus. This protein has the ability to bind Ig in the wrong orientation (i.e. by the end of the antibody which does not enable the link with the antigen), to allow the bacterium disrupt the Ig. This protein forms part of the bacteria arsenal of protection against the immune system. The antigens are immobilized by passive adsorption onto the surface of a SPE. After incubation of the sample containing anti-LAM, the AuNPs labeled protein A are added. The protein A binds the anti-LAM allowing subsequent electrochemical detection. This is achieved by the electrooxidation of AuNPs in 0.1M HCl leading to a strong signal amplification [34].

weeks apart are used to determine whether the measles infection is acute or convalescent [35]. 2.4.2. Human immunodeficiency virus (HIV) A sensor for the determination of anti-HIV antibodies consisting of a β-galactosidase enzyme immobilized onto a disk microelectrode arrays has recently been described. The enzyme is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind to the enzyme, the 3D conformation changes affecting positively the performance of the active site and, consequently, the enzyme activity is stimulated. The enzymatic product p-aminophenol from the substrate p-aminophenyl β-d-galactopyranoside is measured by chronoamperometry [36]. 2.4.3. Pseudorabies virus Pseudorabies virus is responsible of the Aujeszky’s disease which is a serious acute infectious disease for livestock and pets. An immunosensor for Pseudorabies virus antibody in swine serum was developed with magnetic beads as a platform for the immobilization and immunoreaction. Gold nanoparticles served as electroactive label for the electrochemical detection [37]. 2.4.4. Fever virus The antibodies against African swine fever virus can be detected by a quartz crystal microbalance assay with the corresponding antigen immobilized by adsorption on a gold surface of the crystal. The resonance frequency of the piezoelectric crystal is associated with a mass change due to the reaction between the antigen immobilized at the surface and the antibody allowing the detection of the antibody [38]. 2.4.5. Epstein-Barr virus A SPR sensor for antibody against Epstein-Barr virus has been described by Rossi et al. This virus causes infectious mononucleosis and the disease is diagnosed by the corresponding antibody determination. In this work, the antigen used as probe is a special synthetic peptide [13]. 2.4.6. Bovine herpesvirus-1 The Bovine herpesvirus-1 causes an infectious bovine rhinotracheitis which is a highly contagious disease of cattles. In order to determine if the cattles are contaminated, a search of antibodies in milk can be realized. A biosensor assay (Vantix™) has been developed to determine Bovine herpesvirus-1 antibodies in milk samples. The Vantix™ biosensor consists of a working and a reference electrode coated by a conductive polypyrrole polymer and additionally covered by a protective plastic film. The antigen is immobilized onto the working electrode. The test is a sandwich-type immunoassay format with short incubation periods. The sensor detects a change of potential in the presence of 3,3′,5,5′tetramethylbenzidine: positive samples produce a rapid initial increasing of potential difference. This assay comprises two incubation periods of 5 min while still maintaining adequate sensitivity. The short incubation times lead to less non-specific interactions which appeared to be less of a problem compared to ELISA assays. The samples are classified as positive or negative in function of the voltage reading at the end of the test [39].

2.4. Viral infections 2.5. Parasites 2.4.1. Measles The detection of measles-specific IgG using an electrochemical impedimetric immunosensor has been developed. The impedimetric measurement advantages are that the detection is achieved in a labelfree manner and with a limited number of steps. The diagnosis of measles can be done with the determination of IgG. Two tests, two

2.5.1. Echinococcus granulosus Echinococcus granulosus or echinococcus of the dog is a tiny tapeworm which adult parasitizes the small intestine of the dog and whose larval form can develop in humans and causes hydatidosis (echinocossosis). This disease affects the liver but can spread to other

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parts of the body, such as the lungs or the brain. The detection of IgG antibodies specific to Echinococcus granulosus in human serum samples can be performed for the immunodiagnosis of hydatidosis. A microfluidic immunosensor has been developed to determine IgG anti- Echinococcus granulosus. The devise consists of a Plexiglas system with a central channel and a gold electrode. The antigen is immobilized onto the electrode and an HRP labeled anti-IgG and catechol are used for the amperometric detection in the presence of H2O2 [40]. 2.5.2. Babesia bovis Babesia bovis is a protozoan parasite of cattle which occasionally infects humans and causes hemolytic anemia known as babesiosis. An immunosensor with a gold working electrode was proposed. A recombinant version of the C-terminal portion of a protein from Portuguese Babesia bovis is immobilized onto the gold electrode. EIS measurements are performed after incubation of the sample containing the target antibodies [41]. 2.5.3. Schistosoma japonicum Schistosoma japonicum is a parasitic flatworm responsible of schistosomiasis, a disease affecting the urinary tract or the intestines. An amperometric immunosensor has been developed to determine antibodies against Schistosoma japonicum. The immunosensor principle is a sandwich-type format using an HRP labeled anti-IgG and o-aminophenol as substrate [42]. Previously, using the same method, a fluorescent immunosensor has been developed. The substrate of the HRP labeled anti-IgG is 3, 3′, 5, 5′-tetramethylbenzidine. The oxidized product is quantitated fluorimetrically. The detection by fluorescence provides a sensitively ten times higher compared to amperometric detection [43]. 2.5.4. Toxoplasma gondii Toxoplasma gondii, a parasitic protozoan, causes toxoplasmosis. This disease is generally asymptomatic but severe complications are possible in acquired immune deficiency syndrome (AIDS) patients and for the fetus in case of mother contamination. Clinical diagnosis of toxoplasmosis can be realized by detection of antiToxoplasma gondii immunoglobulins. An agglutination-based immunoassay was developed using a piezoelectric quartz crystal as transducer and gold nanoparticles as the support for the antigen immobilization. This point-of care test has shown results comparable to those of the ELISA method [44]. 2.6. Vaccination The presence or absence of antibodies against the pathogen (virus, bacteria, toxin) causing the disease in human serum is useful to determine the need for vaccination or the efficiency of previous vaccination. 2.6.1. Influenza virus An immunosensor based on the modification of a gold microelectrode array by antigens from human influenza virus hemagglutinin was developed. The label free detection is realized by EIS [45]. 2.6.2. Hepatitis B virus A sandwich immunoassay has been realized: the antigen is captured on the surface of magnetic beads. The beads are subsequently incubated with human serum containing the target IgG followed by recognition with AuNPs conjugated anti-IgG. The electrochemical detection (chronoamperometry) procedure is based on the electrocatalytic hydrogen generation from protons. The magnetic beads with immobilized immunocomplex are placed onto the SPE in the presence of 2M HCl. A potential of +1.35V is applied for 1 min

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and then a negative potential of −1.00V for 5 min, and the cathodic current generated is recorded. This procedure allows measuring the AuNPs catalytic properties towards hydrogen evolution [46]. 2.6.3. Tetanus toxin An electrochemical immunosensor for anti-Clostridium tetani antibody determination in serum has been developed. The antigen tetanus toxoid is immobilized on magnetic beads. The sandwich type immunoassay occurs onto the beads in Eppendorf minitubes. The anti-tetani antibody is incubated in the presence of the toxoid functionalized beads and then reacts with HRP-labeled anti-IgG. The beads are spiked onto the SPE for amperometric measurements. HQ served as redox label. The level of anti-Clostridium tetani antibody in guinea pig serum samples is determined with a LOD close to that of ELISA but in a shorter time [47]. Decentralized use outside the laboratory is not feasible because of the many manipulations and successive rinses of magnetic beads. Therefore, another strategy was considered. Rather than immobilizing the toxoid on beads, it can be fixed directly on the surface of the SPE. This second strategy is less sensitive but is easier to be implemented compared to the immunoassay using magnetic beads [48]. 2.6.4. Tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B and hepatitis B A biosensor consisting of an optical array biosensor to determine simultaneously tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B and hepatitis B in sera was developed in order to monitor the efficacy of vaccination. The array utilizes a planar waveguide patterned with an array of immobilized recognition elements (antigens) to capture targets of interest. The antigens are immobilized onto borosilicate microscope slides and the bound targets are detected using labeled “tracer” antibodies and evanescent illumination [49]. 2.7. Cancer Antibodies can be generated due to an immunoresponse against abnormal proteins present in case of cancers. The detection of these proteins can also be made by highlighting of corresponding antibodies. The presence of these antibodies is of importance in tumor diagnosis, prognosis and relapse monitoring [50]. In this context, an immunomagnetic-electrochemiluminescent sensor for p53 antibodies [50] and a photoelectrochemical immunoassay of antibody against tumor-associated carbohydrate antigen [51] have been developed. A mutation of tumor suppressor p53 gene causes the production of an abnormal p53 protein. Antibodies against this protein have been determined according to the following procedure: firstly, a biotinylated antibody anti-p53 is immobilized onto a bead and commercial p53 protein is captured. Then, the sample containing the target antibody is added and the antibody binds the p53 protein at another site. At last an anti-IgG antibody, labeled with ruthenium (II) tris-bipyridal, is added and, when bound to the bead immunocomplex, it generated light in the presence of an excess of tripropylamine [50]. Tumor-associated carbohydrate antigens are often found on the surface of cancer cells. Because of the chemical complexity of glycan chains, it is difficult to determine this antigen. The presence of corresponding antibodies (IgM) can be used for the clinical diagnosis. The sensor is a sandwich-type immunoassay with the use of an anti-IgM HRP conjugated. The graphene/CdSe nanocomposite is used as a sensitive photoelectric interface. The enzyme catalyzes the oxidation of 4-chloro-1-naphthol followed by the precipitation and the photoelectrochemical detection. Ascorbic acid served as a sacrificial electron donor during the photocurrent measurement. The sandwich immunocomplexes increase the steric hindrance and retard the diffusion of ascorbic acid to the photoelectric interface. Finally, after enzymatic biocatalytic precipitation,

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the formation of insoluble and insulating layer introduces a barrier for the electron transfer at the electrode-solution interface and leads to further decrease of the photocurrent [51]. 3. Analytical figures of merit Table 1 summarizes some examples of immunosensors for the detection of antibodies in different samples taking into consideration their analytical characteristics such as the limit of detection (LOD), the analysis time (without antigen immobilization) and the number of steps (without washing steps). As the antibodies are molecules of large size, it is preferable to apply a sandwich-type immunoassay format. Indeed, the steric hindrance is relatively limited. This strategy allows the use of an anti-Ig labeled with an enzyme, usually HRP or AP allowing for high biocatalytic signal amplification. Optical, amperometric or fluorimetric detection is then readily implemented. The test selectivity is increased thanks to the double antigen recognition. The number of steps to perform, however, and the need to add many reagents make sandwichtype immunoassays poor candidates for point of care application. The on-site detection is possible only by applying a cartridge comprising a nanofluidic flow and reservoirs to avoid manipulations by medical doctors or technicians. The repeated incubation steps result in a longer analysis time which implies that the sandwich-type im-

munoassay format should preferably be used in laboratory rather that for point of care situations. Recently, however, D. MartínYerga and A. Costa-García proposed an interesting one-step strategy to perform a sandwich-type immunoassay for the anti transglutaminase determination. The used sensor is an 8-channel SPEs onto which the antigen (transglutaminase) has been immobilized. A mixture of serum containing antibodies of interest, enzyme labeled anti-IgG and the substrate of the enzyme are incubated onto the biosensor. Finally the electrochemical detection is realized by applying amperometry [26]. This strategy has the advantage of limiting the manipulations and enables unqualified personnel to use the test. The principal disadvantage is the long time of incubation (80 min) which prohibits point-of-care use such an immunosensor. Label free immunosensors use generally based on SPR, piezoelectricity or EIS for the detection. If the device is miniaturized, these immunosensors are adapted for on-site applications but they are more sensitive to non-specific adsorptions phenomena particularly if the sample to be analysed is complex (e.g. whole blood). The non-specific adsorption of interfering proteins (e.g. immunoglobulins and other proteins in serum samples) at the surface of the physical transducer is a major trouble causing a non-specific signal which results in a loss of sensitivity. The use of a blocking agent and washing steps are often necessary. The latter can be realized with a buffer solution containing tween 20 [15,39,47], a

Table 1 Selected examples of immunosensors for antibodies determination with electrochemical (EC), optical, or piezoelectricity (piezoelec) detection. The number of the step corresponds to the number of step without the biosensor preparation and washing steps Detection

Sensor

Analyte

Sample

LOD

Time of analysis

Number of step

ref

EC

Ampero Ampero EIS EIS (label free) Ampero Ampero Potentio Ampero Ampero Voltamp Ampero Chrono-ampero SWV DPV EIS DPV Ampero

IgE IgE peanuts

HB HS

30 min 2h

HS HS Milk GPS HS HS HS HS HS HS HS HS HS

1h15min 50 min 15 min 1h 26 min 30 min 30 min 1h 1h 2h 2h 80 min 2h

2 3 3 2 3 2 3 3 3 3 3 3 3 3 3 1 3

[15] [16]

Anti-Salmonella typhi Anti-HIV Anti-Bovine herpesvirus-1 Anti-Clostridium tetani Anti-Echinococcus granulosus Anti-gliadin (IgA, IgG) Anti-gliadin Anti-hepatitis B virus Anti-transglutaminase Anti-transglutaminase Anti-transglutaminase Anti-transglutaminase Anti-transglutaminase

Ampero EIS EIS Ampero (flow) Ampero (flow) SWV DPV EIS DPV Ampero Fluo Fluo UV-vis Fluo

Anti-transglutaminase Anti-Influenza virus Anti-Babesia bovis Anti-Helicobacter pylori Anti-Helicobacter pylori Anti-Helicobacter pylori Anti-Mycobacterium lipoarabinomannan Anti-measles Anti-Pseudorabies virus Anti-Schistosoma japonicum Anti-Schistosoma japonicum Anti-Helicobacter pylori Anti-gliadin Anti-Tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B and hepatitis B Anti-Epstein-Barr virus Anti-Trypanosoma cruzi Anti-Salmonella enteritidis Anti-Salmonella typhi Anti-p53 Anti-Toxoplasma gondii

HS HS BS HS HS HS HS HS/CS SS RS RS HS HS HS

0.09 ng mL−1 0.3 ng mL−1 5 pg mL−1 0.5 ng mL−1 distinguish between + and distinguish between + and distinguish between + and 0.005 IUmL−1 0.091 ng mL−1 9.1 IU mL−1 46 ng mL−1 3 mIU mL−1 distinguish between + and 2.2UmL−1 2.4 IU mL−1 IgG: 2.7 IU mL−1 IgA: 1.7 IU mL−1 390 ng mL−1 1 pg mL−1 0.37 IU mL−1 0.6 IU mL−1 0.5 IU mL−1 5.3 ng mL−1 10 ng mL−1 1/1000 50 ng mL−1 4.5 ng mL−1 0.17 UmL−1 1 μg mL−1 sub-μg mL−1

30 min 20 min 1h 25 min 30 min 25 min 30 min 40 min 1h 80 min 60 min 27 min 1h 78 min

3 2 2 3 3 3 3 2 3 3 3 3 3 3

[25] [45] [41] [28] [27] [29] [34] [35] [37] [42] [43] [30] [21] [49]

HS HS egg yolk HS HS RS

0.2 ng mL−1 distinguish between + and distinguish between + and 1:204800 dilution 10 pg mL−1 1:5500 dilution

15 min 20 min 10 min 31 min 30s

1 1 1 1 3 1

[13] [53] [33] [32] [50] [44]

Optical

Piezoelec

SPR SPR SPR SPR Electro-chem QCM

[31] [36] [39] [47] [40] [52] [20] [46] [18] [24] [17] [26] [23]

Ampero: amperometry; EIS: electrochemical impedance spectroscopy; Potentio: potentiometry; Voltamp: voltamperometry; Chronoamp: chronoamperometry; SWV: square wave voltammetry; DPV: differential pulse voltammetry; Fluo: fluorescence; UV-visible: UV visible; Electrochem: electrochemiluminescence; QCM: quartz crystal microbalance; HB: human blood; HS: human serum; GPS: guinea pig serum; BS: bovine serum; SS: swine serum; RS: rabbit serum.

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Tris–HCl buffer [31] or a phosphate buffered saline [40]. Different blocking agents are traditionally used such as bovine serum albumin (1% [15,40,49], 2% [22,51,50], 3% [31]) or casein 2% [16,52]. Gelatine can be added to the sample dilution buffer to prevent nonspecific signal generation when analyzing serum samples [39]. The blocking agents work by covering the unoccupied areas of the transducer and prevent the adsorption of other proteins for the analyzed sample. The blocking of the surface can be generally realized in advance but the requirement of washing steps make the immunosensor less convenient for a point of care application. It is essential for an immunosensor dedicated to a medical application that the sensitivity is sufficient to quantify antibodies to physiological or pathological levels. Therefore, an application on real samples is recommended. Along with sufficient sensitivity, accuracy must be good. The latter can be evaluated by checking that there is a good correlation between the results obtained with the developed immunosensor and those obtained with an ELISA test. This correlation has been demonstrated by several teams [20,29,52].

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