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Electrochemical magneto immunosensor for the detection of anti-TG2 antibody in celiac disease
Abstract
An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H2O2 as substrates by square wave voltammetry was correlated with the ATG2. Graphite-epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%.
... Another ECIS based on magnetic beads was developed using the same principle as described above [51], only with HRP-labeled anti-human IgA, o-phenilendiamine and H 2 O 2 as substrates, and square wave voltammetric detection [52]. Intra-and inter-assay precisions were 10-13 and 14-16 %, respectively, and recoveries between 94 and 123 %. ...
... Therefore, the assessment of antibodies against oTG2 presents higher diagnostic accuracy, especially to monitor adherence to the glutenfree diet [56]. As opposed to the other ECIS that were based on the closed conformation of TG2 [46][47][48][49][50][51][52][53][54][55], two biosensors used oTG2 as specific antigen [57,58]. A gold nanoparticle-functionalized glassy carbon electrode was modified with an 11-mercaptoundecanoic acid SAM to immobilize oTG2 onto the surface [57]. ...
... Although magnetic particles are not a typical reagent useful for evocation of a measurable signal, they can be easily applied for separation processes and improve analytical properties by, e.g., analyte isolation or the possibility of reusing expensive reagents [72,73]. For example, magnetic beads coated with TG2 were also chosen for the construction of immunosensors for the detection of TGAs [51,52]. The determination of gliadin contaminations in gluten-free foods for CD patients is a further adoption of magnetic immunosensors [74,75]. ...
Celiac disease (CD) is an autoimmune enteropathy initiated and sustained by the ingestion of gluten in genetically susceptible individuals. It is caused by a dysregulated immune response toward both dietary antigens, the gluten proteins of wheat, rye, and barley, and autoantigens, the enzyme tissue transglutaminase (TG2). The small intestine is the target organ. Although routine immunochemical protocols for a laboratory diagnosis of CD are available, faster, easier-to-use, and cheaper analytical devices for CD diagnosis are currently unavailable. This review focuses on biosensors, consisting of a physicochemical transducer and a bioreceptor, as promising analytical tools for diagnosis of CD and other diseases. Examples of recently developed biosensors as well as expectations for future lines of research and development in this field are presented.
... When diagnosing CD clinically, the most common serological test is an enzyme-linked immunosorbent assay (ELISA) with optical detection [4]. In ELISA, antibodies are identified by several steps (incubation, washing, and separation procedures) and require complex equipment [5,6]. ...
... Other attributes include suitable electroanalytical qualities, simplicity and affordability, design versatility, small dimensions, and the potential for inclusion in portable systems [9]. Recently, different surface chemistry methods, mainly anti-gliadin and anti-tTG, have been used to detect autoantibodies specific to CD [3][4][5][6][7][8][9]. ...
A nanohybrid-modified glassy carbon electrode based on conducting polypyrrole doped with carbon quantum dots (QDs) was developed and used for the electrochemical detection of anti-tissue transglutaminase (anti-tTG) antibodies. To improve the polypyrrole conductivity, carrier mobility, and carrier concentration, four types of carbon nanoparticles were tested. Furthermore, a polypyrrole-modified electrode doped with QDs was functionalized with a PAMAM dendrimer and transglutaminase 2 protein by cross-linking with N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC). The steps of electrode surface modification were surveyed via electrochemical measurements (differential pulse voltammetry (DPV), impedance spectroscopy, and X-ray photoelectron spectroscopy (XPS)). The surface characteristics were observed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and contact angle measurements. The obtained modified electrode exhibited good stability and repeatability. DPV between − 0.1 and 0.6 V (vs. Ag/AgCl 3 M KCl reference electrode) was used to evaluate the electrochemical alterations that occur after the antibody interacts with the antigen (transglutaminase 2 protein), for which the limit of detection was 0.79 U/mL. Without the use of a secondary label, (anti-tTG) antibodies may be detected at low concentrations because of these modified electrode features.
Graphical Abstract
... Hereby, this operation had an influence on the reduction in biological matrix effect and the possibility of interferences due to the presence of serum components. Electrochemical magnetoimmunosensors recently became a useful tool for the determination of analytes important in the field of clinical, food and environmental analysis [31][32][33][34][35]. ...
... Thus, it is important to highlight that electrochemical magnetoimmunosensors developed were characterized by high selectivity. Electrochemical magnetoimmunosensors recently became a useful tool for the determination of analytes important in the field of clinical, food and environmental analysis [31][32][33][34][35]. Most of them were constructed in sandwich configuration using the enzyme-labelled antibodies. ...
The simultaneous detection of atherosclerotic cardiovascular disease (ACSVD) biomarkers was recently of great scientific interest. In this work, magnetic beads-based immunosensors for the simultaneous detection of low density lipoprotein (LDL) and malondialdehyde-modified low density lipoprotein (MDA-LDL) were presented. The approach proposed was based on the formation of two types of specific immunoconjugates consisting of monoclonal antibodies: anti-LDL or anti-MDA-LDL, together with redox active molecules: ferrocene and anthraquinone, respectively, coated on magnetic beads (MBs). The decrease in redox agent current in the concentration range: 0.001–1.0 ng/mL for LDL and 0.01–10.0 ng/mL for MDA-LDL, registered by square wave voltammetry (SWV), was observed upon the creation of complex between LDL or MDA-LDL and appropriate immunoconjugates. The detection limits of 0.2 ng/mL for LDL and 0.1 ng/mL for MDA-LDL were estimated. Moreover, the results of selectivity against the possible interferents were good, as human serum albumin (HSA) and high density lipoprotein (HDL), stability and recovery studies demonstrated the potential of platform proposed for early prognosis and diagnosis of ASCVD.
... The GEC-E working electrodes were prepared in our laboratory following a protocol previously reported [38]. Briefly, a copper tube (3 mm internal diameter, 5 mm external diameter and 17 mm height) was inserted into a cylindrical acrylic sleeve body (5 mm internal diameter, 8 mm external diameter and 20 mm height). ...
... Comparison between electrochemical and optical methods[38] based on peroxidase-inhibition for glyphosate detection. ...
Glyphosate is the most widely used herbicide in Argentina. The information about this herbicide is controversial as some organisms classify it as dangerous and others as safety. Moreover, the world regulations are very different depending on country. However, many studies had reported its presence in numerous sample types, and also, had demonstrated adverse effects on environmental and human health. In the present work, two electrochemical screening methods based on horseradish peroxidase (HRP) inhibition have been developed for the glyphosate detection. In one of them, the working electrode was built in our laboratory with a graphite-epoxy compound mixture (GEC-E). While the other is based on a biosensor built with commercial screen-printed electrodes (SPE), for which on the working electrode, the HRP enzyme was immobilized by using the affinity reaction between streptavidin and biotin-peroxidase. The detection electrochemical technique was square-wave voltammetry (SWV). When glyphosate inhibits the activity of the soluble or immobilized enzyme, a decrease in the signal is generated at the electrode or biosensor, respectively, and this decrease is proportional to the amount of glyphosate in the sample. The linear range obtained was from 0.16 to 500 ng L⁻¹ glyphosate (R² = 0.9727) for the soluble HRP-based inhibition method with a limit of detection (LOD) of 0.085 ng L⁻¹. While the range was from 0.08 to 11 μg L⁻¹ glyphosate (R² = 0.9799) for immobilized HRP-based inhibition method with a LOD of 45 ng L⁻¹. For the screening detection of glyphosate, the decision limits (CCα) and detection capabilities (CCβ) were 115 and 130 ng L⁻¹ for the soluble HRP-based inhibition method and 111 and 122 ng L⁻¹ for biosensor, respectively. Recoveries from 71% to 96% and from 79% to 120% were obtained by two electrochemical methods from groundwater and superficial water with high organic matters spiked with glyphosate, respectively. Therefore, both electrochemical methods allow determining the glyphosate concentration at trace levels in accordance with the strictest European Parliament regulation (100 ng L⁻¹). The soluble HRP-based inhibition method shown better performance than biosensor, but the latter due to its simplicity, stability and portability could be used in the field monitoring or detection of this herbicide in the exposed areas.
... Immunosensors, a special type of biosensors based on the interaction between antigens and antibodies, have widespread applications in food security (J. Dong et al. 2013;Chu and Wen 2013), disease diagnosis (Liu et al. 2008;Montrose et al. 2013;Kergaravat et al. 2013), and environmental monitoring (Marzo et al. 2013). Since the first immunosensor was reported in 1959 (Yalow and Berson 1959), large numbers of immunosensors using electrochemical (Kergaravat et al. 2013;Wu, Liu, and He 2011;H. ...
... Dong et al. 2013;Chu and Wen 2013), disease diagnosis (Liu et al. 2008;Montrose et al. 2013;Kergaravat et al. 2013), and environmental monitoring (Marzo et al. 2013). Since the first immunosensor was reported in 1959 (Yalow and Berson 1959), large numbers of immunosensors using electrochemical (Kergaravat et al. 2013;Wu, Liu, and He 2011;H. Dong et al. 2006), optical (Hong and Kang 2006;Ge et al. 2013), and piezoelectric (Guo et al. 2012) methods have been constructed for proteins and biomarkers. ...
A novel amperometric immunosensor was fabricated using an electropolymerized conducting polymer composite of pyrrole and 4-(3-pyrrolyl) butyric acid as the probe immobilization matrix. The biomolecule was immobilized via covalent bonding on the polymer surface with a high density of carboxyl groups. Instead of directly attaching the probe to the polymer surface, the streptavidin-biotin affinity complex was introduced to improve the bioactivity, orientation, and the amount of immobilized material. Polyvinyl alcohol was employed to improve the hydrophilicity of the polymer surface and further decrease the nonspecific protein adsorption. As a result, high sensitivity and specificity were achieved with a wide linear dynamic range. Using mouse immunoglobulin G as a model protein, the immunosensor exhibited a linear relationship between 7 ng/mL and 20 µg/mL with a limit of detection of 7 ng/mL.
... Lately electrochemical immunosensors based on the capture of ATG2 by immobilization of TG2 onto electrode surface and magnetic beads (MB) were reported [11][12][13]. ...
... This value is lower than its corresponding obtained by the mean plus two-fold standard deviation of the negative sample (9.8 U, n = 19) [34] and another the cut-off value obtained by one-tailed t test at a 99% confidence level (11 U) [35]. In addition the cut-off value from ROC analysis is lower than its corresponding (16.95 U) obtained by using electrochemical magneto immunosensor recently reported for ATG2 detection [13]. Taking into consideration these results, a cut-off value of 8.1 U ATG2 from ROC analysis was chosen for further studies. ...
A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit.
... One another group developed an indirect electrochemical magneto immunosensor for anti-transglutaminase enzyme (ATG2) assay in serum samples of patients with celiac disease. First, the TG2 was covalently immobilized on magnetic beads (MBs) and following addition of ATG2, the Peroxidase-labeled antibody (anti Ig-HRP) was incorporated for the revelation and the analytical signal was detected by SWV [50]. ...
This review (with 77 refs.) describes the progress that has been made in biosensors for the detection of autoimmune diseases, mainly via detection of autoantibodies. In addition, specific proteins, cytokines and ions have also been introduced as promising diagnostic biomarkers. Following an introduction into the various kinds of autoimmune diseases, we first discuss the state of the art in respective electrochemical biosensors and nanobiosensors (with subsections on amperometric, impedimetric, voltammetric and photoelectrochemical methods). The next large chapter covers optical methods (with subsections on electrochemiluminescence, fluorescence and surface plasmon resonance). We then make a critical comparison between commercially available kits used for detection of autoimmune diseases with the established biosensors. Several Tables are also presented that give an overview on the wealth of methods and nanomaterials. Finally, in the conclusion part, we summarize the current status, addresse present issues, and give an outlook on potential future opportunities.
Graphical abstractSchematic representation of various developed optical and electrochemical biosensors and nanobiosensors for rapid detection of autoimmune diseases nanobiosensors for rapid detection of autoimmune diseases which could significantly prevent irreversable tissue damages and increse the quality of life in these patients
... In particular, the DL was calculated as DL = 3(SD/m), where SD is the standard deviation of the y-axis intercept and "m" is the slope (sensitivity) of the regression line, giving a result of 1.8 ng mL −1 . The low detection limit, the wide linear range, and excellent reproducibility indicate that the TGNEE immunosensor offers analytical performances that are competitive with those achievable by other electroanalytical sensors developed for the same goals [11][12][13][14]. On the other hand, we wish to point out that the ECL sensor developed recently is characterized by a slightly lower detection limit (0.47 ng mL −1 vs. 1.8 ng mL −1 ), wider linear range, and higher sensitivity; however, it has the disadvantage of relying on a more complex analytical procedure and the use of more sophisticated and expensive equipment [42]. ...
Celiac disease (CD) is a gluten-dependent autoimmune disorder affecting a significant percentage of the general population, with increasing incidence particularly for children. Reliable analytical methods suitable for the serological diagnosis of the disorder are urgently required for performing both the early diagnosis and the follow-up of a patient adhering to a gluten-free diet. Herein we report on the preparation and application of a novel electrochemical immunosensor based on the use of ensembles of gold nanoelectrodes (NEEs) for the detection of anti-tissue transglutaminase (anti-tTG), which is considered one reliable serological marker for CD. To this end, we take advantage of the composite nature of the nanostructured surface of membrane-templated NEEs by functionalizing the polycarbonate surface of the track-etched membrane with tissue transglutaminase. Incubation of the functionalized NEE in anti-tTG samples results in the capture of the anti-tTG antibody. Confirmation of the recognition event is achieved by incubating the NEE with a secondary antibody labelled with horseradish peroxidase (HRP): in the presence of H2O2 as substrate and hydroquinone as redox mediator, an electrocatalytic current is indeed generated whose increment is proportional to the amount of anti-tTG captured from the sample. The optimized sensor allows a detection limit of 1.8 ng mL⁻¹, with satisfactory selectivity and reproducibility. Analysis of serum samples from 28 individuals, some healthy and some affected by CD, furnished analytical results comparable with those achieved by classical fluoroenzyme immunoassay (FEIA). We note that the NEE-based immunosensor developed here detects the IgG isotype of anti-tTG, while FEIA detects the IgA isotype, which is not a suitable diagnostic marker for IgA-deficient patients.
... 29 sera samples from clinically conrmed cases of celiac disease and 19 negative control sera were tested with successful results. 169 Giannetto et al. 170 prepared an amperometric immunosensor based on the covalent immobilization of tTG in its open conformation (open-tTG) for the determination of anti-tTG IgA and IgG antibodies in human serum. A glassy carbon electrode electrochemically functionalized with gold nanoparticles and subsequently modied with an 11-mercaptoundecanoic acid self-assembled monolayer was used for the covalent anchoring of tTG. ...
Autoantibodies (AAbs) are antibodies produced against our own cells or tissues either providing a first defense against infections or indicating the presence of pathological processes. They are not only able to inform on diseases evolution but also to predict some illnesses well in advance. Currently, the evaluation of the number and type of formed AAbs is employed to assess the risk, rate, severity and progression of autoimmune and cancer diseases, and to help to find therapies to prevent or mitigate these illnesses impact. Conventional methods for the determination of AAbs generally suffer from low sensitivity, time-consuming and laborious methodologies and need specialized technicians and well-equipped labs. Consequently, seeking for new methodologies for the rapid and low-cost screening of AAbs is of great interest for global health because it would shorten the delay between sample collection and diagnosis and improve the introduction of modern diagnostics into the developing world. Electrochemical biosensors are considered as a promising alternative to conventional techniques for the determination of clinical biomarkers due to their simplicity of use, low cost, high sensitivity, multiplexing abilities or compatibility with microfabrication and point of care testing. This review is focused on the critical discussion of selected electrochemical biosensors described up to date for the determination of AAbs related to autoimmune diseases and several types of cancer. An overview pointing out future directions in this field is also provided.
... The use of magneto-electrodes for the electrochemical detection of various kinds of compounds was significantly improved as novel sensing strategy. Taking advantage of the ability of magneto electrochemical sensors to capture different functionalized magnetic beads as well as Mag- MIPs from solution makes it an excellent devise for determination of molecules and biomolecules in recent years [69,82,83]. This sensing strategy is based on the impregnation of an external magnet onto the electro- chemical sensors, which leads to easy collection of MagMIPs from the solution containing the analyte as indicated in Scheme 4. ...
This review focuses on the recent achievement during period of 2013–2018 related to the electrochemical sensors based on molecularly imprinted polymers (MIPs) combined with nanomaterials for various kinds of applications. MIPs based electrochemical sensors have found a great interest due to their high stability, short time required for electropolymerization, and high specificity towards the target analyte. The sensitivity is considered as one of the important parameter in electrochemical sensing strategies that should be improved by the combination of highly conductive nanomaterials with selective MIPs. In general, the most employed nanomaterials are magnetic nanoparticles, gold nanoparticles (AuNPs), carbon nanotubes and graphene. This review discusses the main current achievement as well as the current challenges regarding the development of biomimetic sensors in electroanalysis.
... Another ECIS based on magnetic beads was developed, which used HRP-conjugated anti-human IgA as a label, o-phenylenediamine and H 2 O 2 as substrates, and square wave voltammetric studies for detection (Adornetto et al., 2012;Kergaravat et al., 2013). Intra and inter-assay precisions were found to be 10-13% and 14-16%, with recoveries between 94% and 123%, respectively. ...
Celiac disease (CD) is an intestinal issue activated by the inappropriate immune reaction towards gluten protein of wheat, rye, barley, oats, and autoantigen, tissue transglutaminase. Regardless of the accessibility of immunochemical conventions for research facility analysis of CD, there is as yet a need of speedier, less expensive, and simpler devices for diagnosing CD. This review concentrates on progresses in biosensors for diagnosing CD in perspective of the scaled down hardware, multianalyte discovery and low sample volume necessity. Various recently developed biosensors in this field are presented.
... Kergaravat et al. [33] developed an electrochemical magneto immunosensor for the detection of TGA Type 2. TG Type 2 was immobilized covalently on magnetic beads (MB) modified with tosyl groups and the immunological reaction was performed on these MB-TG2 beads as solid support. Anti-human IgA(α-chain specific)-HRP was used as receptor and signal label in a sandwich-type arrangement. ...
Celiac disease is a chronic gluten-initiated autoimmune disorder that predominantly damages the mucosa of the small intestine in genetically-susceptible individuals. It affects a large and increasing number of the world’s population. The diagnosis of this disease and monitoring the response of patients to the therapy, which is currently a life-long gluten-free diet, require the application of reliable, rapid, sensitive, selective, simple, and cost-effective analytical tools. Celiac disease biomarker detection in full blood, serum, or plasma offers a non-invasive way to do this and is well-suited to being the first step of diagnosis. Biosensors provide a novel and alternative way to perform conventional techniques in biomarker sensing, in which electrode material and architecture play important roles in achieving sensitive, selective, and stable detection. There are many opportunities to build and modify biosensor platforms using various materials and detection methods, and the aim of the present review is to summarize developments in this field.
... Electrochemical techniques were performed with a voltammetric analyzer Epsilon BAS, Bioanalytical Systems Inc. (West Lafayette Indiana, USA) with a three electrode system based on graphite-epoxy composite (GEC) as working electrodes [16]; platinum as auxiliary electrode and Ag/AgCl in 3 mol L −1 KCl solution as reference electrode (Eref) (Orion 92-02-00). All potentials are given versus Eref. ...
This work presents the development of a sensitive and friendly environment electrochemical methodology with high analytical performance for quinolone detection. The method is based on square-wave anodic stripping voltammetry which is simple and reliable for determination of quinolone family on the bismuth film electrode (BiFE). The deposition potential, deposition time, buffer solution pH, voltammetry detection technique and bismuth concentration at fixed quinolone concentration (1 μg L⁻¹) were evaluated by fractionated factorial and central composite designs. The high cross-reactivity obtained in the evaluation of different quinolones showed that this methodology could be applied to quinolone family with limits of detection (LOD) close to 0.5 ng L⁻¹. Since is widely known that quinolones can reach aquatic ecosystems, the developed electroanalytical method was applied in the monitoring of moxifloxacin (MOXI) concentrations in ecotoxicity studies by means of acute toxicity test using two cladoceran species as biological models: Daphnia magna and Ceriodaphnia dubia. As a result of ecotoxicity, both species showed comparable sensitivities at MOXI. Toxicity studies of quinolones on aquatic organisms are very limited so the effects of MOXI to cladoceran species are highlighted. The interaction of both disciplines –Chemistry and Ecology- would allow knowing of global behavior of quinolones in natural environments, from an integrative perspective.
... Sensitive and accurate biosensing systems for these biomarkers can not only significantly aid in the early diagnosis and clinical management of AIDs but also help to establish therapeutic strategies [4]. Biosensors for the detection of autoantibodies specific to AIDs, including antiphospholipid syndrome (APS) [91][92][93][94], rheumatoid arthritis (RA) [95][96][97][98][99], systemic lupus erythematosus (SLE) [100][101][102][103][104][105][106][107], multiple sclerosis (MS) [108][109][110][111], and celiac disease (CD) [112][113][114][115][116][117][118][119][120][121][122][123][124], are now constructed based on diverse techniques with many advantages, including high sensitivity and easy operation. ...
Biosensors for proteins have shown attractive advantages compared to traditional techniques in clinical laboratory diagnosis. In virtue of modern fabrication modes and detection techniques, various immunosensing platforms have been reported on basis of the specific recognition between antigen-antibody pairs. In addition to profit from the development of nanotechnology and molecular biology, diverse fabrication and signal amplification strategies have been designed for detection of protein antigens, which has led to great achievements in fast quantitative and simultaneous testing with extremely high sensitivity and specificity. Besides antigens, determination of antibodies also possesses great significance for clinical laboratory diagnosis. In this review, we will categorize recent immunosensors for proteins by different detection techniques. The basic conception of detection techniques, sensing mechanisms, and the relevant signal amplification strategies are introduced. Since antibodies and antigens have an equal position to each other in immunosensing, all biosensing strategies for antigens can be extended to antibodies under appropriate optimizations. Biosensors for antibodies are summarized, focusing on potential applications in clinical laboratory diagnosis, such as a series of biomarkers for infectious diseases and autoimmune diseases, and an evaluation of vaccine immunity. The excellent performances of these biosensors provide a prospective space for future antibody-detection-based disease serodiagnosis.
... Other analytical characteristics such as linear range or precision are similar to those obtained by the sensor developed in this work, but the estimated limit of detection (1.0 U/mL) is lower due to the higher sensitivity and the low non-specific adsorption. There are other works that describe the development of immunosensors for the detection of anti-tTG IgA antibodies [51][52][53][54][55] , but its analytical comparison is complicated due to using different concentrations units. ...
In this work, we describe the use of a magnetoelectrochemical support for screen-printed electrodes to improve the anodic stripping voltammetry of cadmium due to the generated magnetohydrodynamic (MHD) effect. To create a significant MHD effect, Fe(iii) was added at mM concentrations to the solution. The reduction of Fe(iii) simultaneously with the cadmium deposition on the electrode surface allowed the production of a high cathodic current, which generated a large Lorentz force capable of exerting a convective effect on the solution in the presence of the magnetic field. This convective effect allowed the increase in the mass transfer in the quiescent solution, enhancing the deposition of cadmium as observed by an increased stripping peak current. The optimized method was applied to the detection of CdSe/ZnS quantum dots (QDs) in solution. Using the magnetoelectrochemical support, we were able to detect extremely low concentrations of QDs, with a detection limit of 100 amol of QDs (in particle number). The great performance shown by this system was evaluated in biosensing applications. Firstly, detection of biotin was carried out using a competitive bioassay between biotin and QD-labelled biotin, obtaining good analytical results (0.6 × 10(-10) M as the limit of detection). Then, the magnetoelectrochemical support was tested in a more complex biosensor for the determination of anti-transglutaminase IgA antibodies, a celiac disease biomarker. This work shows that the improvement in the metal electrodeposition caused by the MHD effect can be used successfully for the development of disposable electrochemical biosensors with great performance using screen-printed electrodes.
... Esta nueva tecnología, basada en la utilización de nuevos materiales como las nanopartículas y puntos cuánticos, disminuye el tiempo del laboratorista, menor uso de reactivos, son baratos, de fácil interpretación y portabilidad (2) . Los anticuerpos que detectan la IgA ATG tienen sensibilidad 100%, especificidad 84% y excelente correlación con las pruebas de ELISA (39,49) . Se espera en un futuro próximo la desaparición de las pruebas de ELISA. ...
La enfermedad celiaca (EC) es una afección autoinmune multisistémica inducida por el gluten en personas genéticamente susceptibles. La presentación clínica clásica es con un síndrome diarreico crónico, aunque existen formas atípicas y silentes, lo cual dificulta el diagnóstico. El mismo se basa en la detección de diversos autoanticuerpos, sobre todo la IgA contra la transglutaminasa tisular, aunque la prueba de oro es el estudio anatomopatológico de biopsias duodenales obtenidas por endoscopia digestiva alta. La ausencia del gen HLA es una prueba que se utiliza para descartar la EC. La respuesta a la dieta sin gluten confirma esta afección, que de no diagnosticarse oportunamente y tratarse en forma adecuada puede llevar a complicaciones graves.
... Recently, the performance of magnetic electrodes in electrochemical methods has been improved by measuring different combinations. Electrochemical magnetic sensors are capable of capturing different functionalized magnetic beads from a solution, which make them sensors for determining molecules and biomolecules [35][36][37][38]. ...
The electrochemical behavior of tramadol (TRA) on magneto layer double hydroxide (LDH)/Fe3O4@glassy carbon electrode (LDH/Fe3O4@GCE) was evaluated. Some theoretical thermodynamic and kinetic parameters were also determined using chronoamperometric and voltammetric methods. The ability of the modified electrode to determine trace amounts of TRA was studied using differential pulse voltammetry (DPV) as a sensitive electrochemical method. For characterizing and investigating the performance of LDH/Fe3O4@GCE, various methods including scanning electron microscopy (SEM), X-ray diffraction (XRD), cyclic voltammetry (CV), and DPV were used. The effect of pH, scan rate, and time on the voltammetric response of TRA were investigated. Under the optimized conditions, the anodic peak current was linear for the concentration of TRA in the range 1.0–200.0 μmol L−1 with the detection limit of 3.0 × 10−1 μmol L−1. This method was also successfully used to detect the concentration of TRA in human serum and urine samples.
... An electrochemical magnetoimmunosensor was also developed for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease [24]. The approach involved the selective capture of ATG2 onto transglutaminase enzyme (TG2) covalently immobilized on tosyl activated-MMPs and their labeling using HRP-conjugated secondary antibodies. ...
Ultrasensitive biosensing is currently a growing demand that has led to the development of numerous strategies for signal amplification. In this context, the unique properties of magnetic particles; both of nano- and micro-size dimensions; have proved to be promising materials to be coupled with disposable electrodes for the design of cost-effective electrochemical affinity biosensing platforms. This review addresses, through discussion of selected examples, the way that nano- and micro-magnetic particles (MNPs and MMPs; respectively) have contributed significantly to the development of electrochemical affinity biosensors, including immuno-, DNA, aptamer and other affinity modes. Different aspects such as type of magnetic particles, assay formats, detection techniques, sensitivity, applicability and other relevant characteristics are discussed. Research opportunities and future development trends in this field are also considered.
... Celiac disease can be diagnosed in blood serum samples by detecting three types of specific biomarkers, including anti-transglutaminase IgA autoantibodies (anti-TG2), anti-endomysial autoantibodies, and Ab against deaminated forms of gliadin (one of the proteins composing cereal gluten). Kergaravat et al. reported an electrochemical magnetoimmunosensor for the detection of anti-TG2, which is crucial for the diagnosis and monitoring of celiac disease (Kergaravat et al. 2013). In this case, transglutaminase Ag was immobilized on tosyl-activated MP. ...
Electrochemical magnetoimmunosensors combine a number of issues that guarantee extremely high performance and also compatibility with the study of complex sample matrices. First, analyte immunocapture exploits the high affinity and specificity of antibodies. Second, magnetic particles (MP) provide faster and more efficient immunocapture than binding on two-dimensional structures, separation from nontarget sample components, and concentration of the target analyte. Finally, electrochemical detection supplies sensitivity and fast signal generation using robust and potentially miniaturized measurement equipment and transducers. On the contrary, MP handling is slightly more complex for end-users and more difficult to integrate in point-of-care devices than the manipulation of a classical biosensor. Attempts have been made to automate immunomagnetic binding, and the first robotized systems and platforms for the fluorescent and spectrophotometric detection of magnetoimmunoassays have already reached the market. Among the different types of electrodes available, screen-printed electrodes (SPE) stand out because of their low production cost and yet acceptable performance and interdevice reproducibility, which make them an excellent choice for analytical applications. In addition, each SPE entails a whole electrochemical cell stamped on a planar physical substrate, which makes it possible detection in small volumes and is especially favorable for the magnetic confinement of MP and the integration of microfluidic structures. In this article, we discuss the advantages obtained by using SPE and MP for the production of electrochemical magnetoimmunosensors and the clues for the successful development of such devices. We then revise some of the most outstanding works published in the literature.
... 12 From these basis, it is evident that the development of a IgG-based diagnostic method with improved sensitivity and specificity is highly required. Many trials have been made to develop variety of electrochemical methods [17][18][19][20][21][22] but, so far, no unique confirmatory serological method has been adopted for practical use. Accordingly, we propose herein a novel anti-tTG immunosensor utilizing an original electrogenerated chemiluminescence (ECL) detection strategy based on nanoelectrode ensembles (NEEs). ...
We report here the design of a novel immunosensor and its application for celiac disease diagnosis, based on electrogenerated chemiluminescence (ECL) readout and using membrane-templated gold nanoelectrode ensembles (NEEs) as a detection platform. An original sensing strategy is presented by segregating spatially the initial electrochemical reaction and the location of the immobilized biomolecules where ECL is finally emitted. The recognition scaffold is the following: tissue transglutaminase (tTG) is immobilized as a capturing agent on the polycarbonate (PC) surface of the track-etched templating membrane. It captures the target tissue transglutaminase antibody (anti-tTG), and finally allows the immobilization of a streptavidin-modified ruthenium-based ECL label via reaction with a suitable biotinylated secondary antibody. The application of an oxidizing potential in a tri-n-propylamine (TPrA) solution generates an intense and sharp ECL signal, suitable for analytical purposes. Voltammetric and ECL analyses evidenced that the ruthenium complex is not oxidized directly at the surface of the nanoelectrodes, instead ECL is generated only following the TPrA oxidation which produces the TPrA●+ and TPrA● radicals. With NEEs operating under total overlap diffusion conditions, high local fluxes of these reactive radicals are produced by the nanoelectrodes in the immediate vicinity of the ECL labels, so that they efficiently generate the ECL signal. The radicals can diffuse over the short distance (< 200 nm) required for their reaction with the 〖Ru(bpy)〗_3^(2+) label. In addition, the ECL emission is obtained by applying a potential of 0.88 V vs Ag/AgCl, which is about 0.3 V lower than when ECL is initiated by the electrochemical oxidation of 〖Ru(bpy)〗_3^(2+) (i.e. 1.2 V vs. Ag/AgCl). The immunosensor provides ECL signals which scale with anti-tTG concentration with a linearity range between 1.5 ng.mL-1 and 10 µg.mL-1 and a detection limit of 0.5 ng.mL-1. The sensor was finally applied to the analysis of anti-tTG in human serum samples, showing to be suitable to discriminate between healthy and celiac patients.
... Recently, using magneto electrodes in electrochemical by sensing various compounds has been improved. The ability of magneto electrochemical sensors to capture different functionalized magnetic beads from a solution has made these sensors an excellent devise to determine molecules and biomolecules [30][31][32][33]. ...
An electrochemical magneto Au nanoparticles/carbon paste electrodes (MAuNP/CPE) which is used for the determination of acetaminophen (AC) in real samples was developed. Initially, Au nanoparticles were immobilized at the surface of Fe3O4 (AuNPs@Fe3O4), which was used as a sorbent for capturing AC molecules. After adding AuNPs@Fe3O4 to the AC solution and stirring for 20 min, the AuNPs@Fe3O4 was gathered on the magneto electrode based on its magnetic field. The AC molecules which became adsorbed at AuNPs@Fe3O4 were analyzed by differential pulse voltammetry (DPV). For characterization and investigation of the performance of AuNPs@Fe3O4 and MAuNPs/CPE, various methods, including scanning electron microscopy, X-ray diffraction, UV–Vis spectroscopy, electrochemical impedance spectroscopy, cyclic voltammetry and DPV were used. Under the optimized conditions, the anodic peak current was linear to the concentration of AC in the range of 0.1 to 70.0 μmol L− 1 with the detection limit of 4.5 × 10− 2 μmol L− 1. This method was also successfully used to detect the concentration of AC in pharmaceutical formulations and human serum samples. In addition, the proposed magneto sensor exhibited good reproducibility, long-term stability and fast current response.
... Recently, using magneto electrodes in electrochemical by sensing various compounds has been improved. The ability of magneto electrochemical sensors to capture different functionalized magnetic beads from a solution has made these sensors an excellent devise to determine molecules and biomolecules [30][31][32][33]. ...
... These results and analytical characteristics are comparable to those obtained by other electrochemical immunosensors using similar detection platforms but with longer assay times and performing a blocking step [30,40,41]. In other cases, where shorter analysis times are achieved, the procedure is more complicated with several steps [42][43][44]. Only a blocking-free strategy was published by Pividori et al. [45] using graphite-epoxy composite electrodes, but a complex methodology with several time-consuming (20 min) washing steps to prevent nonspecific adsorption is carried out. ...
A blocking-free one-step immunosensing strategy using 8-channel screen-printed arrays for the detection of anti-transglutaminase IgA antibodies, celiac disease biomarkers, was developed. A simple but novel immobilization approach to efficiently modify the surface of screen-printed electrodes with a recognition element was employed in order to minimize the non-specific adsorption on the electrode surface, and the optimization of a methodology without a blocking step was carried out. After the functionalization of the electrode surface with tissue-transglutaminase, two different immunoassays, using multi-step and one-step strategies, were optimized. Serum controls from a commercial ELISA kit, anti-human IgA labelled with biotin and streptavidin labelled with CdSe/ZnS quantum dots were employed as bioreagents for the immunoassay. Screen-printed arrays were used as the solid support for the immunosensor and the detection of Cd(II) was performed in situ by anodic stripping voltammetry after an acid attack of the QDs. The electrochemical response from Cd(II) was correlated with the anti-transglutaminase IgA antibodies concentration. The analytical characteristics obtained for the multi-step and one-step electrochemical immunosensors allow to discriminate between positive and negative sera controls, establishing this biosensor as an useful tool for the determination of celiac disease biomarkers.
... A second electrochemical magneto immunosensor for the detection of TGAs was presented by Kergaravat et al. [21]. Two electrochemical cells were tested. ...
Celiac disease is a permanent intolerance to gluten proteins of wheat, rye, barley, and oats in genetically susceptible individuals. The clinical picture is characterized by inflammation and damage of the small intestinal mucosa and malabsorption of essential nutrients. Therapeutically, a lifelong strict gluten-free diet is necessary. The diagnosis of celiac disease is complex and includes symptomatology, serology, small intestinal histology, and genetic status. Serological testing plays a central role within the diagnostic procedure and is based on the measurement of disease-specific antibodies against gluten proteins (antigen) and tissue transglutaminase (autoantigen). Immunofluorescence detection and enzyme-linked immunosorbent assays are currently most often applied for antibody testing. However, these tests are expensive and time-consuming. Therefore, simple and rapid alternative methods have been developed during the last years, and electrochemical immunosensors seem to be the most promising analytical tools. The architecture of these sensors may comprise the following elements: working and reference electrodes, covalent or noncovalent binding of the antigen to the surface of the working electrode by means of a functional monolayer, and blocking of unreacted binding sites. The analytical procedure is initiated by adding the analyte (serum antibodies) and an analyte-specific second antibody, which is usually labeled with an enzyme. The special reaction of the enzyme with an appropriate substrate results in a product that initiates a current that can be measured by different electrical methods. A number of different electrochemical immunosensors variable in different electrodes, binding systems, secondary antibodies, and current measurements have been developed. Most of them have been tested with real human serum samples of celiac patients and healthy individuals, and some of them reached disease sensitivity and specificity comparable with traditional analytical systems. Thus, electrochemical immunosensors can be promising alternatives to existing diagnostic tests in the future. They are simple, reliable, robust, user-friendly, and cost-effective tools with short operation times.
... Recently, using of magneto electrodes in electrochemical sensing of various compounds was improved. Ability of magneto electrochemical sensors to capture different functionalized magnetic beads from solution makes it an excellent devise for determination of molecules and biomolecules in recent years (Yang and Li, 2001;Conzuelo et al., 2012;Kergaravat et al., 2013). ...
A novel magneto multiwalled carbon nanotube/carbon paste electrode (MMW/CPE) for the determination of mefenamic acid (MFA) was introduced. Magnetic molecularly imprinted polymer nanoparticles (MMIPNPs) were synthesized and then added to the solution of MFA. After stirring for 20 min, the MMW/CPE was immersed in the solution of MFA (contain MMIPNPs) and the MMIPNPs were captured by it. Then oxidation of MFA was analyzed by differential pulse voltammetry (DPV). Electrochemical impedance spectroscopy, cyclic voltammetry, and DPV were employed to characterize the MMW/CPE. The MMIPNPs exhibited a high selectivity and sensitivity toward MFA. The effect of various experimental parameters including pH, MMIPNPs dosage, stirring time, accumulation potential and time on the voltammetric response of MFA were investigated. Under the optimal conditions, selective detection of MFA in a linear concentration range of 2.0–1000.0 nmol L−1 was performed with the detection limit of 1.2 nmol L−1 (3 S/N). To further study the practical applicability of this method, it was applied to the analysis of real samples and the obtained results were satisfactory.
... The detection is carried out subsequently using screenprinted electrodes with the aid of a magnet to preconcentrate the magnetic beads. A similar system using GEC electrodes as electrodic platform and peroxidase as label was developed by Kergaravat et al [11]. ...
... Microfluidic-based analyzers are currently receiving considerable attention in many clinical, diagnostic and pharmaceutical settings [1]. These systems have many potential advantages, including reduced reagent consumption, smaller analysis volumes, faster analysis time, higher levels of throughput and automation, and increased instrument portability [2][3][4]. These advantages provide highly efficient immunoassay platforms based on microfluidics. ...
This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems) was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead–protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.
Celiac disease (CeD) is an autoimmune disorder triggered by sensitivity to gluten, a protein complex found in wheat, barley, and rye. Gliadins, a component of gluten, are proteins that trigger an immune response in individuals with CeD, primarily affecting the small intestine’s inner lining. Despite a 1–1.5% prevalence, only 24% of cases are diagnosed due to non-specific symptoms. Screening is advised for high-risk groups, including first-degree relatives and type 1 diabetes patients. The accurate diagnosis of this condition and the assessment of the patient’s response to the current treatment – a lifelong gluten-free diet – necessitate using dependable, swift, sensitive, specific, uncomplicated, and affordable analytical methods. Detecting CeD biomarkers in whole blood, serum, or plasma provides a non-invasive approach that serves as an ideal initial diagnostic step. Biosensors offer a novel and alternative way for CeD detection, began emerging in 2007, and hold promise for clinical and point-of-care applications. This review explores the use of biomarker-based diagnostic approaches for CeD, with a focus on biosensors. It delves into the progress of biosensors for CeD diagnosis, identifying trends and challenges in this evolving field. Key biomarkers are highlighted, offering insights into the evolving landscape of biosensors in CeD detection.
Over the last decade, autoimmune diseases (ADs) have undergone a significant increase because of genetic and/or environmental factors; therefore, their simple and fast diagnosis is of high importance. The conventional diagnostic techniques for ADs require tedious sample preparation, sophisticated instruments, a dedicated laboratory, and qualified personnel. For these reasons, biosensors could represent a useful alternative to these methods. Biosensors are considered to be promising tools that can be used in clinical analysis for an early diagnosis due to their high sensitivity, simplicity, low cost, possible miniaturization (POCT), and potential ability for real-time analysis. In this review, recently developed biosensors for the detection of autoimmune disease biomarkers are discussed. In the first part, we focus on the main AD biomarkers and the current methods of their detection. Then, we discuss the principles and different types of biosensors. Finally, we overview the characteristics of biosensors based on different bioreceptors reported in the literature.
A nanostructured electrochemical sensors is developed to detect IgA isotypes of anti-tissue transglutaminase (anti-tTG) by recording an anodic electrocatalytic current. To this aim, nanoelectrode ensembles (NEEs) prepared by electroless gold deposition on track-etched polycarbonate, are at first functionalized with tissue transglutaminase (tTG) to capture anti-tTG from serum samples. In the following step, the selective detection of the IgA isotype of anti-tTG is performed by reacting with anti-IgA secondary antibody labelled with glucose oxidase. In case of positive response, the addition of glucose (substrate) and (ferrocenylmethyl)trimethylammonium (redox mediator), produces an electrocatalytic anodic current which scales with the IgA anti-tTG concentration. The optimized immunosensor provides low detection limit (0.7 U mL⁻¹), can be applied in serum samples diluted with buffer solution and show to be suitable for quantifying IgA isotype of anti-tTG in human serum samples from celiac patients.
Celiac disease (CD) is a T cell-mediated inflammatory autoimmune disorder of the upper small intestine caused by the ingestion of gluten. It is increasingly recognized as a global problem by experts and societies. The diagnosis of CD is of crucial importance because its delay strongly affects patient's health and quality of life. The diagnosis of CD is, however, complex and requires reliable, sensitive, specific, rapid, simple, and cost-effective, as well-as non-invasive analytical tools. There is also a high demand to develop simple point-of-care (POC) tests for non-specialists at home or in doctors' offices. Analytical techniques are now moving toward the development of fast, more simple, non-invasive, and POC analyses. The present review focuses on recent advances of CD biomarker detection in body fluids, concerning CD specific autoantibody detection in blood and saliva using electrochemical, optic-fiber, and piezoelectric biosensors and POC finger-prick tests, and identifying CD characteristic volatile organic compounds (VOCs) in urine and feces.
The determination of 1-chloro-2,4-dinitrobenzene (CDNB) was used as a proof-of-concept to a simple analytical practical configuration applying magnetic molecularly imprinted particles (mag-MIPs). Mag-MIPs were captured from an emulsion by a home-made magneto-sensor (where a small magnet was entrapped by a graphite-epoxy composite) and then, this sensor, was transferred to the solution containing the analyte, where, after binding to the mag-MIPs, the analyte was directly analysed using differential pulse voltammetry (DPV) since the magneto-sensor acted as the working electrode. After optimization, a detection limit of 6.0 μmol L⁻¹ with a RSD of 2.7% was achieved along with suitable recoveries and selectivity. This methodology offers a different approach for electroanalytical methodologies using mag-MIPs.
The analysis of dyes in hair samples can provide useful information for forensic purposes. The present work describes a novel method for the extraction of dye from a hair sample and its determination using a composite electrode to pre-concentrate carboxyl-functionalized magnetic nanoparticles (CFMP) employed to collect the dye in solution, hence increasing the sensitivity of the analysis. The Basic Brown 16 dye, which is widely used in temporary hair dying, was chosen as a model compound. After 15 s of reaction between 1.5 × 10− 5 mol L− 1 of dye and 0.1 mg mL− 1 of carboxyl-modified magnetic nanoparticles in phosphate buffer electrolyte at pH 7.0, the derivative dye was collected during 40 s at the graphite-epoxy composite electrode and then transferred to a new solution of phosphate buffer at pH 7.0. The dye presented a peak current at a potential of 0.42 V that was almost 400 times higher than without the preconcentration step, suggesting that the dye was pre-accumulated due to strong magnetic interaction with the composite electrode. Under optimized conditions, the analytical curve constructed using square wave voltammetry was linear for BB16 dye concentrations between 1.00 × 10− 7 and 1.00 × 10− 6 mol L− 1. The limits of detection and quantification were 1.01 × 10− 8 and 2.37 × 10− 8 mol L− 1, respectively. The proposed method was successfully applied in the determination of BB16 dye extracted from a dyed hair strand sample by alkaline digestion.
Autoimmune diseases occur when the immune system generates proinflammatory molecules and autoantibodies that mistakenly attack their own body. Traditional diagnosis of autoimmune disease is primarily based on physician assessment combined with core laboratory tests. However, these tests are not sensitive enough to detect early molecular events, and quite often, it is too late to control these autoimmune diseases and reverse tissue damage when conventional tests show positivity for disease. It is fortunate that during the past decade, research in nanotechnology has provided enormous opportunities for the development of ultrasensitive biosensors in detecting early biomarkers with high sensitivity. Biosensors consist of a biorecognition element and a transducer which are able to facilitate an accurate detection of proinflammatory molecules, autoantibodies and other disease-causing molecules. Apparently, novel biosensors could be superior to traditional metrics in assessing the drug efficacy in clinical trials, especially when specific biomarkers are indicative of the pathogenesis of disease. Furthermore, the portability of a biosensor enables the development of point-of-care devices. In this review, various types of biomolecule sensing systems, including electrochemical, optical and mechanical sensors, and their applications and future potentials in autoimmune disease treatment were discussed.
Celiac Disease and Gluten: Multidisciplinary Challenges and Opportunities is a unique reference work-the first to integrate the insights of the causes and effects of celiac disease from the chemistry of reaction-causing foods to the diagnosis, pathogenesis, and symptoms that lead to proper diagnoses and treatment. With an estimated three million people in the United States alone affected by celiac disease, an autoimmune digestive disease, only five percent are properly diagnosed. Drawing on the connection between foods containing gluten and the resulting symptoms, this resource offers distinctive information that directly explores and links food science, medical diagnostics, and treatment information. A helpful tool for researchers and medical practitioners alike, Celiac Disease and Gluten: Multidisciplinary Challenges and Opportunities helps refine research targets, and provides a comprehensive overview on the multidisciplinary approaches to all crucial aspects related to celiac disease. • Presents key information from medical and food science research, as well as provides clinical insights • Provides direct corollary insights between source and symptom • Written by experts whose detailed experiments and results have shaped our understanding of celiac disease.
Celiac disease (CD) is defined as a chronic immune-mediated enteropathy precipitated by exposure to dietary gluten (storage proteins of wheat, rye, barley, and oats) in genetically predisposed individuals. Recent epidemiologic data suggest a prevalence of approximately 1% in most Western countries. The genetic predisposition includes human leukocyte antigen (HLA) DQ2 and DQ8 genes as major factors and a number of non-HLA genes. In addition to gluten, other environmental triggers such as pathogenic microorganisms, timing of gluten introduction, and breastfeeding may be important for disease development. CD is classically characterized by small intestinal villous atrophy, crypt hyperplasia, and increased lymphocyte infiltration. The clinical features of CD include both intestinal symptoms, such as diarrhea or steatorrhea, as well as extraintestinal symptoms, such as bone pain or osteoporosis. CD is frequently associated with autoimmune diseases, such as type 1 diabetes, and neurological and psychiatric complaints, such as schizophrenia. Further gluten-related disorders are dermatitis herpetiformis, gluten ataxia, wheat-dependent exercise-induced anaphylaxis, non-celiac gluten sensitivity, and irritable bowel syndrome. The diagnostic scheme of CD is usually based on symptoms typical of CD, testing of serum antibodies, and histological judgment of small intestinal biopsies. The complex pathomechanism of CD consists of incomplete digestion of gluten, para- and transcellular passage of gluten peptides through the epithelium, and immune responses in the lamina propria. The adaptive immunity includes the deamidation and transamidation of gluten peptides by tissue transglutaminase, their binding to HLA-DQ molecules, their presentation to T cells, and the subsequent delivery of damaging cytokines and chemokines. Additionally, antibodies against gluten peptides and autoantibodies against transglutaminase are produced by B cells. The adaptive immunity is accompanied by the innate immunity characterized by the production of interleukin 15 and the expansion of intraepithelial lymphocytes.
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.
Transglutaminase is primarily involved with activation and progression of coeliac disease through the ingestion of gluten which initiates the production of antibodies against transglutaminase. This results in the damage of the small intestine reducing the diffusion of digested foods into the blood vessels. The diagnosis of coeliac disease can be done by the measurement of antibodies against transglutaminase. This work involves the development of an electrochemical immunosensor for the detection of anti-transglutaminase antibodies. A platinum modified polypyrrole-cobalt (II) salicyladiimine metallodendrimer composite capped with transglutaminase and bovine serum albumin was used for the determination of anti-transglutaminase antibodies. Surface roughness of 879.24 nm, 76.55 nm and 364.27 nm for polypyrrole, cobalt (II) salicyladiimine metallodendrimer and their respective composite in conjunction with cyclic voltammetry showed that such composites provide a suitable micro-environment for monitoring antibody-antigen interactions. Electrochemical impedance spectroscopy parameters such as exchange current and heterogeneous rate constant are used to evaluate immunosensor where the rate constants were 2.7 x 10-6 cm2 s-1, 4.6 x 10-6 cm2 s-1 and 4.16 x 10-6 cm2 s-1 for cobalt (II) salicyladiimine metallodendrimer, polypyrrole and their composite. Impedimetric immunosensor gave a limit of detection of 201 ng mL-1 and linear range of 10-5 - 10-4 mg mL-1 with correlation coefficient of 0.948.
Screen-printing is one of the most promising approaches towards simple, rapid and inexpensive production of biosensors. Disposable biosensors based on screen printed electrodes (SPEs) including microelectrodes and modified electrodes have led to new possibilities in the detection and quantitation of biomolecules, pesticides, antigens, DNA, microorganisms and enzymes. SPE-based sensors are in tune with the growing need for performing rapid and accurate in-situ analyses and for the development of portable devices. This review (with 226 refs.) first gives an introduction into the topic and then is subdivided into sections (a) on DNA sensors (including methods for the detection of hybridization and damage), (b) on aptasensors (for thrombin, OTA, immunoglobulins and cancer biomarkers), (c) on immunosensors (for microorganisms, immunoglobulins, toxins, hormones, lactoferrin and biomarkers), (d) on enzymatic biosensors (for glucose, hydrogen peroxide, various pharmaceuticals, neurotransmitters, amino acids, NADH, enzyme based sensors).
Figure
This review first gives an introduction into the topic of screen-printed electrodes for biosensing and is subdivided into sections (a) on DNA sensors, (b) on aptasensors, (c) on immunosensors, (d) on enzymatic biosensors. It contains 226 references
A miniaturized electrochemical biosensor array with in situ detection of quantum dots (QDs) was developed for the detection of anti-transglutaminase IgG antibodies (a celiac disease biomarker) in human sera. For the fabrication of the sensor, a 8-channel screen-printed carbon electrochemical arrays were used as transducers and modified with tissue-transglutaminase by adsorption. The immunologic reaction was carried out in a few simple steps: reaction with human serum, which contains the analyte of interest, followed by the immunoreaction with anti-human IgG labeled with CdSe/ZnS QDs and electrochemical detection of Cd(2+) released from QDs. All steps were performed on the screen-printed arrays as the solid support, and the detection of Cd(2+) was performed in situ after acid attack of the QDs without a transfer step by voltammetric stripping. The electrochemical response was correlated with the anti-transglutaminase IgG concentration. The developed electrochemical immunosensor is a trustful screening tool for celiac disease diagnosis discriminating between positive and negative sera samples with high sensitivity.
A piezoelectric immunosensor was developed for the first time for direct detection of anti-tissue transglutaminase antibodies (anti-tTG), very specific biomarkers for reliable and early diagnosis of celiac disease. Since the inflammation processes associated to the pathology's occurrence involve tTG structural changes from closed to open conformation as well the extended structure has been demonstrated to have higher diagnostic accuracy if compared with closed conformation, the new strategy undertaken in this study was based on the immobilization of tTG enzyme in its open conformation as receptor on immunosensor surface. Ten nm-sized gold nanoparticles conjugated with secondary antibodies were exploited for signal amplification. Liquid phase detection conditions using a laminar flow cell were properly selected in order to have a good signal stability both in dynamic and in static modes. Optimization of the operating conditions, by experimental design on mouse anti-tTG antibodies in serum, allowed us to obtain a model for the realization of a reliable piezoelectric immunosensor with high potential as diagnostic device for the determination of human autoantibodies of celiac patients.
Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to “blank” sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r
2 of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.
Immunoglobulin A tissue transglutaminase is the single most efficient serological test for the diagnosis of celiac disease. It is well known that immunoglobulin A tissue transglutaminase levels correlate with the degree of intestinal damage, and that values can fluctuate in patients over time. Serological testing can be used to identify symptomatic individuals that need a confirmatory biopsy, to screen at-risk populations or to monitor diet compliance in patients previously diagnosed with celiac disease. Thus, interpretation of serological testing requires consideration of the full clinical scenario. Antigliadin tests are no longer recommended for the diagnosis of classical celiac disease. However, our understanding of the pathogenesis and spectrum of gluten sensitivity has improved, and gluten-sensitive irritable bowel syndrome patients are increasingly being recognized. Studies are needed to determine the clinical utility of antigliadin serology in the diagnosis of gluten sensitivity.
In this brief review we want to give a guidance to all the researchers who want to challenge themselves in the task of developing electrochemical immunosensors for the first time. We will focus here only on practical aspects trying to give the readers useful insights that should be considered such as the choice of the electrode to be used, the best electrochemical and immunological procedures, the immobilization procedures, the instruments to purchase and other basic aspects of electrochemical immunosensors. In doing so we offer a wide view of the research on electrochemical immunosensor applications that have appeared in the literature in the last 5 years.
Celiac disease is an inflammatory disorder of the small intestine induced by intake of wheat gluten and other prolamines in genetically susceptible individuals. This disease is manifested by an increased number of intraepithelial and lamina propria lymphocytes, villous atrophy, tissue remodeling and the presence of anti-transglutaminase antibodies. The most widely accepted pathogenic model is based on adaptive immunity after T CD4+lymphocyte stimulation by tissue transglutamine-modified gluten peptides and HLA-DQ2/DQ8 restriction, which produce proinflammatory cytokines.Gluten also activates innate immunity and epithelial cytotoxicity mediated by intraepithelial lymphocytes. Although the effect of specific antibodies remains unclear, the availability of serological and immunogenetic markers as diagnostic tools has increased our knowledge of celiac disease and has led to a reevaluation of the diagnostic criteria, especially in adults with minimal or atypical disease expression.
Background
Anti-transglutaminase antibodies have been proposed as a useful tool in the diagnosis and follow-up of celiac disease.
Aim
To compare anti-transglutaminase antibodies with the classical antibodies used to diagnose celiac disease.
Material and method
Patients who underwent duodenal biopsy for suspected celiac disease were selected if they had the following serum antibody samples: antiendomysial IgA, anti-transglutaminase IgA, antigliadin IgG, and antigliadin IgA. A diagnostic value of each of these antibodies was established according to the final diagnosis, taking the duodenal biopsy as the reference.
Results
One hundred twenty-two patients with duodenal biopsy and serologic markers for celiac disease were selected. Thirty-six patients were children (< 14 years-old) and 86 were adults. A diagnosis of celiac disease was made in 41 patients (26 children and 15 adults). Of the 15 adults, only 2 (13%) presented typical malabsorption syndrome. The sensitivity and specificity of anti-transglutaminase antibodies was 100% and 98% respectively compared with values of 97% and 98% for antiendomysial IgA, 85% and 97% for antigliadin IgA, and 97% and 92% for antigliadin IgG antibodies.
Conclusions
The diagnostic value of anti-transglutaminase antibodies is high in celiac disease. These antibodies may be useful as a screening test to select candidates for duodenal biopsy.
Magnetised silica-based microparticles (MMPs) (5 μm) with a high density of nanopores were used for horseradish peroxidase (HRP) immobilization and amperometric biosensor development in batch conditions. The resulting biosensor was applied to study the peroxidation of clozapine (CLZ) which is a dibenzoazepine drug often used in the treatment of neurological disorders. The amperometric response corresponded to the electroreduction of CLZ-oxidized products namely a nitrenium cation and quinoneimine derivative of CLZ. Despite a relatively low amount of immobilized HRP (0.3 μmol/g), clozapine quantification in the micromolar concentration range was achieved by the use of a magnetized solid paraffin carbon paste electrode for microparticles attraction. Diffusion of substrate and products of the enzyme reaction within the nanopores were identified as limiting factors in the biosensor response. This amperometric biosensor configuration has number of interesting advantages such as ease and reproducible microparticle layer renewing, low enzyme consumption, controlled surface immobilization, protective enzyme microenvironment etc.
Celiac disease is an autoimmune disorder that affects the gastrointestinal tract upon ingestion of gluten, which triggers the production of antibodies against gliadin and tissue transglutaminase, activating an inflammatory response and inducing tissue damage in the small intestine resulting in malabsorption. The measurement of these antibodies in an individual's blood can be used to screen for celiac disease and the criteria for definitive diagnosis is currently being revised to be based on serological analysis rather than biopsy. In the work reported here, an electrochemical immunosensor for the detection of human anti-tissue transglutaminase antibodies was developed, consisting of gold-based self-assembled monolayers of a carboxylic group terminated bipodal alkanethiol that is covalently linked to tissue transglutaminase, the antigen for the immunorecognition of circulating autoantibodies. The presence of the autoantibodies was recorded using horseradish peroxidase labeled anti-human antibodies, which provided an enzyme based electrochemical signal. Optimization and characterization of the surface of the sensor was carried out by electrochemical impedance spectroscopy and surface plasmon resonance. The immunosensor gave a stable quantitative response to different antibody concentrations after 30 min with a limit of detection of 390 ng/mL and an RSD of 9%, n = 3. The developed immunosensor was tested with calibrator solutions as well as with real patients’ samples, and the results compared to those obtained from Eurospital's Eu-tTG IgA and IgG ELISA kits, showing an excellent degree of correlation.
A disposable immunomagnetic electrochemical sensor involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed and applied for the detection of polychlorinated biphenyls (PCBs). The sensor was based on a direct competitive immunoassay scheme in which antibody-coated magnetic beads were used as solid phase; then SPCEs were employed as transducers for the evaluation of the extent of the immunochemical reaction using an alkaline phosphatase (AP) labelled tracer. The α-naphthyl phosphate was used as enzymatic substrate and the α-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV).A detection limit of 0.4 ng/mL for Aroclor 1248 PCB mixture was obtained. The performance of the sensor was successfully evaluated on marine sediment extracts and soil samples spiked with different Aroclor mixtures indicating that this new and sensitive technique offers great promise for decentralized environmental applications.
Rigid conducting carbon–polymer composites are ideal for the construction of electrochemical sensors. The plastic nature of these materials makes them modifiable, permitting the incorporation of fillers before they are cured. A great number of biological materials can be immobilised by blending them with these composites to form new biocomposite materials. These biocomposites not only act as reservoirs of the biological material but may also contain catalysts, mediators and cofactors that improve the response of the resulting electrochemical biosensors. One of the outstanding features of these conducting biomaterials is their rigidity, resulting in a high mechanical stability over time. Furthermore, the sensing surface can be renewed by a simple polishing procedure. The proximity of the redox centres of the biological material and the conducting sites on the sensing surface favours the transfer of electrons between electroactive species. This may in some cases permit the regeneration of the biological component without using cosubstrates and mediators. These biocomposites are prepared easily and they show improved electrochemical characteristics. This is very attractive for the mass fabrication of amperometric biosensors.
The development of composites based on conductive phases dispersed in polymeric matrices has led to important advances in analytical electrochemistry, particularly insensor devices. These new materials combine the electrical properties of graphite with the ease of processing of plastics (epoxy, methacrylate, Teflon, etc.) and show attractive electrochemical, physical, mechanical and economical features compared to the classic conductors (gold, platinum, graphite, etc.). The properties of these composites are described, along with their application to the construction of conductometric, potentiometric and amperometric sensors. The chemical modification of the composites by blending fillers that improve the analytical characteristics of the resulting sensors is discussed, particularly for the case of amperometric devices.
The electrochemical detection for horseradish peroxidase-cosubstrate-H(2)O(2) systems was optimized. o-Phenilendiamine, phenol, hydroquinone, pyrocatechol, p-chlorophenol, p-aminophenol and 3,3'-5,5'-tetramethylbenzidine were evaluated as cosubstrates of horseradish peroxidase (HRP) enzyme. Therefore, the reaction time, the addition sequence of the substrates, the cosubstrate:H(2)O(2) ratio and the electrochemical techniques were elected by one-factor optimization assays while the buffer pH, the enzymatic activity and cosubstrate and H(2)O(2) concentrations for each system were selected simultaneously by response surface methodology. Then, the calibration curves for seven horseradish peroxidase-cosubstrate-H(2)O(2) systems were built and the analytic parameters were analyzed. o-Phenilendiamine was selected as the best cosubstrate for the HRP enzyme. For this system the reaction time of 60s, the phosphate buffer pH 6.0, and the concentrations of 2.5×10(-4)molL(-1) o-phenilendiamine and of 1.25×10(-4)molL(-1) H(2)O(2) were chosen as the optimal conditions. In these conditions, the calibration curve of horseradish peroxidase by square wave voltammetry showed a linearity range from 9.5×10(-11) to 1.9×10(-8)molL(-1) and the limit of detection of 3.8×10(-11)molL(-1) with RSD% of 0.03% (n=3).
Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.
Gliadin is a constituent of the cereal protein gluten, responsible for the intolerance generated in celiac disease. Its detection is of high interest for food safety of celiac patients, since the only treatment known until now is a lifelong avoidance of this protein in the diet. Therefore, it is essential to have an easy and reliable method of analysis to control the contents in gluten-free foods. An electrochemical magneto immunosensor for the quantification of gliadin or small gliadin fragments in natural or pretreated food samples is described for the first time and compared to a novel magneto-ELISA system based on optical detection. The immunological reaction was performed on magnetic beads as solid support by the oriented covalent immobilization, of the protein gliadin on tosyl-activated beads. Direct, as well as indirect competitive immunoassays were optimized, achieving the best analytical performance with the direct competitive format. Excellent detection limits (in the order of μg L(-1)) were achieved, according to the legislation for gluten-free products. The matrix effect, as well as the performance of the assays was successfully evaluated using spiked gluten-free foodstuffs (skimmed milk and beer), obtaining excellent recovery values in the results.
Concentration of progesterone in milk may be used to predict pregnancy status of dairy cattle by the 21st day after insemination. However, the accuracy of this method may be affected by fat-solubility of progesterone and sample storage conditions. After coagulation of a milk sample with rennet, an alternative method is to quantify progesterone concentration in whey with a novel, validated EIA. In this experiment, a receiver operating characteristic (ROC) analysis was performed to estimate the optimal discrimination point for whey progesterone concentration, using a sample of 991 Friesian cows evaluated between the 42nd and 44th day after insemination. Cows also were diagnosed for pregnancy by rectal palpation at this time. The overall conception rate at palpation was 57%. ROC analysis indicated that 259 pg/mL progesterone in whey was the most effective cutoff to discriminate correctly between pregnant and non-pregnant cows. Using this point for prediction, sensitivity was 98.2%, specificity was 70.9% and the area under ROC curve was 0.859, levels generally considered to denote moderate accuracy. The negative likelihood ratio at the cutoff of 259 pg/mL was 0.02, indicating satisfactory performance in detecting negative subjects, while the positive likelihood ratio (+LR=3.37) suggested average performance. In conclusion, EIA of progesterone concentration in whey is a viable method for predicting pregnancy status in cows. However, operators should take management objectives for the herd into account in determining the cutoff point and also considering important influencing variables such as conception rate in the herd. This method can provide diagnostic support for efforts to improve reproductive success, especially in low-fertility herds.
The determination of antigliadin antibodies from human serum samples is of vital importance for the diagnosis of an autoimmune disease such as celiac disease. An electrochemical immunosensor that mimics traditional ELISA type architecture has been constructed for the detection of antigliadin antibodies with control over the orientation and packing of gliadin antigen molecules on the surface of gold electrodes. The orientation of the antigen on the surface has been achieved using a carboxylic-ended bipodal alkanethiol that is covalently linked with amino groups of the antigen protein. The bipodal thiol presents a long poly(ethyleneglycol)-modified chain that acts as an excellent non-specific adsorption barrier. The bipodal nature of the thiol ensured a good spacing and hence good diffusion properties of electroactive species through the self-assembled monolayer, which is vital for the efficiency of the constructed electrochemical immunosensor. The electrochemical immunosensor was characterized using surface plasmon resonance as well as electrochemical impedance spectroscopy. Amperometric evaluation of the sensor with polyclonal antigliadin antibodies showed stable and reproducible low limits of detection (46 ng/mL; % RSD = 8.2, n = 5). The behaviour and performance of the electrochemical immunosensor with more complex matrixes such as reference serum solutions and real patient samples was evaluated and compared with commercial ELISA kits demonstrating an excellent degree of correlation in thirty minutes total assay time; the electrochemical immunosensor not only delivers a positive or negative result, it allows the estimation of semi-quantitative antibody contents based on the comparison against clinical reference solutions.
The identification of specific serological algorithms allowing the diagnosis of celiac disease (CD) is a new challenge for both the clinic and the laboratory. We compared the diagnostic accuracy of three new tests proposed for CD screening with that of the well established IgA tTG, and ascertained whether any combination of these tools might enhance accuracy in diagnosing CD.
In sera from 329 CD and 374 control children, the following were assayed: IgA tTG; IgA/IgG, which identify tTG-gliadin complexes (Aeskulisa Celi Check and CeliCheck IgGA); IgA/IgG, which identify deamidated gliadin peptides and tTG (QUANTA Lite(TM) h-tTG/DGP Screen).
When specificity was set at 100%, the most sensitive index of CD was IgA tTG (75.7%, cut-off=100U), followed by QUANTA Lite(TM) h-tTG/DGP Screen (65.3%, cut-off 145U), Aeskulisa Celi Check (62.6%, cut-off 909U/mL) and CeliCheck IgGA (59.6%, cut-off 977U/mL). Three algorithms were obtained by combining IgA tTG with each of the new tests. The algorithm obtained by measuring IgA tTG and QUANTA Lite(TM) h-tTG/DGP Screen allowed the correct identification of CD in 78.7% of cases (negative predictive value=97.3%).
The two-test based strategy could be used for the cost effective diagnosis of CD.
A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL(-1) with a slope value of 7.0 nA mL ng(-1). The limit of detection was 3.74 ng mL(-1). Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.
Okadaic acid (OA), a lipophilic phycotoxin highly toxic to humans is produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, competitive indirect enzyme-linked electrochemical immunosensor based on super paramagnetic nanobeads has been developed for the detection of OA. Streptavidin-coated magnetic beads were used as support to immobilize the biotinylated OA. Preliminary, colorimetric tests were performed in order to optimize different experimental parameters. Electrochemical detection was carried out by differential pulse voltammetry (DPV). The limit of detection (LOD) (0.38 μg L(-1)), the mid point value (IC(50)) (3.15 μg L(-1)) and the time needed (60 min) for analysis of a real sample validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed, showing an excellent percentage of recovery.
A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol-gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO(2)/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO(2)/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO(2)/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12 ng mL(-1) in pH 5.5 HAc-NaAc containing 5mM H(2)O(2). At a signal-to-noise ratio of 3, the detection limit (LOD) is 5 pg mL(-1) CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay.
A comparative study of four different antibody immobilization techniques that are suitable for modification of surface plasmon resonance (SPR) chip (SPR-chip) is reported. Antibodies against human growth hormone (anti-HGH) were used as the model system. The evaluated SPR-chip modification techniques were (i) random immobilization of intact anti-HGH (intact-anti-HGH) via self-assembled monolayer (SAM) based on 11-mercaptoundecanoic acid (MUA); (ii) random immobilization of intact-anti-HGH within carboxymethyl dextran (CMD) hydrogel by direct covalent amine coupling technique; (iii) oriented coupling of intact-anti-HGH via Fc-fragment to protein-G layer assembled on SAM consisting of MUA (MUA/pG); (iv) oriented immobilization of fragmented anti-HGH antibodies (frag-anti-HGH) via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the intact-anti-HGH was chemically "divided" into two frag-anti-HGH fragments by chemical reduction with 2-mercaptoethylamine (2-MEA). Optimal concentration of 2-MEA for preparation of anti-HGH was 15 mM. The surface concentration of immobilized antibodies and the antigen binding capacity for all four differently modified SPR-chips was evaluated and compared. The maximum surface concentration of immobilized intact-anti-HGH was obtained by immobilizing the antibody within CMD-hydrogel. The maximal antigen binding capacity was obtained by SPR-chip based on intact-anti-HGH immobilized via MUA/pG. The immobilization based on application of frag-anti-HGH was found to be the most suitable for design of SPR-immunosensor for HGH detection, due to its sufficient antigen binding capacity, simplicity, and low cost in respect to the currently evaluated techniques.
A reliable and cost-effective electrochemical method for the detection of deoxynivalenol (DON) in cereals and cereal-based food samples based on the use of a novel anti-DON Fab fragment is presented. The analytical system employed, Enzyme-Linked-Immunomagnetic-Electrochemical (ELIME) assay, is based on the use of immunomagnetic beads (IMBs) coupled with eight magnetized screen-printed electrodes (8-mScPEs) as electrochemical transducers. Using standard solutions of DON, a working range between 100 and 4500 ng/ml was obtained with an EC(50) of 380 ng/ml. The ELIME assay was employed to evaluate the cross-reactivity of the Fab fragment towards different trichothecenes revealing a good selectivity towards DON over other trichothecenes with the exception of 3-Ac-DON. The sensor was then applied to cereals and cereal-based food samples (wheat, breakfast cereal and baby-food) and a wide range of sample treatment procedures was tested. Within-laboratory precision (9-24% repeatability for breakfast cereals and 10-33% for baby-food) and recovery data (82-110% for breakfast cereals and 97-108% for baby-food) were calculated by analyzing blank breakfast cereals and baby-foods fortified with DON, demonstrating that the proposed method has the capability for use as a screening assay for DON in such products.
Serum IgA antibodies against tissue transglutaminase (tTG) are reliable markers for celiac disease (CD), still the diagnostic performance of anti-tTG immunoassays can be improved. A novel ELISA, using fibronectin (FN) as tTG binding protein, was evaluated for the detection of anti-tTG antibodies in CD.
Sera from 173 children with untreated CD and 97 controls were analyzed for IgA, IgG, and IgM anti-tTG antibodies with ELISA using human recombinant tTG with or without FN.
The areas under the ROC (receiver operating characteristic) curves were significantly higher for FN/tTG ELISA compared to tTG ELISA for IgG (0.930 versus 0.809; p < 0.001) and IgM (0.850 versus 0.811; p = 0.019), but not for IgA (0.977 versus 0.970; p = 0.356), respectively. At the fixed diagnostic specificity (100% for IgA and IgM, 99% for IgG), the sensitivity of all three FN/tTG ELISA (92.5% for IgA, 60.7% for IgG, 50.3% for IgM) exceeded those obtained in tTG ELISA (89.0% for IgA, 48.6% for IgG, 38.7% for IgM; p < 0.05). The combined use of IgA- and IgG-FN/tTG ELISA resulted in 95.4% sensitivity and 99.0% specificity for CD.
Using FN to bind tTG improves the diagnostic performance of solid phase anti-tTG antibody assays for childhood CD.
A novel electrochemical immunosensing strategy for the detection of antibodies to tissue transglutaminase (tTG) in human serum is presented. The proposed immunosensor consists of the immobilization by physical adsorption of tTG from guinea pig liver on graphite-epoxy composite (GEC) electrodes. After the reaction with the human serum (containing the specific antibodies in the case of celiac disease), the electrode was incubated with different kinds of secondary labeled antibodies, namely, horseradish peroxidase (HRP)-conjugated goat antibodies to human whole immunoglobulins (Igs), to human IgG, and finally to human IgA. Among the different classes of antibodies in human serum toward tTG, the best results were achieved when anti-tTG IgA antibodies were investigated. In total, 10 positive and 10 negative serum samples were processed, obtaining a sensitivity of 70% and a specificity of 100% compared with the commercial enzyme-linked immunosorbent assay (ELISA) method performed in a hospital laboratory. This strategy offers great promise for a simple, cost-effective, and user-friendly analytical method that allows point-of-care diagnosis of celiac disease.
A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e.g., insecticides and chemical nerve agents, by directly detecting organophosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly efficient magnetic separation with ultrasensitive square wave voltammetry (SWV) analysis with quantum dots (QDs) as labels. A pair of antibodies was used to achieve the specific recognition of OP-AChE that was prepared with paraoxon as an OP model agent. Antiphosphoserine polyclonal antibodies were anchored on amorphous magnetic particles preferably chosen to capture OP-AChE from the sample matrixes by binding their phosphoserine moieties that were exposed through unfolding the protein adducts. This was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, antihuman AChE monoclonal antibodies were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes. Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3-300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promise as a simple, selective, sensitive, and field-deployable tool for the effective biomonitoring and diagnosis of potential exposures to nerve agents and pesticides.
A total number of 50 sera from clinically confirmed cases of canine Borrelia (B.) burgdorferi infection and 44 negative control sera were tested with a B. burgdorferi specific antibody ELISA. The data were submitted to the 'two-graph receiver operating characteristic' (TG-ROC) analysis which is a plot of the test sensitivity (Se) and specificity (Sp) against the threshold (cut-off) value assuming the latter to be an independent variable. Thus, in contrast to the conventional ROC analysis, valid pairs of Se and Sp can be read for pre-assigned threshold values directly from the TG-ROC plots. A cut-off that realises equal test parameters (Se = Sp = theta 0 (theta-zero)) can be obtained as the intersection point of the two graphs. Since the value for theta 0 is below a preselected accuracy level (95% or 90%), two cut-off values are selected that represent the bounds of an 'intermediate range' (IR). IR can be considered as a 'borderline' range for the clinical interpretation of test results. The proportion of the measurement range (MR) that gives unambiguous test results can be expressed using IR as the 'valid range proportion' (VRP = (MR-IR)/MR). VRP and theta 0 are useful parameters for test comparison since they do not depend upon the selection of a single cut-off point. In addition, the selection of cut-off values is supported by graphical displays of efficiency, Youden's index and likelihood ratios which can be considered as functions of the pre-assigned cut-off value. TG-ROC was derived as a user-defined template for a commercially available spreadsheet programme (MS-EXCEL, Microsoft).
Novel polishable immunosensors based on rigid biocomposite materials have been constructed. These biocomposites contain graphite powder, rabbit IgG, and methacrylate or epoxy resins. This material acts as a reservoir for the biological molecules and as a transducer at the same time. In order to study the potential analytical properties of this new type of material, a competitive binding assay was developed to determine the RIgG present in a sample with the aid of goat anti-rabbit IgG labeled with alkaline phosphatase. Using phenyl phosphate as a substrate, the phenol produced by the enzymatic reaction was amperometrically detected at 800 mV (vs Ag/AgC1). The surface of the immunosensor can be regenerated by simply polishing, obtaining fresh immunocomposite ready to be used in a new competitive assay.
The American Journal of Gastroenterology is published by Nature Publishing Group (NPG) on behalf of the American College of Gastroenterology (ACG). Ranked the #1 clinical journal covering gastroenterology and hepatology*, The American Journal of Gastroenterology (AJG) provides practical and professional support for clinicians dealing with the gastroenterological disorders seen most often in patients. Published with practicing clinicians in mind, the journal aims to be easily accessible, organizing its content by topic, both online and in print. www.amjgastro.com, *2007 Journal Citation Report (Thomson Reuters, 2008)
We review the principles and practical application of receiver-operating characteristic (ROC) analysis for diagnostic tests. ROC analysis can be used for diagnostic tests with outcomes measured on ordinal, interval or ratio scales. The dependence of the diagnostic sensitivity and specificity on the selected cut-off value must be considered for a full test evaluation and for test comparison. All possible combinations of sensitivity and specificity that can be achieved by changing the test's cut-off value can be summarised using a single parameter; the area under the ROC curve. The ROC technique can also be used to optimise cut-off values with regard to a given prevalence in the target population and cost ratio of false-positive and false-negative results. However, plots of optimisation parameters against the selected cut-off value provide a more-direct method for cut-off selection. Candidates for such optimisation parameters are linear combinations of sensitivity and specificity (with weights selected to reflect the decision-making situation), odds ratio, chance-corrected measures of association (e. g. kappa) and likelihood ratios. We discuss some recent developments in ROC analysis, including meta-analysis of diagnostic tests, correlated ROC curves (paired-sample design) and chance- and prevalence-corrected ROC curves.
Staphylococcal enterotoxin B (SEB) is one of many toxins produced by the Gram-positive bacterium Staphylococcal aureus. While SEB is known as the causative agent of certain food poisonings it is also considered a biological Select Agent. Thus, rapid and accurate identification of SEB during either surveillance or in response to a biothreat is critical to the mitigation of the suspect agent. This report presents an improved method for the detection of SEB based on a SEB-specific, two-antibody system where one antibody was bound to a magnetic bead particle while the other was labeled with Alexa fluor 647. The assay consisted of one incubation period for 30 minutes where all reagents necessary to detect SEB were included. Using this assay 100 pg of recombinant purified SEB, as well as SEB from the culture supernatant of several strains of methicillin-resistant S. aureus were detected with fidelity. This assay presents improvements over current assays in terms of a combination of the reduction in assay time length, assay sensitivity, ease of use, and application to automated high-throughput analysis. Additionally, this assay can be easily modified to detect a wide range of proteins and whole organisms.
Costs can hamper the evaluation of the effectiveness of new biomarkers. Analysis of smaller numbers of pooled specimens has been shown to be a useful cost-cutting technique. The Youden index (J), a function of sensitivity (q) and specificity (p), is a commonly used measure of overall diagnostic effectiveness. More importantly, J is the maximum vertical distance or difference between the ROC curve and the diagonal or chance line; it occurs at the cut-point that optimizes the biomarker's differentiating ability when equal weight is given to sensitivity and specificity. Using the additive property of the gamma and normal distributions, we present a method to estimate the Youden index and the optimal cut-point, and extend its applications to pooled samples. We study the effect of pooling when only a fixed number of individuals are available for testing, and pooling is carried out to save on the number of assays. We measure loss of information by the change in root mean squared error of the estimates of the optimal cut-point and the Youden index, and we study the extent of this loss via a simulation study. In conclusion, pooling can result in a substantial cost reduction while preserving the effectiveness of estimators, especially when the pool size is not very large.
A novel electrochemical immunosensing strategy for the detection of atrazine based on magnetic beads is presented. Different coupling strategies for the modification of the magnetic beads with the specific anti-atrazine antibody have been developed. The immunological reaction for the detection of atrazine performed on the magnetic bead is based on a direct competitive assay using a peroxidase (HRP) tracer as the enzymatic label. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magnetosensor made of graphite-epoxy composite, which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate and mediator for the enzyme HRP. The electrochemical approach is also compared with a novel magneto-ELISA based on optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked real orange juice samples. The detection limit for atrazine using the competitive electrochemical magnetoimmunosensing strategy with anti-atrazine-specific antibody covalent coupled with tosyl-activated magnetic beads was found to be 6 x 10(-3) microg L(-1) (0.027 nmol L(-1)). This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of biological, food, and environmental samples.
Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3',5,5'-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9+/-4.3 pg ml(-1) for boldenone and 120.2+/-8.2 pg ml(-1) for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC(50)=1.0+/-0.3 ng ml(-1)(n=3, N=3), R(2)=0.969, R.S.D.=9.6% for boldenone and 1.5+/-0.3 ng ml(-1), 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of alpha-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.
A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads--such as those based on the use of Protein A or carboxylate modified magnetic beads - ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 microg L(-1) (5.92 nmol L(-1)) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.
The concentration of anti-transglutaminase antibodies in human sera is an important analytical marker for the diagnosis of the autoimmune disorder celiac disease. In this work, an immunosensor for the electrochemical detection of anti-transglutaminase antibodies in human sera was developed. The immunosensor is based on the immobilization of transglutaminase onto screen-printed gold electrodes which were covered with a polyelectrolyte layer of poly (sodium-4-styrensulfonic acid). The antigen-antibody interaction was evaluated using an amplification step: incubation with peroxidase (POD)-labeled immunoglobulins and subsequent biocatalytic oxidation of 3-amino-9-ethylcarbazole (AEC). Changes in the interfacial properties of the sensor electrode were determined by electrochemical impedance spectroscopy (EIS). Impedance spectra could be fitted to a Randles equivalent circuit containing a constant phase element (CPE). Furthermore, it was shown that impedance measurements could be simplified by performing EIS at only two selected frequencies, without loss of reliability. Incubation of these disposable immunosensor chips with various anti-transglutaminase antibody concentrations resulted in changes in their charge transfer resistance (R(ct)). Thereby, a calibration graph could be established. Finally, immunosensors were used for characterizing different human sera with respect to their anti-transglutaminase autoantibody concentration of the IgG and IgA type.
Autoantibody production is an important feature of many autoimmune disorders, signifying a breakdown of immune tolerance to self-antigens. In celiac disease, an autoimmune enteropathy with multiple extra-intestinal manifestations, autoantibody reactivity to transglutaminase 2 (TG2) has been shown to closely correlate with the acute phase of the disease. It serves as a specific and sensitive marker of celiac disease, and is highly useful in aiding diagnosis and follow-up. Immune reactivity to other autoantigens, including transglutaminase 3, actin, ganglioside, collagen, calreticulin and zonulin, among others, has also been reported in celiac disease. The clinical significance of these antibodies is not known, although some may be associated with specific clinical presentations or extra-intestinal manifestations of celiac disease. This review examines the presence of anti-TG2 and other autoantibodies in celiac disease, discussing their diagnostic value, their potential role in disease pathogenesis and current hypotheses that explain how their release may be triggered.
To investigate if the detection of celiac disease (CD) in children was improved by using alternative conjugates for assessment of tissue transglutaminase (tTG) autoantibodies.
Serum samples from 108 biopsy confirmed CD children and 42 control subjects were investigated for the presence of autoantibodies with tTG coated microplates using protein A (PA), protein G (PG), anti-IgG, or anti-IgA as conjugates.
Of the 108 CD children, 86 (80%) were IgG-tTG positive, 91 (84%) were positive with the PA-conjugate, 94 (87%) were positive with the PG-conjugate, and 103 (95%) were IgA-tTG positive. Among the 42 controls, 4 (10%) were IgG-tTG positive, 5 (12%) were positive with both the PA- and PG conjugates, whereas 3 (7%) were IgA-tTG positive. Compared with IgG-tTG the concordance was 93% for PA and 95% for PG, with a positive correlation between antibody levels (r=0.967 and r=0.975, p<0.0001). All but one CD child were found positive by combining IgG-tTG and IgA-tTG detection.
The sensitivity of IgG-tTG detection with ELISA increased by protein A or protein G conjugates, whereas the specificity was reduced as compared with anti-IgG conjugate. The combined measurement of IgA-tTG and IgG-tTG still seems to be the optimal procedure when screening children for CD.
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