An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.     

A carbon nanotube-based high-sensitivity electrochemical immunosensor for rapid and portable detection of clenbuterol

Carbon nanotubes have shown their unique advantages of mechanical, chemical and electronic properties in bioanalysis. We herein report a new method to efficiently and reproducibly prepare multi-walled carbon nanotubes (MWNTs)-protein sensing layers for electrochemical immunosensors. This method employs centrifugation to prepare a conjugate of MWNTs and goat anti mouse-immunoglobulin G (IgG) (secondary antibody). The conjugates were then deposited on screen-printed electrodes to form a nanostructured layer (MWNT-I layer). CLB monoclonal antibody was assembled through its binding to the secondary antibody. The MWNT-I layer-based electrodes were used for rapid and sensitive amperometric immunosensing detection of clenbuterol (CLB) in swine urine samples. Horseradish peroxidase-coupled CLB (CLB-HRP) competed with free CLB in the samples to bind the monoclonal antibody. It has shown significantly higher sensitivity and better reproducibility than the chemical conjugation method. This MWNT-based immunosensor is highly sensitive, leading to a limit of detection of 0.1 ng/mL within a rapid assay time of 16 min. Its sensitivity is at least 1 order of magnitude higher than that of a normal immunosensor (without MWNTs). The sensing device is portable with disposable screen-printed electrode, satisfactorily meeting the requirements for field detection of food security-related species.     

Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.     

A novel reagentless amperometric immunosensor based on gold nanoparticles/TMB/Nafion-modified electrode

A novel reagentless immunosensor was fabricated by immobilization of redox mediator 3,3',5,5'-tetramethylbenzidine (TMB) on the Nafion (Nf) film modified glassy carbon electrode. Gold nanoparticles were assembled onto the TMB/Nafion film modified electrode to provide active sites for the immobilization of antibody molecules. The antibody (anti-MIgG), in the present study, was fixed on the electrode for the rapid detection of antigen molecules (MIgG as a model analyte). The results showed that the immunosensor based on the immobilized TMB redox mediator exhibited good electrochemical response. A good linear relationship between peak current and the concentration of the MIgG was obtained in the concentration range from 4 to 120ng/mL. The detection limit was estimated to be 1ng/ml. Under the optimized conditions, the immunosensor exhibits good sensitivity, reproducibility and stability.     

Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Labeless AC impedimetric antibody-based sensors with pgml(-1) sensitivities for point-of-care biomedical applications

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.     

Disposable immunosensor for the determination of domoic acid in shellfish

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.     

Cross-talk-free multiplexed immunoassay using a disposable electrochemiluminescent immunosensor array coupled with a non-array detector

A disposable electrochemiluminescent (ECL) immunosensor array was fabricated on a screen-printed carbon electrode (SPCE) substrate to perform multiplexed immunoassay (MIA) for the first time. The SPCE substrate was composed of an array of four carbon working electrodes, one common Ag/AgCl reference electrode, and one common carbon counter electrode. The immunosensor array was constructed by site-selectively immobilizing multiple antigens on different working electrodes of the SPCE substrate. With a competitive immunoassay format, the immobilized antigens competed with antigens in the sample to capture their corresponding tri(2,2'-bipyridyl)ruthenium(II)-labeled antibodies. The ECL signals from the immunosensors in this array were sequentially detected by a photomultiplier with the aid of a homemade single-pore-four-throw switch. Due to the ECL readout mechanism and the sequential detection mode, it could avoid the cross-talk between the adjacent immunosensors, which was common in other reported immunosensor array. Human, rabbit and mouse immunoglobulin Gs were near-simultaneously assayed as the model analytes. The linear ranges for them were 10-400, 20-400, and 20-400 ng/mL, with detection limits of 2.9, 6.1 and 6.5 ng/mL (S/N=3), respectively. This novel ECL strategy based on immunosensor array coupled with non-array detector provided a simple, sensitive, low-cost and time-saving approach for MIA. It showed great application potential in point-of-care test and field analysis of bio-agents, with mass production potential and high throughput.     

Design and development of a highly stable hydrogen peroxide biosensor on screen printed carbon electrode based on horseradish peroxidase bound with gold nanoparticles in the matrix of chitosan

The design and development of a screen printed carbon electrode (SPCE) on a polyvinyl chloride substrate as a disposable sensor is described. Six configurations were designed on silk screen frames. The SPCEs were printed with four inks: silver ink as the conducting track, carbon ink as the working and counter electrodes, silver/silver chloride ink as the reference electrode and insulating ink as the insulator layer. Selection of the best configuration was done by comparing slopes from the calibration plots generated by the cyclic voltammograms at 10, 20 and 30 mM K(3)Fe(CN)(6) for each configuration. The electrodes with similar configurations gave similar slopes. The 5th configuration was the best electrode that gave the highest slope. Modifying the best SPCE configuration for use as a biosensor, horseradish peroxidase (HRP) was selected as a biomaterial bound with gold nanoparticles (AuNP) in the matrix of chitosan (HRP/AuNP/CHIT). Biosensors of HRP/SPCE, HRP/CHIT/SPCE and HRP/AuNP/CHIT/SPCE were used in the amperometric detection of H(2)O(2) in a solution of 0.1M citrate buffer, pH 6.5, by applying a potential of -0.4V at the working electrode. All the biosensors showed an immediate response to H(2)O(2). The effect of HRP/AuNP incorporated with CHIT (HRP/AuNP/CHIT/SPCE) yielded the highest performance. The amperometric response of HRP/AuNP/CHIT/SPCE retained over 95% of the initial current of the 1st day up to 30 days of storage at 4 degrees C. The biosensor showed a linear range of 0.01-11.3mM H(2)O(2), with a detection limit of 0.65 microM H(2)O(2) (S/N=3). The low detection limit, long storage life and wide linear range of this biosensor make it advantageous in many applications, including bioreactors and biosensors.     

Development of urinary albumin immunosensor based on colloidal AuNP and PVA

The development of immunosensors with high sensitivity and specificity in detecting the pathogenic or physiologically relevant molecules in the body, offers a powerful opportunity in early diagnosis and treatment of diseases. In this study, we developed a new competitive immunosensor with employing antibody (Ab) labeled AuNP (Ab-AuNP) and PVA modified screen-printed carbon electrode (SPCE) surface to detect the urine albumin. Field emission scanning electron microscopy (FE-SEM) of modified electrode showed a suitable and stable attachment between HSA antigen- mAb and AuNP. Cyclic voltammetric (CV) method demonstrated that modification process was well performed. Electrochemical measurements including differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were employed for quantitative antigen detection. The electrochemical measurements performed with other proteins mixed with samples demonstrated a high specificity and selectivity for this biosensor in detecting the HSA. In optimal conditions, the immunosensor could detect HSA in a high linear range (from 2.5 to 200 μg/mL) with a low detection limit of 25 ng/mL. This new strategy could be improved and applied to detect the other antigen.     

Simultaneous determination of ascorbic acid, dopamine and uric acid using high-performance screen-printed graphene electrode

A disposable and sensitive screen-printed electrode using an ink containing graphene was developed. This electrode combined the advantages of graphene and the disposable characteristic of electrode, which possessed wide potential window, low background current and fast electron transfer kinetics. Compared with the electrodes made from other inks, screen-printed graphene electrode (SPGNE) showed excellent electrocatalytic activity for the oxidation of ascorbic acid (AA), dopamine (DA), and uric acid (UA). Three well-defined sharp and fully resolved anodic peaks were found at the developed electrode. Differential pulse voltammetry was used to simultaneous determination of AA, DA, and UA in their ternary mixture. In the co-existence system of these three species, the linear response ranges for the determination of AA, DA, and UA were 4.0-4500 μM, 0.5-2000 μM, and 0.8-2500 μM, respectively. The detection limits (S/N=3) were found to be 0.95 μM, 0.12 μM, and 0.20 μM for the determination of AA, DA, and UA, respectively. Furthermore, the SPGNE displayed high reproducibility and stability for these species determination. The feasibility of the developed electrode for real sample analysis was investigated. Results showed that the SPGNE could be used as a sensitive and selective sensor for simultaneous determination of AA, DA, and UA in biological samples, which may provide a promising alternative in routine sensing applications.     

Development of a screen-printed amperometric biosensor for the determination of L-lactate dehydrogenase level.

We attempted to develop a screen-printed biosensor for the amperometric determination of L-lactate dehydrogenase (LDH) level on the basis of NAD(+)/NADH-dependent dehydrogenase reaction. The printing ink for the working electrode consisted of L-lactate, NAD(+), composite polymer of hydroxyethyl cellulose with ethylene glycol, 3,4-dihydroxybenzaldehyde (3,4-DHB) as an electron transferring mediator, and graphite as the conducting material. The 3,4-DHB was electropolymerized on the carboneous working electrode by potential cycling between -200 and +300 mV vs. Ag/AgCl reference electrode. Through the electrocatalytic reaction with immobilized 3,4-DHB, the NADH generated by the LDH reaction could be efficiently oxidized at lower potential than the unmodified carbon electrode. The analytical performance of the electrode was characterized in terms of linear sensing range and detection limit for LDH. The response from the developed biosensor was linear up to 500 U/l of LDH, and the detection limit of 50 U/l was observed at the signal-to-noise ratio of 3.     

Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode

A disposable and mediatorless immunosensor based on a conducting polymer (5,2':5'2"-terthiophene-3'-carboxylic acid) coated screen-printed carbon electrode has been developed using a separation-free homogeneous technique for the detection of rabbit IgG as a model analyte. Horseradish peroxidase (HRP) and streptavidin were covalently bonded with the polymer on the electrode and biotinylated antibody was immobilized on the electrode surface using avidin-biotin coupling. This sensor was based on the competitive assay between free and labeled antigen for the available binding sites of antibody. Glucose oxidase was used as a label and in the presence of glucose, H(2)O(2) formed by the analyte-enzyme conjugate was reduced by the enzyme channeling via HRP bonded on the electrode. The catalytic current was monitored amperometrically at -0.35 V vs. Ag/AgCl and this method showed a linear range of RIgG concentrations from 0.5 to 2 microg/ml with standard deviation +/-0.0145 (n=4). Detection limit was determined to be 0.33 microg/ml.     

Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a disposable electrochemiluminescent immunosensors array

Nowadays, β(2)-agonists are abused illegally as "lean meat agents" for food-producing animals, and cause increasing food-safety accidents in some countries. Due to their hazard to the human health, "lean meat agents" are banned in most countries and required to be routinely monitored. We herein report a disposable electrochemiluminescent immunosensors array for near-simultaneous assay of multiple β(2)-agonist residues in swine urine, by using ractopamine and salbutamol as the models. In this investigation, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensors array. Then the immunosensors array was constructed by site-selectively immobilizing the antigens of ractopamine and salbutamol on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the electrochemiluminescent signals of the two β(2)-agonists were sequentially detected using a non-array detector. The limits of detection for ractopamine and salbutamol were 8.5 and 17pg/mL, respectively, which were much lower than those of the most previous reports. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple β(2)-agonists in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.     

Enzyme immunosensors based on electropolymerized polytyramine modified electrodes

Highly sensitive amperometric enzyme immunosensors for human immunoglobulin G (IgG) were prepared on the basis of electrogenerated polytyramine (PTy, tyramine = p-(2-aminoethyl)-phenol) modified electrodes. Properties of PTy films changed depending on electrolysis conditions. On the basis of the found properties of the films, an effective IgG sensor was prepared: a PTy film was formed first from an acid solution on a Pt electrode, and the surface was further covered with a PTy film from an alkaline methanol solution to give a PTy doubly coated electrode on which anti-IgG was then immobilized. This electrode provided a large surface area with little non-specific adsorption of proteins. By means of the competitive enzyme immunoassay technique using glucose oxidase (GOD) labeled IgG conjugates, IgG was determined in the concentration range of c. 10 pg/ml-1 mg/ml from the oxidation current of H2O2 generated by the enzyme (GOD) reaction using the above IgG sensor. Also, an anti-IgG immobilized electrode, prepared by using a Pt electrode singly covered with a PTy film from an alkaline methanol solution, acted as an effective IgG sensor with a detection limit for IgG of c. 100 pg/ml.     

Highly stable electrochemical immunosensor for carcinoembryonic antigen

The long-term stability of sensing interfaces is an important issue in biosensor fabrication. A novel stable gold nanoparticle (AuNP)-modified glassy carbon (GC) electrode interface (GC-Ph-AuNP)-based biosensor for detecting carcinoembryonic antigen (CEA) was developed. GC electrodes were modified with 1,4-phenylenediamine to form a stable layer, and then AuNPs were bound onto the GC electrodes through CAu bonds. Anti-CEA was directly adsorbed on AuNPs fixed on the GC electrode. The linear range of the immunosensor was from 10 fg to 100 ng mL(-1) with a detection limit of 3 fg mL(-1) (S/N=3). The current of the immunosensor was increased by 4% after one month. The GC-Ph-AuNP immunosensor showed high sensitivity, a wide linear range, low detection limit, and good selectivity and stability. The immobilization method of the immunosensor could be widely applied to construct other immunosensors.     

Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.     

Direct electrochemical addressing of immunoglobulins: immuno-chip on screen-printed microarray

An original immobilisation technology is presented for the development of chemiluminescent protein biochips, suitable for measurement in complex matrices. The immobilisation strategy involved is based on diazotated aniline derivatives, which could be electro-addressed, thus creating a covalent linkage with a conducting material surface. The present electrochemical system is a cost effective and mass-produced carbon paste screen-printed (SP) microarray composed of eight 0.2 mm2 working electrodes, one carbon pseudo-reference electrode and one auxiliary electrode. Rabbit immunoglobulins (IgG) were chemically modified with an aniline derivative (4-carboxymethylaniline) in order to be easily electro-grafted to the SP microarray surface. The possibility of successively electro-address the eight sensing layers of a particular array, with a good reproducibility (more than 80%) and without loss of reactivity was demonstrated. Moreover, these immobilised proteins were subsequently used as a capture agent for the determination of rheumatoid factor (RF) in human sera. The absence of non-specific signal or interference problem enabled the detection of RF values in complex samples in the 5.3-485 IU/ml range with a good correlation with the standard Auraflex ELISA test method.     

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

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.