Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori

In this paper, we have developed and characterized a microfluidic magnetic immunosensor coupled to a gold electrode for the rapid and sensitive quantification of human serum IgG antibodies to Helicobacter pylori. This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. The sensor was completely automated and the antibodies detection in serum samples was carried out using a non-competitive immunoassay based on the use of purified H. pylori antigens that are immobilized on magnetic microspheres 3-aminopropyl-modified. The magnetic microbeads were injected into microchannel devices and manipulated for an external removable magnet. The IgG antibodies in human serum sample are allowed to react immunologically with the immobilized antigens, and the bounded antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP and an electroactive product was detected on gold layer electrode at 0.250 V. The response current obtained from the product of enzymatic reaction is directly proportional to the activity of the enzyme and, consequently, to the amount of IgG antibodies to H. pylori in serum samples. The electrochemical detection can be done within 1 min and total assay time was 25 min. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.37 and 2.1 U mL⁻¹, respectively, and the within- and between-assay coefficients of variation were below 5%. Our results indicate the potential usefulness of our fabricated microbiochip for the early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.     

Integrated microfluidic systems with an immunosensor modified with carbon nanotubes for detection of prostate specific antigen (PSA) in human serum samples

This paper describes the development of an immunosensor coupled to glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (CNT-GCE) integrated with microfluidic systems for rapid and sensitive quantification of prostate specific antigen (PSA) in human serum samples. Mouse monoclonal (5G6) to PSA antibodies were immobilized on a rotating disk. PSA in the serum sample are allowed to react immunologically with the immobilized anti-tPSA and horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to PSA. HRP, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on CNT-GCE at -0.15 V. The electrochemical detection can be done within 1 min and total assay time was 30 min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.08 and 0.5 microg L(-1), respectively and the intra- and inter-assay coefficients of variation were below 4.5%. The electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, which shows potential for detecting PSA in clinical diagnosis.     

[AuCl4]− and Fe3+/[Fe(CN)6]3− ions-derivated immunosensing interface for electrochemical immunoassay of carcinoembryonic antigen in human serum

[AuCl₄]⁻ was initially deposited by electrochemical reduction on a glassy carbon electrode (GCE) to form porous nanogold layer, then prussian blue (PB) was electrodeposited onto the as-prepared nanogold layer, and then secondary nanogold particles were fabricated again on the PB surface by electrochemical reduction for the immobilization of anti-CEA antibodies. The presence of double-layer porous gold nanoparticles enhanced the immobilized amount of biomolecules, and improved the sensitivity of the immunoassay. PB, as a good redox probe, was facile to electrochemical analysis and measurement. Under optimal conditions, the developed immunoassay exhibited dynamic range from 3.0 to 80.0 ng/mL with a detection limit of 0.9 ng/mL CEA (S/N = 3). Moreover, the selectivity, reproducibility and stability of the immunosensor were acceptable.     

Conductometric immunoassay for interleukin-6 in human serum based on organic/inorganic hybrid membrane-functionalized interface

Various sensor-based immunoassay methods have been extensively developed for the detection of interleukin-6 (IL6), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive conductometric immunoassay for IL6 in human serum by using an organic/inorganic hybrid membrane-functionalized interface. Initially, thionine-bound 3,4,9,10-perylenetetracarboxylic acid was doped into colloidal alumina, then nanogold particles were immobilized onto the thionine surface, and then horseradish peroxidase-labeled anti-IL6 antibodies were conjugated on the nanogold surface. The organic/inorganic hybrid membrane provides a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. The detection is based on the change in local conductivity before and after the antigen-antibody interaction in 0.02 M phosphate buffer solution (pH 6.8) containing 50 μM H₂O₂, 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 25 to 400 pg/ml towards IL6 with a relatively low detection limit of 5 pg/ml (S/N = 3). The stability, reproducibility and precision of the immunosensor were acceptable. 37 serum specimens were assayed by the developed immunosensor and standard enzyme-linked immunosorbent assay, respectively, and the results obtained were almost consistent. More importantly, the detection methodology provides a promising approach for other proteins or biosecuritys.     

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.     

Sensitive amperometric immunosensor for α-fetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film

A novel strategy for the fabrication of sensitive immunosensor to detect α-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml⁻¹ with a detection limit of 0.6 ng ml⁻¹. The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP-anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.     

Evaluating the Potential of an Antibody Against Recombinant OmpW Antigen in Detection of Vibrio cholerae by Surface Plasmon Resonance (SPR) Biosensor

Surface plasmon resonance (SPR), a highly sensitive and label-free optical biosensing technique, is a powerful tool for studying biomolecular interactions. An immunosensor for rapid, sensitive, and selective detection of Vibrio cholerae on the basis of SPR is reported. Recombinant OmpW antigen (a bacterial outer-membrane protein) of V. cholerae was expressed and purified and raising of polyclonal rabbit anti-OmpW was done. Antibodies were immobilized on a sensor surface and interactions between OmpW protein and the whole cell of V. cholerae with immobilized antibodies were studied in different experiments. The aim of this study was to evaluate the potential of anti-OmpW in detection of V. cholerae by developing an immunosensor based on SPR. The results showed high affinity interaction between OmpW and anti-OmpW (K _D = 2.4 ± 0.07 × 10⁻⁹ M) and SPR signals had a linear relationship with the number of V. cholerae ranging from 1 × 10² to 1 × 10⁷ cells/mL with limit of detection of 50 cells/mL. The specificity of the developed immunoassay was examined using some non-V. cholerae bacteria which did not produce any significant responses. This method is rapid, sensitive, and specific to target V. cholerae with a total analysis time of less than 60 min.

     

Sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film

A novel strategy for the fabrication of sensitive immunosensor to detect alpha-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml(-1) with a detection limit of 0.6 ng ml(-1). The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP-anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.     

Application of a Fab fragment of monoclonal antibody specific to <Emphasis Type="Italic">N</Emphasis>-terminal pro-brain natriuretic peptide for the detection based on regeneration-free electrochemical immunosensor

N-Terminal pro-brain natriuretic peptide (NT-proBNP) is an important marker for heart failure that reflects ventricular volume expansion, ventricular overload and the degree of cardiac injury. To establish a sensitive and simple detection method for it in serum, a regeneration-free immunosensor with novel Fab fragment monoclonal antibodies was prepared. The sensor detected NT-proBNP from 0.04 to 2.5 ng/ml with a limit of 0.03 ng/ml. The immunosensor is therefore a simple, cost-effective method to detect NT-proBNP and the proposed immunoassay system would enable other proteins to be detected and open new opportunities for protein diagnostics.     

Novel amperometric immunosensors based on iridium oxide matrices

Novel immunosensors based on antibodies immobilized in electrochemically grown iridium oxide (IrOx) thin film matrices have been developed. Antibody loading in the oxide was evaluated using a non-competitive electrochemical immunoassay for IgG. Anti-IgG loading in the oxide was found to be dependent on the concentration of anti-IgG present in the oxide growth step, with 400 microg/ml anti-IgG producing maximum amperometric responses.To study the potential analytical properties of the matrix, the dose-response behavior of the sensors was determined using optimized alkaline phosphatase-linked IgG immunoassay. Hydroquinone diphosphate (HQDP) was used as enzyme substrate and the oxidation of hydroquinone was detected amperometrically at +420 mV. The sensors displayed a linear dose-response behavior for IgG concentrations between 10 and 200 ng/ml, saturating above 600 ng/ml, and had a low detection limit of 8 ng/ml.Finally, the method was used to produce sensors containing immobilized anti-transferrin. Using a non-optimized electrochemical immunoassay for human transferrin (HT), dose-response behavior was observed for HT concentrations between 100 and 600 ng/ml.The results presented in this paper show that IrOx matrices represent a new method for immunosensor fabrication. The oxide acts as a hydrophilic, highly porous, three-dimensional matrix that can immobilize antibodies and retain their activity. The method is attractive because it offers the potential for high antibody loadings and is suitable for mass production of sensors in an easy and economical manner.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

An electrochemical enzyme bioaffinity electrode based on biotin-streptavidin conjunction and bienzyme substrate recycling for amplification

A signal amplificatory electrochemical immunoassay with biotin-streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin-streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 10⁻¹⁴ to 10⁻⁵ g ml⁻¹ was acquired with a relatively low detection limit of 3.5 × 10⁻⁵ g ml⁻¹ for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here.     

Electrochemical immunoassay on a microfluidic device with sequential injection and flushing functions

An integrated microfluidic device with injecting, flushing, and sensing functions was realized using valves that operate based on direct electrowetting. The device consisted of two substrates: a glass substrate with driving and sensing electrodes and a poly(dimethylsiloxane) (PDMS) substrate. Microfluidic transport was achieved using the spontaneous movement of solutions in hydrophilic flow channels formed with a dry-film photoresist layer. The injection and flushing of solutions were controlled by gold working electrodes, which functioned as valves. The valves were formed either in the channels or in a through-hole in the glass substrate. To demonstrate the system's applicability to an immunoassay, the detection of immobilized antigens was performed as a partial simulation of a sandwich immunoassay. Human -fetoprotein (AFP) or an anti-human AFP antibody was immobilized on a platinum working electrode in the chamber using a plasma-polymerized film (PPF). By applying a potential to the injection valves, necessary solutions were injected one by one through the channels into a reaction chamber at the center of the chip and incubated for reasonable periods of time. The solutions were then flushed through the flushing valve and absorbed in a filter paper placed under the device. After incubation with the corresponding antibodies labeled with glucose oxidase (GOD), electrochemical detection was conducted. In both cases, the obtained current depended on the amount of immobilized antigen. The calibration curves were sigmoidal, and the detection limit was 0.1 ng. The developed microfluidic system could potentially be a fundamental component for a micro immunoassay of the next generation.     

Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H⁺-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H⁺ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml⁻¹. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis.     

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL?1 levels. MPs were conjugated with ～90,000 antibodies and ～200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL?1 for PSA and 0.30 pg mL?1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.     

Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen

An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP–HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3.     

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.     

Electrochemical enzyme immunoassay for detection of toxic substances.

Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We developed a new biosensor design that combines the advantages of immunoassay with electrochemical response. We established that this enzyme-linked immunosensor measures toxic substances in biological samples. The biosensor consists of two major elements: (1) an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents, and (2) the electronic components used in the signal readout. The result is an amperometric immunoassay based on coupling the immunochemical reaction to the enzyme electrode response by using a soluble, electrochemically active mediator. The specific question addressed was: Does the system's immunochemical detection reliably respond at sufficiently low analyte concentrations? We present our results in these areas: (1) enzyme immobilization on colloidal gold; (2) colloidal gold-enzyme deposition on the electrode surface; (3) mediator-antigen conjugate synthesis; (4) antibody incorporation at the electrode surface; (5) bioelectrode characterization and optimization; and (6) immunosensor demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as blood and urine and in environmental samples such as wastewater and drinking water.     

Carbon nanotube-based ultrasensitive multiplexing electrochemical immunosensor for cancer biomarkers

A multiplexing electrochemical immunosensor was developed for ultrasensitive detection of cancer related protein biomarkers. We employed disposable screen-printed carbon electrode (SPCE) array as the detection platform. A universal multi-labeled nanoprobe was developed by loading HRP and goat-anti-rabbit IgG (secondary antibody, Ab₂) onto multiwalled carbon nanotube (MWNT). This universal nanoprobe was available for virtually any sandwich-based antigen detection and showed superiority in several areas. By using the SPCE array and the universal nanoprobe, we could detect as low as 5 pg mL⁻¹ of prostate specific antigen (PSA) and 8 pg mL⁻¹ of Interleukin 8 (IL-8) with the electrochemical immunosensor. We also demonstrated simultaneous detection of two protein biomarkers with this platform. With these attracted features, our immunoassay system shows promising applications for in-field and point-of-care test in clinical diagnostics.     

Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.