Enzyme Immunosensor Research Articles

Modified exfoliated graphene functionalized with carboxylic acid-group and thionine on a screen-printed carbon electrode as a platform for an electrochemical enzyme immunosensor.

An enzyme immunoassay was developed based on the coulometric measurement of immunoglobulin M (IgM) against Hantaan viruses (HTNV) by using virus-like particles (VLPs) as recognition molecules. The surface functionalization of screen-printed carbon electrodes (SPCEs) was achieved through paste-exfoliated graphene that was modified with a COOH group and a thionine mediator through supramolecular-covalent scaffolds, on SPCEs by using the binder contained in the ink. After the covalent immobilization of the antibody, the sensor was used for the sandwich enzyme immunoassay of IgM against HTNV. By using HTNV VLPs as the second recognization molecules, the resulting sensor efficiently monitored the reaction of IgM against HTNV and anti-IgM antibody with high specificity. By attaching HTNV nucleocapsid protein antibody conjugate with horseradish peroxidase (HRP) onto VLPs, the signal response of the assay was derived from the coulometric measurement of H2O2 reduction mediated by thionine on the electrode surface after the application of a potential (- 0.2V vs. Ag/AgCl). The ratio of charges measured before or after H2O2 addition was used to quantify IgM because these charges could be used as background charges or total charges, respectively. The ratio exhibited good agreement with IgM concentration within a range 0.1 to 1000pgmL-1, and a detection limit of 0.06pgmL-1 was obtained. The assay demonstrated high sensitivity and specificity toward HTNV-specific IgM in serum.

Enzyme-functionalized electrochemical immunosensor based on electrochemically reduced graphene oxide and polyvinyl alcohol-polydimethylsiloxane for the detection of Salmonella pullorum & Salmonella gallinarum

A novel and ultrasensitive electrochemical enzyme immunosensor based on electrochemically reduced graphene oxide (ERGO) and a polyvinyl alcohol (PVA)-multilayer polydimethylsiloxane (PDMS) film modified screen-printed carbon electrode (SPCE) for the rapid detection of Salmonella pullorum (S. pullorum) & Salmonella gallinarum (S. gallinarum) was proposed in this study. The electrochemical characteristics of the stepwise modified electrodes and the determinations of S. pullorum & S. gallinarum were investigated by cyclic voltammetry (CV). PVA-PDMS was characterized by Fourier transform infrared spectroscopy (FTIR). The conductivity of the electrode was promoted by electrochemically reduced graphene oxide, and the sensitivity of the immunosensor was enhanced and strengthened. The binding capacity and biocompatibility of the ERGO modified electrode was elevated considerably by the PVA-PDMS film. Under optimized working conditions, the sensor showed a good performance with a linear response range from 101 CFU mL−1 to 109 CFU mL−1, and the detection limit was 1.61 × 101 CFU mL−1 (S/N = 3). The proposed enzyme immunosensor had high sensitivity, good specificity, acceptable accuracy and reproducibility, and low detection limit characteristics and could be a promising analytical tool in the detection of S. pullorum & S. gallinarum in practical samples.

Biosensors in clinical chemistry: An overview

Biosensors are small devices that employ biological/biochemical reactions for detecting target analytes. Basically, the device consists of a biocatalyst and a transducer. The biocatalyst may be a cell, tissue, enzyme or even an oligonucleotide. The transducers are mainly amperometric, potentiometric or optical. The classification of biosensors is based on (a) the nature of the recognition event or (b) the intimacy between the biocatalyst and the transducer. Bioaffinity and biocatalytic devices are examples for the former and the first, whereas second and third generation instruments are examples for the latter. Cell-based biosensors utilizing immobilized cells, tissues as also enzyme immunosensors and DNA biosensors find variegated uses in diagnostics. Enzyme nanoparticle-based biosensors make use of small particles in the nanometer scale and are currently making a mark in laboratory medicine. Nanotechnology can help in optimizing the diagnostic biochips, which would facilitate sensitive, rapid, accurate and precise bedside monitoring. Biosensors render themselves as capable diagnostic tools as they meet most of the above-mentioned criteria.

Nanotubes and nanoparticles based 3D scaffolds for the construction of high performance Biosensors

ABSTRACT3D scaffolds with different pore sizes, using single-walled carbon nanotubes (SWCNTs) and nanoparticles of different size were constructed. Biotinylated glucose oxidase (GOX-B) and anti-cholera toxin (anti-CT) were immobilized onto the one and two level nanoscaffolds, functionalized with pyrene-β-cyclodextrin for the construction of glucose based enzyme sensors and immunosensors, respectively. For enzyme sensors, highest current density and sensitivity (41.72 μA cm-2, 3 mA M-1 cm-2) were obtained with two level scaffolds made with 100 nm nanoparticles. In contrast to this, for immunosensors, highest current density and sensitivity (11.71 μA cm-2, 116.2 μA M-1 cm-2) were obtained with two level scaffolds made with 500 nm nanoparticles, indicating that the pore sizes can be adjusted using different size of nanoparticles for the respective applications.

Enzyme immunosensor based on gold nanoparticles electroposition and Streptavidin-biotin system for detection of S. pullorum & S. gallinarum

A novel electrochemical enzyme immunosensor based on the electrodeposited gold nanoparticles and the multistage amplification of streptavidin-biotin affinity system for detection of Salmonella pullorum and Salmonella gallinarum (S. pullorum & S. gallinarum) was investigated in this study. The electrochemical characteristics of the stepwise modified electrodes were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), whereas the determinations of the targets of S. pullorum & S. gallinarum were carried out by CV. As shown in the results of this study, the electron transfer was promoted by the electrodeposition of gold nanoparticles, thus the communication of electrons was enhanced and the conductivity of the electrode was strengthened too. Moreover, the number of the conjugated bio-molecules was elevated greatly by the electrodeposited gold nanoparticles and the streptavidin-biotin, which contributed to the integration of the following modifications and amplification of the current response signal. Under the optimized working conditions, the sensor showed a good performance with a linear response range from 102 CFU/ml to109 CFU/ml, the detection limit for S. pullorum & S. gallinarum determination was 1.95×102 CFU/ml (S/N=3). The proposed enzyme immunosensor with high sensitivity, good specificity, acceptable accuracy and reproducibility, and low detection limit characteristics could be a promising analytical tool in detection of S. pullorum & S. gallinarum in practical samples and a model for the development of immunosensor of other bacterium of interests.

Electrochemical immunosensor for alpha-fetoprotein determination based on ZnSe quantum dots/Azure I/gold nanoparticles/poly (3,4-ethylenedioxythiophene) modified Pt electrode

Abstract A novel amperometric enzyme immunosensor with amplified sensitivity for the determination of alpha-fetoprotein (AFP) was constructed with layer-by-layer assembly of ZnSe quantum dots (ZnSe QDs)/Azure I/gold nanoparticles (nanoAu)/poly (3,4-ethylenedioxythiophene) (PEDOT) on Pt electrode. Firstly, citrate coated nanoAu was immobilized on the PEDOT polymer film, which was electrochemically synthesized in ionic liquid electrolyte of 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF 4 ]). Then, Azure I was immobilized on nanoAu/PEDOT composite matrix as a redox probe and was used to immobilize ZnSe QDs. Subsequently, AFP antibody (anti-AFP) was adsorbed onto the surface of ZnSe layer. Finally, horseradish peroxidase (HRP) was employed to block sites against nonspecific binding and amplify the current signal of the antigen–antibody reaction. The modification processes were characterized by cyclic voltammetry, scanning electron microscopy. The factors influenced the performances of the proposed immunosensors were studied in detail. Because of the synergism between Azure I and nanoAu/PEDOT to facilitate electron-transfer process, and the small diameter of ZnSe QDs favorable for stabilization of biological activity to a large extent, the immunosensor displayed a high sensitivity, fast analytical time, a relatively low detection limit of 1.1 fg/mL at 3 times of signal-to-noise ratio ( S / N = 3), and a especially broad linear response to AFP in a ranges from 5 × 10 −5 to 250 ng/mL. Moreover, the selectivity, repeatability, and stability of the proposed immunosensor were acceptable.

A strategy for signal amplification using an amperometric enzyme immunosensor based on HRP modified platinum nanoparticles

A novel sandwich-type electrochemical enzyme immunosensor for sensitive detection of α-1-fetoprotein (AFP) using a signal amplification strategy based on the enzyme and the catalytic activity of Pt nanoparticles (PtNPs) is investigated in this work. Sensitivity was greatly amplified by the detection mode which was based on dual signal amplification: (1) secondary antibody labeled HRP-functionalized PtNPs were introduced on the electrode surface through biotin–avidin interactions; (2) the biotin-labeled HRP was further attached to HRP-functionalized PtNPs to assemble a dense HRP film, which resulted in significant enhancement of the current response. The adjusted (but not random) orientation of anti-AFP on the nanohybrid film of gold nanoparticles/multi-walled carbon nanotubes (AuNP/MWNT) was accomplished using an avidin–biotin immobilization technique. The nanohybrid film AuNP/MWNT can improve the response of the immunosensors due to its higher exposed surface area, high catalytic activity which facilitates electron transfer and excellent biocompatibility. The performance of the amperometric immunosensor was evaluated and optimized for best performance. The detection limit for AFP was found to be 0.08 ng/mL at S/ N = 3 and the detection concentration were in the range between 0.25 and 100 ng/mL.

Fabrication and evaluation of the enzyme immunosensor for rapid detection of Vibrio parahaemolyticus based on chitosan-SiO2 hybrid membrane

To improve the key technology of immunesensors in immobilizing bio-sensitive element and keeping its bioactivity, an enzyme immunosensor based on chitosan-SiO(2) (CS-Sio(2)) hybrid membrane was fabricated. To estimate the new immunosensor Vibrio parahaemolyticus which was the main pathogens of aquatic products. A CS-SiO(2) hybrid membrane was prepared using sol-gel method. The enzyme immunosensor was fabricated by coating the membrane and horseradish peroxidase labeled Vibrio parahaemolyticus antibody (HRP-anti-VP) on the surface of four-channel screen-printed carbon electrode. The immunosensor was characterized by cyclic voltammetry. Vibrio parahaemolyticus could be detected according to the decrease percentage (DP) of peak current before and after immune response, while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction. Seven kinds of bacteria, like Vibrio alginolyticus, were selected for specific experiments. By studying the infrared spectrum of three kinds of films, the CS-SiO(2) hybrid membrane was prepared and HRP-anti-VP was fixed in the hybrid membrane. Under the optimum conditions of immunoreaction and electrochemical detection, the DP of peak current before and after immune response showed a linear relation with lgC in the range of 10(4) - 10(9) cfu/ml, while the linear regression equation was: DP = 6.5 lgC-3.319, the correlation coefficient was 0.9958 and the detection limit was 6.9 x 10(3) cfu/ml (S/N = 3). The immunosensor possessed acceptable specificity, reproducibility (RSD < 6%), stability (the amperometric response was 95% of the initial response after a week) and accuracy (96.7% of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003). The enzyme immunosensor based on CS-SiO(2) hybrid membrane gave a good performance in rapid detection of Vibrio parahaemolyticus.

Comparison of Electrochemical and Surface Plasmon Resonance Immunosensor Responses on Single Thin Film

AbstractThis paper reports results obtained when comparing an electrochemical enzyme immunosensor and a surface plasmon resonance (SPR) based immunosensor on the same gold surface installed in an electrochemical SPR flow cell. Simultaneous electrochemical and SPR measurements were performed on a gold surface modified with multilayers of poly‐L‐lysine and poly‐styrenesulfonate assembled with the layer‐by‐layer method. First, we obtained the SPR response induced by the formation of an immunocomplex from the shift in the SPR angle by injecting an anti tumor necrosis factor‐α antibody solution labeled with alkaline phosphatase into the flow cell containing the multilayer modified with tumor necrosis factor‐α. Then we compared this SPR result with that obtained for the electrochemical oxidation current of p‐aminophenol catalyzed by alkaline phosphatase from p‐aminophenolphosphate on the same gold film. We compared the two immunosensor responses obtained using the different measurement principles and found that there was a high correlation efficient of 0.973 between them. This was because we were able to immobilize the immunoreagents with good stability and without losing the transport of the enzyme product in the multilayer whose thickness we easily controlled with nanometer scale accuracy. We also report that the detection limit of our electrochemical immunosensor after optimization was around 100 pg/mL (0.4 pM), which is one of the lowest values yet reported for an electrochemical immunosensor.

New Antibody Immobilization Strategy Based on Gold Nanoparticles and Azure I/Multi-Walled Carbon Nanotube Composite Membranes for an Amperometric Enzyme Immunosensor

A convenient and effective strategy based on the unique characteristics of Azure I/multi-walled carbon nanotube (Azure I/MWNT) composite membranes and the amplification response of enzymes to the antigen−antibody reaction was used to develop a highly sensitive amperometric immunosensor for the detection of α-fetoprotein (AFP). A novel type of Azure I/MWNT composite membrane was fabricated by two steps: first, acid-pretreated MWNT was modified on the surface of glassy carbon electrode. Then, Azure I was coated on MWNT to obtain an Azure I/MWNT composite membrane. Subsequently, gold nanoparticles were adsorbed onto the Azure I/MWNT composite surface by electrostatic interactions between the negatively charged gold nanoparticle and the positively charged Azure I. Later, AFP antibodies were assembled onto the surface of gold nanoparticles. Finally, horseradish peroxidase (HRP) instead of bovine serum albumin was employed to block sites against nonspecific binding and amplified the current signal of the antig...

A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode

Recent outbreaks of foodborne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A disposable electrochemical immunosensor for detection of Vibrio parahaemolyticus (VP) based on the screen-printed electrode (SPE) coated with agarose/Nano-Au membrane and horseradish peroxidase (HRP) labeled VP antibody (HRP-anti-VP) has been developed in this paper. Then, the immunosensor was characterized by cyclic voltammetry (CV) and laser scanning confocal microscope (LSCM). The immunosensor was incubated with the one-step immunoassay format involving VP for 30 min at room temperature (25 ± 0.5 °C). The access of the active center of HRP catalyzing the oxidation reaction of thionine by H 2O 2 was partly inhibited by VP, which connected on the surface of the immunosensor by immunoreaction. VP could be quantificationally detected according to the shift of reduction current while CV was used as electrochemical means to detect the products of the enzymatic reaction. Under the optimum conditions of immunoreaction and electrochemical detection, VP was rapidly detectable by sigmoidal curve method and form a linear calibration between 10 5 and 10 9 cfu/ml with an associated detection limit of 7.374 × 10 4 cfu/ml (S/N = 3). The immunosensor had acceptable specificity, reproducibility, stability and accuracy, indicating that the immunosensor could satisfy the need of practical sample detection.

A tris(2,2′-bipyridyl)cobalt(III)-bovine serum albumin composite membrane for biosensors

A concept based on a novel redox-biocompatible composite protein membrane fabrication, double enzyme membrane modification technique and antibody immobilization, was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of carcinoembryonic antigen (CEA). In this concept, a solution of bovine serum albumin (BSA) containing horseradish peroxidase (HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved. Then tris(2,2'-bipyridyl) cobalt(III) (Co(bpy)(3)3+), as a mediator, was embedded in BSA-HRP composite membrane vis the electrostatic force and hydrophobe functions. Later a self-assembled conductive nano-Au monolayer was constructed onto the resultant electrode surface by electrostatic interaction between the negatively charged nano-Au and positively charged Co(bpy)3(3+). Protein A is used as a binding material to achieve an adjusted (but not random) orientation of the antibodies surface for efficient combination of antigens. Finally, the HRP, was employed to block the possible remaining active sites and avoid the non-specific adsorption, which acts not only as a blocking reagent instead of the commonly used BSA but also as the conventional enzyme-labeling to amplify the response of the antigen-antibody reaction. The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)(3)3+/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA in the range of 0.50-80.00 ng/mL with a limit of detection of 0.14 ng/mL, as well as good stability and long-term life.

Amperometric enzyme immunosensors based on layer-by-layer assembly of gold nanoparticles and thionine on Nafion modified electrode surface for α-1-fetoprotein determinations

A novel amperometric enzyme immunosensor with amplified sensitivity has been developed for the detection of α-1-fetoprotein (AFP) based on layer-by-layer (LBL) assembly of gold nanoparticles (nano-Au) and thionine (Thi) on Nafion (Nf) modified electrode surface by electrostatic adsorption. The stepwise layer-by-layer adsorption interface morphology was further characterized by means of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The enzyme, horseradish peroxidase (HRP), was employed to block the possible remaining active sites of the nano-Au monolayer, avoid the non-specific adsorption instead of bovine serum albumin (BSA) and amplify the response of the antigen–antibody reaction. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. Tests performed with this immunosensor showed good linearity and specificity when it was evaluated on several standard serum samples and the working range for the system was 1 to 250 ng/ml with a detection limit of 0.56 ng/ml. In addition, the prepared immunosensor could be regenerated 8 times with 4 M urea solution. When the immunosensor was stored at 4 °C and measured intermittently (every 3–5 days), no apparent change was found over 3 months, it yielded a 6.5% R.S.D.

Amperometric Enzyme Immunosensors for Diagnosing Certain Infectious Diseases

Amperometric enzyme immunosensors based on planar electrodes were developed for the rapid determination of some bacterial antigens. Conditions for the operation of analytical devices (matrix component, pH of the buffer solution, and the dilution of antibodies in the biosensing part of the sensor) were selected. The analytical characteristics of enzyme immunosensors (analytical ranges and determination limits) were estimated. The analytical devices were tested in determining pyogenic streptococcus and aurous staphylococcus antigens in the blood serum of patients suffering from infectious diseases. It was shown that the developed sensors can be used for diagnosing diseases caused by these microorganisms.

Development of an immunosensor for the determination of rabbit IgG using streptavidin modified screen-printed carbon electrodes

Voltammetric enzyme immunosensors based on the employment of streptavidin modified screen-printed carbon electrodes (SPCEs) for the detection of rabbit IgG, as a model analyte, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate (3-IP) were used as the enzymatic label and substrate, respectively. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 °C on the pre-oxidized surface of the SPCEs. The analytical characteristics of these sensors were evaluated using biotin conjugated to AP. The immunosensor devices were based on a specific reaction of rabbit IgG with its biotinylated antibodies, which were immobilised on the modified screen-printed carbon electrodes through the streptavidin:biotin reaction. The immunosensors were used for a direct determination of AP labelled rabbit IgG, and for free rabbit IgG detection using a sequential competitive immunoassay. A calibration curve in the range of 5 × 10 −11 to 1 × 10 −9 M of rabbit IgG was obtained with a estimated detection limit of 5 × 10 −11 M (7.0 ng/ml). These immunosensors were stable for 5 months if they were stored at 4 °C.

Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1×3.2 mm, comprising 42 porous flow channels of 235 μm depth and 25 μm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/ p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H 2O 2. All immunosensors were capable of detecting atrazine at the sub-μg l −1 level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 μg l −1, and IC 50 values of 0.096 and 0.130 μg l −1, respectively. The protein G based immunosensors were regenerated with 0.4 M glycine–HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87–102 and 88–124% at atrazine fortification levels of 0.5–3 and 80–240 μg l −1, respectively.

Determination of gentamicin with an amperometric enzyme immunosensor

A new version of enzyme immunoassay with the use of an amperometric immunosensor was proposed for the determination of an aminoglycoside antibiotic gentamicin. The biosensing part of the enzyme immunosensor simultaneously incorporates immobilized enzyme cholinesterase and antibodies against gentamicin. The detection limit for gentamicin in this method is 1 × 10–9 mg/mL. The time of determination is no longer than 20 min. The stability constants of the immunocomplex and the kinetic parameters of the enzymatic reaction in its presence were determined. Gentamicin was determined in samples of pharmaceutical preparations and foodstuffs (milk).

Kinetic Parameters of Choline Esterase-Catalyzed Hydrolysis in the Presence of the Antigen-Antibody Immune Complex

The influence of antigen-antibody immune complexes (with the immune pairs of Candida albicans and Phytophtora infestans antigens and the corresponding antibodies) on the catalytic activity of immobilized choline esterase in the enzyme immunosensor was studied. The antigen-antibody immune complex can act as effector of choline esterase, suppressing or enhancing its catalytic activity depending on the ratio of the components of the biospecific interaction (antibodies, antigen, enzyme, and substrate). The kinetic parameters of hydrolysis of butyryl thiocholine iodide, a specific substrate of choline esterase (apparent Michaelis constants, maximal reaction rates, activation and inhibition constants) were calculated at various concentrations of the substrate and antigen and at various dilutions of the antibodies. The types and kinds of the observed effects were determined. The effect of pH on the kinetic parameters of the enzymatic reaction was studied. Various pathways, depending on conditions, were suggested for choline esterase-catalyzed hydrolysis of butyryl thiocholine iodide in the presence of the antigen-antibody immune complex.

Amperometric enzyme immunosensor for the determination of the antigen of the pathogenic fungi Trichophyton rubrum

An amperometric enzyme immunosensor for the determination of antigen (Ag) of pathogenic fungi Trichophyton rubrum (TR) was developed. The immunosensor is based on a specific immunoreaction of Ag with antibodies which were immobilized together with cholinesterase into a nitrocellulose membrane (biosensitive part of immunosensor). The biosensitive part could be regenerated and is stable for at least 35 days. The developed immunosensor allows the determination of down to 1×10 −15 mg ml −1 of Ag TR. The total time of immunoassay is less than 20 min. The method was applied for the determination of Ag TR in human blood serum.

AC voltammetric carbon paste-based enzyme immunosensors

Carbon paste electrodes, previously anodised in a basic media, are the basis for the development of a new voltammetric immunosensor device. Passive adsorption of the appropriate immunochemical reagent was performed onto the electrode surface. Alkaline Phosphatase labelled immunoglobulin was the tracer used in this work, 3-indoxyl phosphate being a very suitable enzymatic substrate for the electrochemical detection of the corresponding affinity reaction. The hydrolysis of this molecule generates indigo dimmer. This product was detected by alternating current voltammetry taking advantage of the adsorptive and inherent electrodic properties that it exhibits. The same electrochemical anodisation was used at the end of one assay to remove the entire protein layer attached to the carbon paste surface, allowing the formation of a new sensing phase and the use of the same support in several consecutive experiments. The methodology was applied to the design of two different immunoassays for the determination of human IgG. Good reproducibility of the electrodic signal and a limit of detection around 10 −10 M were achieved.