Amperometric Enzyme Sensor-Based Enzyme Immunoassay for Factor VIII-Related Antigen

Abstract

The coupling of an enzyme immunoassay for factor VIII-related antigen with a commercial glucose oxidase based amperometric sensor permits the determination of 1.6 to 16 ng of factor XIII-related antigen in human plasma. Further pure amperometric sensors or amperometric enzyme sensors for determination of the main marker-enzymes of enzyme immunoassays are described.     

Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

Immunoassay is one of the biochemical analytical techniques using the specific antigen antibody com-plexation for analytical purposes. It has extensive ap-plication in clinical diagnostics, prevention and cure of diseases, and virus diagnostics. The presentation and progress of immunoassay methodology are one of the greatest achievements of bioanalytical chemistry. It is estimated that several-hundred millions of immuno-analytical determinations are carried out every year all over the world. E…     

An Electrochemical Determination of Uric Acid in Sera by a Flow-Through Equipment with and Without Uricase

Abstract

A comparison of an enzymeless direct electrochemical oxidation procedure at a platinum electrode for the determination of uric acid, and an enzyme sensor with immobilized urate: oxygen oxidoreductase (uricase), was performed in flow stream systems. The uricase enzyme electrode is based on the H₂O₂ oxidation current. Both amperometric methods were related to the wall-known photometric uricase-catalase-procedure (UCM) as a reference method. The measured values of both methods are of the first derivatives of current change (dI/dt) due to the electrochemical or electrochemical enzymatic reaction, respectively. The analytical quality of the measurements is characterized by: precision s% within run < 2% day to day < 5% accuracy acceptable (control materials) correlation to reference method r >0.93 analysis rate 80 samples/hr     

Development of a heterogeneous chemiluminescent flow immunoassay for DDT and related compounds

A heterogeneous chemiluminescent (CL) flow immunoassay for DDT was optimized comparing different types of immunoaffinity supports: beads, nylon coils and membranes (membranes HyBondN+). In order to characterize solid immunoaffinity supports two basic immunoassay formats were performed, using (1) enzyme-labeled secondary and (2) enzyme-labeled specific monoclonal antibodies (MAbs). In both formats, hapten DDT5 conjugated to ovalbumin immobilized on solid supports according to the appropriate immobilization procedure, enzyme label (horseradish peroxidase, HRP) and luminescent detection (luminol/H₂O₂/p-iodophenol) were used. The lowest limit of detection (LOD), 1 nM p,p-DDT, was obtained with a membrane-based flow immunoassay with HRP-labeled specific antibody. Beads and packed tubing were discarded as appropriate supports because of the difficulties encountered for reproducible packing and the occurrence of light scatterring (beads), which seriously compromised the performance and reproducibility of the flow immunoassay.     

A Novel Fluorescence Immunoassay Based on Two-time Amplified Fluorescence Signal by Hemin and Magnetic Nanoparticles for the Detection of Antigen

Abstract

A novel, selective, and sensitive magnetic-mimetic enzyme fluorescence immunoassay method for antigen detection has been developed by taking advantage of a magnetic separation process and the amplification feature of the hemin label. This method is based on a twice amplified fluorescence signal. The signal is first amplified due to the ultrasmall size and the high surface-to-volume ratio of the silica-coated magnetite nanoparticles, which enable the nanoparticles to carry much more antibodies. Second, the mimetic enzyme (hemin) as a labeling reagent catalyzes the reaction of p-hydroxyphenyl acetic acid and H₂O₂ can further amplify the fluorescence signal. This protocol was also evaluated for a sandwich-type immunoassay of human IgG, and the calibration graph for human IgG was linear over the range of 0–100 ng mL^?1 with a detection limit of 9.8 ng mL^?1. This method can easily separate magnetic nanoparticles from the solution, which simplified the process and played a promising role for various applications in immunoassay.     

Application of a mimetic enzyme for the enzyme immunoassay for α-1-fetoprotein

A new mimetic enzyme immunoassay (MEIA) method for α-1-fetoprotein (AFP) was developed using a mimetic enzyme Mn(III)-tetra(sulfophenyl)porphine (Mn-TPPS₄) as a labelling reagent to catalyze the fluorescence reaction of 4-hydroxy-phenylacetic acid (HPA) and hydrogen peroxide. In the sandwich assay standard AFP solution or AFP containing umbilical blood serum first reacts with antibody (anti-AFP)coated on a 40-well plate (polystyrene), and then further reacts with Mn-TPPS₄-labelled anti-AFP; the Mn-TPPS₄ on the bound fraction, after removing the free fraction was determined by measuring the fluorescence intensity as a result of the reaction between HPA and H₂O₂, catalyzed by bound Mn-TPPS₄ and anti-AFP conjugate, which was proportional to the concentration of AFP. AFP in the range 0.01–10 μg per well can be determined with a detection limit of 1 ng per well. The method has sufficient sensitivity to be applied to the determination of AFP in umbilical blood serum.     

Improved Sensitive Sandwich Enzyme Immunoassay for Secretory Immunoglobulin a in Human Serum

Abstract

To overcome the IgA interference in enzyme immunoassay for serum secretory IgA (SIgA), a new specific, simple and more sensitive sandwich enzyme immunoassay, fully free of the serum IgA interference, was developed. SIgA standards or samples were added into the wells of polystyrene plate coated with rabbit IgG antibody to human secretory component. After incubation, the wells were washed and then, horseradish peroxidase-labeled Fab′ fragment of goat IgG antibody to human α-chain was added and incubated. The wells were washed again to remove the unbound labeled antibody, and the enzyme activity specifically bound to the well was assayed using 3,3′, 5,5′ - tetramethylbenzidine and H₂O₂ as substrate. The enzyme reaction was stopped by addition of 2M H₂SO₄. The SIgA concentration was determined from the absorbance at 450 nm. The minimun detectable sensitivity was 6ng/ml. The assay system had very good selectivity overcoming the interference of IgA. As the result of high sensitivity, only small amount of sample (2 μ1 for serum) was needed for analysis. In this assay, no cross reactivity was found with purified human IgG, mIgA, IgM or free secretory component (FSC). The recovery of SIgA mixed with human sera or biles was 99.6–108.1%. The coefficients of within-assay and between-assay variation were 5.8–9.3% and 6.2–9.2% respectively. It correlated well with a liquid phase competitive radioimmunoassay for human serum SIgA (r=0.96, n=30, P<0.0l). The level of SIgA in normal human serum was 8.04±3.60 (SD) μg/ml (n=117) and increased significantly in patients with choledocho- lithiasis (57.35±49.70 μg/ml, n=15, P<0.0l). SIgA concentrations in bile samples were also determined by the 2 4′ assay under the condition that FSC did not, interfere with the assay.     

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H₂O₂) and H₂O₂-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H₂O₂ and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H₂O₂, as well as the incubation time between H₂O₂ and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10² CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10³ CFU/mL to 1.18 × 10⁶ CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.     

Helical Carbon Nanotubes: Intrinsic Peroxidase Catalytic Activity and Its Application for Biocatalysis and Biosensing

A combined hydrothermal/hydrogen reduction method has been developed for the mass production of helical carbon nanotubes (HCNTs) by the pyrolysis of acetylene at 475 °C in the presence of Fe₃O₄ nanoparticles. The synthesized HCNTs have been characterized by high‐resolution transmission electron microscopy, scanning electron microscopy, X‐ray diffraction analysis, vibrating sample magnetometry, and contact‐angle measurements. The as‐prepared helical‐structured carbon nanotubes have a large specific surface area and high peroxidase‐like activity. Catalysis was found to follow Michaelis–Menten kinetics and the HCNTs showed strong affinity for both H₂O₂ and 3,3′,5,5′,‐tetramethylbenzidine (TMB). Based on the high activity, the HCNTs were firstly used to develop a biocatalyst and amperometric sensor. At pH 7.0, the constructed amperometric sensor showed a linear range for the detection of H₂O₂ from 0.5 to 115 μM with a correlation coefficient of 0.999 without the need for an electron‐transfer mediator. Because of their low cost and high stability, these novel metallic HCNTs represent a promising candidate as mimetic enzymes and may find a wide range of new applications, such as in biocatalysis, immunoassay, and environmental monitoring.     

Homogeneous Amperometric Immunoassay for Digoxin

Abstract

A homogeneous spectrophotometric EMIT immunoassay for the measurement of digoxin has been converted to an amperometric immunoassay format capable of analysing digoxin in diluted serum. NADH produced by glucose-6-phosphate dehydrogenase (G6PDH) labelled digoxin was detected amperometrically by its rapid, efficient oxidation at platinized activated carbon electrodes (PACE) poised at +150 mV vs Ag/AgCl. The new amperometric procedure resulted in a simplified assay protocol with a decreased overall assay time, enhanced NADH detection and minimal sample pretreatment.     

Fast Amperometric Immunoassay Utilizing Highly Dispersed Electrode Material

Abstract

An immunoassay technique based on a high surface area carbon immunoelectrode is described. Dispersed ULTI carbon serves as a carrier for immobilized antibodies and also as an electrode material. ‘Sandwich’ scheme of immunoassay of rabbit IgG (as a model analyte) has been used. Iodine formed as a result of the enzymatic oxidation of iodide by peroxidase-label has been detected amperometrically. Using dispersed carbon material as a solid support for immobilization of immunoagents and as an electrode material improves the efficiency of immuno-interaction and the sensitivity of electrochemical assay due to the high area-to-volume ratio of solid to liquid phases.

The method based on ‘sandwich’ immunoassay is conducted at 21°C. The time of each stage of incubation (with analyte and with peroxidase-antibody conjugate) is 10 min. Electrochemical detection of peroxidase-labeled immuno-complex does not exceed several minutes. The technique allows fast determination of rabbit IgG with a low detection limit, in the nanomolar range. By comparison, the conventional technique based on ELISA, being performed under the same conditions (temperature and incubation time) does not allow any quantitative determination of IgG for concentration lower then 100 nM.     

Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich Transfer Enzyme Immunoassay) for Antigens

Abstract

A novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens is described. Antigens were reacted with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates. The complex formed of antigens with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates was trapped onto affinity-purified rabbit (antidinitrophenyl bovine serum a1bumin) IgG-coated polystyrene balls. After eliminating excess of the conjugates, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. ß-D-Galactosidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed by fluorimetry. Nonspecifically bound ß-D-galactosidase activity considerably decreased with less decrease in specifically bound ß-D-galactosidase activity. As a result, the detection limits of human thyroid-stimulating hormone (0.01 nu, 0.02 amol) and human growth hormone (10 fg, 0.5 amol) by the present enzyme immunoassay were 30-fold lower than those by the conventional enzyme immunoassay, in which antigens were incubated with monoclonal mouse antibody IgG₁-coated polystyrene balls and rabbit antibody Fab′-ß-D-galactosidase conjugates.     

A New Technique for Chemical Deposition of Pt Nanoparticles and its Applications on Biosensor Design

A new glucose biosensor design based on glucose oxidase (GOD) immobilized by polypyrrole has been described in this paper. The polymerization of pyrrole was initiated by a hexachloroplatinate which itself was reduced into Pt nanoparticles and thus served as a catalyst for the H₂O₂ oxidation. Properties of the produced GOD modified electrode were examined and the activity of the entrapped enzyme was determined by basic application on the amperometric detection of glucose. Much better results were found comparatively with the enzyme electrode for which the enzyme was entrapped by the electrochemically polymerized polypyrrole. This kind of technique for Pt nanoparticles deposition can be generalized to many cases where polypyrrole is used.     

Fast Amperometric Determination of Enzymatic Activity of Glutaminase

ABSTRACT

The activity of the enzyme glutaminase has been measured using a glutamate electrochemical biosensor based on H₂O₂ detection. Calibration curves for glutamate detection and for glutaminase activity using standard glutaminase from Escherichia coli demonstrated the high sensitivity and the rapid analysis time of this novel amperometric procedure, which was 100 times more sensitive than that reported in liternature.

Porcine liver and kidney tissue and human kidney tissue samples have been tested for glutaminase activity, demonstrating the possibility to perform measurement directly on whole tissues, with no need of sample extraction and purification.     

A Fast and Direct Amperometric Determination of Hg2+ by a Bienzyme Electrode Based on the Competitive Activities of Glucose Oxidase and Laccase

A new concept of enzyme inhibition‐based biosensor involving the appearance of an amperometric signal for an inhibition by mercury was developed. The bienzyme sensor was composed of two layers of clay materials. The inner layer was constituted of layered double hydroxides entrapping laccase wired by ABTS. The outer laponite layer contained glucose oxidase (GOD). GOD catalyzed the glucose oxidation with the reduction of O₂ into H₂O₂. This induced a drastic decrease of the biosensor response to O₂ by the electrically wired laccase. HgCl₂ inhibited the O₂ consumption by GOD leading to a signal increase of the electroenzymatic reduction of O₂.     

Determination of underivatized amino acids by high-performance liquid chromatography and electrochemical detection at an amino acid oxidase immobilized CuPtCl6 modified electrode

An amperometric sensor for amino acids based on the immobilization of amino acid oxidase on the surface of a CuPtCl₆/GC is described. The amperometric current is due to the oxidation of H₂O₂ liberated during the enzyme reaction on the surface of the enzyme electrode. The electrode response characteristics as well as kinetic parameters have been evaluated. The enzyme electrode was characterized as an electrochemical biosensor, which was used as detector in high performance liquid chromatography (HPLC) for the determination of a mixture of amino acids with satisfactory results. Received: 31 Jaunary 2000 / Revised: 31 March 2000 / Accepted: 3 April 2000     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide

The use of several designs of amperometric enzymatic biosensors based on the immobilized tyrosinase enzyme (Tyr) for determining dichlorvos organophosphate pesticide are described. The biosensors are based on the reversible inhibition of the enzyme and the chronocoulometric measurement of the charge due to the charge-transfer mediator 1,2-naphthoquinone-4-sulfonate (NQS). Tyr becomes active when reducing the quinone form of the mediator molecule (NQS) to the reactive o-diol form substrate of Tyr (H₂NQS) at the working electrode, thus permitting modulation of the catalytic activity of the enzyme and measurement of the inhibition produced by the pesticide. The full activity of the enzyme reversibly recovers after removal of the pesticide and re-oxidation of H₂NQS.Tyr was immobilized onto electrodes using different procedures: (i) entrapment within electropolymerized conducting and non-conducting polymers, (ii) covalent attachment to self-assembled monolayers (SAM), (iii) cross-linking with glutaraldehyde (and nafion covering) and (iv) dispersion within carbon-paste electrodes. The mediator was co-immobilized onto the working electrode next to the enzyme and reagentless biosensors were subsequently constructed. In the SAM design (ii) NQS was added to the solution. The analytical properties of the different biosensors based on the competitive inhibition produced by dichlorvos were then compared. A detection limit of about 0.06 μM was obtained for dichlorvos with entrapment of NQS and Tyr within electropolymerized poly(o-phenylenediamine) polymer (oPPD), which was the design that proved to have the best analytical performance.     

A hydrogen peroxide biosensor based on multiwalled carbon nanotubes-polyvinyl butyral film modified electrode

A novel approach to construct a amperometric biosensor for determination of H₂O₂ is described. Horseradish peroxidase (HRP) as a base enzyme was immobilized into the mixture of multiwalled carbon nanotubes (MWNTs) and polyvinyl butyral (PVB). Taking the classical hydroquinone as mediator, cyclic voltammetry and amperometric measurements were used to study and optimize the performance of the resulting H₂O₂ biosensor. The effect of the concentration of MWNTs, HRP, hydroquinone, solution pH, and the working potential of amperometry on the electrochemical biosensor was systematically studied. The results showed that the fabricated biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with wide linear range from 0.000832 to 0.6 mM, and low detection limit 0.000167 mM (S/N = 3) with fast response time less than 8 s. The apparent Michaelis–Menten constant was determined to be 0.049 mM. Additionally, the biosensor exhibited high sensitivity, rapid response and good long-term stability.     

Electrochemical Immunoassay of Human Chorionic Gonadotrophin Based on Its Immobilization in Gold Nanoparticles‐Chitosan Membrane

A human chorionic gonadotrophin (hCG) doped gold nanoparticles–chitosan membrane was prepared for forming an immunoconjugate of horseradish peroxidase labeled hCG antibody and hCG on glassy carbon electrode. The nanoparticles provided a congenial environment of the adsorbed proteins. Thus, the immobilized HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive mechanism, an amperometric method for immunoassay of hCG up to 30 mIU mL^?1 with a relatively low detection limit of 0.26 mIU mL^?1 at 3σ was developed. The hCG immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility.