Bienzyme sensors based on “electrically wired” peroxidase (original) (raw)
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The Journal of Physical Chemistry B, 2003
A new approach to the description of the steady-state voltammetric behavior of wired enzyme electrodes in the presence of substrate mass transport polarization is presented. Starting from the exact analytical solutions corresponding to two-dimensional mediator-enzyme structures, experimental conditions are identified where the same equations can be applied to the analysis of the more often encountered three-dimensional catalytic films. These conditions are shown to involve a uniform redox conversion throughout the film. Case diagrams have been developed to assess the validity of this approach and to ascertain the influence of mass transport polarization and electron hopping on the voltammetric response. The relevance of the catalytic half-wave potential, as a direct measure of the ratio of the rates of redox mediation and enzyme turnover, is stressed. The kinetic analysis is applied to the electrocatalytic behavior of taurine-modified horseradish peroxidase, entrapped within a polyvinyl pyridine polymer containing osmium redox centers. This integrated electrochemical system is shown to be characterized by an efficient electronic connection between the catalytic and mediator centers, easy permeation of the substrate through the film, and a low value of the enzyme-substrate Michaelis constant. A sensitivity 20% higher than the maximum value previously reported in the literature for polymerbased peroxidase electrodes is obtained, and it appears to be related to a stronger electrostatic interaction between the negatively charged taurine modified HRP and the positively charged redox polymer. A comparison with kinetic parameters obtained in homogeneous solution (J. Am. Chem. Soc. 2002, 124, 240) suggests that further improvement of this electrode configuration would require a higher fraction of the immobilized enzymes to be effectively connected to the redox network.
Peroxidase-modified electrodes: Fundamentals and application
Analytica Chimica Acta, 1996
Peroxidase-modified amperometric electrodes have been widely studied and developed, not only because of hydrogen-and organic peroxides are important analytes but also because of the key role of hydrogen peroxide detection in coupled enzyme systems, in which hydrogen peroxide is formed as the product of the enzymatic reaction. Many important analytes, such as, aromatic amines, phenolic compounds, glucose, lactate, neurotransmitters, etc. could be monitored by using bi-or multienzyme electrodes. In this review the heterogeneous electron transfer properties of peroxidases are discussed as a basis for the analytical application of the peroxidase-modified arnperometric electrodes, and examples are given for various peroxidase electrode designs and their application.
Amperometric Enzyme Electrodes
Journal of the Brazilian Chemical Society, 1997
Neste trabalho de revisão são analisados os avanços mais recentes em eletrodos enzimáticos amperométricos dando ênfase particular aos biosensores baseados na Glucose Oxidase e na Horseradish Peroxidase. A intermediação redox através de mediadores artificiais solúveis ou ligados a polímeros é discutida em termos dos desenvolvimentos teóricos recentes e verificações experimentais. É analisada a dependência da resposta amperométrica com a concentração do substrato, do mediador e da enzima assim como com o potencial do eletrodo e a espessura do filme. São também avaliadas as possíveis aplicações destes sistemas em esquemas multi-enzimáticos. Recent advances on amperometric enzyme electrodes are reviewed with particular emphasis on biosensors based on Glucose Oxidase and Horseradish Peroxidase. Redox mediation by artificial soluble and polymer attached redox mediators is discussed in terms of recent theoretical developments and experimental verification. The dependence of the amperometric response on substrate and mediator concentration, enzyme concentration, electrode potential and film thickness are analyzed. Possible applications in multienzyme schemes are also analyzed.
Horseradish peroxidase-based organic-phase enzyme electrode
Analytical and Bioanalytical Chemistry, 2005
An organic-phase enzyme electrode (OPEE) based on horseradish peroxidase (HRP) immobilized within Nafion on spectroscopic graphite was investigated in acetonitrile. The amperometric electrode response to hydrogen peroxide and cumene hydroperoxide present was found to be the result of the reduction of oxygen, produced upon enzymatic decomposition of both hydroperoxides (i.e., by the catalase-like activity of HRP). The electrode response was found to depend linearly on the hydroperoxide concentration up to 700 lM within the range of potentials from À200 to À400 mV (versus Ag|AgCl). Detection limits of approximately 45 lM for H 2 O 2 and 100 lM for cumene hydroperoxide were determined under the selected experimental conditions. Nernstian dependence (the open circuit voltage of HRPbased electrode versus logarithm of H 2 O 2 concentration) was obtained between 0.2 and 2.0 mM, with a slope of approximately 23 mV per logarithmic unit, suggesting a catalase-like, two-electron disproportionation of the substrate in acetonitrile.
Peroxide Biosensors and Mediated Electrochemical Regeneration of Redox Enzymes
Analytical Biochemistry, 1997
the target analyte with the redox enzyme/biomolecule This article describes the research investigations on followed by the signal processing based on the electrothe development of the amperometric biosensors chemical mode of transduction. The electrochemical based on mediated bioelectrochemistry. The mediated signal may be the function either (i) of direct electron bioelectrochemistry involving horseradish peroxidase exchange from the biomolecule or (ii) through one of and glucose oxidase within the graphite paste is rethe components other than the biomolecules participatported. The enzyme horseradish peroxidase together ing in the biochemical reaction. However, the electrowith electrochemical mediator was incorporated chemical signal resulting from step (i) significantly inwithin the graphite paste electrode. The amperometric creases the sensitivity and selectivity of the analysis response is based on the mediated electrochemical resince the selective inherent properties of the biomolegeneration of peroxidase within the paste. The medicules are monitored directly. On the other hand, the ated electrochemical regeneration of peroxidase and direct electron exchange from the active site of the bioglucose oxidase was studied and compared using three molecule is restricted (19) since, in most of the cases, different electron transfer mediators-tetracyanoquithe active center lies deeply buried in the polypeptide nodimethane (TCNQ), tetrathiafulvalene (TTF), and structure that causes significant reduction in the elecdimethyl ferrocene (dmFc). The mediated electrotron transfer rate. However, the rate of electron exchemistry involving these three mediators was studied change can be facilitated by incorporating electron based on the cyclic voltammetry. The electrochemical transfer relays between the active site of the biomolecmeasurements show that TTF is better mediator for ule and electrode surface (7, 20). Accordingly, a novel 136
Analytical Chemistry, 1995
An osmium poly(viny1pyridine) redox polymer "wired" horseradish peroxidase electrode has been used to detect H202 for the determination of glucose, lactate, and acetylcholine/choline with liquid chromatography (E) and postcolumn immobilized enzyme reactors (IMERs). The redox polymer 6lm containing peroxidase is coated on the surface of a glassy carbon electrode and operated at +lo0 mV vs Ag/AgCl for the reduction of H202. Compared with a conventional platinum electrode oxidizing H202 at $500 mVvs Ag/AgCl, the peroxidase cathode exhibits a 2-10-fold improvement in sensitivity and detection l i m i t. The enzyme electrode also shows better operational stability than the Pt electrode. When the enzyme electrode is coupled to LC oxidase WIER systems, the initial stabilization of the background ament is significantly faster than that for the conventional Pt electrode. The enzyme electrode has been used to determine glucose and lactate concentrations in rat subcutaneous microdialysates. After 8 days of continuous injection of the dialysate samples, the enzyme electrode sensitivity dropped by only 5%.
Biosensors and …, 2003
This study presents the use of complementary colorimetric and amperometric techniques to measure the quantity of protein or enzyme immobilised onto a carbon paste electrode modified with a layer of electrodeposited polyaniline. By applying a solution of bovine serum albumin at 0.75 mg/ml, efficient blocking of the electrode from electroactive species in the bulk solution could be achieved. When the horseradish peroxidase was immobilised on the electrode, optimal amperometric responses from hydrogen peroxide reduction were achieved at approximately the same concentration. The mass of enzyme immobilised at this solution concentration was determined by a colorimetric enzyme assay to be equivalent to the formation of a protein monolayer. Under these conditions, amperometric responses from the immobilised layer are maximised and non-specific bulk solution interactions are minimised. At higher immobilised protein concentrations, diminished amperometric responses may be due to inhibited diffusion of hydrogen peroxide to enzyme which is in electronic communication with the electrode surface, or impeded electron transfer. #
Electroanalysis, 1997
Tailoring of electrically contacted enzyme electrodes provides the grounds for bioelectronic and biosensor systems. Redox-enzymes organized onto electrodes as monolayer assemblies, and chemically functionalized by redox-relay groups, yield electrically contacted enzyme electrodes exhibiting bioelectrocatalytic features. The sensitivity of the enzyme electrode can be enhanced, or tuned, by the organization of multilayer enzyme electrodes and the application of rough metal supports. Enzyme electrodes of extremely efficient electrical communication with the electrode are generated by the reconstitution of apo-flavoenzymes onto relay-FAD monolayers associated with electrodes. The reconstitution process results in an aligned enzyme on the surface, and its effective electrical contact with the electrode yields selective enzyme electrodes of unprecedented high current responses. Integrated electrodes consisting of relay-NAD(P)+-cofactor and enzyme units are generated by the reconstitution of NAD(P)+dependent enzymes onto a relay-NAD(P)+ monolayer assembly followed by lateral crosslinking of the enzyme network.