Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes

A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. The steady-state current of these enzyme electrodes were reproducible within +/-5.0% of the relative error. Response time was found to be 30-50s and upper limit of the linear working portions was found to be 1.2mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 x 10(-6)M substrate concentrations. The apparent Michaelis-Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.9 kJ mol(-1), respectively. The effects of interferents and stability of the enzyme electrodes were also investigated.     

Enzyme electrodes and their application

Starting from the state of the art, principles for improving the analytical characteristics of enzyme electrodes are discussed. Coupling of appropriate amperometric electrode processes with enzyme systems, e.g. urease or aminopeptidases, results in a simplification of operation. Optimal sample frequencies are realized on the basis of enzyme membranes, with both a small characteristic diffusion time and a high enzyme activity, applied in a well-designed sample-processing system. Coupled enzyme reactions of the sequence or competition type are successfully used for extension to new analytes, e.g. inhibitors, cofactors or alternative substrates. Cyclization of the analyte enhances the sensitivity of enzyme electrodes to the nanomolar concentration range. Enzymic anti-interference layers are a tool for improving the sensor specificity. The operational characteristics of enzyme electrodes are thus adaptable to any given analytical problem.     

Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.     

Immobilization of tyrosinase in polysiloxane/polypyrrole copolymer matrices

Immobilization of tyrosinase in conducting copolymer matrices of pyrrole functionalized polydimethylsiloxane/polypyrrole (PDMS/PPy) was achieved by electrochemical polymerization. The polysiloxane/polypyrrole/tyrosinase electrode was constructed by the entrapment of enzyme in conducting matrices during electrochemical copolymerization. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) were investigated for immobilized enzyme. Enzyme electrodes were prepared in two different electrolyte/solvent systems. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate on the enzyme activity and film morphology were determined. Temperature and pH optimization, operational stability and shelf-life of enzyme electrodes were also examined. Phenolic contents of green and black tea were determined by using enzyme electrodes.     

Ethanol biosensors based on alcohol oxidase

The detection and quantification of ethanol with high sensitivity, selectivity and accuracy is required in many different areas. A variety of methods and strategies have been reported for the determination of this analyte including gas chromatography, liquid chromatography, refractometry and spectrophotometry, among other. The use of the enzyme alcohol oxidase (AOX) on the analysis of ethanol in complex samples allows a considerable enhancement in specificity. This paper reviews the state of the art on ethanol determination based on AOX sensors, using either electrochemical electrodes or immobilised enzyme reactors. Almost all AOX-based ethanol sensors developed so far are based on the monitoring of O2 consumption or H2O2 formation. This has been mostly achieved using amperometric electrodes set at appropriate potentials namely, -600 mV for O2 monitoring or +600 mV for H2O2 monitoring. Mediated and non-mediated bienzymatic systems have also been assembled using AOX coupled to horseradish peroxidase (HRP). Different types of electrodes have been proposed for the detection of ethanol, namely, membrane electrode, carbon paste electrodes, screen-printed electrodes and self-assembled monolayers. Another approach to work with this sensitive enzyme is to use high amounts of AOX in order to create an enzyme reservoir, a strategy which can be implemented using immobilised enzyme reactors. These reactors can be combined with a colorimetric detection in a flow-injection analysis system or with electrochemical transducers.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition

Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K'(M)). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. (c) 1992 John Wiley & Sons, Inc.     

Immobilization of glucose oxidase in conducting graft copolymers and determination of glucose amount in orange juices with enzyme electrodes

Glucose oxidase was immobilized in conducting copolymers of three different types of poly(methyl methacrylate-co-thienyl methacrylate). Immobilization of enzyme was carried out by the entrapment in conducting polymers during electrochemical polymerization of pyrrole on the copolymer electrodes. Maximum reaction rate, Michaelis-Menten constants, temperature, pH and operational stabilities were determined for immobilized enzyme. The amount of glucose in orange juices of Turkey was investigated by using enzyme electrodes.     

In vivo calibration of the subcutaneous amperometric glucose sensors using a non-enzyme electrode

A new two-point calibration method for the subcutaneous amperometric continuous glucose sensor is reported. The proposed method is based on direct measurement of the background current (I(o)) using a non-enzyme electrode. For in vivo test, three electrodes were implanted in rabbits. Two of the three were identical needle-type enzyme electrodes with perfluorinated polymer outer layers (Pt/enzyme layer/Kel-F/PTFE/Kel-F/Nafion) that were placed in subcutaneous tissue and in a vessel (ear artery), respectively. And one non-enzyme electrode with exactly the same membrane composition as those of other two was in the subcutaneous layer to measure the background current. Implantation in the subcutaneous layer generated many crevices on the protecting layers of the electrodes. The signals from enzyme electrodes were effectively corrected by the measured background current from the non-enzyme electrode. In addition, a telemetric monitoring system was developed and evaluated for in vivo continuous glucose monitoring in order to alleviate the problems of motion artifact.     

Electrochemical evaluation of glucose oxidase immobilized by different methods

Glucose oxidase electrodes were constructed on a platinum screen using polyacrylamide gel, glutaraldehyde crosslinking, and glutaraldehyde crosslinking with +0.04 volts dc on the platinum screen as the methods of enzyme immobilization. The electrodes were evaluated in an electrochemical cell for the oxidation of glucose at the enzyme electrode and the reduction of oxygen at a platinum auxiliary electrode, using constant current voltametry or under external load operation. The method of immobilization affected the extrapolated opencircuit potential as well as the half-cell potential and the steady current under external load operation. The charged glutaraldehyde electrode gave the best current performance; however, the small output (microamps) indicated that major problems in electron transfer from an enzyme catalyst to an external circuit must be resolved before such electrodes can be used in practical application.     

Fabrication of comb interdigitated electrodes array (IDA) for a microbead-based electrochemical assay system

This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase. Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA.     

Parameters important in tuning the response of monolayer enzyme electrodes fabricated using self-assembled monolayers of alkanethiols

Enzyme electrodes were observed experimentally to have a broad dynamic range, high sensitivity and excellent reproducibility. The theoretically predicted response of the monolayer enzyme electrodes was in good agreement with that observed experimentally over the broad range of experimental conditions tested. The response is limited by the rate of enzyme turnover by a mediating species rather than mass transport. As a consequence of this limitation, the response was very sensitive to the enzyme loading and the concentration of mediator in the sample solution but insensitive to mass transport variables such as solution stirring or the diffusion coefficients of the substrate or cosubstrate.     

Oxygen-stabilized enzyme electrode for d-glucose analysis in fermentation broths

A new principle for the construction of oxygen-dependent enzyme electrodes is presented. The enzyme electrode is based on a galvanic oxygen electrode which is furnished with an electrolysis anode covered by immobilized enzyme and placed close to the oxygen-sensing surface. An ordinary oxygen electrode is used as a reference electrode. The enzymatic consumption of oxygen in the enzyme electrode generates a potential difference between the electrodes which is utilized to control electrolytic generation of oxygen from water in such a way that zero differential potential is maintained. Thus, the enzyme electrode operates under ambient oxygen tension and does not suffer from oxygen limitation. The electrolytic current in this system gives a measure of the concentration of substrate surrounding the enzyme electrode. The electrode has been applied for continuous d-glucose analysis in situ during batch cultures of Candida utilis.     

Enzyme electrode probes

A survey of the field of enzyme electrode probes is presented. Probes for the analysis of over 40 different substrates and 25 different enzymes are described. Some of the basic properties of enzyme electrodes, their response characteristics, sensitivity, lifetime and specificity are likewise discussed, as is the future of such electrodes in the fields of medicine, manufacturing, biology and chemistry.     

Enzyme electrodes based on organic metals

The parameters of enzyme electrodes based on organic metals are presented. Cytochrome b₂ (E.C. 1.1.2.3), glucose oxidase (E.C. 1.1.3.4), xanthine oxidase (E.C. 1.2.3.2) and peroxidase (E.C. 1.11.1.7) were used in electrodes sensitive to l-lactate, glucose, hypoxanthine and hydrogen peroxide. Electrocatalytic oxidation of NADH on organic metals and ethanol and acetaldehyde sensitive electrodes containing alcohol dehydrogenase (E.C. 1.1.1.1) were studied. Biocatalytic charge accumulation, the mechanism of electron exchange between the enzyme active centres and organic metals, and the future application of organic metals are discussed.     

Immobilization of polyphenol oxidase in conducting copolymers and determination of phenolic compounds in wines with enzyme electrodes

Polyphenol oxidase (PPO) was immobilized in copolymers of thiophene functionalized menthyl monomer (MM) with pyrrole. Immobilization of enzyme was performed via entrapment in conducting copolymers during electrochemical polymerization of pyrrole. Maximum reaction rates, Michaelis-Menten constants and temperature, pH and operational stabilities of enzyme electrodes were investigated. Total amount of phenolic compounds in red wines of Turkey were analyzed by using these electrodes.     

Proton pathways in lysozyme.

Protonic conduction studies are reported for lysozyme as a function of the number of bound water molecules. Lysozyme samples employing proton-injecting palladium black electrodes exhibited conductivities up to eight orders of magnitude greater than those retained between control (copper) electrodes. The results indicate that water involved in multiple hydrogen bond contact with the enzyme together with hydrogen bonded segments of the enzyme structure provide a hydrogen bond network which is capable of supporting considerable protonic conduction.     

Cerbinal,a Pseudoazulene Iridoid,as a Potent Antifungal Compound Isolated from Gardenia jasminoides Ellis

d-Gluconate dehydrogenase isolated from Pseudomonas fluorescens was immobilized on the surfaces of carbon and gold electrodes by irreversible adsorption. The electrodes with the adsorbed enzyme produced anodic currents in solutions containing d-gluconate. The currents were attributable to the electro-enzymic oxidation (direct bioelectrocatalytic oxidation) of d-gluconate; the electrochemical system required no external redox molecules serving as mediators of electron transfer between the electrode and the adsorbed enzyme. A model of the direct bioelectrocatalysis at the enzyme-modified electrodes is presented.     

Electron-conducting redox hydrogels: Design, characteristics and synthesis

Redox hydrogels constitute the only electron-conducting phase in which water-soluble chemicals and biochemicals dissolve and diffuse. The combination of solubility and diffusion makes the electron-conducting gels permeable to water-soluble biochemicals and chemicals. The electron-conducting redox hydrogels serve to electrically connect the redox centers of enzymes to electrodes, enabling their use whenever leaching of electron-shuttling diffusional redox mediators must be avoided, which is the case in subcutaneously implanted biosensors for diabetes management and in miniature, potentially implantable, glucose-O2 biofuel cells. Because the hydrogels envelope the redox enzymes, they electrically wire the reaction centers to electrodes irrespective of spatial orientation and connect to electrode redox centers of multiple enzyme layers. Hence, the attained current densities of enzyme substrate electrooxidation or electroreduction are much higher than with enzyme monolayers packed onto electrode surfaces.     

Entrapment of cytochrome P450 BM-3 in polypyrrole for electrochemically-driven biocatalysis

In vitro biocatalysis with cytochrome P450 BM-3 was investigated aiming for the substitution of the expensive natural cofactor NADPH by electrochemistry. The monooxygenase was immobilized on electrodes by entrapment in polypyrrole as a conductive polymer for electrochemically wiring the enzyme. Electropolymerization of pyrrole proved to be a useful means of immobilising an active cytochrome P450 BM-3 mutein on platinum and glassy carbon electrodes without denaturation. Repeatedly sweeping the electric potential between −600 and +600 mV versus Ag/AgCl led to enzymatically-catalysed product formation while in the absence of the enzyme no product formed under otherwise identical conditions.