Lo Gorton

Peroxidase-modified electrodes : Fundamentals and application

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 multi-enzyme electrodes. In this review the heterogeneous electron transfer properties of peroxidases are discussed as a basis for the analytical application of the peroxidase-modified amperometric electrodes, and examples are given for various peroxidase electrode designs and their application.

Amperometric biosensors based on an apparent direct electron transfer between electrodes and immobilized peroxidases. Plenary lecture

An apparent direct electron transfer between various electrode materials and peroxidases immobilized on the surface of the electrode has been reported in the last few years. An electrocatalytic reduction of hydrogen peroxide stars at about +600 mV versus a saturated calomel (reference) electrode (SCE) at neutral pH. The efficiency of the electrocatalytic current increases as the applied potential is made more negative and starts to level off at about –200 mV versus SCE. Amperometric biosensors for hydrogen peroxide can be constructed with these types of peroxidase modified electrodes. By co-immobilizing a hydrogen peroxide-producing oxidase with the peroxidase, amperometric biosensors can be made that respond to the substrate of the oxidase within a potential range essentially free of interfering electrochemical reactions. Examples of glucose, alcohol and amino acid sensors are shown.

Electrocatalytic oxidation of reduced nicotinamide coenzymes by graphite electrodes modified with an adsorbed phenoxazinium salt, meldola blue

Abstract Meldola Blue (7-dimethylamino-1,2-benzophenoxazine) can be adsorbed on graphite to give chemically modified electrodes. The electrochemical redox reactions of the phenoxazine are fairly reversible at low coverages with an E′o of −175 mV vs. SCE at pH 7.0. The electrode was most stable in acid solutions, at pH 6.0 its electrochemical activity decreased by 15% during 2h. The adsorbed compound mediated electron transfer in the electrocatalytic oxidation of the nicotinamide coenzymes (NADH and NADPH). The formation of a charge transfer complex between Meldola Blue and the coenzyme is demonstrated by experiments with a rotating disk electrode. The complex decomposes in a rate limiting step (k+2=30 s−1) to the oxidized coenzyme and the reduced Meldola Blue. The latter can be reoxidized in a fast electrochemical step. The overall result is an electrocatalytic oxidation at a voltage which is about 500 mV lower than at an unmodified electrode.

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

Abstract A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β- d -glucose. The detection limit was 0.25 μM β- d -glucose for 50-μl samples. The electrode was kept at −50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.

Selective detection in flow analysis based on the combination of immobilized enzymes and chemically modified electrodes

The combination of immobilized enzymes and amperometry to build selective detection devices in flow-injection analysis and liquid chromatography is described. The pros and cons of enzyme electrodes and of immobilized enzyme reactors are discussed. The paper concentrates on the use of immobilized dehydrogenases, oxidases, peroxidases, and on electrodes on which these enzyme reactions can be selectively followed. The work in the field by the authors is reviewed.

Kinetic models of horseradish peroxidase action on a graphite electrode

Abstract The direct and mediated mechanisms of the electroreduction of hydrogen peroxide at a graphite electrode modified with horseradish peroxidase (HRP) were studied. The turnover number of the heterogeneous electron transfer between adsorbed HRP and the electrode was found to be equal to 0.66 ± 0.28 s −1 . The rate of the reaction of H 2 O 2 with HRP on the graphite surface was found to be 385 times slower than that in solution. p -Cresol, phenol and p -chlorophenol behaved as efficient mediators in the process of bioelectrochemical H 2 O 2 reduction. From the comparison of kinetically limited currents observed during direct and mediated reduction of H 2 O 2 it was concluded that the population of adsorbed HRP molecules and/or the graphite surface structure cannot be treated as homogeneous. It was found that 42% of the total amount of HRP molecules adsorbed on the electrode were accessible for direct unmediated electron transfer from the graphite electrode.

Electrocatalytic oxidation of NAD(P) H at mediator-modified electrodes.

A review is presented dealing with electrocatalytic NADH oxidation at mediator-modified electrodes, summarising the history of the topic, as well as the present state of the art.

ON THE MECHANISM OF H2O2 REDUCTION AT PRUSSIAN BLUE MODIFIED ELECTRODES

Abstract Prussian Blue deposited on the electrode surface under certain conditions is known to be a selective electrocatalyst of hydrogen peroxide (H 2 O 2 ) reduction in the presence of O 2 . The electrocatalyst was stabilized at cathodic potentials preventing its loss from the electrode surface. Hydrodynamic voltammograms of H 2 O 2 reduction indicated the transfer of two electrons per catalytic cycle. The operational stability of Prussian Blue in H 2 O 2 reduction was highly dependent on the buffer capacity of the supporting electrolyte. Since Prussian Blue is known to be dissolved in alkaline solution, it was confirmed that in neutral aqueous solutions the product of H 2 O 2 electrocatalytic reduction is OH − .

Prussian-Blue-based amperometric biosensors in flow-injection analysis

Optimisation of the electrodeposition of Prussian Blue onto mirrored glassy carbon electrodes yielded a modified electrode practically insensitive to oxygen reduction. At the same time the electrode activity towards hydrogen peroxide reduction was extremely high. This allowed the detection of hydrogen peroxide by electroreduction over a wide potential range. Flow-injection investigations of this electrode inserted into a flowthrough electrochemical cell of the confined wall-jet type showed that the response for hydrogen peroxide is limited by diffusion. Glucose and alcohol biosensors were made by immobilisation of glucose oxidase and alcohol oxidase respectively, within a Nafion layer, onto the top of the Prussian-Blue-modified electrodes. By increasing the density of Nafion and decreasing the measuring potential the glucose biosensor was made completely insensitive to both ascorbate and acetominophes.

Flow-injection analysis of phenols at a graphite electrode modified with co-immobilised laccase and tyrosinase

Abstract The kinetic parameters of the oxidation reaction of phenolic compounds by molecular oxygen catalysed by fungal laccase have been studied. An amperometric biosensor for detection of phenols in environmental analysis is proposed. The enzymes laccase and tyrosinase were co-immobilised by adsorption onto a spectrographic graphite electrode. The bienzyme electrode was used as a sensor in a single-line flow-injection system. The analysis is based on the amperometric detection of the enzymatic reaction products at a potential of -0.05 V vs. Ag AgCl . The experimental parameters were optimised. The joint use of laccase and tyrosinase in the analytical procedure allows the detection of a large group of phenolic compounds.