Jenny Emnéus

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.

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.

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.

The development of a peroxidase biosensor for monitoring phenol and related aromatic compounds

Abstract The possibility of horseradish peroxidase (HRP) modified solid graphite and carbon paste electrodes to perform as biosensors for the determination of phenol and related compounds was studied. Phenoxy radicals, formed during the enzymatic oxidation of phenolic compounds in the presence of hydrogen peroxide, are reduced electrochemically. The reduction current is proportional to their concentration in the solution. From the hydrodynamic voltammograms, calibration curves and performance stability it was concluded that the reduction of phenoxy radicals is more efficient at the solid graphite electrodes in comparison with the carbon paste based sensor. The potentials, at which electrochemical reduction of phenoxy radicals appears, depend on the electron donating properties of the substituent in the phenol molecule. It was found that, in the presence of 10–20 μM of H 2 O 2 in the solution, the responses of HRP-modified solid graphite electrode to p -cresol are rate limited by the enzymatic reaction. The electrode was most stable when the buffer solution contained 5% of methanol. Among 20 phenolic compounds tested, phenol, catechol, resorcinol, p -cresol, 4-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, vanillin and 2-amino-4-chlorophenol can be determined. The greatest sensitivity was obtained for 2-amino-4-chlorophenol (85 nA cm −2 μM −1 ).

Development of enzyme-based amperometric sensors for the determination of phenolic compounds

Abstract The development of biosensor-based analytical techniques for the determination of phenolic compounds in real surface waters is described. The methods start with an enzymes catalytic cycle and then the incorporation of enzyme electrodes into simple flow injection or integrated sample handling units. The catalytic properties of tyrosinases, laccases, and peroxidases are exploited for the construction of sensors with narrow or broad selectivity. Some results on the determination of phenolic compounds in real water samples are presented.

Tyrosinase graphite-epoxy based composite electrodes for detection of phenols

The characterization and analytical performance of a tyrosinase graphite-epoxy electrode for the detection of phenolic compounds are described. The biocomposite configuration is based on the entrapment of commercially available tyrosinase in a graphite-epoxy matrix, and the mixing of the resulting conductive epoxy resin with a hardener. The enzyme electrode is mounted as a working electrode in an amperometric flow cell of the confined wall-jet type and studied in the flow injection mode. The bioprobe is electrochemically characterized by hydrodynamic and cyclic voltammetry for catechol and phenol. An applied potential of −100 mV vs. Ag/AgCl is found to be optimal for electrochemical reduction of the enzyme products (quinone forms) for the biocomposite electrode. The dependence of the response of the biocomposite on the flow rate, the amount of loaded enzyme, the buffer composition, pH, and oxygen is investigated. The response of the biosensor to different phenolic compounds is also evaluated. The limits of detection (S/N = 3) for phenol and catechol were 1·0 μM and 0·04 μM, respectively. No loss in response could be detected after 100 injections of catechol (R.S.D. <2%). Stability of the biocomposite depends on storage conditions. Theoretical advantages described in the literature for biocomposite electrodes, for example, repolishing and bulk modification, are empirically studied in this work.

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 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm 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 H2O2. All immunosensors were capable of detecting atrazine at the sub-microg 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 microg l(-1), and IC50 values of 0.096 and 0.130 microg 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 microg l(-1), respectively.

Electrochemical properties of some copper-containing oxidases

Abstract The electrochemical behaviour of copper-containing oxidases (two laccases, tyrosinase, ceruloplasmin and ascorbate oxidase) has been investigated at graphite and carbon electrodes. These enzymes catalyse the oxidation of organic and inorganic substrates by molecular oxygen in homogeneous solutions. Molecular oxygen is reduced directly to water in a four-electron mechanism. Direct (mediatorless) bioelectrocatalysis of oxygen reduction was observed with electrodes modified with fungal and lacquer tree laccase. It was found that when the electrode modified with fungal laccase was used oxygen electroreduction started at a potential approximately 0.35 V more positive than that observed with the electrode modified with lacquer tree laccase. The effect of different pretreatments of the electrode surface before adsorption of laccase was investigated. It was established that the optimum pH for the electrocatalytic reaction of oxygen reduction in the presence of lacquer tree laccase is shifted towards the alkaline range compared with that with fungal laccase. No redox transformations of the prosthetic groups of the two laccases were revealed in anaerobic conditions and in the presence of various promoters. No bioelectrocatalytic reaction of oxygen was revealed with the other oxidases. An electrochemical response was obtained at electrodes coated with only tyrosinase or ceruloplasmin in the absence of oxygen. The conditions which can influence the bioelectrocatalysis have been discussed.

Phenol oxidase-based biosensors as selective detection units in column liquid chromatography for the determination of phenolic compounds

Abstract Amperometric biosensors of two types based on the phenol oxidase tyrosinase (EC 1.18.14.1, monophenol monooxygenase) are presented. The enzyme was immobilised either on solid graphite electrodes or in carbon paste electrodes. The performance of the two biosensors was investigated with respect to immobilisation technique, pH, flow-rate and oxygen dependence. The use of detergents in the mobile phase was shown to greatly influence activity, selectivity, and operational stability of the biosensors. One of the developed biosensors was further used as a selective and sensitive detector in a column liquid chromatographic system for the determination of phenolic compounds in a spiked wastewater sample.