Mediated enzyme electrodes

Abstract

This review summarizes the long time experience of the team at the Electrochemistry of Biocatalysts and Metal-Air Systems Department in the field of the mediated enzyme electrodes. Investigated is the redox behaviour of a mediated enzyme electrode depending on the kind of carbon materials, needed to produce the electrode (compact carbon material -pyrolytic graphite or dispersed carbon material -carbon black), the type of mediators (ferrocene derivates, nickelocene and benzoqoquinone), and the type of enzyme (glucose oxidase and lactate oxidase). Key words: mediator, enzyme electrode, pyrolytic graphite electrode, carbon black INTRODUCTION The research work in the field of the biosensors has expanded very rapidly since the development of glucose enzyme electrodes, which are of particular interest in the biomedical analysis field The electrochemical biosensor is a complicated device which converts a biological recognition process into an electrical signal, the amplitude of which is related to the concentration of the analyte. The coupling of the electron transfer of the enzyme with the electron transfer at the electrode poses a major problem to the biosensor development. In most cases the enzyme redox centres are essentially insulated within the enzyme molecule so that a direct electron transfer to the surface of the conventional electrode does practically not occur. The electrical communication between the redox centres of the enzyme and the electrode requires either the presence of oxygen and hydrogen peroxide and their diffusion to and from the enzyme redox centres, or the presence of a redox mediator Glucose oxidase (GOD) is one of the first enzymes used in the biocatalytic electrochemical sensing elements The detection principle is based on an electrochemical reaction in which the H 2 O 2 production or the O 2 consumption is detected. The produced H 2 O 2 can be electrochemically oxidised on an electrode at a constant potential, and the generated anodic current is used to measure the glucose concentration. Together with the natural electron acceptor in reaction (1), oxygen, and other low-molecular-weight compounds are used as mediators between the enzyme and the electrode. The mediator is a low molecular weight redox couple, which shuttles electrons from the enzyme redox center to the surface of the electrode. The following scheme describes the reactions which take place on the mediated electrode where GOD ox and GOD red are the enzyme in oxidized and reduced form of its active center, respectively, M ox and M red are the oxidized and the reduced form of the electrochemical mediator. The conjugation of the enzyme catalyzed steps (2) and (3) is accomplished through the electrochemical reaction (4). The use of the electrochemical mediator in the amperometric enzyme electrodes is connected with two basic requirements to these electrodes: the amperometric signal of the electrode must be independent of the concentration of the dissolved oxygen in the electrolyte; and the working potential © 2011 Bulgarian Academy of Sciences, Union of Chemists in Bulgaria *To whom all correspondence should be sent: E-mail: [email protected] 82 of the enzyme electrode must be low enough so that no organic compound in the electrolyte could be oxidized simultaneously with the substrate Quinones, organic dyes such as methylene blue, phenazines, methyl violet, Alizarin yellow, prussian blue, thionin, azure A and C, toluidine blue, iron complexes such as hexacyanoferrat, ferrocene and their derivatives, act as mediators and have been widely used in a number of biosensors The reduced forms of the mediators can be electrochemically oxidized on an electrode at potentials lower than that of the H 2 O 2. This gives a possibility to design biosensors, operating at potentials lower than those based on the natural enzymatic reaction (1). It must be noticed that a low working potential of the biosensor is preferable in order to decrease the rate of oxidation of some compounds, usually present in physiological fluids. The oxidation of this compound results in an electric signal, disturbing the signal, obtained by the glucose oxidation. In this way the possible interference with the amperometric signal of the biosensor is reduced The enzyme electrode is prepared by the application of mediator solution on the activated pyrolytic graphite surface, and after drying in the air, the electrode surface is covered with a GOD or LOD solution of the enzyme in a phosphate buffer (pH 7). After drying at a room temperature, the electrode is washed in a buffer solution. Between measurements the enzyme electrode is kept at 4 ºC. The enzyme electrodes are prepared also by consecutive application of a mediator and an enzyme on the porous supporting layer of carbon black-PTFE (polytetrafluorethylene) material. In other cases, a porous matrix of carbon material, wet proofed with PTFE, is embedded in a plastic tube and covered by a thin layer of a mixture of the same wet proofed material and mediator (p-benzoquinone). This thin layer forms the face of the electrode onto which the enzyme is immobilized. The electrochemical measurements are performed in a two-or three-electrode cell with an Ag/AgCl reference electrode and Pt counter electrode. The electrolytes comprise of a 0.1 M phosphate buffer solution and a 0.1 M phosphate buffer solution, containing 0.1M KCl. RESULTS AND DISCUSSION Mediated enzyme electrodes, prepared from electrochemically activated pyrolytic graphite electrode The quasi-redox potentials of the investigated ferrocene derivatives are obtained from the cyclic voltammograms of an electrochemically activated pyrolytic graphite electrode, modified with the corresponding compound. The obtained experimental values of the quasi-redox potentials of the investigated ferrocene derivatives are in a good coincidence with the literature data, as well as with our data, previously obtained with the same ferrocene derivatives, adsorbed on another type of carbon electrode The methanol-ferrocene possesses a more negative quasi-redox potential than that of the ferrocene, so that it is suitable to be used as a mediator in glucose and lactate electrodes. The R. I. Boukoureshtlieva et al.: Mediated enzyme electrodes 83 cyclic voltammogram is measured in a 0.1M phosphate buffer The steady-state current of the investigated enzyme electrodes, as a function of the substrate concentration, is studied in both, the presence of oxygen dissolved in the electrolyte, and after the significant decrease of the dissolved oxygen concentration by blowing argon through the electrolyte. The behaviour of the lactate oxidase enzyme electrodes with mediator methanolferrocene or butylferrocene is also investigated. In order to check if the substrate (Li L-lactate) is directly oxidized on the electrode surface, electrode is prepared from electrochemically activated pyrolytic graphite, modified with a mediator and with a covering layer of Nafion. The steady-state current of these electrodes at a constant potential of + 350 mV vs. Ag/AgCl is measured in the presence of Li L-lactate in the solution, and no amperometric signal is observed, i.e. the substrate is not directly oxidized on the electrode at this potential. The calibration curve of a lactate electrode with butylferrocene is presented in Application of the mediated enzyme electrode at a low working potential is preferable for the biosensing. The investigations are oriented at selecting those ferrocene derivatives which possess a redox potential lower than that of the ferrocene. Another way to achieve a mediated electrode with a low working potential is to use a structural analog of ferrocene which possesses a redox potential lower than that of the ferrocene. Investigations of the electrochemical behavior of the metallocenes in non-aqueous electrolyte have shown that the redox potential of a nickelocene/nicelocenium couple is ca. 0.4 V more negative than that of the ferrocene/ferrocenium couple The stable cyclic voltammograms of the electrochemically activated pyrolytic graphite electrodes with adsorbed nickelocene and with adsorbed ferrocene are juxtaposed i