Development of a bienzymic graphite–Teflon composite electrode for the determination of hypoxanthine in fish

Abstract

A bienzymic amperometric graphite–Teflon composite biosensor, in which xanthine oxidase and peroxidase, together with the mediator ferrocene, are incorporated into the electrode matrix, was developed for the determination of hypoxanthine in fish samples. These biosensors were fabricated by simple physical inclusion of the enzymes and the mediator in the bulk of the graphite–Teflon matrix. A Teflon content of 60%, a working potential of 0.00 V, a temperature of 30 ± 1 °C and a pH of 7.4 were selected as the optimum working conditions. The composite bioelectrode operated over long periods owing to the renewability of its surface by polishing. Reproducible amperometric responses were achieved with different electrodes fabricated from different composite matrices, and no significant loss of the enzyme activity was observed after more than 6 months of storage at 4 °C. A detection limit for hypoxanthine of 9.0 × 10^–8 mol l^–1 was obtained by amperometry in stirred solutions. An interference study of different substances which may be present in samples together with hypoxanthine demonstrated very good selectivity for the determination of this analyte. The bienzymic composite biosensor was applied to the determination of hypoxanthine in sardine muscle tissue, and the method was validated by comparing the results with those obtained by applying a recommended reference method.