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The use of a laccase biosensor, under both batch and flow injection (FI) conditions, for a rapid and reliable amperometric estimation of the total content of polyphenolic compounds in wines is reported. The enzyme was immobilized by cross-linking with glutaraldehyde onto a glassy carbon electrode. Caffeic acid and gallic acid were selected as standard compounds to carry out such estimation. Experimental variables such as the enzyme loading, the applied potential, and the pH value were optimized, and different aspects regarding the operational stability of the laccase biosensor were evaluated. Using batch amperometry at -200 mV, the detection limits obtained were 2.6 x 10(-3) and 7.2 x 10(-4) mg L(-1) gallic acid and caffeic acid, respectively, which compares advantageously with previous biosensor designs. An extremely simple sample treatment consisting only of an appropriate dilution of wine sample with the supporting electrolyte solution (0.1 mol L(-1) citrate buffer of pH 5.0) was needed for the amperometric analysis of red, rosé, and white wines. Good correlations were found when the polyphenol indices obtained with the biosensor (in both the batch and FI modes) for different wine samples were plotted versus the results achieved with the classic Folin-Ciocalteu method. Application of the calibration transfer chemometric model (multiplicative fitting) allowed that the confidence intervals (for a significance level of 0.05) for the slope and intercept values of the amperometric index versus Folin-Ciocalteu index plots (r = 0.997) included the unit and zero values, respectively. This indicates that the laccase biosensor can be successfully used for the estimation of the polyphenol index in wines when compared with the Folin-Ciocalteu reference method.

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The performance of graphite-70%Teflon-peroxidase-ferrocene composite electrodes, fabricated by simple physical inclusion of the enzyme and the mediator into the bulk of a graphite-Teflon matrix, by using both batch and flow-injection modes, is reported. The electrode responds very rapidly to changes in H202 concentration due to the absence of a membrane barrier on the electrode surface and of a covalent attachment of the enzyme to graphite. The composite enzyme electrode showed a long-term operation due 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 seven months of drystorage at 4°C. A bienzymatic graphite-70%Teflon-peroxidase-glucose oxidase-ferrocene electrode has been developed, too, showing a rapid response to the changes in the glucose concentration. The stability of the bienzymatic composite electrode was similar to that of the peroxidase biosensor. This biosensor was applied to the determination of glucose in must and wine samples by batch amperometry and flow injection with amperometric detection modes.