MEDIATORLESS HORSERADISH-PEROXIDASE ENZYME ELECTRODES BASED ON ACTIVATED CARBON - POTENTIAL APPLICATION TO SPECIFIC BINDING ASSAY

Horseradish peroxidase (HRP) enzyme electrodes based on activated carbon containing various amounts of platinum have been constructed by simple passive adsorption. Direct electron transfer, between the electrodes and HRP resulted in the electroenzymic reduction of H2O2 at potentials of less than +480 mV (vs. Ag/AgCl). The highest cathodic current was obtained using HRP adsorbed to non-platinized activated carbon (NPAC), which gave a current density of 637 nA dm3 mumol-1 cm-2 at +50 mV. A linear calibration curve for H2O2 measurement was obtained over the range 0.2-150 mumol dm-3. Kinetic analyses of these data gave a heterogeneous rate constant k(ME)' of 5.3 x 10(-3) cm s-1 and an enzyme turnover number k(cat,E)' of 2.8 X 10(-2) cm s-1. The HRP-NPAC electrode showed good storage stability in phosphate-buffered saline solution (pH 7.4) at 4-degrees-C, with a calculated half-life of 235 days. The potential for HRP-NPAC electrodes to be applied in specific binding assays, such as immunoassays, was assessed using a biotin binding procedure based on competition with glucose-oxidase-labelled biotin for available avidin binding sites on an immunoaffinity membrane. The detection of H2O2 generated from a specifically bound glucose oxidase label, on the addition of beta-D-glucose, resulted in a cathodic current which was inversely proportional to the biotin concentration over the range 0.1-300 mug dm-3.