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

Horseradish peroxidase (HRP) enzyme electrodes based on activated carb on containing various amounts of platinum have been constructed by sim ple passive adsorption. Direct electron transfer, between the electrod es and HRP resulted in the electroenzymic reduction of H2O2 at potenti als of less than +480 mV (vs. Ag/AgCl). The highest cathodic current w as obtained using HRP adsorbed to non-platinized activated carbon (NPA C), 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 heter ogeneous rate constant k(ME)' of 5.3 x 10(-3) cm s-1 and an enzyme tur nover 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 (p H 7.4) at 4-degrees-C, with a calculated half-life of 235 days. The po tential for HRP-NPAC electrodes to be applied in specific binding assa ys, such as immunoassays, was assessed using a biotin binding procedur e based on competition with glucose-oxidase-labelled biotin for availa ble 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 w as inversely proportional to the biotin concentration over the range 0 .1-300 mug dm-3.