MEDIATOR-MODIFIED ELECTRODES FOR ELECTROCATALYTIC OXIDATION OF NADH

The large class of NAD(P)(+)-dependent dehydrogenases can be utilized for the construction of amperometric enzyme sensors if a suitable redox mediator catalyzes the electron transfer from NAD(P)H to the electrode. Among several compounds tested, the quinonoid cationic redox dyes N-methyl-phenazinium, 1-methoxy-N-methyl-phenazinium and Medola Blue exhibit the best performance for NADH oxidation. These ionic redox dyes can be immobilized as tetrarhodanato-diammine-chromates (Reineckates) in graphite-epoxy composite electrodes. The slow release of the cations has been monitored using scanning electrochemical microscopy (SECM). The results support strongly a mechanism of mediated electron transfer via dissolved mediator species. The kinetic properties of the composite electrodes regarding heterogeneous and homogeneous rate constants were studied by cyclic voltammetry and chronoamperometry. The remarkable stability of the composite electrode allowing more than 9500 measurements results from the large amount of incorporated Reineckates. The suitability of the composite electrodes for detection of NADH at concentrations between 0.5 mu M and 3 mM at potentials close to 0 mV (vs. Ag\AgCl) has been demonstrated. Based on the composite electrode as transducer, an amperometric enzyme sensor for ethanol was constructed using a novel method for coimmobilizing dehydrogenase and coenzyme in a polyurethane hydrogel. The sensor shows responses to ethanol in the concentration range 0.03-9.5 mM within 1-2 min (90%) even in the absence of dissolved coenzyme.