Potentiometric method for substrate analysis using immobilized NAD + -dependent oxidoreductase enzymes

Two coenzyme-dependent oxidoreductases, glucose dehydrogenase and alcohol dehydrogenase, were immobilized in polyacrylamide gel over a platinum grid matrix and used as enzyme electrodes to measure their substrate concentrations in buffered aqueous solutions. The immobilized enzymes were used to oxidize their substrates in the presence of NAD +. Ferricyanide was used as the redox mediator and electroactive species. The determinations of glucose and ethanol were utilized to demonstrate and evaluate the performance of the system. The described methodology should be readily applicable to the analysis of numerous other substrates of coenzyme-dependent oxidoreductases.