AMPEROMETRIC ENZYME ELECTRODES FOR NAD(P)H AND UREA

Abstract

Amperometric enzyme electrodes for NAD(P)H as well as bienzyme electrodes for dehydrogenase substrates have been developed on the basis of the horseradish peroxidase catalyzed aerobic oxidation of reduced pyridine nucleotides. Limits of detection are: 20 ymol/l NADH or 30 umol/1 NADPH in the HRP electrode and 0.8 umol/1 NAD(P)H, 80 pmol/l glucose, 100 ywmol/l ethanol and 200 umol/l isocitrate in HRP-dehydrogenase bienzyme electrodes with cofactor recycling. Relative standard deviations are 4 %, measuring frequencies 6-8 samples/h. Other types of amperometric biosensors are based on the electrochenical hydrazine oxidation. The dependence of the anodic current on the hydrazine concentration at constant pH values was used to determine enzyme activities of human serum and bovine eye lens leucine aminopeptidase (LAP) and of human serum alanine aminopeptidase (AAP). Detection limits were 5 units/l, the correlation of the results in serum with the respective optical method was better for AAP then for LAP. Kinetic constants of bovine lens LAP were found in the same range as with the optical method. At constant hydrazine concentration its oxidation current is a linear function of the hydroxyl ion concentration. This dependence was used to develop an amperomeric urea electrode. Typical parameters are: linear range 0.8-35 mmol/l, response time 20s, relative standard deviation 1%, frequency 40 samples/h and operational stability two weeks. The urea content in pure solutions, in dialysates of artificial kidneys and in human serum was determined in good correlation with Berthelot’s method. Buffer influences were eliminated by two electrode difference measurements.