NADH and glutamate on-line sensors using Os-gel-HRP/GC electrodes modifiedwith NADH oxidase and glutamate dehydrogenase

We have developed a highly sensitive and selective on-line biosensor for de tecting the reduced form of nicotinamide adenine dinucleotide (NADH) produc ed by the enzymatic reactions of dehydrogenases with Various substrates suc h as glutamate. The sensor consists of a glassy carbon electrode modified w ith an osmium-polyvinylpyridine-based bottom layer containing horseradish p eroxidase, and a bovine serum albumin (BSA)-gluteraldehyde (Glut) top layer containing NADH oxidase (NOX) or glutamate dehydrogenase (GluDH) and NOX. We assembled the modified electrode in a thin-layer radial flow cell and sa mple solution was continuously introduced into the cell with a syringe pump . We optimized the sensitivity of the NADH sensor by adjusting the glutaral dehyde amount in the immobilized layer, the applied potential and the pH of buffer solution. We examined the flow-rate effect on the current response and the conversion efficiency of NADH at the modified electrode. As a resul t, we achieved a sensitivity of 48.8 nA cm(-2) mu M-1, a detectable concent ration range of 25 nM similar to 10 mu M and a detection limit of 20 nM (S/ N = 3) for the NADH sensor. The interference from ascorbic acid and other e lectroactive interferents can be greatly reduced since the sensor can be op erated below 0 mV versus Ag/AgCl. The NADH sensor is relatively stable sinc e it retains 70% of its original response after I month if stored at 2-8 de grees C in a dry state after use. Furthermore, we fabricated a glutamate se nsor by coimmobilizing GluDH and NOX in the BSA-Glut top layer. The detecta ble glutamate concentration range is from 0.1 to 10 mu M and the detection limit is 0.1 mu M (S/N = 3). Our glutamate dehydrogenase-based sensor offer s good selectivity as regards other amino acids. (C) 1999 Elsevier Science S.A. All rights reserved.