We have developed a highly sensitive and selective on-line biosensor for detecting the reduced form of nicotinamide adenine dinucleotide (NADH) produced by the enzymatic reactions of dehydrogenases with various substrates such as glutamate. The sensor consists of a glassy carbon electrode modified with an osmium-polyvinylpyridine-based bottom layer containing horseradish peroxidase, 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 sample solution was continuously introduced into the cell with a syringe pump. We optimized the sensitivity of the NADH sensor by adjusting the glutaraldehyde 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 result, we achieved a sensitivity of 48.8 nA cm-2 μM-1, a detectable concentration range of 25 nM ~10 μM and a detection limit of 20 nM (S/N = 3) for the NADH sensor. The interference from ascorbic acid and other electroactive interferents can be greatly reduced since the sensor can be operated below 0 mV versus Ag/AgCl. The NADH sensor is relatively stable since it retains 70% of its original response after 1 month if stored at 2-8°C in a dry state after use. Furthermore, we fabricated a glutamate sensor by coimmobilizing GluDH and NOX in the BSA-Glut top layer. The detectable glutamate concentration range is from 0.1 to 10 μM and the detection limit is 0.1 μM (S/N = 3). Our glutamate dehydrogenase-based sensor offers good selectivity as regards other amino acids.
- Glutamate dehydrogenase
- NADH and glutamate on-line sensor
- NADH oxidase
- Osmium-poly(vinylpyridine) wired horseradish peroxidase/glassy carbon electrode
ASJC Scopus subject areas
- Biomedical Engineering