Glucose Sensor Using NAD-Dependent Glucose Dehydrogenase, Diaphorase, and Osmium Complex

摘要

It is still challenging to detect glucose selectively in serum and blood, and quickly using nicotinamide adenine dinucleotide (NAD-GDH). In this paper, we report a selective and rapid glucose sensor, based on electrochemical-enzymatic-enzymatic (ENN) redox cycling involving bis(2,2-bipyridyl)dichloroosmium(II)[Os(bpy)_2Cl_2], diaphorase (DI), NAD~+, NAD-GDH, and glucose. DI and Os(bpy)_2Cl_2 are used to obtain fast mediated oxidation of NADH that is generated as a result of glucose oxidation by NAD-GDH. DI and NAD-GDH are co-immobilized via affinity binding on an avidin-modified indium tin oxide electrode to obtain fast and stable ENN redox cycling. Two enzymes (DI and NAD-GDH) and two electron mediators [Os(bpy)_2Cl_2 and NAD~+] are efficient. The reason is that they are insensitive to oxygen. The applied potential (0.0 V vs Ag/AgCl) is low enough to minimize interfering electrochemical reactions, and the redox reactions of Os(bpy)_2Cl_2 with interfering species are slow. NAD-GDH is much less reactive to problematic monosaccharides such as xylose, fructose, galactose, and mannose than glucose. Artificial serum containing 5% (w/v) human serum albumin shows a similar electrochemical back-ground level in serum. All results enable us to obtain selective and reproducible glucose detection. The fast ENN redox cycling allows sensitive glucose detection with a wide range of concentrations in artificial serum with a short measuring time (5 s) without an incubation period.