Bioelectrochemical detection systems for substrates of dehydrogenases

Abstract

In the context of the development of enzyme electrodes for the determination of the substrates of dehydrogenases, different systems for the oxidation of NADH were tested. In a homogeneous system with the dehydrogenase, NAD+ and phenazine methosulphate, the oxygen consumption caused by oxidation of the substrate was determined with a Clark electrode. Satisfactory sensitivity and reproducibility were obtained in the range 0.04–1 mM for several substrates, but the enzyme was rapidly denatured by the mediator. The physical entrapment of dehydrogenases together with a water-forming NADH oxidase on a Clark electrode yielded enzyme electrodes for different substrates with linear responses in the range 0.1–10 mM and a response time of 2 min. Owing to the instability of the NADH oxidase, the half-life of the electrodes was less than 5 h. The most promising systems were obtained by the coupling of the dehydrogenases to 3-β-naphthoyl-Nile Blue-modified graphite electrodes leading to oxygen-independent catalysed electrochemical NADH oxidation. The systems were optimised with regard to the NAD+ concentration in the solution and the pore size of the membranes covering the electrodes. By immobilisation of the enzymes, mainly by chemical binding to Eupergit, a considerable increase in their stability could be achieved, and electrodes were obtained with a half-life of 8.5 d, a response time, τ½, of 3.5 min and linear responses over four orders of magnitude.