engleski

Development of Electrodes for In-Situ Regeneration of NADH: The Kinetics of Electrochemical Reduction of NAD^+ on a Ru-modified Glassy Carbon Electrode

Nicotinamide adenine dinucleotide NAD(H) is a cofactor that is involved in a large number of biochemical processes. The high cost of NAD(H) is one of the major limitations for its large-scale industrial use. Therefore, it is of great importance to develop systems that could regenerate NADH in-situ and allow its catalytic quantities to be used. Electrochemistry offers a number of advantages compared to (bio)chemical processes for NADH regeneration. However, the use of unmodified metal electrode surfaces is not possible due to the formation of an inactive dimer (NAD2). The research in this area performed in our laboratory has focused on the development of direct and enzyme-mediated electrode systems for NADH regeneration. The present work describes the development of a ruthenium modified glassy carbon electrode (RuGC) for the direct electrochemical reduction of NAD^+ (i.e. regeneration of NADH). The kinetics of NAD^+ reduction reaction and the optimization of the NADH regeneration process was investigated on the RuGC electrode in a wide polarization rate, concentration, temperature, and pH range using electrochemical potentio-dynamic/-static dc, ac and spectroscopic techniques. It was shown that the modification of GC by a sub-monolayer of Ru can provide an electrode surface capable of reducing NAD+ directly to NADH, avoiding the formation of a dimer. The reaction is highly irreversible, and occurs at high cathodic overpotentials, where the reaction rate is controlled by the surface diffusion of electroactive species. The reaction is pH independent. It was postulated that Ru sites have a bifunctional role, serving as both proton-providing sites, and as a physical barrier for dimerization of NAD-free radicals. A set of kinetic and thermodynamic parameters was calculated and verified independently using various experimental techniques: the apparent standard heterogeneous electron-transfer rate constant, the apparent transfer coefficient, the diffusion coefficient, and the standard Gibbs free energy of activation. The regeneration of NADH using the RuGC electrode resulted in a high yield (96%) of enzymatically active NADH. Fig. 1: Absorption spectra recorded at various times during the electrolysis of 220  M NAD^+ solution in phosphate buffer (PB) pH 7.0 at -1.2 V vs. SCE. The curves shift to higher absorbance values with increase in time ; 20, 40, 60, 90, 120, 180, 240, 310, and 370 minutes. Inset: (a) Cyclic voltammograms of a RuGC electrode in PB pH 7.0 containing 725  M of NAD^+ recorded at various scan rates. The scan rate increases in the direction of the peak increase as 30, 50, 70, 100, 150, 200, and 300 mVs^-1. Dependence of the (b) peak potential, and (c) peak current on scan rate.

Ru(GC) electrode; NAD(H) regeneration; Electrochemical methods; Kinetic and thermodynamic parameters.

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