Catalytic Oxidation of NADH on Gold Electrode Modified by Layer-by- Layer Self-assembly of Thermostable Diaphorase and Redox Polymer

摘要

A redox polymer, poly(ethylenimine)ferrocene (PEI-Fc) was synthesized by attaching ferrocene groups to the backbone of water soluble poly(ethylenimine), and multilayer film in nano-scale was assembled on gold electrode by alternate layer-by-layer adsorption (LBL) of the positively charged PEI-Fc and the negatively charged thermostable diaphorase (DI) from B.Stearothermophilus. The LBL process was monitored and analyzed by quartz crystal microbalance (QCM) technique, which confirmed the formation of the multilayer structure. The electrochemical oxidation of coenzyme (reduced nicotinamide adenine dinucleotide, NADH) was observed on the electrode fabricated with PEI-Fc/DI multilayer film, and the influence of layer number on current response was investigated. The modified electrode retained ca. 65% relative response after storage in buffer for two months and 50% relative response after incubation at 80 ℃ for 3 min, which inferred that the multilayer structure was unique stable. A NAD-dependent glucose-6-phosphate dehydrogenase (G6PDH) was also immobilized via the same LBL technique, and electrode modified with PEI-Fc/DI/G6PDH film exhibited current response to glucose-6-phosphate in the presence of free NAD~+.