A spontaneous growth of a diaphorase enzyme layer over a gold electrode for the catalytic reduction of NAD⁺

A diaphorase enzyme electrode for the catalytic reduction of NAD⁺, the oxidized form of nicotinamide adenine dinucleotide, has been prepared. The enzyme layer grew spontaneously over an aminoethanethiol self assembled monolayer on a gold plate electrode. The growth was accomplished by simply dipping the electrode covered by the aminoethanethiol monolayer into a solution containing both glutaraldehyde and diaphorase. We suggested that the glutaraldehyde as a cross-linking reagent was attached to the amino groups of the aminoethanethiol monolayer and the diaphorase enzyme molecules were bound to free aldehyde groups of the glutaraldehyde. Further attachments of the enzyme molecules over the bound enzyme molecules continued with the bridging of the glutaraldehyde. In frequency measurements with a quartz crystal microbalance, the frequency decrease was much more than it was for that of the enzyme monolayer formation, and an enzyme layer thicker than a monolayer was formed. The modified electrode was employed to reduce NAD⁺, using diffusional methyl viologen as an electron transfer mediator. The NAD⁺ was electrocatalytically reduced, and the catalytic current was almost equivalent to that with the multilayered electrode of ten enzyme layers.