The formation of a diaphorase enzyme multilayer bound to a self-assembled monolayer for the mediated enzyme-catalyzed reduction of NAD⁺

A diaphorase enzyme multilayer was constructed over an aminoethanethiol self-assembled monolayer on a gold electrode for the enzyme-catalyzed reduction of NAD⁺. Methyl viologen dissolved in a solution was used as a mediator to transfer electrons between an electrode surface and the immobilized enzyme, and the reduced enzyme consequently reduced the NAD⁺ substrates. As the layers of the enzyme were assembled, stepwise enhancements of the catalytic reduction currents were observed in the cyclic voltammetric measurements. It is supposed that more enzyme was accumulated with the multilayer formation than with a monolayer and the rate of reaction between the mediator and the enzyme was increased. The height of the catalytic wave could be controlled by the number of enzyme layers and the multilayered-enzyme electrode is considered to be used for an efficient catalytic NAD⁺ reduction. The concentration of glutaraldehyde used as a crosslinking reagent for the attachment of the enzyme layer affected the current heights and its density around the enzyme seemed to be related to the catalytic reactivity. An optimal temperature maintained during the voltammetric measurements was necessary to obtain a better catalytic reduction wave.