NAD⁺-dependent enzyme electrodes: Electrical contact of cofactor-dependent enzymes and electrodes

NAD⁺-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD⁺ monolayer. The redox active monolayer is assembled via covalent attachment of pynoloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au electrode, followed by covalent linkage of N⁶-(2-aminoethyl)-NAD⁺ to the monolayer. The surface coverage of PQQ and NAD⁺ units is ca. 1.2 x 10-¹⁰ mol cm^-2. The surface coverage of LDH bound to the redox active monolayer is ca. 3.5 x 10^-12 mol cm^-2. The assembled LDH monolayer is active in the bioelectrocatalytic oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH in the presence of Ca²⁺ ions. The LDH associated with the PQQ-NAD⁺ monolayer assembled on the electrode surface exhibits moderate stability, and the biocatalyst dissociates to the electrolyte solution. Dissociation of LDH is enhanced in the presence of solubilized NAD⁺. Cross-linking of the monolayer-bound LDH with glutaric dialdehyde yields an integrated stable enzyme electrode for the bioelectrocatalyzed oxidation of lactate. The electrode acts as an amperometric biosensor for lactate. Affinity binding of NAD⁺-dependent alcohol dehydrogenase to the PQQ-NAD⁺-monolayer-modified Au electrode, followed by cross-linking of the enzyme, yields an enzyme electrode for the bioelectrochemical detection of ethanol.