NAD(-DEPENDENT ENZYME ELECTRODES - ELECTRICAL CONTACT OF COFACTOR-DEPENDENT ENZYMES AND ELECTRODES())

NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrro loquinoline quinone-NAD(+) monolayer. The redox active monolayer is as sembled via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au electrode, followed by coval ent linkage of N-6-(2-aminoethyl)-NAD(+) to the monolayer. The surface coverage of PQQ and NAD(+) units is ca. 1.2 x 10(-10) moi cm(-2). The surface coverage of LDH bound to the redox active monolayer is ca. 3. 5 x 10(-10) mol cm(-2). The assembled LDH monolayer is active in the b ioelectrocatalytic oxidation of lactate. The bioelectrocatalyzed proce ss involves the PQQ-mediated oxidation of the immobilized NADH in the presence of Ca2+ ions. The LDH associated with the PQQ-NAD(+) monolaye r 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-linkin g of the monolayer-bound LDH with glutaric dialdehyde yields an integr ated 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 t he enzyme, yields an enzyme electrode for the bioelectrochemical detec tion of ethanol.