Electrical contacting of flavoenzymes and NAD(P)⁺-dependent enzymes by reconstitution and affinity interactions on phenylboronic acid monolayers associated with Au-electrodes

The preparation of integrated, electrically contacted, flavoenzyme and NAD(P)⁺-dependent enzyme-electrodes is described. The reconstitution of apo-glucose oxidase, apo-GOx, on a FAD cofactor linked to a pyrroloquinoline quinone (PQQ) phenylboronic acid monolayer yields an electrically contacted enzyme monolayer (surface coverage 2.1 × 10^-12 mol cm^-2) exhibiting a turnover rate of 700 s^-1 (at 22 ± 2 °C). The system is characterized by microgravimetric quartz-crystal microbalance analyses, Faradaic impedance spectroscopy, rotating disk electrode experiments, and cyclic voltammetry. The performance of the enzyme-electrode for glucose sensing is described. Similarly, the electrically contacted enzyme-electrodes of NAD(P)⁺-dependent enzymes malate dehydrogenase, MaID, and lactate dehydrogenase, LDH, are prepared by the cross-linking of affinity complexes generated between the enzymes and the NADP⁺ and NAD⁺ cofactors linked to a pyrroloquinoline quinone phenylboronic acid monolayer, respectively. The MaID enzyme-electrode (surface coverage 1.2 × 10^-12 mol cm²) exhibits a turnover rate of 190 s^-1, whereas the LDH enzyme-electrode (surface coverage 7.0 × 10^-12 mol cm²) reveals a turnover rate of 2.5 s^-1. Chronoamperometric experiments reveal that the NAD⁺ cofactor is linked to the PQQ-phenylboronic acid by two different binding modes. The integration of the LDH with the two NAD⁺ cofactor configurations yields enzyme assemblies differing by 1 order of magnitude in their bioelectrocatalytic activities.