Crosslinked integrated enzyme electrodes for bioelectronic applications

Affinity interactions between electron-relay-functionalized electrodes and redox enzymes form stable protein-monolayer assemblies that upon crosslinking yield integrated, electrically contacted, enzyme electrodes. A functionalized monolayer consisting of pyrroloquinoline quinone, covalently linked to N-6-(2-aminoethyl)-NAD(+) (PQQ-NAD(+) monolayer), was assembled on a Au electrode. The NAD(+)-cofactor units form an affinity complex with the NAD(+)-dependent enzyme lactate dehydrogenase (LDH). Crosslinking of the affinity complex with glutaric dialdehyde yields a stable crosslinked two-dimensional array of the enzyme on the electrode surface. The resulting PQQ-NAD(+)/LDH crosslinked protein array is electrically contacted and reveals bioelectrocatalytic features. The electrocatalyzed oxidation of biocatalytically generated NADH by the PQQ components yields an amperometric response whose magnitude is related to the concentration of lactate. Similarly, an integrated, electrically contacted, enzyme electrode was generated by the crosslinking of a microperoxidase-ll (MP-11) and nitrate reductase (NR) affinity complex on a Au electrode with glutaric dialdehyde. Bioelectrocatalyzed reduction of nitrate (NO3-) to nitrite (NO2-) by the crosslinked enzyme electrode proceeds by the MP-ll-mediated reduction of NR. The enzyme electrode stimulates the electrocatalyzed reduction of NO3- with a current yield corresponding to ca. 85%.