FULLY INTEGRATED BIOCATALYTIC ELECTRODES BASED ON BIOAFFINITY INTERACTIONS

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affin ity matrices and further cross-linking of the enzyme monolayers. A cat alyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependen t enzymes are used to construct integrated electrically contacted bioc atalytic systems. NAD(+)-dependent lactate dehydrogenase (LDH) is asse mbled onto a pyrroloquinoline quinone-NAD(+) monolayer. The redox-acti ve monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N-6-(2-aminoethyl)-NAD(+) to the mono layer. The interface modified with the PQQ-NAD(+)-dyad provides tempor ary affinity binding for LDH and allows cross-linking of the enzyme mo nolayer. The cross-linked LDH is bioelectrocatalytically active toward s oxidation of lactate. The bioelectrocatalyzed process involves the P QQ-mediated oxidation of the immobilized NADH. Integrated, electricall y contacted bioelectrodes are produced by the affinity binding and fur ther cross-linking of nitrate reductase (NR) (cytochrome-dependent, E. C. 1.9.6.1 from E. coli) or Co-II-protoporphyrin IX reconstituted myog lobin (Co-II-Mb) atop the microperoxidase-11 (MP-11) monolayer associa ted with a Au-electrode. The MP-11 monolayer provides an affinity inte rface for the temporary binding of the enzymes, that allows the cross- linkage of the enzyme molecules. The MP-II assembly acts as electron t ransfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and Co-II-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogen ation, respectively. Two Fe-III-protoporphyrin IX units are reconstitu ted into a four alpha-helix bundle de novo protein assembled as a mono layer on a Au-electrode. Vectorial electron transfer proceeds in the s ynthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the Fe-III-protoporphyrin IX reconstituted de novo p rotein monolayer and NR yields an integrated, electrically contacted e nzyme electrode that stimulates the bioelectrocatalyzed reduction of n itrate. (C) 1998 Elsevier Science S.A. All rights reserved.