Electrical contact of redox enzymes with electrodes: Novel approaches for amperometric biosensors

Electrical communication of the redox-active center of enzymes with an electrode surface is a fundamental element for the development of amperometric biosensor devices. Different methods to assemble enzyme-electrodes exhibiting electrical contact between the redox protein and electrode surface are discussed with specific examples for tailoring glucose sensing electrodes. By one approach, a multilayer array of glucose oxidase is assembled on a Au-electrode. The number of enzyme layers is controlled by the synthetic methodology to assemble the electrode. Electrical contact between the enzyme array and the electrode is established by chemical modification of the protein layer with N-(2-methyl-ferrocene)-caproic acid, acting as an electrical wiring redox mediator. The number of enzyme layers associated with the electrode allows one to control the sensitivity of the resulting enzyme electrode. A further means of enhancing the sensitivities of enzyme electrodes involves the application of rough Au-electrodes as base-support to assemble the enzyme network. The high surface area of these electrodes (roughness factor = 20) allows the increase of the biocatalyst content in a single monolayer, and the resulting amperometric responses of the electrodes are ca 8-fold enhanced compared to enzyme layers assembled on smooth electrodes of identical geometrical areas. A novel method to electrically wire flavoenzymes with electrode surfaces was developed by reconstitution of the apo-flavoenzyme with a ferrocene-tethered FAD diad. Reconstitution of ape-glucose oxidase with the ferrocene-FAD diad yields an active bioelectrocatalyst of direct electrical communication with the electrode, 'electroenzyme'. The reconstitution methodology was further applied to tailor enzyme-electrodes of superior properties for electrical contact with the electrodes. A pyrroloquinoline quinone-FAD diad monolayer was assembled on a Au-electrode. Ape-glucose oxidase was reconstituted on the surface with the FAD-cofactor site to yield the aligned biocatalyst on the electrode. The pyrroloquinoline quinone, PQQ, redox unit acts as an electron relay that electrically contacts the FAD redox-site of the enzyme with the electrode. The surface reconstituted enzyme exhibits direct electrical communication with the electrode and acts as bioelectrocatalyst for the oxidation of glucose. The electrical communication of the reconstituted glucose oxidase on the PQQ-FAD monolayer is extremely efficient. The experimental current density at a glucose concentration of 80 mM is 300 +/- 100 mu A.cm(-2). This value overlaps the theoretical current density of glucose oxidase electrode (290 +/- 60 mu A.cm-(2)) taking into account the limiting turnover-rate of the enzyme, 900 +/- 150 s(-1) (at 35 degrees C). The extremely efficient electrical contact of the reconstituted enzyme and the electrode yields an enzyme-electrode that is insensitive to oxygen and is not affected by glucose-sensing interferants such as ascorbic acid. The application of the different enzyme-electrode configurations as bioelectronic devices for the determination of glucose is addressed. (C) 1997 Elsevier Science S.A.