Design of amperometric biosensors and biofuel cells by the reconstitution of electrically contacted enzyme electrodes

The electrical contacting of redox enzymes with electrodes is the most fundamental requirement for the development of amperometric biosensors and biofuel cell elements. For the effective electrical communication of redox enzymes with electrodes the use of electron relay units that transport the electrons between the enzyme redox center and the conducting surface is essential. Also, the structural alignment of the redox enzyme units in respect to the electrode in a configuration where the enzyme redox center is in close proximity to the conductive surface is needed. The present report summarizes the reconstitution paradigm developed by our laboratory in the last decade as a versatile method to electrically contact redox enzymes with electrodes, and as a generic approach to develop amperometric biosensors and biofuel cell elements. The process is based on the reconstitution of the apo-enzyme on a relay-cofactor monolayer on thin film-functionalized electrode. Different relay units were used to electrically communicate flavin adenine dinucleotide (FAD)-containing enzymes (flavoenzymes) or pyrroloquinoline quinone (PQQ)-containing enzymes with electrodes. This included molecular redox-active relays, molecular redox-active 'shuttles', redox-active polymers (e.g., polyaniline), An nanoparticles, and carbon nanotubes. The reconstitution of different apo-enzymes on these relay-cofactor-functionalized electrodes led to unprecedented efficient electrical contacting between the redox centers of the enzymes and the electrodes. Besides very sensitive amperometric biosensors that emerged from this method, the resulting amperometric biosensors revealed high selectivity and specificity. A related approach to establish electrical contact between redox enzymes dependent on diffusional cofactors and electrodes and to develop an integrated bioelectrocatalytically active enzyme electrode was developed. The method involved assembly of a relay-cofactor diad on the electrode, and the surface crosslinking of an affinity complex generated between the enzyme and the surface-confined cofactor units. This method was successfully applied to electrically contact nicotinamide adenine dinucleotide (phosphate) NAD(P)(+)-dependent enzymes and cytochrome c-dependent enzymes. For example, enzyme-modified electrodes for the bioelectrocatalyzed oxidation of alcohol, lactate and malate were fabricated by the electrical contacting of the respective NAD(P)(+)-dependent dehydrogenases. Similarly, the bioelectrocatalytic reduction of O-2 was accomplished by an integrated cytochrome c/cytochrome oxidase-functionalized electrode. The electrically contacted enzyme electrodes were also used to develop noncompartmentalized biofuel cell elements. Biofuel cell elements consisting of electrically contacted reconstituted enzyme electrodes were constructed. Glucose or alcohol were used in these systems as fuel substrates and O-2 as oxidizer.