Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of dehydrogenase enzymes and gold (Au) nanoparticle is described. The Au nanoparticles (AuNPs) have been self-assembled on a thiol-terminated, sol-gel-derived, 3-D, silicate network and enlarged by hydroxylamine seeding. The AuNPs on the silicate network efficiently catalyze the oxidation of NADH with a decrease in overpotential of approximately 915 mV in the absence of any redox mediator. The surface oxides of AuNP function as an excellent mediator, and a special inverted "V" shape voltammogram at less positive potential was observed for the oxidation of NADH. The AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. The nanostructured electrode shows high sensitivity (0.056 +/- 0.001 nA/nM) toward NADH with an amperometric detection limit of 5 nM. The electrode displays excellent operational and storage stability. A novel methodology for the fabrication of a NADH-dependent dehydrogenase biosensor based on the integration of dehydrogenase enzyme and AuNPs with the silicate network is developed. The enzymatically generated NADH is, in turn, electrocatalytically detected by the AuNPs on the silicate network. The integrated assembly has been successfully used for the amperometric biosensing of lactate and ethanol at a potential of -5 mV. The biosensor is very stable and highly sensitive, and it has a fast response time. The excellent performance validates the integrated assembly as an attractive sensing element for the development of new dehydrogenase biosensors.