A novel electro-optical sensor format with generic applicability for exploitation with NAD(P) dependent enzymes.

Abstract

This paper describes the development of a novel optically interrogated enzyme electrode with generic applicability for NAD(P) dependent enzymes. The example reported here employs a multi-enzyme pathway comprising the enzymes pyruvate kinase, hexokinase, glucose-6-phosphate dehydrogenase and diaphorase. The final substrate of this pathway, dichlorophenol indophenol (DCPIP), was immobilised within an ultra-thin polymer film of o-phenylenediamine, itself electrochemically polymerised onto a conductive gold coating on the surface of a support polyethylene sheet. Dichlorophenol indophenol (DCPIP) absorbs within the visible region of the spectrum with a lambda(max) approximately 600 nm. When reduced, the molar absorption coefficient at this wavelength decreases significantly and DCPIP effectively becomes colourless (DCPIPH(3)). Ultra-thin layers of gold (<10 nm thickness) exhibit an optical absorption minimum at wavelengths of approximately 520 nm and therefore light within this region of the spectrum may be transmitted with relative ease through the polymer/gold/polyethylene optrode. Results presented within this paper show how this electro-optical sensor may be used to determine concentrations of adenosine triphosphate (ATP) within a sample. In the presence of ATP a colour change from blue to colourless was observed for DCPIP when the assay was performed in solution. However, when DCPIP was immobilised within a polymeric film onto the surface of gold coated electrodes, a colour change from blue to red was observed corresponding to a third redox state of DCPIP (DCPIPH).