A novel electro-optical sensor format with generic applicability for exploitation with NAD(P) dependent enzymes.
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).
Law
Karen A
KA
Institute of Bioscience and Technology, Cranfield University at Silsoe, Bedforshire MK45, 4DT, Silsoe, UK.
Warrington
Rachael J
RJ
McGurk
Antony
A
Higson
Séamus P J
SP
D015086:2,6-Dichloroindophenol / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D009249:NADP / Q000737:chemistry*; D055095:Optics and Photonics / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009249:NADP; D000255:Adenosine Triphosphate; D015086:2,6-Dichloroindophenol