An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration.
A biosensor for detection of formate at submicromolar concentrations has been developed by co-immobilizing formate dehydrogenase (FDH, E.C. 1.2.1.2), salicylate hydroxylase (SHL, E.C. 1.14.13.1) and NAD(+) linked to polyethylene glycol (PEG-NAD(+)) in a poly(vinyl alcohol) (PVA) matrix in front of a Clark-electrode. The principle of the bi-enzyme scheme is as follows: formate dehydrogenase converts formate into carbon dioxide using PEG-NAD(+). Corresponding PEG-NADH produced is then oxidized to PEG-NAD(+) by salicylate hydroxylase using sodium salicylate and oxygen. The oxygen consumption is monitored with the Clark-electrode. The advantages of this biosensor approach are the effective re-oxidation of PEG-NADH, and the entrapment of PEG-NAD(+) resulting in avoiding the addition of expensive cofactor to the working medium for each measurement. This bi-enzyme sensor has achieved a linear range of 1-300 microM and a detection limit of 1.98 x 10(-7) M for formate (S/N=3), with the response time of 4 min. The working stability is limited to 7 days due to the inactivation of the enzymes. Only sodium salicylate was needed in milli-molar amounts.
Mak
Karen K W
KK
Sino-German Nano-Analytical Lab (SiGNAL) and Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. karen@ust.hk
Wollenberger
Ulla
U
Scheller
Frieder W
FW
Renneberg
Reinhard
R
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D003067:Coenzymes / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004793:Enzyme Reactivators / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017743:Equipment Reuse; D005240:Feasibility Studies; D005560:Formate Dehydrogenases / Q000737:chemistry*; D005561:Formates / Q000032:analysis* / Q000737:chemistry; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry; D009243:NAD / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D003067:Coenzymes; D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D005561:Formates; D009097:Multienzyme Complexes; D009243:NAD; C030544:formic acid; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; D005560:Formate Dehydrogenases