An amperometric biosensor for acetylcholine determination prepared from acetylcholinesterase-choline oxidase immobilized in polypyrrole-polyvinylsulpfonate film

摘要

In this paper, a novel amperometric acetylcholine (ACh) biosensor with immobilization of acetylcholinesterase-choline oxidase (AChE-ChO) on polypyrrole-polyvinylsulphonate (PPy-PVS) film has been successfully developed. AChE and ChO enzymes were immobilized on PPy-PVS film by cross-linking with glutaraldehyde (GA) and bovine serum albumine (BSA). Determination of ACh was carried out by oxidation of enzymatically produced H_2O_2 at 0.3 Ⅴ vs Ag/AgCl. The effects of pH and temperature were investigated and optimum parameters were found to be 9.0 and 65 ℃, respectively. There were two linear parts in the region between 1.0 × 10~(-8)-1.0 × 10~(-7) M (R~2 = 0.994) and 1.0 × 10~(-7)-1.0 × 10~(-6) M (R~2 = 0.994). The detection limit of the biosensor was 5.0 × 10~(-9) M and response time of the biosensor was 200 s. Storage stability and operation stability of the enzyme electrode were also studied. The results showed that 83.1 % of the response current was retained after 18 activitiy assays. ACh biosensor gave perfect reproducible findings with 4.4% relative standard deviation. The prepared ACh biosensor retained 31% of its initial activity after 35 days storage in 0.1 M glycine buffer solution at 4℃.