Acetylcholinesterase-choline oxidase-based mini-reactors coupled with silver solid amalgam electrode for amperometric detection of acetylcholine in flow injection analysis

Abstract

A stable and simple amperometric biosensor based on the separated bienzyme (acetylcholinesterase (AChE) – choline oxidase (ChOx)) part has been successfully developed for the determination of acetylcholine (ACh) using flow injection analysis for the first time. The bienzyme part consists of two consecutively connected enzymatic mini-reactors filled by mesoporous silica powders (SBA−15) covalently covered by AChE and ChOx, respectively. The oxygen consumption as the result of two sequential enzymatic reactions was monitored amperometrically. The wall-jet cell with working silver solid amalgam electrode covered by mercury film was used for four-electron oxygen reduction at the highly negative potential. The experimental parameters including the amounts of immobilized enzymes, pH of the carrier solution, the detection potential, flow rate, and injection volume of ACh were optimized. The detection limit was found to be 4.1 μmol L−1. The interfering effect of 1.0 mmol L−1 glucose, 0.1 mmol L−1 uric acid, and 0.1 mmol L−1 ascorbic acid to 0.1 mmol L−1 ACh was 0.6%, 2.2%, and 2.8%, respectively. The developed amperometric ACh biosensor showed 59.8% of its initial response after >400 measurements over 100 days.