Amperometric Biosensor Based on Enzymatic Reactor for Choline Determination in Flow Systems

A simple, selective and stable biosensor with the enzymatic reactor based on choline oxidase (ChOx) was developed and applied for the determination of choline (Ch) in flow injection analysis with amperometric detection. The enzyme ChOx was covalently immobilized with glutaraldehyde to mesoporous silica powder (SBA-15) previously covered by NH₂-groups. This powder was found as an optimal filling of the reactor. The detection of Ch is based on amperometric monitoring of consumed oxygen during the enzymatic reaction, which is directly proportional to Ch concentration. Two arrangements of an electrolytic cell in FIA, namely wall-jet cell with working silver solid amalgam electrode covered by mercury film and flow-through cell with tubular detector of polished silver solid amalgam were compared. The experimental parameters affecting the sensitivity and stability of the biosensor (i. e. pH of the carrier solution, volume of reactor, amount of the immobilized enzyme, the detection potential, flow rate, etc.) were optimized. Under the optimized conditions, the limit of detection was found to be 9.0×10⁻⁶ mol L⁻¹. The Michaelis-Menten constant for covalently immobilized ChOx on SBA-15 was calculated. The proposed amperometric biosensor with the developed ChOx-based reactor exhibits good repeatability, reproducibility, long-term stability, and reusability. Its efficiency has been confirmed by the successful application for the determination of Ch in two commercial pharmaceuticals.