Affinity Methods to Immobilize Acetylcholinesterases for Manufacturing Biosensors

Abstract

Two new affinity methods to immobilize acetylcholinesterase (AChE) onto screen‐printed electrodes (SPE) are described and compared for the fabrication of SPE: one based on metal chelate affinity (MCA) and one based on the Concanavalin A (Con A), a sugar residue binding lectine. The manufacturing and the optimization procedure for each type of biosensor are discussed with respect to no‐specific binding, functionalization of the SPE surface and the amount of enzyme used. A linear response range to acetylthiocholine substrate between 10 and 100 µmol L−1 was obtained for Con A sensors and between 1 and 60 µmol L−1 with the MCA sensors. The possibilities to reuse the sensors fabricated using both affinity immobilization chemistries were also discussed. The optimized sensors were used to study the inhibitory effects of organophosphorus pesticides on immobilized AChE activity. A detection limit of 5 × 10−7 mol L−1 chlorpyrifos methyl‐oxon (ee AChE sensor) and 2 × 10−9 mol L−1 paraoxon (dm AChE sensor) was achieved for the sensors with the enzyme attached via Con A and NTA–Ni, respectively.