Acetylcholinesterase biosensor based on prussian blue-modified electrode for detecting organophosphorous pesticides

In this study, a novel acetylcholinesterase (AChE) biosensor was developed based on dual-layer membranes (chitosan membrane and prussian blue membrane) modifying glassy carbon electrode (GCE). A chitosan membrane was used for immobilizing AChE through glutaraldehyde cross-linking attachment to recognize pesticides selectively. A prussian blue (PB) membrane was electrodeposited on the surface of GCE to enhance electron transfer. Before the detection, the chitosan enzyme membrane was quickly fixed on the surface of PB/GCE with O-ring to prepare an amperometric AChE-PB/GCE sensor for organophosphorus (OP) pesticides. The electrochemical behaviour of AChE-PB/GCE was studied, and the results showed that the chitosan membrane as carrier can absorb a large amount of enzyme, and PB has a significant synergistic effect towards enzymatic catalysis. As a result of these two important enhancement factors, the proposed biosensor exhibited extreme sensitivity to OP pesticides compared to the other kinds of AChE biosensor. The influences of phosphate buffer pH, substrate concentration, incubation time of pesticide on the response of the fabricated biosensor were investigated. Under optimum conditions, the inhibition rates of these pesticides were proportional to their concentrations in the range of 0.01–10 μg l−1, 0.05–10 μg l−1, 0.03–5 μg l−1, and 0.05–10 μg l−1, respectively. The detection limits were found to be 2.5 ng l−1 for dichlorvos, 15 ng l−1for omethoate, 5 ng l−1 for trichlorfon and 10 ng l−1 for phoxim. Moreover, the biosensor exhibited good reproducibility and stability, and it was suitable for trace detection of OP pesticide residue.