Flow-Injection System with Site-Specific Immobilization of Acetylcholinesterase Biosensor for Amperometric Detection of Organophosphate Pesticides

Abstract

ABSTRACTA flow-injection system with integrated amperometric biosensor featuring an easily replaceable immobilzed acetylcholinesterase (AChE) membrane was studied. The amperometric biosensor was constructed on the basis of site-specific immobilization of AChE on a hybrid polymer membrane with integrated multi-walled carbon nanotubes. Multistage modification of the membrane and immobilization of the enzyme was proved by Fourier transform infrared spectroscopy. The optimum flow-rate of the flow-injection analysis (FIA) system was 0.5 mL/min. It gave a linear response to acetylthiocholine chloride from 2 μM to 100 μM, with an average RSD of 3.0% (n = 6). The sensitivity of the constructed biosensor was 0.093 μA/μM·cm2. The Kmapp value of the immobilized AChE was 1.15 mM and the linear correlation coefficient R2, 0.9949. The method had a low detection limit for three organophosphorus pesticides (OPs) in model pesticide solutions—paraoxon ethyl (0.9×10−12 M), monocroptophos (1.8×10−12 M) and dichlorvos (2.0×10−12 M). This indicated that the action of multi-walled nanotubes and controlled site-specific enzyme immobilization ensured high electrocatalytic activity and selectivity of the biosensor towards pesticides. It was found that the biosensor can be reused 15 operation cycles. After storage for 30 days the enzyme membrane retained over 80% of its initial response. The FIA system was used for detection of anti-cholinesterase activity of two binary OP mixtures. The results for paraoxon + monocroptophos and paraoxon + dichlorvos showed that the total inhibition activity was not simply additive, but was lower than the sum of the individual inhibition values. Moreover, the difference between the sum of the individual inhibition values and the real results for the mixture was bigger for the binary system paraoxon and dichlorvos (7-10%) compared with that for paraoxon and monocroptophos (5-7%). The developed biosensor system is an ideal tool for monitoring of organophosphate pesticides.