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Electrochemical Detection for Acetylcholine Esterase Inhibitor Using Multienzyme-modified Microchip

Han, Yong duk; Yoon, Jae Ho; Yoon, Hyun C.
Institute for Medical Sciences
A microchip-based electrochemical biosensing system for acetylcholine esterase (AchE) inhibitor detection was developed. The AchE inhibitor molecules represented as organophosphorus (OP) compounds are widely used as pesticide, insecticide and chemical warfare agent. Due to its toxicity to human, development of accurate and convenient biosensing system for detection of remained OPs in food or environment is highly required. In this work, we employed bienzyme bioelectrocatalysis involving AchE and choline oxidase (ChOx) reactions. Acetylcholine (substrate of AchE) is degraded into choline (Ch) by AchE, and Ch is then used as ChOx substrate, which generates an amplified electrochemical signal based on its bioelectrocatalytic reaction. By the irreversible inhibition reaction of OP to the AchE, AchE activities appeared as electrochemical signals are inversely proportional to the OP concentrations. Based on the measurement of OP-induced electrochemical signal alterations, we can quantify the amount of OP. To develop an on-site biosensor, this bienzyme reaction was accomplished in single microchip containing microfluidic channel and micro-patterned electrode. For the construction of multienzyme layers in microchip, two different enzyme immobilization methods were employed. ChOx was immobilized on the electrode using poly-L-lysine (PLL), glutaraldehyde, and amine-rich interfacial surface. AchE was immobilized on the aldehyde group-functionalized magnetic particle (MP) via Schiff’s base formation, and the resulting AchE-modified MPs were deposited on the electrode using a magnet installed under the microfluidic channel. Using the developed OP biosensing chip, detection of diazinon (a model OP compound) at various concentrations was successfully carried out (from 0.05 ppm to 0.9 ppm). The self-contained microchip structure of the developed biosensor is easy to operate. We expect that the developed system can be practically applied in various fields where the OP detection system is required such as food industry and national defense.
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