An acetylcholinesterase (AChE) purified from rat brain was immobilized onto gold nanoparticles (AuNPs) assembled on the surface of porous calcium carbonate (CaCO(3)) microsphere. The resulting AChE-AuNPs-CaCO(3) bioconjugate was mounted on the surface of Au electrode with the help of silica sol-gel matrix to prepare the working electrode. This electrode was connected to Ag/AgCl (3M/saturated KCl) as standard and Pt wire as an auxiliary electrode through a potentiostat to construct an organophosphorus (OP) biosensor. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The biosensor showed optimum response at pH 7.0, 30°C, when polarized at +0.2V. Two OP compounds, malathion and chlorpyrifos could be detected in the range of 0.1-100 nM and 0.1-70 nM, respectively at 2.0-3.0% inhibition level of AChE. The sensor was reactivated by immersing it in 0.1 mM 2-pyridine aldoxime for 10 min. The detection limit of the sensor was 0.1 nM for both malathion and chlorpyrifos. The biosensor exhibited good reusability (50 times without considerable loss) and storage stability (50% within 60 days, when stored at 4°C).
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