SiO2 nanosheets (SNS) have been prepared by a chemical method using montmorillonite as raw material and were characterized by scanning electron microscopy and X-ray diffraction. SiO2 nanosheet–Nafion nanocomposites with excellent conductivity, catalytic activity, and biocompatibility provided an extremely hydrophilic surface for biomolecule adhesion. Chitosan was used as a cross-linker to immobilize acetylcholinesterase (AChE), and Nafion was used as a protective membrane to efficiently improve the stability of the AChE biosensor. The AChE biosensor showed favorable affinity for acetylthiocholine chloride and catalyzed the hydrolysis of acetylthiocholine chloride with an apparent Michaelis–Menten constant of 134 μM to form thiocholine, which was then oxidized to produce a detectable and fast response. Based on the inhibition by pesticides of the enzymatic activity of AChE, detection of the amperometric response from thiocholine on the biosensor is a simple and effective way to biomonitor exposure to pesticides. Under optimum conditions, the biosensor detected methyl parathion, chlorpyrifos, and carbofuran at concentrations ranging from 1.0 × 10−12 to 1 × 10−10 M and from 1.0 × 10−10 to 1 × 10−8 M. The detection limits for methyl parathion, chlorpyrifos, and carbofuran were 5 × 10−13 M. The biosensor developed exhibited good sensitivity, stability, reproducibility, and low cost, thus providing a new promising tool for analysis of enzyme inhibitors.
