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Acetylcholinesterase (AChE) biosensors based on the catalytic activity of AChE in the detection of carbamate pesticides and the organophosphate pesticides is extensive. The modification of electrode surface represents a crucial step in the preparation of biosensor, for one hand it could increase the stability of enzyme immobilization onto the electrode surface, for the other hand it could improve effectively the sensitivity of signal.In this paper three kinds of amperometric AChE biosensors were developed based on nanomaterials, and several kinds of methods were used to modify the electrode surfaces, such as self-assembly, electrodeposition and covalent bonding. The properties of AChE biosensors were characterized by methods of scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. As tests demonstrated, the developed AChE biosensors had high sensitivity and stability, and were suitable for trace detection of pesticide residues in actual samples of vegetables and fruits.(1) Studies on AChE biosensor based on O-carboxymethyl chitosan-gold nanoparticles nanocompositeO-carboxymethyl chitosan-gold nanoparticles nanocomposite was prepared by in-situ synthesis method. Because O-carboxymethyl chitosan is water soluble, and has better biocompatibility, the prepared O-carboxymethyl chitosan-gold nanoparticles nanocomposite had high stability, and was more suitable for the immobilization of AChE. The nanocomposite was firstly dropped onto the surface of the glassy carbon electrode (GCE), and in order to increase the stability of AChE immobilization onto the electrode, the surface carboxyl groups were activated by the co-addition of N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Under optimized conditions, using chlorpyrifos as the model compound, the results showed that the inhibitory effect of chlorpyrifos was proportional to its concentration ranging from0.1to20μg/L and from20to100μg/L in different slopes, with a detection limit of0.07μg/L.(2) Studies on AChE biosensor modified with hollow gold nanosphersHollow gold nanospheres (HGNs) have several advantages over the solid counterparts such as higher specific surface, lower density and reduction of costs and better biocompatibility. In addition, hollow gold nanospheres as tiny conductive center can promote the electronic transfer in the electrochemical reaction because of the adequate exposure of active sites. The amperometric biosensor based on the HGNs modified electrode was constructed, using chlorpyrifos and carbofuran as model compounds, under optimized conditions, the inhibition rates of pesticides were proportional to their concentrations in the ranges of0.1-150and0.1-200μg/L for chlorpyrifos and carbofuran, respectively, the detection limits were0.06μg/L for chlorpyrifos and0.08μg/L for carbofuran. Compared with gas chromatograph, this biosensor had the advantages of shorter detection time and lower detection limit, and their recoveries were both higher than90%.(3) Studies on AChE biosensor based on one-step electrodepositionIn this work, chitosan (Chit), prussian blue (PB), multiwall carbon nanotubes (MWNTs), HGNs were cleanly and quickly fabricated onto the gold electrode based on one-step electrodeposition method. The synergistic effects of Chit, PB, MWNTs and HGNs could highly enhance the electrochemical response, thus improved the sensitivity of biosensor. After the immobilization of AChE, it was then covered with Nafion film, which could prevent the loss of the enzyme molecules, enhance the stability of biosensor. The electrochemical properties of AChE biosensors were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. Based on the inhibitory action of pesticides on the AChE activity, using malathion, chlorpyrifos, monocrotophos and carbofuran as model compounds, the prepared biosensor showed a high sensitivity, wide range, low detection limit, and could also be used for direct analysis of actual samples.Through the comparison between three methods of preparation of AChE biosensors, HGNs have excellent electrical conductivity over the solid counterparts, the prepared biosensor based on HGNs showed wide range and low detection limit, the synergistic effects of Chit, PB, MWNTs and HGNs could highly enhance the electrochemical response, this feature can be used to preparation acetylcholinesterase biosensor of more superior performance.
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