Study On The Rapid Detection Of Pesticide Residues With Cholinesterase From A Serum

Up to now, chemical prevention has still been a main method for controlling plant diseases and insect pests. Organophosphate and carbamate pesticides are extensively used in agricultural products such as vegetables and grain etc, although they are very toxic pesticides. The pesticide residues in agricultural products become much serious due to using more and more pesticides to control the plant diseases and insect pests. It is often happened that people are poisoned for eating some vegetables and fruits including excessive pesticide residues. So it is imperative to develop a simple and rapid detection method for pesticide residues. In current research, an enzyme inhibition method of pesticide residues has been developed.The toxic action of organophosphate and carbamate pesticides is based on the rapid irreversible inhibition of the cholinesterase (ChE). There is a good relationship between pesticides exposure and ChE activity. So the pesticide residues in agricultural products can be detected by analyzing the change of ChE activity. In the project, an appropriate ChE has been got, and its characterization has been studied. On the basis of these works, two rapid detection methods of pesticide residues, colorimetry and rapid test paper, were developed.A BChE from A serum was selected to determinate pesticide residues by comparison of many BChE from different materials. The BChE was purified to electrophoretic homogeneity by salting out, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. Finally the enzyme was purified 278.5 folds and the enzyme activity recovery ratio was 17.8%. The properties of the BChE from A serum has been studied, and the results showed that the enzyme km value was 2.4×10^-5, the optimum reaction temperature was 37℃ and the optimum reaction pH was between 7.5⁸.0; and the enzyme activity was not inhibited by excess butyrylthiocholine iodide.Two rapid detection methods were developed. â‘ Colorimetry: in the reaction system, S-butyrylthiocholine iodide was substrate and 5,5'-dithiobis(2-nitrobenzoic acid) was chromogenic agent. By calculating inhibition rate of ChE activity, the condition for pesticide residues could be estimated. The standard of detection was a dosage of inhibiting 35% of ChE activity. When the inhibition rate was higher than 35%, it indicatedthat the sample had excessive pesticide residues. The detection time was 20min⁴0min, and several samples can be determined at one time. The coincidence extent of colorimetry and gas chromatography is above 85%. ?Test paper: The substrate was indophenol acetic ester. Accordingly, the condition of pesticide residues was estimated by chromogenic reaction. If the test paper became white or Cambridge blue, it was positive reaction indicating that the sample has excessive pesticide residues; otherwise, the sample was safe. The detection time was 15min. It was found that this ChE was very sensitive to Dichlorvos, but not to Methamidophos. The minimal detected concentration of some commonly used pesticides was 0.001mg/kg¹0mg/kg.