Research Of Novel Detection Methods For Profenofos, Malathion And Acetamiprid

The issue of pesticide residues is one of the widely concerned food and environmental safety problems all over the world. Effective pesticide residue detection systems are the first requirement for survey and control the issue of pesticide residue. Presently, the desired detection system should including rapid pesticide residue detection methods as local screening tools and standard pesticide residue detection methods as final qualitative and quantitative confirmation tools. Under such background, this dissertation surrounding the topic of development of novel pesticide residue detection methods and has carried out the following three research work.(1) The pesticide residue detection methods specified in each kind of standards (such as CAC standard, national standard, industry standard, and so on) trend to upgrade to chromatography and mass spectrometry coalition technology. Hence, a novel qualitative and quantitative confirmation method for profenofos that based on the LC-MS/MS (liquid chromatography-mass spectrometry) technology was developed in the chapter 2 of this dissertation, first of all. Then, supporting pre-treatment methods to a series kind of samples were also developed, and those methods were further applied to studying the residual behavior of profenofos under paddy field conditions. The following results were obtained in this chapter. (a) Under the arranged experimental condition, m/z 302.8 and m/z 344.8 can be utilized as qualitative and quantitative ions for profenofos detection, respectively. (b) The recoveries of the developed method in rice plant, paddy soil, paddy water, brown rice, and rice hull were stabilized (RSD ranged form 1.05⁹.56%) and higher then 80%, and the detection limits were reached to 10^-9 (i.e.μg/kg) level. (c) Under paddy field conditions, the degradation of profenofos in rice plant, paddy soil, and paddy water were all fitted to the first-order kinetic equation (C = C₀e^-λt) and the half-lives (t_1/2) were calculated to be 5.47, 3.75 and 3.42 days, respectively. (d) If applying profenofos 40% EC to paddy field according the recommend dosage (600 g.ai/hm²), an interval period about 21 days was needed to guarantee the eaten safety of rice.(2) Immunoassay is the most widely used rapid pesticide residue detection method. In traditional pesticide immunoassay, competitive assay model was usually applied. However, the detection sensitivity, precision, and linear range of this model were typically inferior to its noncompetitive counterparts. Therefore, a noncompetitive pesticide immunoassay method of O,O-dimethyl organophosphorus pesticides that based on anti-idiotype antibodies (AId or Ab2) was developed in the following chapter of this dissertation. Here, phage display technology was applied to preparation anti-idiotype antibodies. The following results were obtained in this chapter. (a) The carbodiimide method can applied to immobilizing the generic hapten of O,O-dimethyl organophosphorus pesticides to EAH Sepharose 4B, and the coupling products can applied to purifying the class-specific antibody of O,O-dimethyl organophosphorus pesticides. (b) Using the purified antibody as immobilized antigen, twelve clones with anti-idiotype properties were successfully obtained from human single fold scFv libraries I + J (Tomlinson I+J) by phage display technology, and further identification results indicated that clones B9 and D11wereα-AId andβ-AId with relative high activity, respectively. (c) Based on clones B9 and D11, a noncompetitive immunoassay was successfully developed, and the IC₅₀ value and detection limit (ratio of single to noise is 3) of this method for Malathion were calculated to be 113.7±34.18μg/L and 10.54μg/L, respectively.(3) Another bottleneck for developing pesticide immunoassay method is the difficulty inherent in the preparation of high quality specificity antibody. Aptamer is a novel type of bio-recognition molecule with antibody substitution potential, and have become the hotspot in relevant research field. For this reason, aptamer isolation and identification work were carried out and rapid screening method of acetamiprid that based on aptamer was explored in the last part of this dissertation. The following results were obtained in this chapter. (a) Each key steps of the applied non-immobilized aptamer screening method were effective. (b) After 18 round repeat selection, fourteen acetamiprid specific aptamer sequences were obtained, and aptamer S18 was the most active one. The apparent dissociation constant (Kd) of aptamer S18 was calculated to be 4.98μmol/L, and the specificity of S18 was satisfactory. (c) Based on aptamer S18, a rapid screening method of acetamiprid wad developed, and its detection limit was estimated to be about 0.05 mmol/L (11.1 mg/L).