有机磷农药快速检测试纸的初步研究
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摘要
目的：依据酶抑制原理建立有机磷(Organic phosphorus, OP)农药残留快速检测方法,制备快速检测试纸,建立用于普通民众日常检测水果、蔬菜中的微量农药残留,以及为农田、市场现场快速检测农药含量的方法。为今后进一步开发和利用胆碱酯酶检测OP农药残留污染打下良好的基础,提供理论依据。方法：采用硫酸铵沉淀法,普鲁卡因胺(Procainamide)-ECH琼脂糖4B亲和层析纯化胆碱酯酶。以壳聚糖膜为载体,采用戊二醛交联法固定胆碱酯酶,制备胆碱酯酶试纸。设计正交试验考察牛血清白蛋白(Bovine serum albumin, BSA)百分含量,戊二醛(Glutaraldehyde, GA)浓度,BSA%与GA浓度二者间的交互作用及固定化所采用何种载体材料等因素对固定化的影响,确定固定化条件。对胆碱酯酶试纸进行灵敏度和差异性分析。结果：各条件对固定化的影响大小依次为：固定化载体>戊二醛浓度>戊二醛×BSA>BSA浓度。在5.3×10-3-5.3μg/L范围内,胆碱酯酶抑制率与敌敌畏质量浓度的回归方程为y=12.799Ln(x)+72.384(R=0.9724),最低检出浓度为0.17μg/L。固定化酶在15天内活力保持稳定。结论：分离、纯化胆碱酯酶采用条件：缓冲为含0.5% TritonX-100,20mmol/L(pH7.0)的磷酸缓冲,用40%硫酸铵沉淀。固定化采用条件：壳聚糖膜载体,0.01%戊二醛,2%牛血清白蛋白,0.05mol/L(pH6.0)磷酸缓冲液。通过对胆碱酯酶分离、纯化及固定化条件的研究,探讨了各因素之间的影响和关系,建立了胆碱酯酶分离、纯化和试纸制备的方法。该固定化方法易重复,酶的稳定性高。
Objective:Organphosphorus pesticides rapid test strip is studied, which based on enzyme inhibition by organic phosphorus pesticide. The method is established for routine pesticide residues testing for fruits, vegetables, and rapid detection of pesticide in the market, as well as in farm land. Good foundation is laid and theoretical basis is provided, for the further exploiture and utilization cholinesterase to detect OP pesticide. Methods: Ammonium sulfate precipitation, procainamide-ECH Sepharose 4B affinity chromatography were used to separate and purify cholinesterase. Chitosan membrane as a carrier, cholinesterase was immobilized by glutaraldehyde cross-linking. Immobilization factors was studied by orthogonal experiment, which were bovine serum albumin (Bovine serum albumin, BSA) percent content, glutaraldehyde (Glutaraldehyde, GA) concentration, interaction between BSA% and the GA concentration, and membrane materials used in immobilization. Sensitivity and diversity of cholinesterase test strip was analyzed. The market for sale dichlorvos EC was calibrated. Differences and sensitivity of test strip were analyzed. Results:affect of conditions on the immobilization:vector> glutaraldehyde>glutaraldehyde×BSA>BSAconcentration. From 5.3×10-3-5.3μg/L, enzyme inhibition rate and the concentration of dichlorvos follow the regression equation:y =12.799Ln(x)+72.384(R=0.9724). Its minimum detectable concentration was 0.17μg/L. Immobilized enzyme activity did not change within 15 days. Conclusions:Separation and purification cholinesterase conditions:extraction buffer was containing 0.5% TritonX-100, 20mmol/L (pH7.0) phosphate buffer, precipitation with 40% sulfate ammonium. Immobilization conditions:chitosan membrane carrier,0.01% glutaraldehyde,2% bovine serum albumin,0.05mol/L (pH6.0) PBS. Conditions of separation, purification cholinesterase was Studied, and factors of various immobilization was explored. Methods of separation, purification cholinesterase and enzyme strip preparation were established. The immobilization method was reproducible. Immobilized enzyme was stable.
Objective:Organphosphorus pesticides rapid test strip is studied, which based on enzyme inhibition by organic phosphorus pesticide. The method is established for routine pesticide residues testing for fruits, vegetables, and rapid detection of pesticide in the market, as well as in farm land. Good foundation is laid and theoretical basis is provided, for the further exploiture and utilization cholinesterase to detect OP pesticide. Methods: Ammonium sulfate precipitation, procainamide-ECH Sepharose 4B affinity chromatography were used to separate and purify cholinesterase. Chitosan membrane as a carrier, cholinesterase was immobilized by glutaraldehyde cross-linking. Immobilization factors was studied by orthogonal experiment, which were bovine serum albumin (Bovine serum albumin, BSA) percent content, glutaraldehyde (Glutaraldehyde, GA) concentration, interaction between BSA% and the GA concentration, and membrane materials used in immobilization. Sensitivity and diversity of cholinesterase test strip was analyzed. The market for sale dichlorvos EC was calibrated. Differences and sensitivity of test strip were analyzed. Results:affect of conditions on the immobilization:vector> glutaraldehyde>glutaraldehyde×BSA>BSAconcentration. From 5.3×10-3-5.3μg/L, enzyme inhibition rate and the concentration of dichlorvos follow the regression equation:y =12.799Ln(x)+72.384(R=0.9724). Its minimum detectable concentration was 0.17μg/L. Immobilized enzyme activity did not change within 15 days. Conclusions:Separation and purification cholinesterase conditions:extraction buffer was containing 0.5% TritonX-100, 20mmol/L (pH7.0) phosphate buffer, precipitation with 40% sulfate ammonium. Immobilization conditions:chitosan membrane carrier,0.01% glutaraldehyde,2% bovine serum albumin,0.05mol/L (pH6.0) PBS. Conditions of separation, purification cholinesterase was Studied, and factors of various immobilization was explored. Methods of separation, purification cholinesterase and enzyme strip preparation were established. The immobilization method was reproducible. Immobilized enzyme was stable.
引文
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[15]赵为武.农产品农药残留问题及治理对策[J].植物医生,2001,14(3)：10-13.
[16]崔洪力,李强,刘美良.药残留及监控对策[J].农业与技术,2002,2(5)：74-76.
[17]何文初.加强中国农药污染环境法治思考[J].湖南农业大学学报,2002,(9)：33.
[18]Perez-Ruiz T, Martinez-Lozano C, Garcia MD. Determination of N-methylcarbamate pesticides in environmental samples by an automated solid-phase extraction and liquid chromatographic method based on post-column photolysis and chemiluminescence detection[J].Chromatogr A,2007,1164(1-2):174～180.
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[22]Hiemstra M, de Kok A. Comprehensive multi-residue method for the target analysis of pesticides in crops using liquid chromatography-tandem mass spectrometry [J]. ChromatogrA,2007,1154(1-2):3-25.
[23]张丽,杨迎春,吉爱军等.鲁米诺-过氧化氢体系流动注射化学发光法测定乐果[J].四川师范大学学报(自然科学版),2008,31(2)：250-252.
[24]魏福祥,王振川,王金梅.乙酰胆碱酯酶生物传感器法测定蔬菜水果中有机磷农药残留[J].食品科学,2007,28(2)：229-231.
[25]赵肃清,孙远明,张春艳.甲胺磷单克隆抗体制备及鉴定[J].免疫学杂志,2003,19(2)：142-145.
[26]邰春娇,吴晓芳.快速检测蔬菜中农药残留的方法探讨[J].中国卫生检验杂志,2007,17(6)：1124.
[27]高晓辉.蔬菜上农药残留快速检测势在必行[J].农药科学与管理,2000,21(1)：16-20.
[28]万圣,吕延成.有机磷及氨基甲酸酯类农药残留检测研究进展[J].广东农业科学,2009,3：110-112.
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