Immunoassay Of Nine Typical Pesticides In Plant Derived Food

The problem of excessive pesticide residues in food occurs frequently,which seriously threatens people’s health.Along with the continuous expansion of the scope of food safety supervision,traditional instrument detection methods are difficult to meet the testing requirements of massive samples.It is urgent to develop efficient,portable and low-priced detection methods to provide effective tools for food safety monitoring at the grass-roots unit level.Therefore,nine pesticides with high detection frequency in agricultural products（triadimefon,triadimenol,myclobutanil,azoxystrobin,kresoxim-methyl,dicofol,chlorpyrifos,pyraclostrobin and lambda-cyhalothrin）were taken as research objects in this study to synthesize artificial antigens,develop monoclonal antibodies,establish colloidal gold immunochromatographic assay,and successfully apply them to the on-site rapid detection of the above pesticide residues in tomatoes,cucumbers,celery and wheat.1.Due to the simple structure and low molecular weight of pesticides,it is impossible to cause immune response.According to the structural characteristics of target pesticides,chemical transformation is carried out from the parent nucleus structure of pesticides and the intermediates for synthesis to prepare highly selective artificial antigens.（1）The haptens of triadimefon,triadimenol,myclobutanil,azoxystrobin,kresoxim-methyl,dicofol and chlorpyrifos were designed and synthesized from the parent nucleus structure.Triadimefon and triadimenol selectively recognized hapten was obtained by substitution reaction of hydroxy group of triadimefon with sodium chloroacetate;The cyano group in myclobutanil reacts with di-tert butyl dicarbonate to obtain specific hapten;The hapten of azoxystrobin was obtained by acid hydrolysis of the ester group of azoxystrobin;The specific hapten was prepared by oximation reaction of carbonyl group in the structural analog of kresoxim-methyl with carboxymethyl hydroxylamine hemihydrochloride;The hydroxyl group of dicofol was substituted with 3-bromopropane,and the carbonyl group of dicofol analog was oximized with carboxymethyl hydroxylamine hemihydrochloride to obtain two haptens;The chlorine atom at C3 position of chlorpyrifos and 3-mercaptopropionic acid undergo sulfydryl substitution reaction to modify the structure,introduce the active group carboxyl with linker arm,and obtain hapten.（2）The haptens of pyraclostrobin and lambda-cyhalothrin were designed and synthesized from pesticide intermediates.Using 1-（4-chlorophenyl）-1H-pyrazole-3-ol,an intermediate of pyraclostrobin,as raw material,the hapten of pyraclostrobin was synthesized by hydroxy activaion,bromination with 2-Nitrobenzyl bromide,a linker arm with ester group was introduced,which was converted into carboxyl group by acid hydrolysis to synthesize the hapten of pyraclostrobin.Using 3-（3-hydroxyphenyl）propionic acid,an intermediate of lambda-cyhalothrin,as raw material,benzyl alcohol was introduced to protect the carboxyl group,and added with trimethylsilyl cyanide to form cyanalcohol,and then dehydrated and condensed with lambda cyhalothric acid to prepare hapten.（3）Preparation of artificial complete antigensThe active carboxyl groups of the above haptens were activated by carbodiimide method,and coupled with the free amino groups of carrier protein keyhole limpet hemocyanin（KLH）,bovine serum albumin（BSA）,and ovalbumin（OVA）to prepare immunogens and coating antigens.The conjugates were characterized by ultraviolet and visible spectrophotometry and denaturing electrophoresis.The UV spectrum contained the characteristic absorption peak of hapten and protein,or showed a red shift or blue shift of the spectral peak relative to the carrier protein,or its electrophoresis band lagged relative to the carrier protein,which proved that the complete antigen synthesis was successful.Carboxyl groups of haptens were activated by carbodiimide method,and then coupled with KLH,BSA,OVA to prepare immunogen and coating antigen.The conjugates were characterized by UV spectrophotometry and denaturing electrophoresis.The conjugates were characterized by UV spectrophotometry and denaturing electrophoresis.The conjugates contained characteristic spectral absorption peaks of haptens and proteins or showed red shift or blue shift of spectral peaks relative to carrier proteins,and their electrophoresis bands lagged relative to carrier proteins,which proved that complete antigen synthesis was successful.2.Preparation of monoclonal antibodiesImmunize the mice with the synthetic immunogens.After multiple immunizations,select the mice with high titer and high inhibition rate to the target pesticide for cell fusion.Hybridoma cells were screened by limited dilution technology.After several cloning,the hybridoma cell lines secreting specific monoclonal antibodies were obtained:4A11（triadimefon and triadimenol）,4B1（myclobutanil）,4B7（azoxystrobin）,6B1（kresoxim-methyl）,4H9（dicofol）,3G9（chlorpyrifos）,3B1（pyraclostrobin）and 1E7（lambda-cyhalothrin）.The half inhibition rate(IC₅₀)of the monoclonal antibody is 0.029～9.98ng/m L,and the antibody affinity constant K_Dof the monoclonal antibody is 10⁷～10¹²L/mol,with high affinity.The subtypes of antibody 4A11 are Ig G1 and Ig G2a,the subtype of antibody 3G9 is Ig G1,and the other antibodies are both Ig G2b.Cross reaction experiment showed that the cross reaction rate of antibody 4A11 to triadimefon and triadimenol was100%and 50%respectively.The others were highly specific antibodies with cross reaction lower than 1%.3.Development of colloidal gold strip detection method（1）Single test stripsBy optimizing the p H and antibody concentration of colloidal gold labeled antibody,the strip was prepared and applied to the detection of tomatoes,cucumbers,celery and wheat.Tomato samples were extracted with methanol/water（v/v=1/4）solution and then adjusted p H to neutral for test.Cucumber and celery samples were extracted with methanol/water（v/v=1/4）solution and directly used for detected.Wheat was crushed and through 40 mesh screen,and then extracted with methanol/water（v/v=2/3）solution.The supernatant was centrifuged and diluted twice with 0.01 M PBS（p H 7.4,containing 1%ON-870,1%PEG）for detection.The visual limit of detection（v LOD）of 9 pesticides in tomatoes is 1～20 ng/g.The v LOD in cucumbers is 1～10 ng/g,and that in wheat is 5～100 ng/g.The v LODs of dicofol,chlorpyrifos and lambda-cyhalothrin in celery were 10,20 and 5 ng/g,respectively.All meet the requirements of national food safety limit standards.In the detection of actual samples,the colloidal gold strip detection method was confirmed to be consistent with the LC-MS/MS method,indicating that the developed strip detection method is accurate and feasible,and can meet the actual testing requirements.（2）Multiple test stripsIn order to improve the on-site detection capability,3 test lines are set in the detection area of test strip to improve the detection flux.The p H,antibody concentration and gold labeled antibody diluent of colloidal gold labeled antibody against pyraclostrobin,kresoxim-methyl and myclobutanil were optimized.Under the optimal conditions,the v LOD values of the test strip corresponding to pyraclostrobin,kresoxim-methyl and myclobutanil are respectively 10,10 and 50 ng/g.With the help of colloidal gold test strip reader,three pesticides in wheat can be quantitatively analyzed.The linear ranges are 5.6～116.5 ng/g,4.2～74.4 ng/g and 23.4～383.3 ng/g respectively.And the total detection time is less than 10minutes,which provides a powerful means for on-site detection of food safety.