The Studies On The Detection Of 13 Typical Chemical Residues In Food Samples

In recent years,food safety problems have occurred frequently.In particular,a variety of toxic chemical residues often were detected in bulk foods of citrus,tea,soybeans,and corn,including clomazone,flumioxazin,nitrofen,bifenox,mesotrione and its metabolites,sultrione and its metabolites,dicamba,glyphosate,fenpyroximate and fenazaquin.With the improvement of my country’s food safety testing system and the expansion of testing scope,the number of food samples sampled is increasing year by year.Traditional instrument testing methods are gradually unable to meet the sampling needs of grassroots samples.There is an urgent need to develop more efficient and convenient testing methods.Therefore,in this study,the haptens of the 13 chemical residues were designed and screened by computational simulation technology,and the antigens were synthesized,10 monoclonal antibodies developed.Finally a colloidal gold rapid detection method was established.1.The designed,screened and synthesized of haptensThe chemical residues structure is too simple to cause an immune response.To coupled to carrier proteins,they need to be modified with obtain active groups.The haptens with a functional structural similarity of>50%to the target molecule is analyzed by quantum computing after selecting the active group with RDkit calculation.After Gaussian calculation and Discovery Studio positioning analysis,the potential distribution diagram of the molecule was obtained on the plane with an electron density of 0.001 a.u..（1）For the purpose of screening highly specific antibodies,the haptens were screened and synthesized that were similar to target molecule of overall spatial structure,atoms electrical properties and characteristic groups,including the haptens of clomazone,flumioxazin,dicamba,glyphosate,fenpyroximate and fenazaquin.（2）For the purpose of screening group-selective antibodies,the haptens were screened and synthesized that were similar to target molecule of parent core structure,including the haptens of nitrofen,bifenox,mesotrione and its metabolites,sultrione and its metabolites.2.Preparation of monoclonal antibodies.Bovine serum albumin（BSA）and ovalbumin（OVA）was conjugated with hapten.Mice were immunized with BSA conjugates,hybridoma cell technology was used to screen cell lines secreting high-performance antibodies,and monoclonal antibodies were prepared by inducing ascites.Preparation of highly specific antibodies（crossover rate<5%with structural analogs of the target chemical residue）:CLO-m Ab 4C4,FLU-m Ab 1A7,DIC-m Ab 1B5,GLY-m Ab 1B9,FEP-m Ab 2G9 and FEA-m Ab 3F10,the IC₅₀ values for clomazone,flumioxazin,dicamba,glyphosate,fenpyroximate and fenazaquin are 2.47 ng/m L,0.59 ng/m L,39.41 ng/m L,and1160 ng/m L,206.88 ng/m L,103.61 ng/m L respectively.In addition,the group-selective antibody NIT-m Ab 5G7 was also screened,which can simultaneously recognize nitrofen(IC₅₀value is 1.54 ng/m L)and bifenox(IC₅₀ value is 2.20 ng/m L);the antibody BIF-m Ab 3E3 can also simultaneously recognize nitrofen(IC₅₀ value:73.14 ng/m L),bifenox(IC₅₀ value:5.76ng/m L);the antibody MES-m Ab 4G7 can simultaneously recognize mesotrione(IC₅₀ value:4.21 ng/m L)and its metabolites 4-methylsulfonyl-2-nitrobenzoic acid(IC₅₀ value is 9.11ng/m L)and 2-amino-4-methylsulfonylbenzoic acid(IC₅₀ value is 9.47 ng/m L),sulfotrione(IC₅₀ value is 8.14 ng/m L)and its metabolite 2-chloro-4-methanesulfonylbenzoic acid(IC₅₀value is 12.5 ng/m L);the antibody MES-m Ab 3H7 can simultaneously recognize mesotrione(IC₅₀ value is 3.76 ng/m L)and its metabolites 4-methylsulfonyl-2-nitrobenzoic acid(IC₅₀value:10.81 ng/m L)and 2-amino-4-methylsulfon-ylbenzoic acid（IC50 value:7.36 ng/m L）.All antibody affinity constants are greater than 1×107L/mo L and are high-affinity antibodies.3.Development of colloidal gold test strip detection method for chemical residues in foods.The p H of the colloidal gold-labeled antibody,the amount of antigen and antibody used,and the type of gold-labeled antibody diluent were optimized,and test strips were prepared and applied to the detection of citrus,tea,soybeans,and corn.Chemical residues other than glyphosate were extracted using a mixed solvent of acetonitrile/acetic acid.The extract was centrifuged and the supernatant was concentrated.The residue was resuspended in 0.01 M phosphate buffer（p H 7.4）containing 10%acetonitrile and then detected.For samples containing glyphosate,the extraction solution and resuspension solution need to be changed to acidic ultrapure water and 0.01 M borate buffer（p H 8.8）containing 10%acetonitrile.The visual detection limit（v LOD）of 5 chemical residues in citrus is 1～100 ng/g;the v LOD of 4chemical residues in tea is 2～200ng/g;the v LOD of 9 chemical residues in soybeans is 2respectively～500 ng/g;the v LOD of 6 chemical residues in corn is 10～20 ng/g,all of which can fully meet the limit requirements of national standards.Verified by addition recovery experiments,the recovery rate of this method is between 70%and 120%.Compared with the instrument detection method,the detection results are consistent and the detection time is greatly shortened.The detection results can be obtained within 20～40min.At the same time,a multiple colloidal gold test strip immunochromatographic detection method was developed based on the single contaminant test strip detection method,which can simultaneously detect the contents of mesotrione,clomazone and propargyl flufenate in soybeans.The test results are consistent with the single test strip test method and can meet the limit requirements of national standards.To sum up,this project has innovated the specific regulation method of small molecule hapten recognition,developed a highly sensitive monoclonal antibody that can recognize 13typical chemical residues in food,and established a colloidal gold rapid detection of chemical residues in food.This method can fully meet the limited requirements of national standards and the needs of rapid on-site testing at the grassroots level.