Immunoassays For Clothianidan And Imidacloprid Based On Phage-Displayed Peptides

Clothianidin and imidacloprid are neonicotinoids that cause insect death by binding to their nicotinic acetylcholine receptors and interfering with their normal nerve conduction.Due to their high efficiency,wide spectrum and good inhalation,clothianidin and imidacloprid are widely used in cabbage,pear and other crops for pest control.A number of studies have shown that clothianidin and imidacloprid have chronic toxicity to mammals,birds and aquatic organisms,and are highly toxic for non-target insects such as honeybees,resulted in that clothianidin and imidacloprid has been banned for outdoor use in various EU countries.Thus,the detection of clothianidin and imidacloprid residues in agricultural and environmental samples are of great significance to guarantee the quality and safety of agricultural products,ecological and environmental safety and human health.Instrumental analysis is the standard method for the detection of clothianidin and imidacloprid,which has high sensitivity and good stability.However,the pretreatment of instrumental analysis is complicated,which could not be used for the rapid detection in the field.Immunoassays based on the specific reaction of antibody-antigen have the advantages of rapid,simple,sensitive and low cost,which is suitable for high throughput and field detection.As a supplement to instrumental analysis,immunoassays for pesticide residues have a broad application prospect in the field of pesticide residue detection.However,the existing immunoassay methods of clothianidin and imidacloprid still have the following problems:（1）The sensitivity of the existing immunoassay methods of clothianidin still needs to be improved.（2）Due to the relatively similar chemical structure,current immunoassay methods of clothianidin and imidacloprid generally have cross-reactivity（CR）with other neonicotinoid pesticides,which limit their practical application.In respect of the issues above,pⅧdisplay phagemid p Comb-pⅧand phage display random cyclic 8,9,10-mer peptide libraries were constructed through phage display technology.Peptidomimetics and anti-immunocomplex peptides that could replace artificial antigens in immunoassays were screened by solid phase panning.Competitive and non-competitive phage enzyme-linked immunosorbent assay（phage-ELISA）for clothianidin and imidacloprid was established respectively,which improved the sensitive and specificity of immunoassays.The main results were summarized as follows:1.Based on phagemid p Comb3xss,phagemid p Comb-pⅧwas constructed,and the restriction nuclease site which could not cut the coding sequence of random polypeptide was introduced to avoid the loss of random polypeptide diversity.The phagemid p Comb-pⅧallows foreign protein displayed on the N-terminus of M13 filamentous phage pⅧprotein through linker GGGSS.Based on the phagemid,8,9,10-mer cyclic peptide libraries were constructed,and the practical sizes were 9.17×10⁸、4.38×10⁸and 6.97×10⁸,respectively.DNA sequencing results showed that the actual distribution frequencies of deoxynucleotides and amino acids in the peptide library were consistent with theoretical frequencies,indicated that the library are fully random and have great diversity,which could meet the needs of subsequent panning research.2.The constructed libraries were panned by the antibodies and immunocomplexes of clothianidin and imidacloprid.Twenty eight peptidomimetics with the common sequence of DPXG,two anti-immunocomplex peptides of CAVFTDQWWTGC（2-9-D）,CSPESHWPQVLC（2-9-G）for clothianidin,thirty peptidomimetics with the common sequence of TPAG and two anti-immunocomplex peptides of CWCIEDCSNC（2-1-A）,CVWDGDVGIMYC（2-1-H）for imidacloprid were isolated from the phage display peptide libraries,which provided reagents for the development of competitive and noncompetitive immunoassays for clothianidin and imidacloprid.3.Competitive and noncompetitive phage-ELISAs for clothianidin were developed by using the most sensitive peptidomimetic 2-5-G（CWLTPVGELVC）and anti-immunocomplex peptide 2-9-D（CAVFTDQWWTGC）.Under optimum conditions（0.3mol/L Na⁺,p H=6.0 and 5%methanol）,the competitive phage-ELISA had half maximal inhibition concentration(IC₅₀)of 3.83 ng/m L,linear range(IC₁₀-IC₉₀)of 1.11-13.20 ng/m L and limit of detection（LOD）of 1.11 ng/m L.The sensitivity of the competitive phage-ELISA was improved 11.6-fold than traditional indirect competitive ELISA(IC₅₀=44.6 ng/m L).The assay had CRs of 2.6%,18.2%and 3.61%with imidacloprid,imidaclothiz and nitenpyram,respectively.Under optimum conditions（1.2 mol/L Na⁺,p H=7.4 and 1.25%methanol）,the noncompetitive phage-ELISA had half maximal saturation concentration(SC₅₀)of 0.45ng/m L,linear range(SC₁₀-SC₉₀)of 0.26-0.76 ng/m L and LOD of 0.26 ng/m L.Compared with the traditional indirect competitive ELISA(IC₅₀=27.7 ng/m L),the sensitivity was improved 61.6-fold.The assay has assay had no CR with other neonicotinoids,which was the most sensitive and specific immunoassay for the detection of clothianidin by far as we know.The average recoveries of the competitive Phage-ELISA and noncompetitive Phage-ELISA in paddy water,soil,cabbage,pear and orange were 73.8%-104.1%and 76.6%-102.2%,with the relative standard deviations（RSDs）of 1.5%-11.0%and 2.9%-10.7%,respectively.The results for the detection of unknown concentrations of paddy water and soil samples had good correlations between high perform liquid chromatography（HPLC）and the competitive and noncompetitive Phage-ELISAs,which indicated that the two immunoassays could meet the requirements of the detection of clothianidin residues in agricultural and environmental samples.4.Competitive and noncompetitive phage-ELISAs for imidacloprid were developed by using the most sensitive peptidomimetic 1-9-H（CVPTPAGDFC）and anti-immunocomplex peptide 2-1-H（CVWDGDVGIMYC）.Under optimum conditions（0.14 mol/L Na⁺,p H=6.0and 2.5%methanol）,the competitive phage-ELISA had half maximal inhibition concentration(IC₅₀)of 0.55 ng/m L,linear range(IC₁₀-IC₉₀)of 0.30-1.00 ng/m L and limit of detection（LOD）of 0.30 ng/m L.The assay had CRs of 70.5%,105.8%,27.1%and 11.6%with clothianidin,imidaclothiz,thiacloprid,acetamiprid and nitenpyram,respectively.Under optimum conditions（0.07 mol/L Na⁺,p H=8.0 and 2.5%methanol）,the noncompetitive phage-ELISA had half maximal saturation concentration(SC₅₀)of 0.35 ng/m L,linear range(SC₁₀-SC₉₀)of 0.15-0.80 ng/m L and LOD of 0.15 ng/m L The assay had CRs of 112.9%,21.7%and 6.8%with imidaclothiz,thiacloprid and acetamiprid,respectively,which eliminated the cross reaction with clothianidin and nitenpyram and improved the specificity of method.The average recoveries of competitive Phage-ELISA and noncompetitive Phage-ELISA in paddy water,soil,wheat,brown rice,cabbage,potato,pear and orange were 75.1%-103.5%and73.8%-101.0%,with the relative standard deviations（RSDs）of 1.7%-7.8%and 2%-9.3%,respectively.The results for the detection of unknown concentrations of paddy water and soil samples had good correlations between high perform liquid chromatography（HPLC）and the competitive and noncompetitive phage-ELISA,which indicated that the two immunoassays could meet the requirements of the detection of imidacloprid residues in agricultural and environmental samples.