Study On Competitive Ratiometric Fluorescent Lateral Flow Immunoassay For Rapid Detection Of Chlorothalonil In Cucumber

Chlorothalonil（CTN）is a broad-spectrum organochlorine fungicide,which can effectively prevent and control deseases of fruit rot,anthrax,powdery mildew,grape downy mildew,et,in plants.However,chlorothalonil has good adhesion and can be adhered on the surface of plants for a long time.Residues of chlorothalonil in food could pose serious risk to human health.Therefore,it is particularly important to establish a highly sensitive and rapid method for the detection of chlorothalonil.In this study,the specific monoclonal antibody（m Ab）produced to against CTN.Futhermore,a Competitive ratiometric fluorescence based lateral flow immunoassay（CRF-LFIA）was developed and sucessfuly used for the visual,quantific and rapid detection of CTN.The main studies and results are as follows:（1）Production and characterization of anti-CTN m Ab:Antigen was designed and synthesized via 6-aminocaproic acid method,and CTN complete antigen was synthesized via carbonized diimide method by conjugating hapten to bovine serum protein（BSA）and chicken ovasmin（OVA）,respectively.CTN-BSA was used for mice immunization for 4 times.Serum titer and inhibition rate of mice were monitored by Indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）.The No.8 mice with high titer and inhibition rate were subjected to booster immunization.After cell fusion and subcloning,two cell lines（8D2 and 6F1）were obtained.After extended culture,ascites production,and purification,specific anti-CNT m Ab were achieved.The Half inhibitory concentration(IC₅₀)of CTN m Ab-8D2 and CTN m Ab-6F1 for CNT binding were characterized to 1.89 and 2.49ng/m L,respectively.CTN m Ab-8D2 with high sensitivity was selected for surther study.The results showed that the affinity constant of CTN m Ab-8D2 was 1.65×10⁸L/mol,and it’s cross-reaction to other structural analogues and potential interferors was less than 0.02%.The above results indicated that monoclonal antibody with high specificity and sensitivity against CTN was successfully achieved.It was further used for the development of immunochromatographic approach.（2）Method development of CRF-LFIA:First,magnetic ferroferric oxide（Fe₃O₄）nanoparticles were prepared by solvothermal method,then red emitted fluorescent magnetic nanobeads（FMNBs）with typical core-shell-satellite structure were prepared with TEOS and APTES hydrolysis,FMNBs is coupled with the CTN m Ab to prepare a probe with characterism of both fluorescence and magnetism.Aggregation-induced emission fluorescent microsphere（AIEFM）was immobilized on T-line as green fluorescence reference signal.Based on the quenching of AIEFM by FMNBs and superposition of the two fluorescent signals,A ratio fluorescence immunochromatography method was constructed to detect CTN visually and quantitatively.With the CTN concentration increasing,the T-line color changes from red to green.The change of hue intensity is more easily perceived by the naked eye.The condition of p H,antibody concentration,CTN-BSA concentration and probe dosage of antibody-coupled nanomaterials are optimized.The standard curve for CTN detection in PBS was y=-0.5085 x+2.0644（R²=0.9939）,the linear range was0.1-2 ng/m L,LOD was 0.152 ng/m L,and v LOD was 2 ng/m L.Method validation was conducted by spiked cucumber.The results showed that the recovery ranged from 82.00%to 119.00%and CV from 8.51%to 11.63%,which is in consistent with the results of instrument method.It was revealed that the developed method has great reliability and potential to be used for the real sample testing.In summary,in this study a highly sensitive and specific monoclonal antibodies against CTN was achieved.A competitive ratio fluorescence immunochromatography was developed based on FMNBs fluorescent substance for the detection of CTN.This approach provided alternative methodological support for the rapid detection of CTN in agricultural products.