Study Of Lateral Flow Assay For Rapid Detection Of Florfenicol And Escherichia Coli O157:H7

In recent years,food safety issues,including residues of pesticides and veterinary drugs,prohibited additives,and pathogenic microorganisms,have attracted global attention.The related detection methods have also been widespread concerned.Compared with some common methods,such as traditional culture-based method,molecular biology method and instrumental method,lateral flow assay（LFA）presents the benefits such as simplicity,sensitive,rapidity and low cost.LFA is based on the principle of antigen-antibody specific reaction.Results of qualitative or quantitative analysis are generally obtained by naked-eye and test strip reader,respectively.In order to extend the application,a variety of nano-labels have been applied in LFA to obtain many better results.In the second chapter,the competitive inhibition LFA based on colloidal gold as the label was developed for the qualitative and quantitative detection of florfenicol in chicken.The effect of different sample pretreatment on experimental results was compared.The result of qualitative analysis was based on the color in test line and control line by naked eye.The result of quantitative analysis was obtained by establishing a standard curve using test strip reader.In order to get better detection results,some important parameters had been optimized,including the pH value and concentration of the antibody in the process of conjugation between the antibody and colloidal gold,the spray volume of gold-labelled antibody on conjugated pad,concentration of the antigen on test lines,and detection time.The results showed that the limit of detection of qualitative analysis was 25 ng/m L.The linear range and limit of detection of quantitative analysis was 0.125².50 ng/mL and 0.115 ng/m L,respectively.The detection time was 15 min.The extracting solution of chicken samples obtained by boiled method could be directly used in quick analysis without dilution.This colloidal gold based LFA is one of promising candidates for screening of relevant analytes and on-site detection because of its simplicity and rapidness.In the third chapter,Pd–Pt nanozyme was used as the label,and a sensitive LFA based on sandwich format was developed for the detection of Escherichia coli O157:H7 in milk.The signal amplification is owing to the Pd-Pt nanoparticles possessing excellent peroxidase-like activity toward 3,3′,5,5′-tetramethylbenzidine（a chromogenic reagent）,which can produce visual color band.Qualitative analysis was also achieved by observing the test line and control line.Results of quantitative analysis could be obtained by calibration curve.In this study,a series of important experimental parameters was optimized,including the pH value of the solution in the process of conjugation between the antibody and Pd–Pt NPs,the related concentrations of antibody and Pd–Pt NPs,the amount of NPs-labelled antibody complex,and detection time.The results indicated that the sensitivity of qualitative analysis was 1.0×10³ cfu·m L^-1.The linear range and sensitivity of quantitative analysis was 1.0×10³¹.0×10⁶ cfu·mL^-1and 9.0×10² cfu·m L^-1,respectively.The sensitivity of quantitative analysis was 111-fold higher than that of traditional colloidal gold-based LFA.The proposed lateral flow assay has remarkable potential in the detection of various pathogens in food samples.