Study On Rapid Detection Of Furazolidone Contamination In Food Samples By Lateral Flow Immunoassay

Furazolidone has been widely used as a broad-spectrum antibiotic for the treatment of gastrointestinal infections caused by Escherichia coli and Salmonella spp.and as a growth promoter in cattle,pigs and poultry.However,previous research has revealed that the drug’s metabolites have some side effects such as mutagenic,carcinogenic,and teratogenic effects on human health.Such evidences have been taken as the basis to forbid the use of nitrofuran antibiotics in food animal production in the European Union,USA,and China.However,driven by profit,nitrofuran antibiotics are still often used illegally.Thus,it is of great practical significance to establish a highly sensitive on-site rapid detection method to monitor the abuse of furazolidone antibiotics.Due to the short half-life,the FZD parent drug is extremely unstable in vivo,while its metabolite,3-amino-2-oxazolidinone（AOZ）,can bind tightly to the protein in the tissue matrices and exist stably in body.So the residual of FZD is generally determined by detecting its metabolite AOZ.CPAOZ,the derivative of AOZ,was used as analyte to determinate the residues of AOZ in various food systems.In this work,to realize the rapid analysis of AOZ in various food samples,three lateral flow immunoassays（LFAs）for CPAOZ detection were constructed.The main research contents and results of this thesis are as follows:1.Preparation and characterization of the monoclonal antibody（McAb）against CPAOZ.The anti-CPAOZ McAb ascite was collected by intraperitoneal injection of mice,and the anti-CPAOZ McAb was purified.The identification results showed that the McAb was of high purity.The McAb titer was 1:128000,indicating that the high affinity of the McAb.The IC₅₀ of the McAb to CPAOZ was 2 ng·mL^-1,indicating that the high sensitivity of the McAb.2.Construction and characterization of a novel LFA based on double gold nanoparticles（GNPs）labeled antibodies and signal amplification for CPAOZ detection.Firstly,the traditional GNPs based LFA was constructed.Using GNPs as the McAb labeling material,the optimal preparation conditions of Au labeled McAb（Au-Ab）were determined.The visual detection limit（VDL）of the test strip to CPAOZ was 5 ng·mL^-1.The new developed biosensor was designed by utilizing the specific binding of McAb and anti-antibody（anti-Ab）,Au-Ab and Au labeled anti-Ab（Au-anti-Ab）as common detection probe and signal booster were prepared respectively.When the two complexes were eluted successively from the same conjugation pad by the sample solution,a powerful network structure could generate to accumulate numerous GNPs and thus significantly strengthen the signal intensity of detection line owing to the recognition of Ab and anti-Ab both labeled on GNPs.On the other hand,the enhanced signal can reduce the amount of antibody required in the assay,making more intense competitive reaction between the antigen and the small molecules,further improving the sensitivity of the assay.Under the optimal conditions,the VDL of the new assay for the residual marker of CPAOZ was 1ng·mL^-1,which was at least 5-fold improved over that of the traditional GNPs based LFA.Furthermore,this strategy was successfully applied in the residual monitoring of AOZ in five different food samples（milk power,shrimp,chicken,fish and pork）.Altogether,application results exhibited that the improved LFA has great potential for on field detection of AOZ residues in food samples.3.Construction and characterization of a novel LFA based on crystal violet staining for CPAOZ detection.In this study,McAb can be directly marked with crystal violet by one-step staining and the selectivity of LFA can be guaranteed by the specificity combination of McAb and antigen.After the optimization of experimental conditions,the optimal concentration of crystal violet was selected as 0.05%,the volume of crystal violet solution was 100μL,the optimal incubation time was 4 min and the reaction time was set at 10 min.Under optimal conditions,the VDL of the new protocol for the residual marker of CPAOZ was 7 ng·mL^-1.Although the analytical sensitivity was not significantly improved,the method provides a signal by simple crystal violet staining instead of complex nanomaterial labeling.Compared with the traditional method,the crystal violet staining strategy not only avoided the synthesis of signal materials,but also simplified the preparation process of the signal probe.Furthermore,the assay simplified the construction process of the method,reduced analysis cost and at the same time ensured the detection performance.Besides,the test strip has no cross-reaction with other CPAOZ structural analogs and can be successfully applied to the detection of AOZ contamination in shrimp,fish and pork samples.