Study On Peroxidase-like Nanozyme Catalyzed Signal Amplification Dip-stick Immunoassay For Food Contaminant Detection

Chemical and biological contaminants in food greatly affect human health.17β-estradiol（E2）,as a female hormone,is used to accelerate animal growth,increase lean meat proportion and increase milk production.However,the improper use of E2 may lead to residues in the edible parts of animals.After being enriched in the human body through the food chain,it will harm the reproductive system and immune system.In addition to chemical contaminants,food raw materials can also be directly or indirectly contaminated with biological contaminants during acquisition,processing,and transportation.Foodborne pathogens are a kind of biological pollutants that cannot be ignored in food.Among them,Salmonella typhi（S.typhi）and Escherichia coli O157:H7（E.coli O157:H7）are two kinds of highly infectious and harmful foodborne pathogens.After infection with foodborne pathogens serious threats to life and health may occur,including vomiting,diarrhea,serious sepsis,and visceral damage.Therefore,to realize food safety supervision and management,it is very necessary to construct a fast and sensitive detection method to detect contaminants in food.In this study,E2 was used as a representative of chemical pollutants,E.coli O157:H7 and S.typhi were used as representatives of biological pollutants.Two kinds of dip-stick immunoassay（DSIA）based on signal amplification by peroxidase-like nanozymes were developed to achieve rapid detection of harmful substances in food.The main research contents and results are as follows:1.Development of DSIA based on flake vanadium disulfide（VS₂NS）peroxidase-like nanozymes for chemical contaminant detection of E2 in food:First,VS₂NS was synthesized by hydrothermal method.Compared with natural horseradish peroxidase,VS₂NS showed better catalytic performance and stability.VS₂NS were bound to antibodies by electrostatic adsorption to form VS₂NS-m Ab probes.Under optimized conditions,the computational detection limit of E2 is 0.065 ng/m L for VS₂NS-DSIA.With 3,3’,5,5’-tetramethylbenzidine（TMB）as the substrate,at the participation of hydrogen peroxide（H₂O₂）,the linear detection range can be expanded by 1.5 times compared with that of the original reaction.The VS₂NS-DSIA has no cross-reaction with Levofloxacin,Streptomycin sulfate,Tobramycin,Kanamycin,Albuterol,and Clenbuterol,showing a strong specificity.Finally,VS₂NS-DSIA was applied for the detection of E2 in pork and beef samples.The cut-off of pork was 14 ng/g,the cut-off of beef was 20 ng/g,and the recovery rate of E2 in food samples was 92.42-104.98%,which proved that the test paper has good practicability in food samples.2.Development of DSIA based on CoFe₂O₄（CFO）peroxidase-like nanozymes for biological contaminants detection of E.coli O157:H7 and S.typhi in food:First,CFO were synthesized by hydrothermal method.In addition to the colorimetric properties of its own color,the material has excellent peroxidase activity and magnetic separation properties.The CFO is bound to the antibody by electrostatic adsorption to form the CFO-m Ab probe.Under optimized conditions,the visual detection limits（v LODs）for E.coli O157:H7 and S.typhi are10⁴ CFU/m L and 10² CFU/m L,respectively.Using TMB and H₂O₂ as the substrate,the v LOD of the CFO-DSIA for E.coli O157:H7 is 10³ CFU/m L and the quantitative detection range is10³-10⁸ CFU/m L.The v LOD of the CFO-DSIA for S.typhi is 10² CFU/m L,and the quantitative detection range is 10²-10⁸ CFU/m L.The CFO-DSIA shows no cross-reaction to Salmonella London,Salmonella enteritidis,Listeria monocytogenes,Staphylococcus aureus,and Shigella fowleri,showing a high specificity.Finally,the CFO-DSIA was used for the detection of E.coli O157:H7 and S.typhi in milk and beef samples.In milk samples,E.coli O157:H7 had a v LOD of 5×10³ CFU/m L and S.typhi had a v LOD of 5×10² CFU/m L.In beef samples,E.coli O157:H7 had a v LOD of 5×10³ CFU/g and S.typhi had a v LOD of 5×10²CFU/g.In dual-reading recovery rate of E.coli O157:H7 was 94.51-114.24%,and the average recovery rate of S.typhi was 89.20-113.45%,which proved that the test paper also has good practicability in food samples.