Development Of LFIA And ELISA Based On Tyramine Signal Amplification Strategy For Sensitive Detection Of Danofloxacin

Danofloxacin（DAN）,the third generation of fluoroquinolones derivative,is widely used for livestock,poultry,and aquatic products treatment because of its broad-spectrum activity against bacteria.DAN can kill bacteria by blocking DNA replication via inhibiting the activity of gyrase in Gram-positive bacteria and topoisomerase IV in Gram-negative bacteria.However,the abuse of DAN can lead to allergy,headache,diarrhea,liver injury,and antimicrobial resistance,pose threats to human health.Therefore,the United States,the European Union and China have established strict limits of DAN in animal derived food.Tyramine signal amplification（TSA）is an enzyme-mediated signal amplification technology.The principle of TSA:in the presence of hydroxyl radical（·OH）produced by horseradish peroxidase（HRP）catalyzing H₂O₂,tyramine（TYR）can be transformed into a short-lived intermediate,and the intermediate can covalently bind to each other or adjacent protein residues including tryptophan,histidine,and tyrosine residues.TYR was prebinding labeled with fluorescent dyes or other signal materials,and signal amplification can be triggered by phenol polymerization reaction.In this study,TYR-Au NPs was formed by labeling TYR on the Au NPs,and the signal changes caused by Au NPs aggregation were used as the signal output for the highly sensitive visualization detection of DAN.In this work,TSA was introduced to lateral flow immunoassay（LFIA）for detection of DAN.HRP can be captured on the T or C line via immunochromatography through antigen-antibody specific reaction.After adding TYR-Au NPs and H₂O₂,the HRP catalyzed H₂O₂ to produce·OH.The·OH can convert TYR-Au NPs and tyrosine residue-HRP into radical intermediates.These radical intermediates can be conjugated via covalent bonds.Consequently,the Au NPs-TYR-HRP complex was deposited on the T or C line.When there was DAN in the sample to be tested,the binding of HRP on the T-line would be inhibited or completely blocked.Some key parameters including the concentration of DAN-BSA,the amount of monoclonal antibody（m Ab）,the volume of HRP-Ig G,the volume of TYR-Au NPs,and the concentration of H₂O₂ were optimized as 1 mg/m L,40 ng,3μL,40μL,and 0.1 M.By plotting the kinetics of the reaction,the optimal reaction time of Au NPs aggregation was 23 min.Under the optimal conditions,the standard curve of TYR-Au NPs LFIA was y=0.43 lg x+0.66（R²=0.995）with the limit of detection（LOD）at 0.032 ng/m L and the linear range between 0.25-5 ng/m L.The proposed LFIA could be used with the naked eyes to qualitatively detect DAN with a cut-off limit（COL）of 2.5 ng/m L.The LOD and COL of TYR-Au NPs LFIA was 7and 3-fold lower than that of the traditional Au NPs LFIA.The recovery rates of the chicken and pork samples spiked with DAN by the proposed TYR-Au NPs LFIA was86.04%–105.14%and 92.41%–110.19%,with the coefficient of variation（CV）of1.71%–2.05%and 4.42%–6.37%,respectively.In this work,TSA was introduced to surface plasmon resonance enzyme linked immunosorbent assay（p ELISA）to establish an ELISA detection method for DAN.The polymerization of TYR could be induced by·OH produced by HRP catalyzing H₂O₂,results in the aggregation of TYR-Au NPs,with the color of Au NPs turning red to blue.Ascorbic acid,which was produced by alkaline phosphatase catalyzing ascorbic acid-2-phosphate,can reduce the·OH,then prevent tyramine from polymerization and Au NPs from aggregation,with the color of Au NPs retaining red.When DAN was in the sample,the color of Au NPs solution changed from red to blue.Some key parameters including the concentrations of m Ab,ALP-Ig G,H₂O₂,and time of ALP catalyzing ascorbic acid-2-phosphate were optimized as 0.2μg/m L,1.0μg/m L,0.5 mmol/L and60 min.Under the optimal conditions,DAN was detected by the proposed p ELISA with the naked eyes at a cut-off limit of 0.4 ng/m L,which was 24-fold lower than that of the traditional ELISA（10 ng/m L）.Chicken and pork samples spiked with DAN has been detected,and the results exhibited good accuracy and stability of the proposed p ELISA.In summary,based on TSA,LFIA and p ELISA were established in this study for sensitively qualitative and quantitative detection,and could provide new perspectives for the detection of food hazard.