Study On Time-resolved Fluorescence Immunochromatography Based On Eu³⁺ Fluorescent Probe For Eugenol

Eugenol（2-methoxy-4-propenylphenol,Eugenol）is a biologically active compound with strong clove aroma,which has a strong anesthetic effect on fish and is widely used in fishery anesthesia with the advantages of wide source,low cost,good effect and short residual period.However,some studies have pointed out the risk of carcinogenicity and liver damage to humans.China has not yet issued clear regulations on the use of fish anesthetics eugenol,there is confusion and abuse of its use in the market,so the development of rapid,efficient and accurate detection method can provide technical support for the normal circulation and quality assurance of aquatic products.In this study,based on the immunological properties of eugenol,eugenol colloidal gold immunochromatography and time-resolved fluorescence immunochromatography test strips were developed by preparing eugenol antigen and its monoclonal antibody.The main research contents are as follows:（1）Synthesis and identification of eugenol artificial antigenIn this study,eugenol hapten was synthesized by nucleophilic substitution method,and the synthesized structure was identified by mass spectrometry and nuclear magnetic resonance.The hapten was coupled with carrier proteins BSA and OVA by carbodiimide method to synthesize Eugenol-COOH-BSA and Eugenol-COOH-OVA,which were identified by UV-scan spectroscopy and protein gel electrophoresis.（2）Preparation and identification of anti-eugenol monoclonal antibodyThe mice were immunized by Eugenol-COOH-BSA to obtain a highly sensitive and pure anti-eugenol monoclonal antibody at a concentration of 4.05 mg/m L,which was obtained by optimizing the working concentration of the antigen and antibody.The optimal antigen and antibody concentration were 2μg/m L and 0.63μg/m L,respectively.Finally,the competition inhibition standard curve equation of anti-eugenol monoclonal antibody is determined as:y=9.671lnx+10.539,R²=0.998,and IC₅₀ is 0.0592μg/m L,and there were almost no cross-reaction with eugenol structural analogs（methyl eugenol,methyl isoeugenol,eugenol acetate and acetyl isoeugenol）except isoeugenol with the cross-reaction value of 7.4%,indicating that the The prepared antibody has strong specificity.（3）Development of test strips for eugenol detectionFirstly,test strips were prepared with colloidal gold as the signal output carrier,and the colloidal gold and labeled probes were characterized by transmission electron microscopy and ultraviolet-visible spectrophotometer.The relevant parameters of the colloidal gold labeling p H,amount of labeling antibody,labeling antibody time,T-line and C-line buffer system and blocking solution,sample pad treatment solution,T-line and C-line coating concentration and colloidal gold were optimized.The amount of labeled antibody was preliminarily developed,and the colloidal gold immunochromatographic test strip of eugenol was initially developed.Secondly,the time-resolved fluorescent microspheres were used as markers to prepare test strips,and the optimal selection of different fluorescent microspheres and nitrocellulose membranes were compared,and the preparation process of the test strips was optimized,including the activation time of the microspheres,the p H of the labeling buffer,the amount of fluorescent microsphere-labeled antibody added,etc.,according to the final optimization results,the standard curve of the fluorescent test strip was prepared as y=-2.415lnx+2.5894,R²=0.994,and the elimination concentration was 1.0μg/m L.The limit of detection was 0.3μg/m L,and the detection limit of the instrument was 0.1μg/m L.The performance of the prepared fluorescent test strips was tested,and low,medium and high concentrations of eugenol were added to the transfer water samples.The recovery rate of the test strips was basically stable between 94%and 109%,and the intra-and inter-assay coefficients of variation were observed.All are less than 8%,showing good precision.The test strip results and HPLC results are linearly fitted to obtain the equation:y=-1390.1x+1767.9,R²=0.995,showing good accuracy;high temperature destructive Experiments show that the test strip can be stored for at least 1 year under normal temperature and sealed conditions,showing good stability.The results showed that the established colloidal gold immunochromatography method can be used for qualitative and semi-quantitative detection,while the time-resolved fluorescence immunochromatography method can meet the needs of on-site quantitative detection.The sample testing requirements have laid the foundation for the research and development of commercial eugenol rapid test strips.