大位阻农药三唑锡ScFv识别机制及其纳米生物传感器的研究

Immunoassay and effective detection for small molecule sterically hindered pollutants have become an important topic of scientific research in food safety. Sterically hindered Azocyclotin was selected as the object in this project. The hapten towards Azocyclotin was designed by molecular simulation technology and conjugated with hapten-carrier to synthesize artificial antigen. Balb/c mice were immunized to obtain monoclonal antibody which specificity and affinity were comparative analyzed comparatively. The single chain variable fragment antibody (ScFv) was prepared by constructing antibody library by the phage display technology. Directed evolution of ScFv was realized by the method of errorprone PCR. Selective variation of ScFv was investigated while recognition mechanism of Azocyclotin hapten and ScFv antibody was clarified by the methods of homology modeling, dynamic simulation and molecular docking. ScFv substrate with controllable and sensitive performance was prepared by surface modification and nanotechnology. Fast detection of Azocyclotin in food with high-sensitivity and specificity was realized by the PAMAM-MWCNTs immune sensor. This project provides valuable information about preparing ScFv of small molecule sterically hindered pollutants and understanding its immune recognition. The project also has an important theoretical and practical significance for clarifying structure-activity relationship of hapten-antibody and preparation of highly sensitive and selective immunoassay instrument.

对食品中大位阻小分子污染物进行免疫分析和有效检测是食品安全的重要研究课题。本项目以大位阻农药三唑锡为研究对象，利用分子模拟技术设计半抗原，将其与载体偶联制备人工抗原后免疫动物，获取单克隆抗体，比较分析抗体的特异性、亲和性，通过噬菌体表面展示技术构建抗体库制备单链抗体（ScFv），采用错配 PCR 方法对 ScFv 定向进化，结合同源模建、动力学模拟和分子对接手段，分析 ScFv 选择性变化规律，阐释三唑锡半抗原结构与抗体识别的机制。通过表面修饰技术和纳米技术，制备性能可控的纳米增效ScFv 抗体敏感膜基片，设计、构建三唑锡检测PAMAM-MWCNTs免疫传感器，实现对食品中三唑锡残留的高灵敏度、高特异性快速检测。本项目的开展将为大位阻小分子污染物ScFv 制备及阐明其免疫分子识别机制提供理论支撑，对深入理解半抗原-抗体构效关系，制备高灵敏、高选择性免疫分析仪器具有重要的理论和实践探索意义。

对食品中大位阻小分子污染物进行免疫分析和有效检测是食品安全的重要研究课题。本项目以大位阻农药三唑锡为研究对象，成功合成并鉴定了三唑锡的半抗原及人工抗原，免疫BALB/c小鼠、细胞融合后得到1株稳定分泌三唑锡抗单克隆抗体的杂交瘤细胞，命名为 6D2。并以此作为反应核心技术制备了三唑锡传感器并对反应条件进行了优化，确定了0.75%作为免疫传感器中Nafion的最佳浓度；1:400为nano-Co3O4/Pan溶液最佳稀释比；1:1600作为人工偶联抗原的固定浓度稀释比；1:400作为三唑锡抗体的最佳稀释比；pH=4的PBS（1/15 mol/L）作为基体溶液。毒死蜱传感器反应条件的优化，确定了1.25%作为免疫传感器中Nafion的最佳浓度；1:800为最佳稀释比；1:1600作为人工偶联抗原的固定浓度稀释比；1:1000作为毒死蜱抗体的最佳稀释比；pH=4的PBS（1/15 mol/L）作为基体溶液。将传感器与高效液相色谱方法进行了比较，传感器方法的检出限、重现性、稳定性、回收率和高效液相色谱方法相比，无显著差异，且具有方便快捷、适于现场检测等优点，比高效液相色谱方法的应用更广。