食品中小分子化学污染物新型肽配体筛选策略及非竞争检测模式研究

In the field of rapid detection small chemical contaminants in food, the current research is focused on developing affinity ligand assisted biodetection, biosensor or biochip methods. The bottleneck for developing such methods include the tedious work for molecular modification and immobilization in affinity ligand preparation and the inherent difficulties related to singleness epitope of small chemicals for develop non-competitive assay model. Inspired by the capsulated theory of cyclodextrin to organic small molecules and technology of heavy metal immunoassay, and combined with our previous foundation work of phage display technology, the current proposal is designed to overcome the above bottlenecks. Chlorpyrifos and chloramphenicol were selected as typical targets for developing novel strategy that without the need of molecular modification and immobilization for peptide ligand screening and developing non-competitive detection model for small chemicals. For detail, the binding features of cyclodextrin to chlorpyrifos and chloramphenicol will be firstly investigated by molecular simulation and spectroscopy method and the optimized binding condtions were screened for further research. Then, cyclodextrin is linked to carrier protein and utilized for indirect immobilizing the small chemicals. After this, phage display technology is applied for special peptides preparation, i.e. the prepared peptide ligands can specially recognize the small chemical-cyclodextrin complex, but not recognize the small chemical and cyclodextrin solely. At last, non-competitive detection model will be developed by utilizing these active materials. Novel approach and new theory for small chemical contaminants rapid detection would be explored by carry out these works, and technological and theoretical foundation would be provided for optimizing the food safety detection system.

基于亲和配体构建生物检测及生物传感器或生物芯片等技术是小分子化学污染物快速检测领域当前研究与探索的重点。亲和配体制备需繁琐的分子改造和固定等环节，小分子表位单一难以建立非竞争检测模式，是此类技术发展的两大瓶颈。本项目在环糊精包合有机小分子的相关理论和重金属免疫分析技术的启发下，结合前期噬菌体展示技术研究的工作基础，拟以毒死蜱和氯霉素为典型对象探索免除小分子改造和固定的肽配体筛选策略，并建立基于肽配体的小分子非竞争检测模式。项目首先利用分子模拟和光谱学方法研究小分子与环糊精的结合特性并确定最佳包合条件；然后借助环糊精的桥梁作用间接固定小分子，并利用噬菌体展示技术筛选针对“环糊精-小分子”复合物的亲和肽配体；最后再探讨利用所获肽配体构建小分子化学污染物非竞争检测模式。通过本项目的开展，将探索出小分子化学污染物生物检测的新途径和新理论，为食品安全检测体系的完善提供技术依据和理论参考。

项目针对食品中小分子化学污染物亲和肽配体制备难，难以构建非竞争检测模式等技术瓶颈，设计利用环糊精对疏水性小分子的包合特性，构建一种可用于食品中小分子化学污染物非竞争检测的肽配体筛选策略并进一步构建相应检测模式。项目以毒死蜱和氯霉素作为模式小分子，首先利用分子模拟和光谱学方法确证了β-环糊精对其二者的包效应；然后将β-环糊精与牛血清蛋白进行偶联，并证实偶联状态的环糊精分子仍能包合毒死蜱和氯霉素。在此基础上，以β-环糊精-牛血清蛋白偶联产物为核心材料，从商品化的噬菌体展示线性十二肽库中筛选获得了5 个可以特异识别环糊精-毒死蜱包合物的肽配体，8个可以特异识别环糊精-氯霉素包合物的肽配体。最后，利用亲和力最强的肽配体（8#克隆）构建毒死蜱的非竞争检测模式，该检测模式能够特异性地识别毒死蜱，而对丙溴磷、除线磷和溴硫磷等结构类似农药分子无识别；但在利用肽配体构建氯霉素的非竞争检测模式时检测效果不佳，其原因可能是化学合成的肽配体与噬菌体展示状态下的肽配体具有不同的构想。本项目所构建的检测模式无需对小分子物质进行改造和固定，可为小分子化学污染物的快速检测提供新的参考。