农兽药残留多元快速检测的新型适配体上转换发光生物传感技术研究

Environmental pollution and food safety have become focuses of global attention, Specially the pesticides and veterinary residues is a very big issue in China now.They not only are harmful to people's health, but also imped the economy development which are severe and prominent in our country. How to expeditiously measure these pesticides and veterinary residues in environment and food are urgently needed to be solved. The existing methods, however, cannot meet thes requirement. In the light of the imperative demands, a new type of detector for pesticides and veterinary residues measurement would be designed and developed.In this study, Some typical pesticides and veterinary residues, such as organic phosphorus, estradiol and diethylstilbestrol are chosen as targets, and ready to establish a multiplex and rapid detection for pesticide and veterinary drug residues by novel upconverting phosphor biosensing technology based on aptermer modification. Firstly ,Nucleotide probe sequence designed by computer-aided molecular simulation, then this study intends to carry out discrepant and enrichment screening by systematic evolution of ligands by exponential enrichment technology to prepare high-affinity aptamers of organic phosphorus, estradiol and diethylstilbestrol. The aptamers are aminationlized and coupled with organic linking arms. Hydrothermal method is employed for the synthesis of the upconverting phosphor particles (UCP) with the functional groups being coupled and activated. And then, the aptamer-modified UCP is synthesized by mild carbodiimide method. We developed a aptamer-modified UCP multiplex detection disk for the establishment of novel upconverting phosphor biosensing technological platform The series of novel detecting technologies of environmental pollution and food safety risk monitoring will be established, and the sensitive, precise, trace and quantitative portable detection methods of the multiple pesticide and veterinary drug residues is achieved. It can be applied in the fields of environmental pollution and risk of food safety assessment and control, criminal inspection and entry-exit inspection and quarantine, etc. with theoretical significances and practical prospects, and to promote the development for food safety detection technologies.

环境污染与食品安全已成为全球关注的焦点，其中环境与食品中农兽药残留尤为突出，不仅严重危害人们的身体健康，而且阻碍经济发展，已成为我国严峻和突出的问题。本项目针对我国环境与食品中农兽药残留快速检测方法不足的问题，以久效磷、雌二醇和己烯雌酚等典型农兽药为代表，根据靶标的结构特点，通过计算机分子模拟设计不同的核酸探针，采用指数扩增配体的系统进化技术差异和富集筛选，制备有机磷、雌二醇和己烯雌酚的高亲和力核酸适配体并进行氨基化和偶联有机连接臂；建立制备上转换发光（UCP）纳米颗粒技术方法和功能基团的偶联活化；制备适配体修饰的UCP颗粒，构建基于核酸适配体修饰的新型上转发光多通道检测的新型生物传感技术平台，实现对多种农兽残留的高通量痕量定量检测。该项目研究成果可用于环境监测、食品安全风险监控、刑事侦检与出入境检验检疫等领域，具有很好的理论价值和应用前景。

环境污染物（如：农药残留、环境内分泌干扰物等）是危害人体健康的重要因素之一，本项目以几种典型农兽药污染物（雌二醇、双酚A、己烯雌酚、多氯联苯）为靶标，根据靶标的结构特点，通过计算机分子模拟设计、选择相应的核酸序列，制备了雌二醇双酚A、己烯雌酚和多氯联苯的高亲和力核酸适配体；制备了980nm和808 nm激发的上转换纳米颗粒，并对其合成条件进行探索优化，在其表面进行多种功能修饰偶联了不同的靶标适配体作为生物检测探针。通过探索上转换探针的合成和修饰方法，与金纳米颗粒结合制备了AuNP-AuNP-UCNP三联体结构，并利用荧光共振能量转移技术实现了对双酚A和雌二醇的快速同时检测；探索了免疫层析试纸条结合上转换纳米材料用于高灵敏快速检测的可行性，实现了对雌二醇、己烯雌酚、四环素等5种农兽药污染物的快速定量检测；采用一步法在UCNPs表面修饰烯基并在表面接枝分子印迹法合成UCNP表面上的印迹层，印迹层可以选择性地和敏感地识别目标，实现了对己烯雌酚的免标记定量检测；并且，提出了一种用于检测PCB72 / 106的新型双扩增策略，第一次将HCR，UCNPs和切口内切核酸酶结合起来进行检测，依靠双重信号放大策略，实现了对多氯联苯的超灵敏检测；最后，将上转换颗粒结合多巴胺自聚合的黑磷纳米片表面，设计了竞争免疫荧光猝灭传感器，实现了对己烯雌酚的快速定量检测；进一步构建了新型黑磷-金复合纳米猝灭基质材料，目标物适配体的互补链设计的正四面结构捕获探针自组装于基质表面，实现了双重检测信号放大，结合808 nm激发的双色上转换颗粒实现了对双酚A和雌二醇的同时快速定量检测。. 在本项目支持下，发表文章13篇，其中SCI论文7篇，中文核心期刊论文2篇，在投的SCI文章4篇，主编专著1部；申请国家发明专利4项，部分技术参与科技奖申报，获得国家科技进步二等奖1项、中国分析测试协会科技进步一等奖1项；培养硕士生4名，博士生3名；参加国际学术交流会议4次。