基于工程化适配体的水中抗生素快速定量倏逝波传感检测技术

Remarkable water safety issues and environmental pollutions caused by antibiotic residues in water have attracted increasing social attention. There is an urgent need to develop novel monitoring theories and relative sensing technologies for ultrasensitive detection of antibiotic residues in water bodies, which possess prospective and strategic significance. This project aims at filling the technological gaps in aptamer-based optical sensors for rapid detection of antibiotic residues in water bodies. Rational truncation and split of antibiotic-specific aptamers are investigated, which lay the foundation to reveal the universal principles and general approaches towards the engineering modification of antibiotic-specific aptamers. Using the split aptamer as antibiotic-specific report molecule, a novel "nucleic acid-protein" conjugate-based sandwich-type evanescent wave sensing technology is proposed. The advantages of such an evanescent wave-based sensing system include ultrasensitivity and rapid detection. Both reaction conditions and detection performances of the proposed sensors are optimized by studying the surface chemical modification and regeneration strategies of the evanescent wave chip, as well as the interfacial self-assembly behaviors and response kinetics of split aptamers. Moreover, by investigating the influence of several typical environmental matrices on the biosensing effects, an anti-environmental matrix effects biosensing strategy is proposed. Ultimately, the rapid, sensitive and quantitative analysis against biological contamination is achieved. The anticipated results can enrich the aptamer engineering theories, expand the existing engineering aptamer library, and provide new technologies in the need for major national environmental issues involving the environmental behavior, risks, and early warning of antibiotic residues.

我国环境水体抗生素污染形势不容忽视，及早开展水中抗生素类新兴痕量污染物检测技术和方法研究具有前瞻性和战略意义。本课题针对目前抗生素残留尚缺乏核酸适配体光学传感器快速检测技术这一现状开展基础研究。研究抗生素特异性适配体的工程化截短和裂分，揭示适配体工程化改造原理与共性准则；以裂分适配体为抗生素特异性报告分子，结合倏逝波传感系统超灵敏快速等优势，创新性提出一种基于“核酸-蛋白质”缀合物的夹心法倏逝波核酸传感分析技术；研究倏逝波芯片表面化学修饰及稳定再生方法，裂分适配体界面自组装行为、反应动力学和调控方法，并优化反应条件及检测性能；研究典型水环境要素对生物传感分析过程的影响机制，提出抗基质效应方法；最终实现对抗生物污染物的快速高灵敏定量分析。预期成果能够丰富适配体工程化改造技术理论，充实现有工程化适配体材料库，为研究抗生素环境行为和风险以及抗生素残留监测预警等国家重大需求问题提供崭新的技术手段。

本课题针对目前抗生素残留尚缺乏核酸适配体光学传感器快速检测技术这一现状开展基础研究，取得的主要成果如下：1）首次打破“维持母适配体靶标结合口袋完整性”的传统假设，提出在适配体功能环结构中部截断的新型裂分方法。新获得四环素、妥布霉素与新霉素三种裂分适配体，解离常数分别为101.5±0.7nM、52.3±10.3μM与414.2±8.5μM；分子动力学模拟与实验结果表明四环素裂分适配体体系中有新结合口袋形成，上述工作扩充了工程化核酸材料库，为下游传感应用奠定基础；2）基于碱基互补配对原理构建单链核酸传感界面，首次开发等电点牛血清蛋白界面封闭剂，将单链核酸传感界面检测互补序列信噪比由1.6提升至14.1，同时界面稳定再生次数可达百次，有力促进了可再生倏逝波核酸传感技术的实用化；3）集成抗生素高灵敏检测的倏逝波荧光生物传感器，建立了以功能核酸为报告分子的抗生素高灵敏微量污染物分析方法，实现了对链霉素微量抗生素的特异性可再生检测，检出限达到33nM (19µg/L)，线性范围为60~526nM。此外，基于新型裂分适配体设计酶联吸附检测方案，对妥布霉素裸眼检出限达1μM (0.47mg/L)。本项目培养博士生一名、硕士生一名、博士后一名，在国际环境及传感技术知名期刊发表SCI论文10篇（含Analytical Chemistry封面文章1篇），平均影响因子6.52。申请人以第一发明人申报发明专利5项，授权1项。依托本项目，申请人获批1项NSFC-RS（中英）国际合作与交流项目（项目号：21677082）。项目在研期间，组织承办三届水监测相关国际研讨会（2017年，2018年，2019年）。任务负责人及课题骨干以邀请报告形式展示任务成果5次，显著提升了团队在本领域的国际影响力。