基于锁核酸适体的水中抗生素的便携检测研究

In recent years, the water environment and drinking water pollution accidents caused by antibiotics occurred frequently. Although conventional methods for detection of antibiotics can provide accurate information about environmental samples, they require the use of expensive instruments, careful calibration and a relatively long time to obtain results, making it difficult for on-site and real-time monitoring. Therefore, it is of critical importance to develop real-time and portable detection methods to ensure public health. Portable detection methods for antibiotics in water environment and drinking water based on locked aptamer will be developed in this project. Natural aptamer usually has some limitations, such as the week binding caused by restriction of functional groups, the low stability, et al. The introduction of locked nucleic acids during the in vitro selection process help us to obtain locked aptamer for antibiotics with high affinity, high specificity and high stability. Afterwards, truncation of the selected locked aptamers will be conducted to get much shorter aptamer, which will make sensing application easier. Conformational and structural characterization of the selected aptamer will also be studied to provide fundamental recognition for aptasensing. Finally, portable detection methods for antibiotics will be developed based on locked aptamer and a personal glucose meter. Effects of different testing conditions and environmental background matric on the sensing performance will also be studied to help establish the standard detection methods based on our developed technology.

近年来，水环境及饮用水中抗生素污染事故爆发频繁。传统的抗生素检测方法不仅需使用昂贵的大型仪器，而且检测费时费力，难以实现实时便携检测。因此，开发抗生素的实时便携检测技术对保障公众健康至关重要。本项目拟开发基于锁核酸适体的抗生素便携检测技术，以满足我国水环境及饮用水中抗生素的现场、快速、超灵敏、高特异性等多种监测和预警应用需求。针对天然核酸适体识别靶标有限、亲和力低、稳定性差等问题，通过筛选过程中锁核苷酸的引入，进行锁核酸适体的筛选，以期获得高亲和力、高特异性、高稳定性的抗生素锁核酸适体。而后，对上述抗生素锁核酸适体进行合理截短和结构解析，以获得更利于传感应用的短序列锁核酸适体，并为锁核酸适体传感技术提供理论基础。最后，开发基于抗生素锁核酸适体和血糖仪的便携检测技术，考察不同测试条件及不同环境基质因素对测试性能的影响，建立用所开发的便携检测技术进行实际水样中抗生素浓度的标准测试方法。

本课题针对我国日益严峻的抗生素污染以及目前缺乏对抗生素进行实时便携分析方法的问题，通过集成化学生物学、物理化学、材料学、环境科学等学科的先进技术和理论，开发基于核酸适配体的水中抗生素的便携检测技术。首先，用结构转换指数富集型系统进化技术进行了抗生素核酸适配体的筛选，发现该方法存在中间序列易发生碱基突变或缺失从而导致筛选无法进行的问题。其次，对现有的抗生素核酸适配体（卡那霉素核酸适配体）进行结构性能表征，利用圆二色谱、荧光染料染色和传感器验证等手段来解读卡那霉素核酸适配体的结构，及其在溶液中的存在方式，发现卡那霉素核酸适配体在溶液中会发生聚集，卡那霉素能够裂解核酸适配体之间形成的聚体结构。在此基础上，开发了一种基于靶标结合诱导荧光猝灭的倏逝波核酸适配体生物传感器，在液相中完成反应后，可在不需信号分离步骤前提下实现在传感器上对卡那霉素的定量检测，检测区间为 0.2-200 μM，检测限为 26 nM，并可应用于地表水中卡那霉素的检测。该传感器首次实现了对卡那霉素氨基糖苷类结构类似物的定量检测，其他种类抗生素对检测结果无干扰，所构建传感器在中性再生液中（pH 为7的0.5% SDS）即可完成多次再生，信号保持稳定。最后，通过等温热滴定验证了可卡因核酸适配体与奎宁的亲和力，并在此基础上开发了一种基于核酸适配体和血糖仪的水中奎宁含量的检测方法，其对缓冲液中奎宁的检测限为 0.14 μM，对 80%（V/V）再生水出水水样中奎宁的检测限为0.32 μM。