基于“freeze-thaw”诱导的新型无试剂化纳米粒子负载适体探针高效快速检测中药中真菌毒素污染研究

Mycotoxins in traditional herbal medicine are serious threats to the health of human, and if screening method that features good sensitivity, rapidness and high capability of anti-interference is developed, it will be of great significance for ensuring drug safety. The reported rapid detection technologies based on the antigen-antibody reaction, are limited by some factors such as characteristics of raw materials, chemical conditions, labeling technique, which leading to difficulties to break through bottleneck caused by complex matrices of traditional herbal medicine. Biosensoring systems based on nanoparticles and aptamer is an attractive strategy for detecting trace chemical hazards, due to its high sensitivity and specificity. However, complex and time-consuming construction has limited its application in real sample analysis. This work will be performed on the basis of the previous exploratory studies, and the mycotoxins which is easy to be contaminated in traditional herbal medicine and with high toxicity such as AFB1 and OTA will be as the subjects. The conception and design involves that the novel landmark “freeze-thaw” directed labeling strategy acts as a key role for faster preparation of nanoprobes and the subsequent construction of nanoflare, which focuses on overcoming the rate-limiting step in the construction of the existing system; in addition, this labeling strategy is reagentless which indicates the concept of green analytical chemistry; and then fluorescence resonance energy transfer between the manageable nanoparticles will be used to improve sensitivity and specificity, and finally tackling the problem of matrix interference from traditional herbal medicine. The current study will aim to create a breakthrough for the application of the aptamer based technique for screening of mycotoxin in traditional herbal medicine, and provide powerful technical support for monitoring and early-warning of the threat caused by mycotoxins.

真菌毒素是威胁中药安全性的严重隐患，建立灵敏、快速、强抗基质干扰的筛查体系对保障临床用药安全具有重要意义。目前，已报道的免疫快检技术受原料特性、检测化学环境、标记技术等制约，难以突破中药复杂基质干扰难题。基于纳米材料和适配体的传感技术灵敏度高、专属性好，在复杂体系痕量分析领域中有良好发展前景，但繁琐、耗时的检测体系构建极大限制了其实际应用。本项目拟在前期开展的探索性研究基础上，以中药易污染、高毒性的AFB1和OTA为模式研究对象，创新性地提出采用基于“freeze-thaw”诱导的无试剂化里程碑式新型快速标记策略，简化适体功能化纳米探针及纳米耀斑构建程序，解决传统检测的限速瓶颈，过程符合绿色分析化学理念；利用可调控纳米材料的荧光共振能量转移，提高检测灵敏度与专属性，攻克中药复杂基质干扰难题，预期为适配体在中药真菌毒素筛查中的应用提供突破口，为中药中真菌毒素危害监测与预警提供有力技术支撑。

真菌毒素在自然界中分布广泛，毒性较强，严重危害人体健康。中药材作为一类特殊的农产品，在种植、采收、加工和流通等环节均可能由于操作不当而污染真菌毒素，从而影响中药的用药安全性，因此中药中真菌毒素快速检测方法的研究对真菌毒素污染的监测和防控具有重要意义。本研究聚焦中药中常见真菌毒素的简便、高效、快速检测方法的研究：（1）适配体是真菌毒素快速检测中常用的识别元件，其常与纳米金、量子点等纳米材料相结合，并通过比色、荧光等检测模式进行检测，但适配体功能化纳米材料的制备往往涉及极其复杂的标记方法，耗时且不利于重复。冻融（freeze-thaw）技术无需任何试剂辅助即可实现纳米金与单链核苷酸分子的快速偶联，是一种绿色、简便的偶联方法。本项目主要研究了基于freeze-thaw技术构建功能化纳米探针的方法，并利用纳米金与量子点之间的荧光共振能量转移（FRET），建立真菌毒素的均相快速检测方法。（2）本项目还针对中药真菌毒素快速检测中常常遇到的中药复杂基质干扰难题，研究了简单的样品前处理方法，以提高检测准确度。（3）以纳米金为载体，构建双功能探针，以提高检测灵敏度，并成功将直接竞争模式应用于中药材样品检测中。本研究所建立的检测方法，可为中药中真菌毒素的高效、高通量、快速检测提供新途径。