基于核酸编码及数字滚环扩增的真菌毒素小分子高通量计数分析

Mycotoxin, widely found in natural foods, is a kind of low-molecular-mass secondary metabolites produced by fungi, some of which present carcinogenic, teratogenic and mutagenic effects. Due to the strictness of limited standards of the state, the diversity of mycotoxin and the complexity of food sample matrix, there is a high demand for building mycotoxin analysis method possessing high precision, sensitivity, high-throughput and robustness. To address these challenges, this project will utilize recognition-enhanced aptamer probe, combining with the nucleic acid amplification technology fit for highly multiplex single-molecule detection, to realize high-throughput digital counting of mycotoxin molecules in complex samples. Firstly, new strategies by the design of thermodynamics, kinetics of nucleic acid hybridization and the utilization of nucleic acid photochemical reaction, were proposed to improve the binding efficiency of aptamer, enabling highly efficient recognition of target low-affinity small molecule. Then, the visualization and digital detection of molecular recognition events are realized by adopting the stoichiometric nucleic acid amplification technology. Finally, sequence-encoded fluorescence tags based on the programmability of the nucleic acid sequence were built to achieve highly multiplex detection of target molecules, enabling high-throughput digital quantification of mycotoxin molecules. These strategies would confer sufficient accuracy and throughput for mycotoxin analysis, and provide analysis platforms for the prevention and control of food contamination.

真菌毒素是由真菌产生的一类次级代谢小分子，部分种类具有致癌、致畸和致突变效应，并广泛存在于天然食物中。由于国家限量标准的严格性、真菌毒素种类的多样性和食物样品基质的复杂性，目前亟需发展精准定量、高灵敏、高通量及抗干扰的真菌毒素分析方法。本项目拟采用识别增强设计的核酸适配体探针，结合单分子高通量的核酸扩增技术，实现对复杂样品内真菌毒素的高通量计数分析。首先，提出增强适配体识别效率的新策略，通过核酸杂交热力学、动力学及核酸光化学反应设计，构建适配体探针，实现对弱结合力的小分子的高效识别；然后结合化学计量比型的滚环扩增技术，实现分子识别事件的可视化、数字化检测；最后利用可编程的核酸序列，设计核酸序列编码的荧光标记方法，实现对目标分子高通量的检测，从而构建复杂样品内真菌毒素高通量计数分析方法，以有效解决真菌毒素分析目前面临的定量准确性和通量方面的问题，为食品污染物的防控提供有效的技术平台。

本项目针对真菌毒素分析的需求和核酸适配体应用的关键问题，探索发展提高核酸适配体识别效率的策略，开发复杂食品样品中真菌毒素的精准定量方法，主要成绩有：1）构建了核酸适配体固有构象诱导的真菌毒素均相快速检测方法；2）提出了核酸适配体识别增强策略，提升对低亲和力靶标分子的变构响应，实现了包括真菌毒素在内的小分子计数分析；3）构建了结构分辨的核酸适配体纳米探针，实现了复杂活细胞环境中真菌毒素分子的高信噪比成像，构建了单细胞水平真菌毒素动态示踪方法。在本基金的资助下，项目负责人以第一/通讯作者在包括Anal. Chem., ACS Sensors和Chem. Commun.等期刊发表论文8篇，获得国家授权发明专利5项。该项目可为食品真菌毒素污染速测和毒理分析提供新思路和解决方案。