基于“流式微球-适体芯片”的中药真菌毒素现场高通量“掌上检测”体系构建

Traditional Chinese medicines (TCMs) are prone to contamination by multi-mycotoxins in the process of storage and circulation, affecting the quality and safety of them, so, on-site detection of mycotoxins with low time, effect and cost has been the focused issue of the TCM research fields and the supervision departments. Aptamer can identify and adsorb the targets with good specificity and high affinity. Cytometric bead assay (CBA) technique has the advantage of simultaneously and quickly detecting the targets in a small amount of sample. Based on the established method for screening aptamer, and the mycotoxins with high toxicity and incidence in TCMs as the objects, we aimed to firstly screen aptamers for specially detecting mycotoxins in TCMs using the modified capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) technique, then, together with CBA technique to establish the novel "cytometric bead-aptamer chip" composite system being applied for the small-sized, portable flow cytometer for on-site high-throughput detection of multi-mycotoxins in TCMs. Through comparing the results of on-site analysis using the novel established system and indoor detection using UPLC-MS/MS by statistical analysis, the accuracy and universality of the novel system for more TCMs would be confirmed. Our study would provide references and supports for on-site "handheld detection" of mycotoxins in TCMs. In summary, this project has important theoretical and practical significance for eliminating potential security risks of mycotoxins in TCMs, and for breaking foreign trade barriers, further for increasing exports of more TCMs.

中药材在储藏和流通过程中易污染多种真菌毒素，节时、省力、低成本的现场快速检测已成为中药安全研究领域和监管部门密切关注的议题。借助适体(Aptamer)能实现对待测目标的特异识别，流式微球技术具有仅需少量样品即可实现多种待测目标实时、快速检测的优势。本项目拟在前期建立的适体筛选评价技术基础上，重点针对毒性大、易污染中药材的真菌毒素，首先采用改良的毛细管电泳-指数富集的配基系统进化技术(CE-SELEX)筛选适合中药检测的适体，配以流式微球技术，构建基于"流式微球-适体芯片"的中药真菌毒素现场、高通量快速检测的便携式流式细胞仪分析体系。同时佐以UPLC-MS/MS的实验室检测结果，确证新体系在中药材上的准确性和普适性，为实现中药真菌毒素现场"掌上检测"提供科学依据和技术支持。该项目的实施对消除中药真菌毒素的潜在安全隐患、突破国外贸易壁垒、增加我国中药材的出口创汇，具有重要的理论价值和现实意义。

近年来，真菌毒素污染已成为影响中药材质量和安全的重要因素之一，在世界范围内引起广泛关注，建立高效灵敏的痕量真菌毒素污染水平的检测手段已成为中药材安全性监控的迫切需求。常规的液相色谱-荧光检测法和液质联用技术因样品前处理复杂、成本较高、仪器笨重和操作繁琐，很难满足中药材现场大批量样品快速监测的市场需求。酶联免疫吸附法(ELISA)因稳定性和灵敏度较差，假阳性率偏高，实际应用中较难普及。为此，本项目拟建立基于“流式微（磁）球-免疫悬浮芯片”的便携式流式细胞仪分析体系，用于中药真菌毒素污染水平和质量监控的现场高通量快速检测，结合UPLC-MS/MS的实验室检测确证新体系的准确性，为实现中药中真菌毒素的现场检测提供数据支撑和技术支持。.本项目主要从以下三个方面展开。（1）建立了基于间接竞争原理的流式微球技术快速检测中药（以麦芽为例）中赭曲霉毒素A（OTA）的残留水平，其灵敏度（检测限IC10）为0.12 ng/mL，并用于16批麦芽样品中OTA的定量检测，并经UPLC-MS/MS确证，保证该方法的准确、可靠；（2）基于磁性微球易分离富集、操作快速简便的优势，建立新型、绿色环保的基于“流式磁球-免疫探针”的中药中OTA灵敏快速检测方法，利用单克隆抗体和包被抗原的间接竞争原理，实现了在复杂样品基质中OTA的超灵敏准确检测，该方法对麦芽中OTA的检测限（IC10）为25 ng/kg；（3）为最终实现高通量的POC（point-of-care）检测目标，建立了流式磁球悬浮芯片的微阵列检测技术，并用于莲子中黄曲霉毒素B1（AFB1）和薏苡仁中玉米赤霉烯酮（ZEN）的高通量快速检测，该技术对基质中AFB1和ZEN体现了较高的灵敏度，检测限（IC10）分别为7.8125 pg/kg和15.35 ng/mL，借助传统方法比对和单细胞成像仪表征，仅需50 μL样品溶液即可在30 s内完成100个样品的同时分析。.本研究较好地满足了大批量中药材中多种真菌毒素的现场超灵敏检测需求，为复杂基质中痕量目标物的准确、超灵敏检测提供了新思路，也为中药材安全监管提供技术支持，以保证药材质量和安全，以期推动中药材产业化的快速发展。