基于“纳米金-亲和素双重信号放大系统”的多元微流控SPR传感器在线检测中药中多农药残留

Based on previous research, the project aimed at multi-residual pesticides for different purposes containing carbendazim (fungicide), chlorpyrifos (insecticide), atrazine (herbicide) and dicofol (acaricide), which were very hazardous and heavily polluted in Traditional Chinese Medicines (TCMs). In the project, we will design and prepare a dual amplified surface plasmon resonance chip (SPR chip) combined the advantages of gold nanoparticles and biotin-avidin system, and adopt a cross-technology of multi-subject such as materials, analysis, optics as well as immunochemistry. The biosensor will be fabricated with a four-channel microfluidic platform. The research will carry out self-assembly of SPR chip, competitive immunoreaction, real-time SPR monitoring throughout the whole process, together with performance optimization on sensing platform. The established system will be applied in real TCMs screening and verified by LC-MS/MS and GC-MS/MS. The project was trying to improve the SPR intensity on the interaction of protein with small target and adapt to the sensitivity requirement for pesticides screening in real-time testing. It will contribute to the fast identification of pesticides problem in TCMs industry. The project will have an important theoretic value and practical significance in curtailing the excessive residue at the beginning, guaranteeing medical quality and safety, and breaking through numerous barriers to international trade.

本课题拟在前期研究基础上，重点针对杀菌剂多菌灵、杀虫剂毒死蜱、除草剂阿特拉津和杀螨剂三氯杀螨醇等毒性危害大，易污染中药的多种类型混合农药残留问题，联合纳米金和生物素-亲和素双重信号放大策略，采用材料、分析、光学和免疫化学等交叉学科的技术手段，设计合成等离子体共振免疫芯片（SPR chip），构建能同时多检测的四通道微流控传感平台。研究将开展自组装SPR芯片、免疫竞争反应、全流程实时SPR信号监测及生物传感平台性能优化等研究，同时应用于大宗中药样品在线监测，高通量液质气质技术对比验证SPR传感器实际应用中的准确性。本课题的实施旨在通过改善蛋白-小分子作用时的信号强度，以适应农残在线快速筛查中的灵敏度需求，为实现中药产业中农药残留问题的灵敏快速鉴别，从源头遏制农残超标，保障中药质量和安全，突破国外贸易壁垒，具有重要的理论价值和现实意义。

本课题聚焦高污染高残留农药，针对中药农残控制不同应用场景需求，（1）建立了同时多种高污染农药联合检测的多元SPR芯片体系，实时在线同步定量多菌灵、毒死蜱和阿特拉津残留，其该自组装SPR芯片可重复利用，有助于节约快检成本；（2）富含淀粉、高多糖基质、色素的中药在SPR芯片体系中存在粘度、堵塞、影响灵敏度等难题，研究利用磁性微球悬浮阵列法均相检测优势，建立了超灵敏阿特拉津和毒死蜱悬浮阵列检测方法，检测限低至ppb级，并在常用山药、人参、三七药材中准确检测出毒死蜱和阿特拉津残留；（3）为满足普遍药材源头农残污染检测的问题，项目进一步研制了便携式多菌灵胶体金免疫层析快检产品，在包含根、茎、叶、花、果实、种子等不同药用部位合计56种药材中进行适用考察，含人参、三七、金银花等常用80%以上药材均适用，该快检产品经第三方验证性能优异；（4）除快速检测技术外，课题以柴胡等大宗药材入手，优化前处理方法和色谱质谱条件，建立了中药材中GC-MS/MS法高通量残留农药检测方法，并对大量药材的残留水平进行分析。通过该课题的实施，共发表论文14篇，其中SCI 10篇，中文4篇；申报专利1项；研制团体标准“T/CATCM 010-2019植物性中药材及饮片多菌灵的快速检测-胶体金免疫层析法”1项，项目研究成果在中药应用基层进行示范推广。上述研究成果为解决中药质量安全中关键的农药残留问题提供了技术支撑，也促进科研人员如何在理论基础研究中深入思考对中药农残中卡脖子难题分层解决，促进基础技术研发实现成果转化，助力中药质量安全控制。