基于“纳米晶-重组抗体”微阵列免疫芯片的中药材上农药多残留检测新方法研究

Long-term and numerous use of triazine herbicides on traditional Chinese medicines (TCMs) in the process of planting can easily cause multi-pesticide residues, affecting the quality and safety of them. Therefore, on-site detection of triazine herbicides with little time and low cost has been the urgent requirement of the TCM research fields and the supervision departments. Recombinant antibody has the ability of identifying the targets with good specificity and high affinity. Microarray immunochip technique labeled by nanocrystal has the advantage of simultaneously and sensitively detecting the targets in a small amount of sample. Based on the preparation of controlled nanocrystal, and triazine herbicides with high toxicity and long half-time in TCMs as the objects, we aimed to design and synthesize the selected hapten by using the computer-aided 3D molecular molecular modeling technology, and to screen the recombinant antibody for specially detecting triazine herbicides in TCMs through phage antibody library technology, then, together with microarray immunochip technique based on "nanocrystal-recombinant antibody" to establish a novel method suitable for the small-sized, portable microarray chip scanner for on-site high-throughput detection of triazine herbicides in TCMs. Through comparing the results of on-site analysis using the novel established method and indoor detection using UPLC-MS/MS system by statistical analysis, the accuracy and universality of the novel method for more TCMs would be confirmed. The study would provide data and technique supports for on-site rapid detection of multi-pesticide residues in TCMs. In summary, this project express important theoretical and practical significance for eliminating potential security risks of pesticide residues in TCMs, and for breaking the foreign trade barriers, further for increasing exports of more TCMs.

中药材种植过程中三嗪类除草剂的长期大量使用易导致多农药残留，高效、低成本的现场检测已成为中药安全研究领域和监管部门的迫切需求。借助重组抗体能特异识别待测目标，基于纳米晶标记的微阵列免疫芯片技术仅需少量样品即可实现多目标成分的实时、灵敏检测。本项目拟在前期合成可调控纳米晶的基础上，重点针对毒性大、半衰期长的三嗪类除草剂，采用计算机辅助3D分子模拟技术设计并合成群选性半抗原，通过噬菌体抗体库技术筛选适合中药材检测的重组抗体，配以微阵列免疫芯片技术，建立基于“纳米晶-重组抗体”的中药材三嗪类除草剂现场高通量检测的便携式微阵列芯片扫描仪分析新方法。同时佐以UPLC-MS/MS 的实验室检测结果，确证新方法在中药材上的准确性和普适性，为中药材农药多残留的现场检测提供数据和技术支持。该项目的实施对消除中药材农药残留的潜在安全隐患、突破国外贸易壁垒，增加我国中药材出口创汇，具有重要的理论价值和现实意义。

本课题重点针对三嗪类除草剂，通过系统和深入的研究，（1）应用UFLC-MS/MS建立了检测中药中三嗪类农药多残留的测定方法；（2）应用计算机模拟技术，设计制备了三嗪类农药半抗原结构，建立 QSAR 模型研究抗原抗体识别与分子结构之间的定量构效关系；（3）运用基因重组技术，构建了三嗪类农药单链抗体（ScFv）核苷酸序列，制备获得了三嗪类农药ScFv抗体。此外，针对莠去津、扑草净、多菌灵、毒死蜱四种应用广泛、易污染中药材的农药，制备了其单克隆抗体，并应用酶联免疫吸附测定法（ELISA）、胶体金免疫层析法、表面等离子共振技术（SPR）多种免疫检测技术和精密分析仪器LC-MS/MS、GC-MS/MS，构建了中药中农药残留的快速高通量分析体系，具体如下：（1）制备了莠去津、扑草净、多菌灵和毒死蜱四种农药的单克隆抗体；（2）以制备的四种农药单克隆抗体为基础，分别建立了中药中农残检测的ELISA方法；（3）基于制备的莠去津、扑草净单克隆抗体，建立了中药中莠去津、扑草净快速、现场、低成本的胶体金免疫层析法；（4）以制备的扑草净、多菌灵和毒死蜱单克隆抗体为基础，构建了SPR生物传感器用于检测中药中农药残留，所建方法具有灵敏度高、检测快速的优点；（5）基于LC-MS/MS和GC-MS/MS分析仪器和QuEChERS 样品前处理技术，构建了中药中400种农药多残留检测平台等。通过该项目的实施，共发表文章17篇，其中SCI文章12篇（影响因子大于5的5篇），中文文章5篇。培养和正在培养博士、硕士研究生8名。申报专利2项。荣获3等奖一项，研制团标2项（一项已发布，一项已立项）。项目完成人荣获“2018年海南省领军人才”称号。项目成果在相关学术会议以及中药行业推介会上进行了科普性介绍，且在国内外研讨会进行了研究成果的推广与介绍。上述研究成果为实现中药中农药残留现场、高通量、快速的筛查和确证奠定了良好的基础，对中药种植过程中防止农药残留及污染，保障中药材质量，突破国际贸易壁垒，具有重要的理论和实际应用的双重意义。