脱氧雪腐镰刀菌烯醇和T-2毒素纸基微流控芯片检方法研究

Abstract: Trichothecenes are one of the most serious toxic mycotoxin, causing significant safety hazards to both human and animal husbandry. Deoxynivalenol (DON) and T-2 toxin are two major mycotoxins belong to the wide family of trichothecenes due to their frequent occurrence in toxicologically relevant concentrations worldwide. Nowadays, the use of ELISA and colloidal gold-based immunochromatographic assay as main analytical tools for the detection of DON and T-2 toxin. However, the colloidal gold-based immunochromatographic assay is difficult to meet the requirement of high-thoughput detection and the ELISA exits shortcomings, such as inconvenience for on-site test. Therefore, in the project proposed here, the microfluidic paper-based analytical devices (μPAD) is used as the tool to breakthough the bottleneck. In this study, we try to prepare anti-T-2 monoclonal antibodies(mAbs) and anti-DON mAbs. And then, we intend to use those mAbs to construct a μPAD based on the time-resolved fluorescent immunoassay (TRFIA) to detect both DON and T-2 toxin in samples. The implement of the project should be useful and significant to the advance of analytical tools for determination of DON and T-2 toxin.

摘要：单端孢霉烯族毒素是对人类健康和畜牧业生产造成严重危害的一类真菌毒素，其中T-2毒素和脱氧雪腐镰刀菌烯醇(DON)因其高毒活性及污染的严重性广受关注。目前，免疫分析方法（酶联免疫法，ELISA；胶体金免疫层析法，LFIA）是对此类毒素进行快速分析的主要手段。但LFIA不能满足高通量筛选的需要；ELISA需在实验室进行，且操作复杂，因此发展快速、简单、高通量的在线检测分析方法具有重要意义。本项目拟制备T-2毒素与DON的单克隆抗体，同时，以纸基微流控芯片技术作为突破口，构建基于镧系元素(Sm3+、Eu3+)的时间分辨荧光免疫分析纸基微流控芯片，在此基础上，建立竞争性的、快速、简单、高通量的纸基微流控芯片检测方法，优化各种参数，并以此对谷物及其制品中T-2毒素和DON进行分析。本项目的实施对于发展T-2毒素和DON的高通量在线生物分析技术具有重要的理论意义和实际应用价值。

单端孢霉烯族毒素中的T-2毒素（T-2 toxin）和脱氧雪腐镰刀菌烯醇(Deoxynivalenol，DON)因其高毒活性及污染的严重性广受关注。纸基微流控芯片技术为二者的现场高通量筛选提供了重要的技术手段。本项目制备T-2毒素单克隆抗体四株（7D1，4D11，5F8，11B4）和DON单克隆抗体四株（4E6，7A9，3H6，3E11）。以7D1和4E6为材料，制备了时间分辨荧光纸基芯片（TRFIA-μPAD）和超薄核壳表面增强拉曼纸基芯片（uts-SERS-μPAD）并在此基础上建立了谷物样本检测方法。TRFIA-μPAD方法（玉米、小麦、大米）的检测限为：T-2毒素（2.70 μg kg-1、13.82 μg kg-1、1.73 μg kg-1），DON（8.15 μg kg-1、16.07 μg kg-1，5.33 μg kg-1）；uts-SERS-μPAD检测方法对玉米样本中DON的检测限为3.0 ng kg-1。TRFIA-μPAD和uts-SERS-μPAD方法灵敏度达到了国家对T-2毒素和DON的残留限量要求。本项目的研究成果对发展T-2毒素和DON的高通量现场分析技术具有较高的理论意义和应用价值。本项目共计培养3名研究生，发表3篇SCI文章，申报国家发明专利1项。