果蔬中链格孢毒素微流控芯片在线萃取、浓缩及多元定量检测方法研究

The current detection methods and sample preparation procedures of Alternaria toxins in fruits and vegetables are often complex, time-consuming, toxic and high consumption of organic reagents. To address the problem, the microfluidic chips will be constructed in this project, integrating the functions of the ionic liquid-aqueous two-phase extraction (IL-APTE), a ZigZag micro-mixing technology and a magnetic immunoassay based on H2O2-mediated fluorescence quenching of CdTe QDs, for realizing the on-line extraction, concentration and multivariate analysis of Alternaria toxins. The main research contents include: the reusable the microfluidic chips which will be prepared consist of three modules in series (an ionic liquid-aqueous two-phase extraction (IL-ATPE) zone，a ZigZag micro-mixing zone and a magnetic immunoassay fluorescent quantitative zone). Secondly, the IL-ATPE extraction and concentration mechanism of microfluidic chips will be investigated using three Alternaria toxins. Next, we will prepare the antigen and monoclonal antibody of alternaria toxins to construct the microfluidic magnetic immunoassay chips based on H2O2-mediated fluorescence quenching of CdTe QDs. Additionally, the quantitative detection of three alternaria toxins in fruits and vegetables will be realized, and the parameters of sensitivity and analysis time will be compared with that of ELISA and LC-MS. This project could provide an important technical support for protecting the health of consumers.

本项目针对果蔬中链格孢毒素现有检测方法及其样品前处理技术（off-line）所存在的复杂、费时、毒性、有机试剂消耗量大等问题，旨在搭建一种集离子液体双水相萃取技术、ZigZag微混合技术、基于H2O2介导CdTe QDs荧光淬灭的磁免疫定量检测技术为一体的并具有实际应用价值的微流控芯片，实现链格孢毒素在线（on-line）绿色萃取、浓缩及多元定量检测。具体研究内容包括：制备含三个串联功能区域（离子液体双水相萃取区、ZigZag微混合区和磁免疫多元定量检测区）并可重复利用的微流控芯片；以链格孢毒素为分析对象，研究微流控芯片离子液体双水相萃取、浓缩机理；研制链格孢毒素抗原及单抗，首次建立H2O2介导CdTe QDs荧光淬灭的微流控磁免疫定量检测方法；实现果蔬中三种链格孢毒素的定量检测，并在灵敏度、分析时间等参数上与ELISA、LC-MS进行比较。本项目的实施为保障消费者健康提供重要的技术支撑。

链格孢毒素具有基因毒性、诱变性以及致癌性。在番茄，小麦，葡萄，葡萄酒，燕麦片和大麦等食品中都已检测到不同浓度的链格孢毒毒素。因此，以液相色谱-质谱联用（LC-MS）为主的多种分析方法已被用于链格孢毒素的定量检测中。然而，这些大型仪器设备的成本高，并且需要专业的技术人员。微流控芯片作为一种快速、便携、低成本的检测分析平台，具有流体的精准控制、样品和试剂的低消耗、检测时间短以及分析平台的微型化特性而受到人们的广泛关注。此外，微流控芯片与免疫分析的结合被认为是高灵敏、快速、高通量和自动化即时检测（POCT）最有前景的分析平台之一。基于以上研究背景，本项目制备了含三个串联功能区域（离子液体双水相萃取区、ZigZag微混合区和磁免疫多元定量检测区）并可重复利用的微流控芯片；以链格孢毒素为分析对象，首次建立H2O2介导CdTe QDs荧光淬灭的微流控磁免疫定量检测方法，得到链格孢毒素AME的最低检测限为0.25 pg/mL；建立了一种基于智能手机APP的微流控磁免疫比色传感快检方法，利用UV-Vis检测方法得到链格孢毒素AME的最低检测限为12.5 pg/mL，利用智能手机APP得到的最低检测限为200 pg/mL；最后利用上述新型快检技术实现了多种果蔬实际样本中链格孢毒素的定量检测。该方法可用于食品样品中其他真菌毒素或半抗原的快速、高灵敏、低成本和实时实地检测。