一步式电化学快速免疫检测纸基芯片研究

The traditional electrochemical immunoassay has the disadvantages such as cumbersome steps，time consuming and low automation. Furthermore, the potential hazard during toxic substance detection process demands for reducing manual involvement. Considering these problems, by the combination of paper-based microfluidic technologies and electrochemical immunoassay methods, the sensitive microelectrodes will be integrated in paper-based microfluidic system to achieve the rapid electrochemical immunoassay detection for aflatoxin in this proposal. Among the entire steps of testing process, only dropping sample requires manual operation. The nucleation mechanism of metal nanomaterials on fiber structure will be researched for the preparation method to obtain paper-based microelectrode. The methods to control the morphology of microelectrodes interface will be studied based on the sensitizing effect of micro and nanostructures on the electrode surface. The reaction and signal transduction mechanism between the target molecule and sensing surface will be analysed to improve the sensing properties of the sensitive electrode. Immune paper-based microfluidic system will be rationally designed. The correspondence between the analyte concentration and electrochemical signal will be established and the electrical response signal will be effectively extracted. By testing and optimizing chip detection performance，the portable electrochemical immunoassay paper-based chips, with high sensitivity, good specificity and easy opration will be eventually developed for the trace analysis of toxic materials. Especially it will provide an effective method for on-site rapid detection in the field of environmental protection and food safety.

针对传统电化学免疫传感器操作繁琐、过程耗时、自动化程度低等缺点，以及毒害物检测过程中潜在危险性所导致的应尽量减少人工参与的需求，本项目提出将纸基微流控技术和电化学免疫分析方法相结合，在纸基微流控系统中集成敏感微电极，实现对黄曲霉毒素的一步式电化学快速免疫检测，整个检测过程中仅“滴加样品”一步人工操作。研究金属纳米材料在纤维结构表面的成核、成膜机理，获得纸基衬底材料上的微电极制备方法；基于电极表面微纳结构增敏效应，获得微电极敏感界面的形貌调控方法；深入分析靶标分子与敏感表面之间的反应机理和信号传递机制，提高电极敏感特性；合理设计纸基免疫微流控系统；建立待测物浓度与电化学信号的对应关系和对电响应信号的有效提取方法。通过测试并优化芯片性能, 最终研制出灵敏度高、特异性好、操作简易的便携式电化学免疫检测纸基芯片，为毒害物的痕量分析，特别是环境保护与食品安全领域的现场快速检测提供有效方法。

本项目针对毒害物检测过程中应尽量减少人工参与并使用一次性探头的应用需求，基于纸基材料的多孔结构和亲水特性，开展纸基微传感电极芯片研究。该研究采用硝酸纤维素膜作为衬底材料，利用磁控溅射技术在纸基的微观纤维状结构表面构建具有三维结构的微电极，并研制出适合金电极表面修饰的AFB1抗体/壳聚糖-纳米金复合膜,可用于黄曲霉毒素的便携式低成本现场快速检测。.研究了金、铂、银等多种金属纳米材料在纸基纤维状结构表面的成核和成膜方式，探索了磁控溅射技术的工艺参数，制备出具有良好电化学特性和三电极体系的纸基电极芯片。基于蜡封技术实现纸基电极芯片封装及微流路设计，并采用3D打印技术加工出配套固定测试装置和引线设计。设计敏感膜的微观结构，通过在含有壳聚糖和氯金酸的混合溶液中电沉积制备壳聚糖-纳米金复合膜；利用壳聚糖氨基的共价键合作用成功将黄曲霉毒素单克隆抗体固定在壳聚糖-纳米金复合膜表面；研究了黄曲霉毒素检测过程中靶标分子与敏感膜的反应机理，以及生物反应信号到电信号的转换机制；结合参数优化实验，基于AFB1抗体/壳聚糖-纳米金复合膜可实现对黄曲霉毒素样品溶液分段线性检测，检测下限可以达到0.12 ng/mL，体现了较高的灵敏度，以及较好的特异性和重复性。本研究有利于推动痕量毒害物快速检测技术的发展，特别是对环境保护与食品安全领域的现场快速检测提供有效解决方案。.本项目共发表论文8篇，其中SCI期刊论文4篇，EI期刊论文2篇，中文核心期刊论文1篇，会议论文1篇；申请发明专利1项；项目资助3位研究生获得博士学位、1位研究生获得硕士学位。