基于纳米抗体建立赭曲霉毒素A与玉米赤霉烯酮超灵敏FRET均相免疫检测方法的研究

Ochratoxin A (OTA) and zearalenone (ZEN) are the common mycotoxins in food chain, which pose serious health hazards to humans and to domestic animals. The enzyme-linked immunoassay (ELISA) and gold immunochromatographicassay (GICA) are widely used for OTA and ZEN routine screening determination because of its sensitivity or rapid and simpe operation. But, the antigen-antibody interaction in the above methods is performed in heterogeneous reaction system. Among of them, the operational process of ELISA is complicated and time consuming, whereas the sensitivity of GICA is relatively low. FRET-based immunosensors are more advantageous with higher sensitivity, fast liquid-phase binding kinetics, and homogeneous immunoassay with no washing and separation steps. Increasing energy transfer efficiency between donor and acceptor is a key factor for further improving the sensitivity of FRET based homogeneous immunoassay. In this proposal, a homogeneous FRET immunosensor will be develpoed for ultrasensivity detection of OTA/ZEN, in which the nanobodies with smallest size will be used as conjugate component between donor and acceptor, while flower-shaped gold nanoparticles (AuNFs) will be used as energy acceptor for improving FRET efficicency because of its large surface area and high molar extinction coefficients. Taken together, this proposal will discuss the feasibility of implementing an efficient energy thansfer by shortening the distence between donor and acceptor, increasing the mole extinction coefficients of the acceptor and optimzing the experimental conditions of immunoassay. The final goal of this proposal is to establish a platform based on FRET immunosensor for ultrasensitivity detection of samll molecular antigen in homogeneous system.

赭曲霉毒素A（OTA）和玉米赤霉烯酮（ZEN）是食物链中常见的污染物，对人畜健康危害极大。现例行筛查检测中最常用方法为酶联免疫吸附与胶体金免疫层析法。以上两种方法为非均相免疫学反应，其中前者操作繁琐耗时，而后者虽然操作简单快速，但检测灵敏度偏低。基于荧光共振能量转移（FRET）的均相免疫检测法具有操作简单、反应速度快、检测灵敏度高的特点。实现高效的FRET效率是进一步提高均相免疫检测方法灵敏度的关键。本研究拟以OTA、ZEN为检测对象，采用具有大比表面积及高摩尔消光系数的花状纳米金为能量受体，水溶性量子点为能量供体，以新型抗体识别元件-纳米抗体作为供/受体之间连接臂，实现量子点和花状纳米金之间的高效能量转移。总之，本项目旨在探讨通过提高能量受体摩尔消光系数，缩短供/受体连接臂距离以及优化免疫学反应条件，实现供/受体之间高效荧光能量转移及小分子抗原超灵敏检测的可行性，并为其提供理论依据。

基于荧光共振能量转移（Flurescence Resonance Energy Transfer，FRET）理论的均相免疫分析法在液相中实现抗原/抗体反应，具有反应速度较快、操作相对简单，灵敏度较高等特点。本项目以真菌毒素为检测对象，以量子点作为能量供体，采用具有较大的比表面积和摩尔消光系数的花状纳米金为能量受体，以新型抗体识别元件-驼源纳米抗体（VHH）作为供/受体之间连接臂，利用QDs 和AuNFs 的均具有的波长可调优势，构建了光谱匹配、重叠度高的FRET 免疫学检测体系，主要研究内容包括：抗真菌毒素纳米抗体的制备与鉴定；真菌毒素抗独特性纳米抗体的制备与鉴定；基于纳米抗体的真菌毒素免疫学检测方法的建立；花状纳米金和球状纳米金的合成、表征及免疫学检测性能的比较；基于纳米抗体和花状纳米金FRET均相免疫检测方法的优化与构建。研究结果表明，以驼源性纳米抗体缩短供/受体连接臂距离和以具有较大摩尔消光系数的花状纳米金作为能量受体，可以显著提高FRET 均相免疫检测方法的灵敏度，扩大检测线性范围，为食品安全领域的小分子超灵敏FRET均相免疫检测法的建立提供了理论依据。