新型纳米标记探针免疫生物传感对食品中黄曲毒霉素检测新方法研究

Accompanying with the development of nanotechnology and biotechnology, nanolabel holds great potential as the next-generation detection strategy of succesful immunoassay because of its quantum size effect and high surface-to-volume ratio.The rapidly emerging research field of nanolabeling technology, and the process used to generate, manipulate and deloy nanomaterials, provides excitingly tools and instrumentation for bioanalytical and biotechnological appplication. One major merit of using nanolabeling is that one can control and tailor their properties in a very predictable manner to meet the needs of specific applications. Furthermore, this method is not only simple to perform, rapid, sensitive and feasible, but also easily multifunctionalized and selectively determined. The aim of this project is to explore novel methods and strategies for the analysis and detection of aflatoxin in foodstuff by coupling a series of labeling systems with a highly sensitive immunoassay protocol. Detailedly, this project consists of the following issues: (i) to prepare and screen polyclonal antibodies with high affinity by using bovine serum albumin (BSA) or ovalbumin (OVA)-aflatoxin B1, B2, G1, and G2 conjugates as immunogens in the rabbit or mouse,repectively; (ii) to fabricate a highly sensitive voltammetric immunosensing for detection of aflatoxin in foodstuff using inorganic ions-doped nanostructures as labels; (iii) to set up novel multiple signal-amplified immunoassays coupling nanolabel with biologically amplified technique; (iv) to construct nove glucometry-based immunoassay methodologies by ultilizing the competive reaction between macromolecule-based nano-polymers, target analyte with the corresponding antibody immobilized on the mesoporous nanostructures; (v) to exploit nove digital multimeter-based immunosensing strategies by coupling an external capacitor with an enzymatic catalysis; and (vi) to explore novel dual-color colorimetric immunoassays based on quantum dot labeling strategy. More importantly, this project could open a new avenue in the magneto-controlled electrochemical immunoassays and provide a convenient platform for analysis and detection of food safety.

纳米标记是纳米技术与生物技术交叉渗透形成的新技术，由于其量子尺寸和表面效应可将免疫分析性能提高到新的水平，使其不仅检测速度快、精度高、可靠性好，还能实现多功能化和选择检测。本项目拟制备一系列纳米标记探针，构建新型免疫分析模式，建立对食品中黄曲毒霉素检测的新方法和新技术。具体内容：（1）制备和筛选高亲和力黄曲霉毒素多克隆抗体；（2）建立以无机金属离子掺杂的纳米颗粒作为信标探针伏安免疫分析新方法；（3）建立以纳米标记与生物学放大技术相结合多重信号放大免疫分析新技术；（4）以多孔材料作为传感探针，利用聚合物纳米颗粒对目标抗体竞争替代反应，建立以血糖仪作为检测手段免疫分析新策略；（5）利用生物活性酶在催化过程中引起膜电位变化，建立以万用表作为检测手段免疫分析新模式；（6）建立以量子点作为信标抗体双色复合光比色免疫分析法。本研究成果有望对食品中低含量生物毒素检测以及食品安全分析等方面提供技术平台。

本项目拟制备一系列纳米标记探针，构建新型免疫分析模式，建立对食品中黄曲毒霉素检测的新方法和新技术: (1) 以黄曲霉毒素B1、B2、G1、G2 为研究对象，根据它们的分子特点，制备出交叉反应小、亲和力高的黄曲霉毒素B1、B2、G1、G2 多克隆抗体；(2) 通过脂质体包裹电子供体多巴胺，利用多巴胺对Mn2+掺杂的Zn3(OH)2V2O7·2H2O纳米带的光电流的增大效应使电极上的电流增大，构建以脂质体作为信号放大、基于Mn2+掺杂的Zn3(OH)2V2O7•2H2O纳米带的电化学传感器，实现对食品中黄曲霉毒素的电化学检测；(3) 设计两条功能化单链DNA，利用抗体(Ab)与半抗原(AFB1-BSA)的特异性反应，组建成类似于Ω的DNA交联体，利用了外切酶III实现对亚甲基蓝标记DNA发夹探针的重复利用，建立以分子生物学为放大技术的免疫分析方法，实现对食品中黄曲霉毒素的一步检测；(4) 以多孔二氧化硅为载体，利用聚乙烯亚胺(PEI)修饰与目标分析物与anti-AFB1抗体的竞争替代反应，建立一种以竞争-替代反应模式用于对食品中黄曲毒霉素一步快速血糖仪检测新方法; (5) 利用单克隆黄曲霉毒素抗体或生物活性酶标记纳米金作为信号来源，利用蛋白分子表面电荷或酶催化底物引起膜电位的改变，建立以生物纳米标记作为信号放大电位仪或万用表作为检测手段的免疫分析新模式；(6) 利用标记BSA-AFB1的葡萄糖氧化酶对底物葡萄糖氧化生成过氧化氢，结合过氧化氢对MnO2纳米片的刻蚀效应，将负载在纳米片上的CQDs释放下来，从而导致MnO2-CQDs复合物光电流的减少，建立一种以刻蚀效应为策略的免疫分析新技术，实现对食品中黄曲霉毒素的光电化学检测，实现样品在复杂体系中灵敏、快速的检测，为食品安全预测、预警等重要领域提供技术平台。研究成果将在蛋白质组学研究、临床疾病的诊断、环境分析、食品安全分析等与人类健康和公共安全密切相关的科学领域发挥重大的作用，具有广泛的应用前景和潜在的巨大经济效益。. 在本项目资助下，已在国际核心期刊上发表研究论文近50多篇，与本项目直接相关的论文16篇（包括：Anal Chem 4篇），影响因子大于5.0的14篇，申请发明专利6项，培养硕士研究生3名，博士研究生8名，并邀请德国慕尼黑工业大学(Prof. Dietmar Knopp)来校交流指导。