表面增强电化学发光免疫法检测食品中的环境雌激素类物质

Environmental estrogen (EE) can interfere with the normal hormone system of animals, which are closely related to many diseases and adversely affect the health of human beings. They are found just about everywhere and actually, they are difficult to avoid. Especially, the existence of Environmental estrogens (EEs) in food shows the emergency threatens to everyone in this world. As a result, developing simple, rapid and sensitive analytical methods for EEs detection is of great importance in food safety and environmental monitoring. Combining the effects of surface enhancement by metallic nanoparticles (MNPs) with the electrochemiluminescence immunoassay (ECLIA), the proposed project is aiming to develop a new ECLIA method for the detection of EEs in food. Firstly, the interaction between the surface effects by MNPs on ECL emission was investigated by immobilizing the MNPs or ECL emitters on the surface of working electrode and monitoring the changes of ECL intensity, spectrum and efficiency. The influencing factors on this interaction involved in the separation distance between MNPs and ECL emitters where the distance can be controlled by introduction of different size of biomolecules and related immunoreactions, and the characteristics of MNPs and ECL emitters. After clarified the interaction, the mechanism of surface enhancement can be explained completely, and the optimization of influencing factor can lead to the maximum surface enhancement on ECL intensity. Therefore, the sensitivity of ECLIA with MNPs should be improved reasonably and greatly. Based on these considerations, a higher sensitive ECLIA is established and can be applied to detect ultra-low level of EEs in food. We lay emphasis on the study of interaction between MNPs and ECL emission. The main innovation of this project is to combination of effects of surface enhancement with ECLIA to improve the sensitivity from the view of analytical methodology. Finally, this project may provide a rapid, simple but sensitive method for more than EEs detection, which is in urgent need in monitoring the food safety.

建立简便、快速、灵敏的环境雌激素分析方法对于环境健康预警具有重要意义。本项目拟结合金属纳米粒子的表面增强效应和电化学发光免疫分析技术，发展一种新型的分析方法用于食品中环境雌激素的检测。具体方案为通过考察金属纳米粒子的表面效应对电化学发光强度、发光光谱、发光效率等方面的影响，以及电化学发光对金属表面等离子体共振产生的影响，研究纳米粒子表面效应与电化学发光辐射之间的相互作用，揭示作用机理，并利用表面效应增强电化学发光信号。在此基础上，与免疫分析相结合，进一步提高电化学发光免疫分析方法的灵敏度。项目的重点是调控表面效应与发光辐射之间的相互作用，建立表面增强的电化学发光免疫分析方法。主要创新点是将利用表面增强效应改善电化学发光免疫方法的分析特性，提高检测灵敏度。本课题有望为环境雌激素在食品安全中的超低含量检测提供一种灵敏、简便的新方法，还可能推广到其它可用于免疫分析的物质的检测与监控。

建立简便、快速、灵敏的环境雌激素分析方法对于环境健康预警具有重要意义。将金属纳米粒子的表面增强效应和电化学发光免疫分析技术结合，可发展一种新型的分析方法用于食品中环境雌激素的检测。本项目首先以纳米金和Ru(bpy)32+/TPA电化学发光体系为研究对象，引入免疫反应调控二者之间的距离，考察了纳米金的表面效应对发光行为的影响。结果发现只有将发光分子固定在电极上，纳米金通过标记抗体经过免疫反应连接到电极后，可以使电化学发光信号增强约2倍。同时我们还考察了纳米粒子的粒径和组成对表面增强作用的影响：（1）表面增强效应随着纳米金粒径的增大而增加，43 nm时增强作用最大；当继续增大纳米金粒径，表面增强作用反而减弱。（2）核壳型双金属纳米球（Ag100-xAux，x=0-40）的表面增强作用随着Au壳包裹而增加，x=30达到最强；当Au壳厚度继续增加表面增强作用开始减弱；双金属间的协同作用导致该类纳米粒子对电化学发光信号增强高于Au、Ag单金属纳米粒子。在此基础上，建立了一种纳米金表面增强电化学发光免疫分析法检测肉制品和奶制品中雌二醇含量的分析方法。在最佳测量条件下，定量雌二醇的线性范围为1-500 ng/mL，计算得到该分析方法的检测限为0.21 ng/mL。交叉反应说明该方法具有良好的选择性，加标回收实验说明该方法具有良好的准确性。对实际样品的检测结果表明，上述检测体系与传统仪器检测其成本却大大低于传统仪器，操作也更简便快捷；与经典的电化学发光方法相比具有更高的灵敏度，展现了良好的实际应用前景。为了进一步提高金属纳米粒子增强电化学发光分析方法的灵敏度，我们还系统的研究了纳米金的电催化效应、淬灭等作用对发光信号的影响，考察了空间距离等因素对纳米金的各种效应的影响。在此基础上，建立了几种纳米金增强的电化学或电化学发光分析新方法。该项目的实践，有望为环境雌激素类内分泌干扰物在环境监测、食品安全、检验检疫等领域的检测提供一种快速、灵敏、牢靠的新方法。