量子点标记小分子有机污染物多组分均相免疫分析原理与技术研究

After the development on pesticide residue immunoassays in many years and the investigation on quantum dots (QDs) labeled heterogeneous immunoassays in recent years, the main kinds of synthetic organic pesticides and environmental endocrine disruptors(EEDs) which are obviously harmful to environment, food safety and human health will be continually chosen as new research objects to synthesize optimally designed haptens and artificial antigens, and to prepare specific antibodies by immune animals or hybridoma technique. This research project will mainly focus on the development of QDs labeled homogeneous immunoassays. The water soluble QDs group with high fluorescence quantum yield, wide exciting spectrum, narrow and non-interference fluorescence spectra will be optimized, the nonradiative water soluble fluorescence quenching reagents (FQR) will be prepared and optimized. The different antibodies and haptens will be conjugate to different QDs and FQR to develop fluorescence quenching based homogeneous immunoassays. The optimized antibodies and the corresponding QDs labeled haptens will be employed to develop fluorescence enhanced homogeneous immunoassays. The investigation will be conducted to understand the principles and laws of fluorescence enhancement and fluorescence quenching in homogeneous immune reaction, and to achieve new breakthroughs on the development of specific, sensitive, convenient and express QDs- labeled multicomponent homogeneous immunoassays for small molecular organic pollutants monitoring.

在本课题组多年来农药残留免疫分析和近年来量子点标记非均相免疫分析技术研究的基础上，继续以对环境和食品安全及人类健康有较大影响的主要有机合成农药和环境内分泌干扰物（EEDs）为研究对象，进行半抗原和人工抗原的优化设计与合成，以人工抗原免疫动物或采用杂交瘤技术制备对目标分析物具高特异性的抗体。重点研究并优化量子产率高、激发光谱宽、荧光光谱窄且互不干扰的水溶性量子点组合，研制并优选非辐射型水溶性纳米荧光淬灭剂。以不同荧光发射光谱的量子点、非光辐射型水溶性纳米荧光淬灭剂分别标记不同目标分析物半抗原和抗体，建立基于荧光淬灭的量子点标记多组分均相免疫分析技术。优化抗体与对应量子点标记半抗原组合，建立基于荧光增强的量子点标记多组分均相免疫分析技术。研究均相免疫反应中量子点标记物荧光强度变化的原理与规律，在建立特异性强、灵敏度高、简便快速的量子点标记小分子有机污染物多组分免疫分析技术方面取得新突破。

在课题组多年来小分子有机物免疫分析技术研究的基础上，以我国目前大量生产或频繁使用、对环境和农产品安全及人类健康有较大负面影响的重要农药、药物的主要品种为目标分析物，通过计算机辅助进行半抗原、人工抗原的分子设计，合成目标分析物半抗原。将半抗原分别与蛋白质和辣根过氧化物酶共价偶联制备人工抗原和酶标半抗原，以人工抗原免疫动物制备出3种新抗体（抗拟除虫菊酯共同结构1种、抗克伦特罗2种）并建立了相应的酶联免疫吸附分析技术。优化筛选出荧光光谱窄、量子产率高的水溶性量子点共6种。以不同荧光发射波长的量子点与不同抗体共价偶联，分别制备荧光发射波长不同的量子点标记抗体。在适当浓度的量子点标记抗体溶液中加入对应的目标分析小分子有机物（农药或药物），与量子点标记抗体进行均相免疫反应，改变了量子点标记抗体的表面性质，产生荧光强度的变化，量子点标记抗体的荧光淬灭程度在一定范围内与体系中对应目标分析物的含量成正比，据此建立单一小分子有机物的量子点标记抗体均相免疫分析技术。将适当浓度的不同量子点标记抗体溶液混合，加入各对应目标分析小分子有机物同时进行均相免疫反应，改变了各量子点标记抗体的表面性质，分别产生荧光强度的变化，各量子点标记抗体的荧光淬灭程度在一定范围内与体系中各对应目标分析物的含量成正比，据此建立对不同目标分析物同时进行高灵敏度快速定性、定量检测的量子点标记抗体多组分均相免疫分析技术。发表相关SCI收录论文2篇、核心期刊论文1篇，获国家发明专利授权2项。