一种新型量子点均相荧光免疫分析方法的建立及其初步应用

A novel method of quantum-dots-based homogenous fluoroimmunoassay is to be established and applied in the one-step detecting AFP in human serum. The phthalocyanine derivative shall be synthesized and used to dye the carboxyl-modified polystyrene microspheres(CPS).This phathalocyanine-dyed CPS will be modified with aminodextran in order to eliminate the non-specific absorption. Such modified CPS works as the donor in the developed system. The phathalocyanine doped in the CPS would activate ambient oxygen in the solution once sensitized by the near-infrared light source. Thereby the singlet oxygen is produced from the donor spheres and diffuses around in the solution. On the other side, thioxene and quantum dots shall be synthesized, respectively and will be doped in the CPS with 1,10-phenanthroline and trioctylphosphine oxide. Such doped modified CPS works as acceptor, where thioxene shall react with the singlet and emits the light with around 340nm. Thereby QDs around the thioxene will emit with around 605nm due to the F?rster resonance energy transfer (FRET) from the thioxene. The donor and acceptor spheres shall be conjugated with two anti-AFP monoclonal antibodies respectively. Then, with double antibodies sandwich mode the mechanism of the efficiencies of singlet oxygen and the FRET are mainly investigated by interactions between two monoclonal antibodies and their corresponding antigen between the donor and acceptor spheres. The issues of highly efficient transfer of singlet oxygen, high efficient RT-FRET, non-specific absorption and spectroscopy between donor and acceptor spheres are solved and optimized by sensitivity, specificity, precision, stability and linear working scales. The preliminary results show that the novel QDs-based immunoassay is feasible and worthy of investigating scientifically with realistic meaning. The most advantages of this QDs-based immunoassay are that the one-step quantitative screening is carried in the homogenous solution without rinsing the free labels and that the signal-noise ratio be enhanced much. This novel method would pave a scientific road for modern immunoassay technology with high speed, high sensitivity, miniaturization and high throughput screening and would be alternative of the traditional clinic diagnosis.

通过检测人血清中甲胎蛋白(AFP),建立一种新型量子点均相荧光免疫分析方法。合成光敏性酞青红、噻吩嗪化合物以及荧光量子点，并将其与天线材料一起分别包埋入微球，制备出感光微球和量子点发光微球。重点研究感光微球与发光微球的单线态氧信号传递效率以及量子点发光微球内部的荧光共振能量转移机制，解决获得高效单线态氧信号传递效率、荧光共振能量转移效率、消除非特异性吸附表面化学等科学技术问题。两微球再分别偶联上AFP的两株单克隆抗体，采用双抗体夹心法模式，从灵敏度、特异度、精密度、线性范围和稳定性等性能指标进行评估优化。初试结果表明，该量子点均相荧光免疫分析方法确实可行，具有极大的理论和现实研究价值。其最大优势是，不需冲洗掉游离标记物，信噪比高。为下一步开发快速便捷、高灵敏度、微量化、多通道、自动化及高通量化的新型免疫学检测试剂提供科学依据，促进现有临床免疫学诊断技术的升级换

目前广泛应用于临床的免疫学检测技术如酶联免疫吸附试验、时间分辨荧光免疫分析等，以微孔板为反应载体，需要洗涤来分离结合标记与游离标记，整个操作过程步骤多、周期长、样品消耗大、不易自动化等缺点。并且该类分析方法因受到背景底色或激发光源的影响，其检测灵敏度并不高，而且检测过程很难实现小型量化、自动化和高通量化。国外厂家凭借其封闭系统或者知识产权等限制使得国内在高端定量免疫学检测技术的发展进展缓慢。为此，本课题建立了一种新型量子点均相荧光免疫分析方法，以适应现代免疫分析技术的发展需求。研究内容主要包括：发光微球的制备；发光微球内部的荧光能量转移；感光微球制备与感光/发光微球的表面化学；新型量子点均相荧光免疫分析方法的建立。本课题成功建立了一种量子点均相免疫分析方法，并用于AFP的检测。经过优化后，该方法分析灵敏度达到0.0911 IU/mL,线性范围为1~1000 IU/mL，精密度分析内、分析间CV均小于10%。发表SCI文章3篇，申请专利2项，培养研究生2名。课题的研究为下一步开发快速便捷、高灵敏度、微量化、多通道、自动化及高通量化的新型免疫学检测试剂提供科学依据，促进现有临床免疫学诊断技术的升级换代。