基于能量转移的新型免疫荧光传感器研究及其在高通量分析蛋白-蛋白相互作用中的初步应用

The study of protein-Protein interactions (PPI) plays important roles in protein functions and life evolution and is a key part of proteomics. However, the current related assays are time-consuming, highly professional or expensive equipment-reqiured. Our previous studies have shown that homogeneous fluorescent protein analysis has a great advantage. A novel energy transfer-based biosensor for homogenous fluoroimmunoassay is to be established and applied in the one-step analysis of protein-protein interaction (PPI). The singlet oxygen-sensitive probe of time-resolved fluorescent europium chelate is synthesized as energy acceptor. The phthalocyanine derivative shall be synthesized as energy donor and used to dye the carboxyl-modified polystyrene nanoparticles. As-prepared biosensor is developed for general immunoassays and applied in high-throughput analysis of interactions between ErbB2 and involved proteins from the pathway of receptor tyrosine kinase introduce. The project focuses on the highly efficient energy transfer of singlet oxygen and on the highly efficient Förster resonance energy transfer between donor spheres and acceptor probes. The scientific issues of efficiency of energy transfer and sensitivity enhancement of fluorescence immunoassays are to be solved. The preliminary results show that the novel energy transfer-based biosensor is feasible and worthy of investigating scientifically with realistic meaning. The developed biosensor is design for generally application in PPI and optimized by sensitivity, specificity and etc. The advantages of the developed biosensor are no-washing, easy protocols, high throughput screening, high sensitivity, feasible minimization and general application. The investigation of the novel biosensor would pave a scientific road for modern immunoassay technology and enhance the PPI study. The completing of the project would strengthen our country’s record of proteomics study and possesses strong scientific research value.

蛋白-蛋白相互作用（PPI）研究是揭示蛋白功能阐释生命现象奥秘的重要手段，是蛋白质组学重要内容。但其现有研究方法较繁琐、专业性强、或需昂贵仪器。我们前期研究表明均相免疫荧光蛋白分析具有较大优势。本项目拟基于能量转移机制研究新型均相荧光分析方法及其常规通用型传感器，通过合成单线态氧敏感性荧光探针和光敏性微球，并初步应用于高通量分析酪氨酸激酶受体介导通路中的靶蛋白ErbB2与直接相互作用蛋白间的PPI。重点研究传感器能量供体与受体之间能量传递以及探针分子内荧光共振能量转移机制，解决能量转移效率和免疫荧光分析灵敏度等科学技术问题。初始实验表明其方案可行，并将从灵敏度和特异度等性能指标评估。该新型传感器无需洗涤、操作简便、通量高、灵敏度高、可微量化、常规通用型等优势。本项目的完成将为新型免疫学研究方法提供科学依据，促进PPI研究升级，保持我国蛋白质组学研究国际先进地位，具有较强的科学研究价值。

蛋白-蛋白相互作用（PPI）研究是揭示蛋白功能阐释生命现象奥秘的重要手段，是蛋白质组学重要内容。但其现有研究方法较繁琐、专业性强、或需昂贵仪器。我们前期研究表明均相免疫荧光蛋白分析具有较大优势。本项目基于能量转移机制研究新型均相荧光分析方法及其常规通用型传感器，通过合成单线态氧敏感性荧光探针和光敏性微球，并初步应用于高通量分析酪氨酸激酶受体介导通路中的靶蛋白ErbB2与常见的直接相互作用JAK2，STAT3和IL6ST蛋白间的PPI。解决了传感器能量供体与受体之间能量传递、探针分子内荧光共振能量转移和蛋白相互作用的免疫荧光分析灵敏度等科学技术问题，细胞裂解液中其他蛋白干扰小，灵敏度高。该新型传感器无需洗涤、操作简便、通量高、灵敏度高、可微量化、常规通用型等优势。本项目的完成将为新型免疫学研究方法提供科学依据，促进PPI研究升级，保持我国蛋白质组学研究国际先进地位，具有较强的科学研究价值。