基于单分子成像的增强型TIRF-SPR双模并行检测方法及其应用研究

单分子检测可以揭示出被大量分子系统平均而抹去的重要信息，而单分子动力学研究，可以揭示生化反应的内在机理，实现特异性检测。本项目针对单分子检测的需要，结合全内反射荧光(TIRF)技术的空间特异性与表面等离子体谐振(SPR)检测的实时全局性，实现双模信号的同时同位检测。本项目重点解决表面等离子体(SP)增强TIRF信号，TIRF-SPR双模检测，单分子动力学研究等关键科学问题。研究出基于棱镜耦合原理的增强型TIRF-SPR双模检测方法与系统样机，实现实时、快速、定量检测生物分子的动力学反应过程。空间分辨率达到单分子成像水平，折射率检测灵敏度达到1e-7RIU，TIRF信号增强10倍以上。结合传感芯片生物修饰技术及探针固定技术，开展无扩增条件下检测单个DNA/RNA分子及其杂交动力学特性的应用研究，为探索分子诊断新技术提供新的方法，为重大疾病的筛查诊断、疗效监测及预后评估提供有利工具。

Single-molecule detection can reveal the important information that has been averaged by a large number of molecular systems, especially single-molecule dynamics studies, which can achieve specific detection and reveal the inherent mechanism of biochemical reactions. Based on the need of single molecule detection, this project combines the spatial specificity of total internal reflection fluorescence (TIRF) technology and the real-time global character of surface plasmon resonance (SPR) detection to realize simultaneous detection and same position analysis of these two signals. This project focuses on the key scientific issues such as surface plasmon wave (SP) enhancement of TIRF signal, TIRF-SPR dual-mode detection, single-molecule kinetics research and so on. An enhanced TIRF-SPR dual-mode detection method and system prototype based on prism coupling principle are developed to realize real-time, rapid and quantitative detection of the dynamic reaction process of biological molecules. The spatial resolution reaches the level of single molecule imaging, the sensitivity of refractive index detection reaches 1e-7RIU, and the TIRF signal is enhanced more than 10 times.Combined with biosensor chip bio-modification technology and probe immobilization technology, the detection of single DNA/RNA molecule without amplification and its hybridization kinetics were studied. It provides new ideas for exploring new molecular diagnostic techniques and powerful tools for screening, clinical diagnosis, therapeutic effect monitoring and prognosis evaluation of major diseases.