基于新型纳米孔阵列-消逝波激发模式的活细胞单分子检测研究

In this project, new matrix composed of nanopore array structure is assembled based on the interaction of surface molecules in the nanoparticles using noble metal nanoparticles as building units. Based on these noble metal nanopore-enhanced fluorescence (NEF) effect and the introduction of evanescent wave as excitation mode, a novel, highly spatial and temporal fluorescence correlation spectroscopy (FCS)-single molecule detection method is developed for single cell analysis. Local surface plasmon resonance (LSPR) enhanced fluorescence effect of noble metal nanoparticles and the nanopore-enhanced fluorescence (NEF) mechanism is investigated. The novel optical configuration induced by nanopore structure and evanescent wave excitation mode can remarkably reduce the detection volume, improve the ratio of signal to background noise and enhance the spatial and temporal resolution. The diffusion behaviors of biomolecules within nanopore excited in the evanescent wave field are investigated and its mathematical model is simulated. The novel FCS-single molecule detection platform for single living cells is constructed and the fine structure of cell membrane is investigated such as dynamic behavior and distribution of the membrane proteins in the cell membrane. And the interaction of ligand with the receptors in the cell membrane is studied.

本项目旨在以贵金属纳米粒子为结构单元，基于纳米粒子表面分子相互作用，组装具有纳米孔阵列结构基质。基于贵金属纳米孔荧光增强效应，建立一种具有高时空分辨的，消逝波激发场的荧光相关光谱单分子探测新方法。研究贵金属纳米粒子具有局域表面等离子体共振（LSPR）荧光增强效应，探究贵金属纳米孔结构增强荧光（NEF）新机理。采用纳米孔结构和全内反射产生消逝波的光学构型显著减小单分子探测方法的检测体积，提高信背（噪）比和单分子探测时空分辨率。探讨纳米孔中分子在消逝波能量场作用下的扩散动力学及理论模型。构建活细胞中荧光相关光谱单分子探测新平台，研究细胞膜的精细结构如膜蛋白在细胞膜上扩散行为和分布等，研究配体与细胞膜上受体相互作用等。

荧光相关光谱(FCS)及其相关技术由于具有检测体积小、灵敏度高、空间分辨率高、非侵入性检测等优点，是目前非常重要的一种单分子检测方法，非常适合用于活细胞内蛋白浓度、扩散行为、寡聚化和相互作用等研究。本项目尝试了在传统的FCS方法基础上，通过构建纳米孔结构进一步约束FCS方法的检测体积，并实现活细胞的原位分析研究。研究内容包括：建立了模板消蚀法和基于点击化学反应构建纳米孔和SPR结构的方法；这些方法可以实现纳米孔结构的构建和荧光增强效果。荧光探针的单分子检测证实其在纳米孔内的扩散时间比开放体系中减小了近8倍，可以增强荧光约200%。建立了细胞原位单分子探测新平台，包括荧光-散射互相关光谱系统和基于光片激发构型的相关光谱系统，这些系统可用于活细胞内单分子检测和酶的定量分析研究。建立了活细胞原位分析新策略，建立了细胞内PTEN蛋白浓度和扩散行为、抑癌蛋白P53和MDM2相互作用分析新方法，揭示发现P53蛋白的TAD结合区的螺旋性和多聚化对两者的相互作用有着决定性作用。建立的这些研究平台和实验方法等对活细胞的原位分析研究开拓了新思路，将促进单细胞分析工作的开展。