基于平板型等离激元微纳结构的单分子FRET调控及其机制研究

Cellular activity is driven by dynamics and interactions of biomolecules, which are often difficult to experimentally quantify as limited in resolution or accuracy. Single-molecule Förster resonance energy transfer (sm-FRET) is one of the most powerful nanoscopic tools to directly measure conformational changes even in live cells. However, the lack of cell-friendly, bright, photostable probes of small size with excellent FRET efficiency remains a challenge. Here, the overall objective is to develop FRET-controlling technique by straight-forward optoplasmonic approaches that do not interfere with cellular processes. Specific goals are 1) an increased FRET efficiency ─ which can directly translate into faster or more precise measurements─ and 2) an option to probe phenomena even above FRET’s blind distance of 10nm to make fluorescence labelling and assay development more versatile for bigger biomolecular complexes. We propose specially designed plasmonic substrates with desired key features and study the quantitative mechanism of photonic environment (local density of optical states-LDOS, mode distribution and polarization) control of FRET theoretically and experimentally. Then the first biomolecular application will be explored on the FRET-boosting substrate. The structures represent relatively simple coatings that are nano-layered on typical glass coverslips, ready to use in traditional fluorescence microscopes and combine with other single molecule/wide field fluorescence measurement techniques. Beyond, the cross-disciplinary project bridges biology, physics and material sciences and thus will facilitate the knowledge transfer.

生物分子结构-功能的动态关联与其对生命活动调控息息相关，其实验测量常受限于分辨率和精确度。单分子Förster共振能量转移是能在体外/活细胞直接测量分子纳米尺度构象变化的有力技术之一，但实现高细胞亲和度、高亮度、光稳定性好、FRET效率高的小尺寸荧光探针及标记仍面临许多技术挑战。本项目旨在现有荧光标记体系下发展生物兼容性高的平板型表面等离激元衬底技术以提高：1）单分子FRET效率-更高时空分辨率；2）FRET作用距离至盲区以上（>10nm）以适用于大空间尺寸分子内（间）构象测量。我们将设计特殊平板型等离激元衬底结构，理论结合实验研究其微纳尺度电磁环境（态密度、模式分布、偏振）对单分子FRET速率/效率的量化调控机制，并基于此探索FRET增强衬底的生物单分子应用。衬底主要由普通玻片镀金属/介质纳米薄膜构成，可与传统荧光显微测量技术兼容。项目有机结合物理、生物和材料学，益于各领域知识有效互递。