單分子量測系統與關聯分析之應用

Applications of Single-Molecule Spectroscopy and Correlation Analysis

楊禮綾(Li-Ling Yang)

指導教授 ： 董成淵

國立臺灣大學/理學院/物理研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.01575

摘要

單分子測量術近年來受到廣泛的應用，尤其在生物分子的構形、動態與其動力學的研究上。系綜測量下無法獲得的資訊，如:總體分佈及變異，則可藉由單一分子的觀察而得。本篇論文的工作旨在建立單分子光學實驗系統及方法，及其相關應用，主要涵蓋兩大部分：

1. 泛素之動態構形研究 – 單分子螢光能量移轉光譜學
2. 時間相關螢光光譜學與單光子探測技術之應用 – 共軛高分子、螢光奈米鑽石、生物分子與微流體混合器

單分子螢光能量移轉光譜學為一奈米尺度下丈量尺規，其能量移轉效率與兩標定染間的距離變化有極大的關聯。透過染料分子的標定，生物分子的構形變化與動態則會反映在螢光能量移轉效率的變化。觀測系統主要為共焦顯微鏡，根據實驗的需要將生物分子固定與玻片表面或於溶液中直接觀測。另一方面，我們亦由螢光光子資料的擷取與分析上著手，透過螢光關聯分子與光子-光子間時距的紀錄獲得額外的資訊，並成功的應用於其他相關實驗上。這些努力主要是爲蛋白質早期摺疊過程之研究而準備，希望能結合快速混合微流體元件、螢光關聯分析與螢光能量移轉光譜以解析摺疊動態。

關鍵字

單分子量測 ； 泛素 ； 螢光能量轉移

並列摘要

Single-molecule detections have been known for the potential to provide additional information beside ensemble measurements. Population heterogeneities and synchronous (or asynchronous) reaction pathways pertaining to conformational dynamics and molecular interactions are veiled by ensemble averaging. The scope of this thesis is to establish general experimental methodologies on the basis of single-molecule detection, and the applications have been covered as well. The frame of this work is mainly composed of two parts:

1. Conformational dynamics of ubiquitin are investigated by spFRET (single-paired Förster Resonance Energy Transfer).
2. Applications of TCSPC (Time-Correlated Single Photon Counting) and FCS (Fluorescence Correlation Spectroscopy) on conjugated polymers, FND (Fluorescence Nano Diamond), biomolecules and micro-fluidics.

FRET serves as a distance ruler in close proximity (~ 1 nm to ~ 10 nm), and dynamic changes in structure of proteins can be thus recorded accurately. We have established optical methods to observe ubiquitin molecules either immobilized on the cover-slip surface or in free solution. On the other hand, detections based on single photon counting and correlation analysis has also been applied for further information aside from intensity analysis. At the same time, a continuous-flow micro-fluidic mixer is under development, which has claimed sub-ms mixing dead time. Cooperating FCS, TCSPC, microscopy, micro-fluidic mixer and FRET provides us with the perspective on investigations population evolution and structural variations in along the kinetics of biomolecules.

並列關鍵字

single molecule ； FRET ； ubiquitin ； TCSPC ； FCS

參考文獻

52. (劉慶川), C.-C.Liu., Investigations on Hydrodynamic Focusing by Con-focal Microscopy and Fluorescence Correlation Spectroscopy (共焦顯微鏡與螢光相關光譜分析於微流體聚焦之研究), in Institute of Applied Mechanics. 2008, National Taiwan Unversity: Taipei.

41. Y.-R. Chang, H.-Y.L., K. Chen, C.-C. Chang, D.-S. Tsai, C.-C. Fu,T.-S. Lim, Y.-K. Tzeng, C.-Y. Fang, C.-C. Han, H.-C. Chang, W. Fann, Nature Nanotech, 2008. 3: p. 284-288.

1. Moerner, W.E. and L. Kador, Optical detection and spectroscopy of single molecules in a solid. Physical Review Letters, 1989. 62(21): p. 2535.

2. Weiss, S., Fluorescence Spectroscopy of Single Biomolecules. Science, 1999. 283(5408): p. 1676-1683.

4. Adachi, K., et al., Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(13): p. 7243-7247.