利用全反射螢光顯微系統量測DNA於液態環境中不同固化方式之動態行為

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研究生：	徐聖傑
論文名稱：	利用全反射螢光顯微系統量測DNA於液態環境中不同固化方式之動態行為 The Dynamic behavior study of DNA under different immobilization methods in liquid environment by Total Internal Reflection Fluorescence Microscopy
指導教授：	許志楧
口試委員:	崔豫笳楊自森
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2012
畢業學年度：	100
語文別：	中文
論文頁數：	70
中文關鍵詞：	量子點、全內反射式螢光顯微鏡、單分子
外文關鍵詞：	quantumdot, TIRF, single molecule
相關次數：	點閱：1 下載：0
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固化於基因晶片上的DNA 探針之動態行為，是以基因晶片做為研究工具的科學家們所感興趣的問題。本實驗透過biotin-streptavidin 的非共價鍵結使DNA一端固化於BSA以及PEG玻片上，希望能夠了解DNA是像海草般在溶液中搖擺；或是像繩索般平躺於玻片表面，因為這兩種不同運動模式將影響DNA雜和之效率。
在螢光標定上，選用了量子點，因為量子點具有螢光亮度強、光穩定性佳無光漂白之特性。我們以TIRFM偵測長度為2834 base pairs且已標定量子點的DNA分子，DNA以biotin-streptavidin方式固化於玻片表面，並透過即時偵測螢光訊號強度隨時間變化建立出單分子追蹤技術，並藉由TIRFM產生的漸逝波，估計螢光於Z軸深度上的位移，並以此推測DNA在玻片表面上之動態行為。
研究發現，以biotin-streptavidin方式鍵結固化於玻片表面之DNA，觀察到如類似海草，靜止於溶液中，並發現少數來回擺動於特定二個位置之情況，對於擺動之現象，可能是利用BSA方式固化後，表面修飾不完全，而殘留些缺陷，使dsDNA附著於此，因此嘗試以PEG固化方式取代BSA修飾表面，排除DNA來回擺動於特定二個位置之情況，此外兩種不同表面固化，得到之dsDNA晃動程度也略些差異。

The dynamic behavior of DNA molecules , when immobilized on
DNA microarrays , has puzzled the gene chip researchers .In this study,
we used biotin-streptavidin interaction binding to the glass surface at the one end of DNA. Finally, we'll try to distinguish that whether DNA is like seaweed wavering in the solution or like a rope sticking on the surface of glass. The two modes have quite different hybridization efficiency.
For fluorescence labeling, quantum dot(Qdot) is a novel fluorescent platform that can emit the fluorescence continually and stably.The TIRF is used to measure the vertical location of fluorescence dye. We measure the TIRF signal of Qdot labeled 2834 bp DNA molecules that were immobilized on the coverslip surface via biotin-streptavidin, and we can estimate the position of Qdot by Gaussian distribution.
The result shows that DNA molecules behave like seaweeds and static in the solution, or swing in the particular two position on bsa-biotin coated surface. This phenomenon maybe be caused by hot spot on BSA-biotin coated surface. Therfore, we use PEG-biotin to coat surface to prevent DNA back and forth swing in the particular two position.

摘要.......................................................I
Abstract..................................................II
致謝.....................................................III
章節目錄...................................................IV
圖目錄....................................................VI
表目錄...................................................VII
第一章 序論.................................................1
1-1 前言...................................................1
1-2 文獻回顧................................................3
1-3 研究動機................................................5
第二章 系統原理與方法........................................7
2-1 螢光發光原理............................................7
2-2全內反射螢光顯微技術.....................................11
2-2-1全反射(Total Reflection ) ...........................11
2-2-2漸逝波(Evanescent wave)............................. 12
2-3偵測極限...............................................13
2-4 物鏡式全內反射螢光顯微系統..............................15
2-4-1 系統架構............................................15
2-4-2 儀器設備介紹.........................................17
2-5 單分子追蹤技術.........................................20
第三章 實驗方法與設計.......................................22
3-1 螢光影像實驗注意事項與準備工作...........................22
3-2 實驗材料...............................................22
3-2-1螢光分子-量子點.......................................22
3-2-2兩端不同之修飾 biotin -DNA-Amine 的製備方法.............23
3-2-3波片之清洗............................................23
3-2-4微流道之製作..........................................25
3-3 實驗設計...............................................25
3-3-1 DNA固化於玻片表面之方法...............................26
3-4 以單分子技術觀察DNA....................................28
3-4-1單分子影像擷取方法.....................................28
3-4-2單分子追蹤方法........................................28
3-4-3系統飄移.............................................29
第四章 實驗結果與討論.......................................31
4-1 固化於表面之單分子螢光影像訊號...........................31
4-2 dsDNA以BSA-biotin方式固化於之動態行為...................33
4-2-1 dsDNA動態行為之Single-State與Multi- states...........34
4-2-2 Single-state........................................39
4-2-3 Multi-states........................................43
4-3 BSA-biotin表面修飾對dsDNA之影響.........................45
4-4利用PEG固化方式排除multi-states現象......................47
4-5系統量測之優化.........................................56 第五章 結論與未來展望......................................60
參考文獻..................................................61

                                

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