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博碩士論文 etd-0801107-210454 詳細資訊
Title page for etd-0801107-210454
論文名稱
Title
B-phycoerythrin 單分子在膠體溶液電泳環境之螢光行為
The fluorescence behavior of B-phycoerythrin single molecules in colloid surrounding
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
104
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2007-07-17
繳交日期
Date of Submission
2007-08-01
關鍵字
Keywords
螢光相關光譜、共焦顯微鏡、介電泳
dielectrophoretic, FCS, confocal microscope
統計
Statistics
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中文摘要
本論文研究單分子狀態之螢光分子運動,以及應用於電泳及介電泳之靈敏觀察。在無外加電場狀況下,我們觀測B-phycoerythrin在膠體溶液中之運動行為;接下來探討外加電場下所驅使帶電螢光分子B-phycoerythrin之驅動現象。
膠體溶液是藉著洋菜交連結構形成多孔性骨架,將溶液(水)包含在內,螢光分子也就可以在孔洞內自由活動,同時受到孔洞大小之限制,因此不會移出顯微鏡清楚聚焦範圍。實驗結果顯示,使用的螢光分子容易黏著於骨架上,僅有極少數分子可觀測到微米尺度的運動。我們記錄這些分子的運動狀態,及加以分析。
更進一步,我們利用電解方式製作微小電極結構。藉著曲率半徑為次微米尺度之電極尖端所造成之強大電場梯度,藉此克服分子在溶液中因熱產生之無秩序性布朗運動,藉以捕捉及控制微小介電物件,甚至到單一螢光分子。
Abstract
The thesis aims to study the motion of individual fluorescent molecules in the gel environments, and applies to the sensitive electrophoretic and dielectrophoretic studies. Firstly, dye molecules (Rhodamine B and DiI) are dispersed, and investigate the individual fluorescent spots. Later on, we study the motion of individual B-phycoerythrin molecules under external electrical field driving.
Due to the porosity, agarose gel is used to contain the liquid, and the dye molecules can freely move within the pores. Thus, one can easily observe the motion of individual dye molecules under high numerical aperture objectives. B-phycoerythrin is chosen for the high extinction coefficient, native charge, and good fluorescent properties. Our results indicate most dye molecules are attached in the rigid structure of the gel. Only very limited molecular motions are observed.
Moreover, we study the dielectrophoretic interaction of the dye molecules. Nano-electrodes are fabricated by electrolysis to have sub-micron aperture silver tips. Due to the high gradient of the electric field, it is used to have strong enough attractive forces around the apex of the tip, to overcome the thermal fluctuations. It allows us further trapping and manipulating small non-charged objects, up to single dye molecules.
目次 Table of Contents
中文摘要....................................................................................................i
英文摘要………………………………………………………………...ii
誌謝……………………………………………………………………..iii
目錄...........................................................................................................iv
圖目錄......................................................................................................vii
第一章 緒論........................................................................................1
第二章 實驗原理......................................................................................3
2.1 節 : 電泳的基本原理...................................................3
2.2 節 : 介電泳力之產生原理...........................................5
2.3 節 : 產生介電泳現象的差異比較...............................8
2.4 節 : 介電泳力的情況下分子大小之侷限...................9
2.4-1節:利用介電泳力將溶液中的膠體
濃縮至某個區域…………………………………9
2.4-2節:單一分子被侷限之研究概況……………………..10
2.4-3節:粒子分離…………………………………………..12
2.4-4節:影響介電泳之
其他因素-布朗運動、導電度、對流性…………12
2.5節:粒子在介電泳動之觀察……………………………..14
2.6節:螢光及磷光簡介…………………….…………..15
2.7節:發生螢光的現象.....................................................17
2.8節:螢光量子效率……….…..……………..………...20
2.9節:螢光生命期…........................................................21
2.10節:激發光譜與放光光譜……….…………………..21
2.11節:螢光量測架構…………………………………..22
2.12節:螢光相關光譜(FCS)簡介……………………….23
2.13節:螢光相關光譜(FCS)工作原理………………….24
2.14節:螢光相關光譜(FCS)之應用...................................25
第三章 實驗架構及樣品..................................................................27
3.1 節 : 實驗架構..............................................................27
3.2 節 : 實驗樣品..............................................................28
第四章 實驗方法及步驟..................................................................32
4.1 節 : 實驗方法..............................................................32
第五章 實驗結果與未來展望……..................................................40
5.1 節:B-phycoerythrin在膠體溶液之實驗結果………40
5.2 節:Ag針尖電極之製作結果與討論…………….......51
5.3 節:FCS實驗結果與討論…………………………….55
5.4 節:結論與未來展望…………..…………………….84
參考文獻.................................................................................................87
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