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研究生：	謝耀郁 Hsieh, Yao-Yu
論文名稱：	侷限於次微米薄膜內DNA分子之泳動及鬆弛行為以及蒸發作用所致之DNA組裝 Motion and Relaxation Behavior of DNA Molecules Confined within Submicron Film and Evaporation-Driven DNA Assembly
指導教授：	魏憲鴻 Wei, Hsien-Hung
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	189
中文關鍵詞：	電泳、侷限效應界面薄膜、DNA拉伸以及鬆弛、表面蠕動、界面活性劑鉤附作用、自組裝、極化
外文關鍵詞：	polarization, interfacial confinement, DNA stretching and relaxation, electrophoresis, anchoring by surface surfactants, surface reptation, self-assembly
相關次數：	點閱：84 下載：2
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本論文在微尺度下以實驗研究DNA分子運動情形。本論文可分成四部份。前三部份(第三，第四以及第五章)探討以電場驅動DNA分子以及其動力行為。我們在微流道內製造一個幾乎貼近管壁的扁長型油珠或氣泡，藉此來產生一個次微米薄膜對DNA分子造成阻礙以及侷限效應，由此效應我們觀察DNA的形態變化和泳動行為。最後一部份(第六章)，我們以蒸發溶液驅使DNA分子沉積和自組裝。
本論文第三章，我們藉由產生一個幾乎貼近管壁的扁長型油珠來研究DNA分子被侷限在此薄膜內之運動情形。我們首先以電場實現DNA之拉伸，並且發現拉伸DNA分子因為與基板表面之界面活性劑鉤附而呈現不同型態。此外，我們藉由測量DNA分子的鬆弛時間來探討DNA大小和基板表面條件如何影響其鬆弛動力。
本論文第四章，我們使用相同於第三章的實驗架構。而此部分是以直流電場驅動DNA進入薄膜後並馬上切換至交流電場，我們發現這些DNA分子會往其載入的反方向泳動並有像是蠕動的行為。我們測量泳動速度的時間平均，其結果顯示較大的DNA分子泳動較慢於小DNA分子，這樣的非零時間平均之泳動現象並不是來自於介電泳，然而，他們可能來自於電雙層中相反電荷離子之電場誘發極化效應所產生的淨偶極電荷，並且產生非零時間平均之庫倫力。
本論文第五章，我們延續第三章的實驗架構來產生兩個不同的系統。第一個系統，我們添加帶高正電之奈米片板或次微米膠體粒子於DNA溶液中，並觀察DNA分子於電場下受這些粒子影響的行為。我們觀察到DNA分子不僅會更靠近，而且會與奈米片板形成聚集。這個現象可能來自於類似同性電相吸引作用造成奈米片板吸附在帶負電的DNA上。如同膠體粒子的效應我們發現DNA分子可被鉤附於奈米片板上，並於通過薄膜後有拉伸、纏繞的行為。第二個系統中，我們以氣泡取代油珠來觀察DNA分子穿過或是繞過氣泡邊緣的現象。
本論文第六章，我們將DNA液珠置於基材上，並觀察DNA分子於蒸發後的沉積和組裝現象。當氣泡完全蒸發並退水後，我們觀察DNA分子於基材上形成環狀沉積。甚至於特定基材以及界面活性劑濃度條件下，DNA於此環狀沉積內有聚集成分子束的情形。
本論文描述由上述各種效應所產生極為豐富的DNA分子之運動行為，這些現象包含因為侷限效應造成DNA分子構形的改變 (第三、第四以及第五章)、DNA分子與界面活性劑或膠體粒子之間複雜的交互作用(第三、第四、第五以及第六章)、DNA分子極化效應造成不尋常的泳動(第四章)、DNA分子的自組裝(第六章)。藉由以上的發現，我們提供了DNA的分離、單一高分子的操控技術以及巨分子的直接組裝等應用上的可能性。

Abstract
In this thesis, we conduct experimental study on the motion of DNA molecules at microscales. The main body of this thesis consists of four parts. The first three parts (Chapters 3,4, and 5) are devoted to the electric-field-driven motion of DNA molecules and their chain dynamics. In this case, we use closely fitting oil slugs or air bubbles in microchannels to create confinement effects or obstacles to DNA molecules. We observe how DNA molecules deform and drift under the influence of these effects. In the last part (Chapter 6), we report evaporation-driven deposition and self assembly phenomena of DNA molecules.
In Chapter 3, we study the motion of DNA molecules confined within the film created by a closely fitting oil slug. We first reveal stretching of these confined DNA molecules by electric fields. We also find that stretched DNA molecules can exhibit various chain configurations due to their anchoring to surface surfactants. In addition, we measure the relaxation time of these DNA molecules and discuss how their relaxation dynamics are influenced by the size of DNA and surface conditions.
In Chapter 4, we employ the same setup as in Chapter 3. Here DNA molecules are subjected to an ac field right after being injected into the film with a dc field. We find, surprisingly, that these DNAs can drift toward the injection end of the slug and exhibit reptation-like behavior. The time-averaged drift velocity is also measured. The result reveals that larger DNAs drift slower than smaller ones. Such non-vanishing time-averaged phenomena are not due to dielectrophoresis. Rather, they could be resulted from the net dipole charge due to field-induced polarization of counterions within the electric double layer and hence from the non-vanishing time-averaged Coulomb force created by this charge.
In Chapter 5, we extend our setup in Chapter 3 to two different systems. In the first system, we add highly positively-charged Nano Silicate Platelets (NSP) or submicron colloids to the DNA solution and observe how DNA molecules behave in electric fields under the influence of these particles. In the presence of NSP, we observe that DNA molecules not only become more compact but also form aggregates with NSP. The phenomena are perhaps attributed to like-charge attraction rising from the absorption of NSP onto negatively charged DNA. As for the effect of colloidal particles we find that DNA molecules can be hooked by the particles, undergo stretching, or exhibit reptation as moving through the film. In the second system, instead of oil slug, we employ an air bubble to observe the dynamics of DNA molecules when they are crossing or passing around the bubble.
In Chapter 6, we place a droplet of a DNA solution on a substrate and observe how DNA molecules are deposited or assembled on the substrate when the droplet is evaporating. We observe that DNA molecules can forms rings after droplet retreats and is completed evaporated, inside these rings, DNA molecules can aggregate into bundles, depending on the condition of the substrate and the surfactant concentration.
This thesis basically reveals rich phenomena of DNA molecules due to various effects. These phenomena involve conformation changes of DNA due to confinement effects (Chapters 3, 4, and 5), complex interactions between DNA and other substances such as surfactants and colloids (Chapters 3, 4, 5 and 6), the unusual drift of DNA due to polarization effects (Chapter 4), and assembly of DNA molecules (Chapter 6). Our finding could have potential applications in DNA fractionation, single-molecule manipulation of polymers, and direct assembly of macromolecules.

目錄
摘要…………………………………………………………………………… ………i
Abstract…………………………………………………………………………...…..iii
致謝………………………………………………………………………………...… .v
目錄…………………………………………………………………………… ….......vi
表目錄……………………………………………………………………….… …......xi
圖目錄………………………………………………………………………...……...xii
符號說明………………………………………………………………………..... .xxiii
第一章緒論................................ ................................ ................................ ......... i
1.1 研究背景................................ ................................ ................................ ... 1
1.2 DNA 分子結構................................ ................................ .......................... 2
1.3 文獻回顧................................ ................................ ................................ ... 4
1.3.1 電泳分離DNA 之應用................................ ................................ ... 4
1.3.2 DNA 拉伸、分子梳以及鬆弛時間之研究................................ ....... 5
1.4 研究動機................................ ................................ ................................ ... 8
第二章基本原理................................ ................................ ................................ ....19
2.1 電雙層(Electrical double layer) ................................ ................................ . 19
2.2 電泳(electrophoresis) ................................ ................................ ................ 21
2.3 電滲流(Electro-osmosis, EO) ................................ ................................ .... 22
2.4 DNA 高分子特性(Static Properties of DNA Polymers) ............................. 23
2.5 DNA 分子電泳................................ ................................ .......................... 25
2.6 界面活性劑................................ ................................ ............................... 26
vii
第三章侷限DNA 分子鏈於次微米薄膜內之鬆弛現象................................ ......33
3.1 延展DNA 分子鏈的鬆弛現象................................ ................................ . 34
3.2 實驗................................ ................................ ................................ .......... 36
3.2.1 微流道裝置................................ ................................ ................... 36
3.2.2 實驗溶液................................ ................................ ....................... 37
3.2.3 硬體架構................................ ................................ ....................... 39
3.2.4 實驗步驟................................ ................................ ....................... 39
3.2.5 相關實驗細節................................ ................................ ................. 41
3.2.6 影像處理軟體與拍攝................................ ................................ .... 43
3.2.6.A 影像擷取軟體Image-Pro Plus 之介紹................................ 43
3.2.6.B 曝光時間(Exposure time)的選擇................................ ......... 44
3.2.6.C 100 倍數物鏡之觀測方法................................ ................... 45
3.3 實驗觀察與紀錄................................ ................................ ....................... 47
3.3.1 侷限DNA 分子於薄膜內的鬆弛行為之觀測............................... 47
3.3.2 DNA 鬆弛時間(relaxation time)之量測................................ .......... 50
3.4 實驗結果與討論................................ ................................ ...................... 51
3.4.1 界面活性劑的作用與影響................................ ............................ 51
3.4.2 DNA 鬆弛時間(relaxation time)之測量結果................................ .. 53
3.4.3 DNA 鬆弛機制的推導................................ ................................ ... 55
3.4.3A 完全不受界面活性劑影響之鬆弛過程............................... 56
3.4.3B 受界面活性劑鉤連、不受界面活性劑拖曳之鬆弛過程..... 58
3.4.3C DNA 受界面活性劑鉤連以及拖曳之鬆弛過程................... 59
3.4.3D 總結三種鬆弛機制並與實驗結果比較.............................. 60
3.5 結論................................ ................................ ................................ .......... 62
第四章 DNA 以電泳方式進入次微米薄膜層後於AC 電場下運動行為之探討91
4.1 DNA 分子於AC 電場下的極化現象................................ ....................... 92
viii
4.2 實驗................................ ................................ ................................ .......... 92
4.2.1 實驗溶液................................ ................................ ....................... 92
4.2.2 硬體架構................................ ................................ ........................ 94
4.2.3 實驗步驟................................ ................................ ....................... 94
4.2.4 相關實驗細節................................ ................................ ................. 95
4.3 DNA 於次微米薄膜內受AC 電場作用運動行為之觀測......................... 96
4.3.1 以氧電漿改質玻璃表面1 hr 並於薄膜內觀察DNA 分子之泳動. 96
4.3.2 以氧電漿改質玻璃表面3 hr 並於薄膜內觀察DNA 分子之泳動. 97
4.3.3 觀測時間效應................................ ................................ ................. 98
4.3.4 實驗討論與結果................................ ................................ ............. 98
4.4 DNA 於DC、AC 電場切換下之泳動機制................................ .............100
4.5 驗證DNA 之極化作用................................ ................................ ............101
4.5.1 改變緩衝溶液對於DNA 移動速度之影響................................ ...101
4.5.2 改變AC 電場強度，對於DNA 淨移動速度之影響...................102
4.5.3 改變載入DC 電壓對於DNA 淨移動速度之影響.......................103
4.5.4 以直流電將DNA 載入薄膜後，靜置一段時間..........................103
4.6 將不同大小DNA 分子混合並於AC 電場下淨移動之觀測...................104
4.7 結論................................ ................................ ................................ ..........104
第五章結合液-液界面及奈米/次微米粒子所形成的侷限空間，對DNA 泳動行為
之探討................................ ................................ ................................ ........123
5.1 實驗................................ ................................ ................................ .........124
5.1.1 實驗溶液................................ ................................ ......................124
5.1.2 實驗步驟................................ ................................ ......................125
5.1.3 相關實驗細節................................ ................................ ...............127
5.2 添加膠體或奈米粒子對侷限於次微米薄膜內DNA 動態泳動之影響..127
5.2.1 添加膠體粒子的影響................................ ................................ ...127
ix
5.2.2 添加帶正電奈米片板粒子的影響................................ ................128
5.3 泳動DNA 分子穿越微米氣泡之現象................................ ....................129
5.4 結論................................ ................................ ................................ ..........130
第六章以蒸發方式驅動DNA 分子並結合與界面活性劑作用之沉積與組裝現象
................................ ................................ ................................ ...................137
6.1 DNA 於玻璃表面之沉積現象................................ ................................ .137
6.2 實驗................................ ................................ ................................ .........138
6.2.1 實驗溶液................................ ................................ ......................138
6.2.2 硬體架構................................ ................................ ......................139
6.2.3 實驗步驟................................ ................................ ......................139
6.2.4 相關實驗細節................................ ................................ ................139
6.3 探討添加界面活性劑以及氧電漿處理對表面DNA 沉積作用的影響..139
6.3.1 DNA 分子於親水性以及疏水性表面型態之觀測.....................140
6.3.2 實驗結果與討論................................ ................................ ...........141
6.4 結論................................ ................................ ................................ .........142
第七章結論、未來展望................................ ................................ ......................148
7.1 結論................................ ................................ ................................ .........148
7.2 未來展望................................ ................................ ................................ .149
參考文獻................................ ................................ ................................ ...............151
附錄A................................ ................................ ................................ ...................157
A.1 光罩設計................................ ................................ ................................157
A.2 光微影 (Photolithography) 製程................................ ...........................158
A.2.1 晶片清洗................................ ................................ .....................158
A.2.2 塗佈光阻 (Spin Coat)................................ ................................ ..158
A.2.3 軟烤 (Soft Bake) ................................ ................................ .........159
A.2.4 曝光 (Exposure) ................................ ................................ ..........160
x
A.2.5 曝後烤 (Post Expose Bake) ................................ .........................161
A.2.6 顯影 (Development) ................................ ................................ ....162
A.2.7 硬烤 (Hard Bake) ................................ ................................ ........162
A.2.8 測量厚度................................ ................................ .....................163
A.3 微流道製作................................ ................................ ............................163
A.3.1 材料................................ ................................ .............................163
A.3.2 微流道模型製作................................ ................................ ..........164
A.4 微流道裝置組裝................................ ................................ ....................165
A.4.1 接合................................ ................................ .............................165
A.4.2 管件組裝................................ ................................ .....................166
A.4.3 PDMS 表面改質................................ ................................ ...........166
A.5 實驗設備................................ ................................ ................................167
附錄B ................................ ................................ ................................ ...................176
附錄C ................................ ................................ ................................ ...................181
附錄D................................ ................................ ................................ ...................184
附錄E ................................ ................................ ................................ ...................186
自述................................ ................................ ................................ ...................189
                                    

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校內：2012-06-02公開
校外：2012-06-02公開

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