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研究生:劉貞汝
研究生(外文):Chen-Ju Liu
論文名稱:結合圓柱陣列與漸擴微流道以電泳分離DNA之研究
論文名稱(外文):DNA Separation by Electrophoresis in Microchannel with Expansion and Sparse Post Arrays
指導教授:謝之真
指導教授(外文):Chih-Chen Hsieh
口試日期:2017-07-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:122
中文關鍵詞:DNA電泳分離漸擴微流道圓柱陣列
外文關鍵詞:DNA electrophoresismicroexpansionpost arrays
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過去的研究中,採用具有六角圓柱陣列的恆截面(constant cross-sectional area)微流道分離大片段的DNA,在高電場下往往會受限於通道效應的影響,使得DNA與圓柱陣列的碰撞機率不高,無法使分離效率提升。本研究結合了過去文獻中用於拉伸DNA的漸擴漸縮微流道以及用於分離DNA之圓柱陣列,希望能藉由DNA在漸擴區受y方向電場梯度的影響進行拉伸、增加其在y方向的投影面積,得以改善高電場下不利碰撞的分子型態、提高在高電場下的分離解析度。我們先利用電腦模擬T4及λ-DNA在具有圓柱陣列之漸擴微流道中的電泳行為及分離解析度,測試不同設計並取最可行的進行實驗。我們使用微影製程及電漿蝕刻在熔融石英基板上製作包含圓柱陣列之微流道,其尺寸皆與模擬相符。我們藉由單分子影像觀察證實DNA的確會進行y方向的延展拉伸,與模擬的預期相符,同時T4 DNA及λ-DNA的移動路徑也與模擬路徑一致。在電場50V/cm下,經歷分離長度10mm後,能達到1.08的分離解析度,在各電場下之分離結果也都能達到基線分離解析度。同時經由實驗驗證的方式,我們亦探討DNA走不同路徑下以及其在實驗過程中產生的碎片對分離解析度的影響。本實驗中藉由與過去文獻中,在恆截面六角圓柱陣列微流道施加連續式及間歇式電場的分離結果比較,我們發現漸擴微流道能在保有一定分離解析度的前提下,大幅縮減分離所需時間。同時,我們將實驗結果與模擬的結果相互比較,發現T4 DNA在近似電場下的脫鉤時間、分子移動路徑以及DNA移動時間與電場的關係等,皆與模擬所預測高度相似。儘管實驗中仍有一些結果未如模擬預期,如DNA寬度分布之標準差的反轉以及兩者在分離解析度上的差異,但我們以現有的實驗數據推測產生誤差的原因,並試著以模擬或實驗的方式進行驗證。我們希望未來藉由持續地修正這些導致兩者產生誤差的因素,能使實驗的分離解析度大幅提升,亦使往後模擬的預測能更貼近真實實驗的結果。
致謝 I
摘要 II
Abstract IV
目錄 VI
圖目錄 IX
表目錄 XVIII
第 1 章 、緒論 1
1.1 前言 1
1.2 研究動機與目的 2
第 2 章 、文獻回顧 4
2.1 DNA介紹 4
2.1.1 DNA的化學結構 7
2.1.2 DNA的高分子性質 11
2.1.3 DNA與染劑分子 13
2.2 電動力學 15
2.2.1 電雙層 15
2.2.2 電泳 17
2.2.3 電滲流 18
2.3 分離原則 19
2.4 現行DNA分離技術 21
2.4.1 凝膠電泳(gel electrophoresis,GE) 22
2.4.2 脈衝場凝膠電泳(pulsed field gel electrophoresis,PFGE) 25
2.4.3 毛細管電泳(capillary electrophoresis,CE) 26
2.5 微流道電泳 27
2.5.1 微流體驅動力 28
2.5.2 以特殊結構微流道電泳分離DNA 29
2.6 電流體動力不穩定性(Electrohydrodynamic instability) 45
2.7 實驗設計構想 48
第 3 章 、設備、材料與方法 52
3.1 實驗設備 52
3.2 實驗材料 54
3.3.1 通道製作 56
3.3.2 溶液配置 65
3.3.3 電場施加裝置 68
3.3.4 結果收集與分析 71
第 4 章 、結果與討論 73
4.1 DNA於微流道中之動、靜態性質 73
4.1.1 鬆弛時間(Relaxation time) 73
4.1.2 特徵脫鉤時間(Unhooking time) 75
4.1.3 泳動率(Mobility) 78
4.1.4 無因次電場Pe 80
4.2 DNA在漸擴微流道中的分析 81
4.2.1 參照模擬結果選擇實驗參數 81
4.2.2 DNA的行為分析 82
4.3 結合圓柱陣列與漸擴微流道之分離結果 88
4.3.1 分離解析度 88
4.3.2 探討不同路徑下對分離解析度的影響 95
4.3.3 DNA碎片對分離解析度的影響 98
4.3.4 與不同通道設計、電場施加模式之分離結果比較 102
4.4 討論實驗與模擬預期結果之相似、相異處 108
4.4.1 不同起始點對解析度之影響 112
第 5 章 、結論 116
第 6 章 、參考文獻 118
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