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研究生:張皓婷
研究生(外文):Hao-Ting Chang
論文名稱:拉伸並固定DNA分子於電滲流微流道生醫晶片之研究
論文名稱(外文):Study of Stretching and Immobilizing DNA Molecules on Electro-osmotic Flow Microfabricated Fluidic Bio-chip
指導教授:林裕城林裕城引用關係
指導教授(外文):Yu-Cheng Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:奈米科技暨微系統工程研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:105
中文關鍵詞:微機電系統技術電滲流DNA 拉伸表面親疏水性改質
外文關鍵詞:Hydrophilic and Hydrophobic ModifiedMEMSEOFDNA Stretching
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本研究主要設計兩種生物晶片,分別是單一微流道與多重微流道晶片,藉由電滲流作用於不同的親疏水性表面,使DNA拉伸並固定於晶片上,改變傳統只能操作小分子DNA及複雜的蛋白修飾晶片表面來達到固定的效果。實驗晶片主要利用微機電系統技術(Micro-Electro-Mechanical-System, MEMS)製作雙層結構於晶片上(包含金電極層與SU- 8微流道層),搭配電滲流操作系統與螢光顯微鏡設備作為實驗平台。實驗主要分成三個部份來探討:第一部份是利用DNA本身呈親水性的特性,搭配不同親疏水性的晶片表面,達到較佳的固定DNA環境;第二部份是最佳電壓測試,測試電壓範圍為從0 V開始直到DNA被拉斷為止,電流保持1 mA,以得到拉伸極限電壓;第三部分是拉伸DNA在微流道中,搭配第一與第二部份測試出的拉伸環境進行實驗。實驗結果顯示,以丙酮清洗的晶片最能表現SU-8與玻璃晶片固定DNA的能力,而於單一微流道中施以20 V可拉伸DNA至230 μm;於多重微流道中,DNA會沿著流道拉伸100 μm。由本研究證明利用電滲流拉伸並固定在疏水性的晶片表面,可有效增加拉伸的長度並應用於大型生物分子的操作。
This study designs two types of DNA stretching biochips. One type is single channel microfluidic chip and the other one is microfluidic chip of multiple channels. DNA can be successfully stretched and immobilized on the surface of proposed microfluidic chips. The chip system is fabricated using Micro-Electro-Mechanical-Systems (MEMS) technology, and DNA is stretched utilizing mechanism of Electro-Osmotic Fluid (EOF). The chips were designed by two sides electrodes, between which the gap is 10 mm, which were located under the microchannel to drive EOF and increase the immobilize potential of biochip.The experiment includes three parts: first, the best voltage was determined to stretch DNA. The initial applied voltage is 5 V, and then increase voltage value until DNA break. Second, stretching DNA on the chip surface modified with different hydrophilic materials. Third, stretch DNA on single microfluidic channel chip and multiple channels. The results indicate that the best environment to stretch and immobilize DNA is that use 20 V to drive EOF with the chip washed by Acetone. The maximum stretching length on a single microchannel chip is 230 μm and that on microfluidic chip of multiple channels is 100 μm. The research successfully designed two microfabricated fluidic bio-chips for DNA stretching and immobilizing
目錄
中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 X
第一章 緒論 1
1-1 生物晶片 2
1-2 生物分子DNA介紹與操作 4
1-2-1 DNA操控 6
1-3 電滲流簡介 11
1-4 微流體生物晶片之發展 14
1-5 表面修飾 16
1-6 研究動機與目的 18
1-7 研究架構 19
1-8 論文架構 21
第二章 微流體晶片的設計與製作 22
2-1 微流體晶片設計 22
2-1-1 電極層設計 22
2-1-2 微流道層設計 24
2-1-3 承載晶片台設計 26
2-2 微流體晶片製作 28
2-2-1 電極製作 32
2-2-2 微流道製作 35
2-2-3 承載台製作 37
2-3 雙股DNA即時觀測系統 43
2-3-1 螢光顯微鏡原理 43
2-4 計算軟體(ImageJ)介紹 46
第三章 實驗與研究方法 52
3-1 實驗儀器 52
3-2 實驗材料與藥品 58
3-3 細胞培養 62
3-4 染色體DNA萃取 64
3-5 實驗方法 66
3-5-1 親水性晶片改質 66
3-5-2 疏水性晶片改質 67
3-5-3 單一流道拉伸實驗 67
3-5-4 多重流道拉伸實驗 68
第四章 結果與討論 69
4-1 晶片製作結果分析 69
4-2 DNA萃取結果分析 72
4-3 晶片改質分析 73
4-4 拉伸DNA於不同親疏水性晶片實驗 76
4-4-1 不同親疏水性晶片拉伸結果 76
4-5 DNA拉伸分析 79
4-5-1 單一流道拉伸結果 79
4-5-2 多重流道拉伸結果 88
第五章 結論 91
5-1 結論 91
5-2 建議 94
參考文獻 96
自述 105

圖目錄
圖1-1 利用電泳操作DNA結果圖 8
圖1-2 T-junction示意圖 8
圖1-3 拉伸T-4 DNA於T型微流道 9
圖1-4 利用奈米流道拉伸T5 phage DNA(103 kb = 35 μm)圖 10
圖1-5 DNA長度統計圖 10
圖1-6 外加電場於晶片中,流體的離子變化示意圖 12
圖1-7 電滲流拉伸DNA示意圖 13
圖1-8 研究架構圖 20
圖2-1 電極層設計圖 23
圖2-2 多重流道電極層設計圖 23
圖2-3 SU-8光罩設計與標示圖 24
圖2-4 金電極與SU-8微流道分解圖 25
圖2-5 多重電極設計圖 25
圖2-6 承載台設計圖 27
圖2-7 承載台分解圖 27
圖2-8 電極與微流道製程示意圖 36
圖2-9 雷射加工機 38
圖2-10 雷射加工系統 40
圖2-11 受激發物體電子能階變化示意圖 43
圖2-12 螢光顯微鏡光學原理示意圖 44
圖 2-13 雙向反射鏡構造 45
圖2-14 由即時影像擷取系統擷取的原始圖檔 47
圖2-15 將圖片改為16位元的圖檔與Image J增強對比值之功能表 47
圖2-16 使用ImageJ增強即時影像對比值圖 48
圖2-17 左圖為原圖檔的灰階值統計圖 48
圖2-18 ImageJ分析目錄中設定比例尺功能視窗圖 49
圖2-19 ImageJ校正比例尺與原始比例尺設定視窗圖 50
圖2-20 ImageJ量測DNA拉伸長度視窗圖 51
圖3-1 接觸角量測系統 54
圖3-2 紫外光-可見光分光光譜儀。 54
圖3-3 即時觀測系統圖 57
圖3-4 U-937細胞圖 65
圖4-1 金電極經微機電製程製作結果圖 69
圖4-2 單一微流道於SEM下觀測圖與α-step量測圖 70
圖4-3 多重微流道於光學顯微鏡及SEM下觀察圖 71
圖4-4 DNA紫外光可見光分光光譜儀量測結果圖 72
圖4-5 晶片表面親疏水性接觸角量測結果圖 74
圖4-6 以氧電漿修飾過的晶片表面拉伸與固定DNA結果 77
圖4-7 以奈米材料YCC-805修飾過的晶片表面拉伸與固定DNA結果 77
圖4-8 以丙酮修飾的晶片表面拉伸與固定DNA結果 78
圖4-9 電滲流拉伸DNA測試電壓圖 81
圖4-10 以5 V拉伸DNA實驗結果圖 84
圖4-11 以10 V拉伸DNA實驗結果圖 84
圖4-12 以15 V拉伸DNA實驗結果圖 85
圖4-13 以20 V拉伸DNA實驗結果圖 85
圖4-14 10 V、15 V與20 V拉伸長度統計圖 88
圖4-15 DNA於10 μm微流道中拉伸結果 90


表目錄
表2-1 雷射加工機規格 38
表4-1 不同親疏水性表面量測數據表 75
表4-2 10 V拉伸DNA長度量測表 86
表4-3 15 V拉伸DNA長度量測表 86
表4-4 20 V拉伸DNA長度量測表 87
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