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學校名稱輔仁大學
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學號493336082
研究生(中)蕭智弘
研究生(英)Hsiao Chih Hung
論文名稱(中)高效能電泳晶片之研究：速度及效率系統之改善
論文名稱(英)The Study of High Performance Microchip Capillary Electrophoresis：Enhancement of Speed and Efficiency
其他題名
指導教授(中)陳壽椿
指導教授(英)Chen Shou Chun
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電子全文送交國圖.同意
國圖全文開放日期.2007.02.10
檔案說明電子全文
電子全文01
學位類別碩士
畢業學年度94
出版年
語文別中文
關鍵字(中)電泳晶片、安培法偵測、碳纖維電極、神經傳導物質、表面修飾、高分子電解質、羥丙基纖維素、pH修飾、抑制擴散。
關鍵字(英)Electrophoresis microchip; Amperometric detection; Carbon fiber electrode; Neurotransmitters; Surface modification; Polyelectrol
摘要(中)高效能電泳晶片之研究：速度及效率系統之改善 摘要 微電泳晶片的製作、應用及微流道修飾一直以來都是本實驗室研究的重點。微型化學分析系統(m-TAS)不但具有價廉、靈敏、快速及溶劑低耗量等優點，在結合高選擇性與高靈敏電化學後還具備有晶片實驗室(Lab-on-a-chip)的條件。 有鑑於微電泳晶片檢測系統的優越性，如將其應用在講求時效性的疾病診斷方面，將有很大的幫助。本論文將針對DNA組成份中的鹼基片段進行探討，結合2公分短流道晶片及間接安培法進行偵測與分離，並探討電活性試劑的選擇，以達到系統最佳化的選擇。 在改善晶片的解析度與功能性方面，第一部份：成功將碳纖維(7 μm)材質移轉於微電泳晶片上分離神經傳導物質，也提高晶片使用壽命與偵測樣品多元化。第二部份：藉由流道表面的改植而邁向快速分離與抑制樣品擴散的目標。據文獻指出，在溶液中添加微量高分子可大幅改變流變性其拖曳現象能降低擾流達80 %，間接改善樣品於電泳中的擴散。所以此部份主要是研究添加微量高分子對電泳分離的效應，探討微電泳晶片流道經過修飾後，對分析樣品滯留時間、訊號?寬、解析度等影響，接著由逆相層析管柱加尾罩(endcap)的概念進而添加小分子試劑於高分子中來改善管柱的平衡，希冀藉此來提昇晶片使用效能，目前發現以羥丙基纖維素結合丙三醇來改質管柱有不錯的效果。第三部分藉由探討不同pH溶液修飾微流道管壁，來觀察其表面帶電荷及電滲透流(EOF)所受影響，進而改善樣品分析效果，以達快速分離的目標。
摘要(英)Abstract The merits inherent with micro capillary electrophoresis of sensitivity, speed and versatility have echoed through the years. Its potential to annex the necessary functional parts for the utopian aim of Lab-on-a-chip is of particular interest to all the workers. Ironically, the tongues that spread the gospels also demand it to be fully-fledged for routine use. Its coupling with amperometric detection is sensitive, which is beneficial to applications in bioanalysis. However, its innate selectivity impedes the detection of the base fragments from DNA, for example, which calls for indirect amperometric detection. In addition, this investigation employs a 2-cm chip to shorten the analysis to a couple of minutes, as opposed to 20 to 30 minutes described in the literature. The study falls into three parts：the use of 7-micron carbon fiber as the electrode to extend its service life and increase sensitivity；surface modification with a trace amount of polyelectrolyte to adjust the flow rheology and to suppress solute diffusion in the channel；modification of surface pH to accelerate the migration and to escalate its resolution.
論文目次目錄 中文摘要……………………………………………………………………......Ⅰ 英文摘要………………………………………………………………………..Ⅱ 目錄……………………………………………………………………...……...Ⅲ 圖目錄……………………………………………………………...…...............Ⅵ 表目錄……………………………………………………..……….…………...Ⅹ 第一章 緒論…………………………………………….……………….........…1 1-1 研究背景與目的………………………………………………..….......…1 1-2 毛細管電泳技術的探討………………………………………………….3 1-2-1 毛細管電泳發展的歷史.……………………………………………3 1-2-2 毛細管電泳之基本組成.……………………………………………6 1-2-3 毛細管電泳分離原理….……………………………………………7 1-2-4 毛細管電泳電泳的分離模式…………………………………...…11 1-2-5 毛細管電泳常用之偵測方式…………………………………...…14 1-3 DNA介紹……………………………………………………………......18 1-3-1 DNA組成………………………………………………………...…18 1-3-2 DNA檢測方法…………………………………………………...…19 1-4 晶片微流道修飾的探討…………………………………………...........21 第二章 實驗部份…………………………………………………………...….23 2-1 微電泳晶片之微機電製程……………………………………………...23 2-1-1 製程設備………………………………………………………...…23 2-1-2 材料與試劑……………………………………………………...…24 2-1-3 微電泳晶片製作流程…………………………………………...…25 2-1-3-1 流道基板製作……………………………………………...….25 2-1-3-2 感應電極基板製作………………………………………...….29 2-1-4 高溫熔融接合…………………………………………………...…30 2-1-5 微電泳晶片組裝………………………………………………...…31 2-1-6 外接式電極晶片………………………………………………...…32 2-1-6-1 一般金屬電極晶片……………………………………………32 2-1-6-2 碳纖維電極晶片………………………………………………34 2-2 微電泳晶片實驗………………………………………………………...36 2-2-1 儀器設備………………………………………………………...…36 2-2-2 試劑……………………………………………………………...…38 2-2-3 實驗操作………………………………………………………...…41 2-2-3-1 微電泳晶片之清洗步驟與維護…………………………...….41 2-2-3-2 微電泳晶片進樣與分離…………………………………...….42 2-2-3-3 微電泳晶片效能檢測……………………………………...….43 第三章 結果與討論………………………………………………………...….48 3-1 碳纖維電極之電泳晶片………………………………………………...48 3-1-1 碳纖維電極效能檢測…………………………………………...…48 3-1-2 神經傳導物質於碳纖維電極晶片檢測………………………...…49 3-2 微電泳晶片檢測與分離DNA鹼基片段……………………………….50 3-2-1 DNA之電化學探討……………………………………………...…50 3-2-2 電活性試劑濃度的探討………………………………………...…51 3-2-3 DNA偵測與分離於膠體電泳槽………………………………...…53 3-2-4 DNA偵測與分離於微電泳晶片………………………………...…54 3-3 微電泳晶片流道改質的研究…………………………….…………......56 3-3-1 樣品電流訊號隨時間變化的探討……………………….……..…56 3-3-2 高分子羥丙基纖維素對樣品影響之研究……………………...…58 3-3-2-1 HPC對樣品峰寬與解析度的影響…………………………….59 3-3-2-2 HPC穩定性的探討…………………………………………….61 3-3-2-3 HPC結合小分子試劑及最佳化的探討……………….…...….62 3-4 酸鹼pH修飾管壁之探討……………………………………….…...….65 3-4-1 修飾晶片穩定性探討…………………………………...................66 3-4-2 修飾時間對樣品滯留的影響…………………………………...…68 3-4-3 不同pH修飾對管壁之影響……………………………………….69 第四章 結論………………………………………………………………...….70 第五章 參考文獻…………………………………………………………...….72 圖目錄 圖1-1. 傳統毛細管電泳系統………………………………………………………...6 圖1-2. 毛細管內壁的電雙層結構與Zata電位的分佈.……...……………………...8 圖1-3. 流體壓力推動與電驅動平面流動示意圖…………………………………...9 圖1-4. 毛細管電泳分離系統中粒子移動速度與電滲透流及電泳動之關係圖….10 圖1-5. 毛細管區帶電泳法示意圖………………………………………………….11 圖1-6. 毛細管等速電泳法示意圖………………………………………………….12 圖1-7. 毛細管等電聚焦示意圖…………………………………………………….13 圖1-8. 雷射誘導螢光偵測法裝置簡圖…………………………………………….15 圖1-9. 毛細管電泳-質譜儀離子化裝置界面模型…………………………………16 圖1-10. 結合直接與間接安培法偵測兒茶酚纇與胜?…...………………………17 圖1-11. DNA的構造……………………………..….…………………….………...18 ?1-12. DNA之安培法感測機制……………………………..…………………….20 圖2-1. 熱蒸鍍系統示意圖………………………………………………………….26 圖2-2. 顯影成效圖………………………………………………………………….27 圖2-3. 微流道基板的蝕刻圖……………………………………………………….27 圖2-4. 表面輪廓儀量測其深寬值………………………………………………….28 圖2-5. 微放電加工穿孔系統配置示意圖………………………………………….28 圖2-6. 微放電加工所得之?道通孔……………………………………………….29 圖2-7. 蒸鍍後的微感應電極基板…………………………………………...……..29 圖2-8. 高溫熔融接合方式示意圖………………………………………………….30 圖2-9. 微感應電極與微流道之相關位置………………………………………….31 圖2-10. 微電泳晶片截面圖外接式金屬電極：(A)佈放前；(B)佈放後……………33 圖2-11. 線上佈放電極晶片的示意圖，流道為5公分直線型……………………..33 圖2-12. 在不同的支持電解質中，三種形式電極的電位範圍…………………….34 圖2-13. 顯微鏡下的碳纖維電極俯視圖…………………………………………...35 圖2-14. 微電泳晶片結合電化學檢測系統概圖…………………………………...37 圖2-15. 微電泳晶片及實驗用平台結構…………………………………………...37 圖2-16. MES結構圖………………………………………………………………...39 圖2-17. 標的分析物結構…………………………………………………………...39 圖2-18. 電活性試劑結構……………………………………………………..…….40 圖2-19. 微流道修飾劑……………………………………………………………...40 圖2-20. 十公分繞線微電泳晶片設計圖……………………………………….…..41 圖2-21. 微電泳晶片進樣與分離模式示意圖……………………………………...42 圖2-22. 分析物多巴胺和兒茶酚的氧化還原反應機制…………………………...43 圖2-23. 標的分析物多巴胺與兒茶酚之循環伏安法測試圖……………………...44 圖2-24. 標的分析物5-羥基引朵醋酸之循環伏安法測試圖……………………...45 圖2-25. 微電泳晶片結合白金電極效能測試圖…………………………………...46 圖2-26. 微電泳晶片分離電壓與樣品滯留時間的關係圖………………………...47 圖3-1. 線上碳纖維電極晶片K3Fe(CN)63- 的循環伏安法測試圖………………...48 圖3-2. 微電泳晶片結合碳纖維電極效能測試圖………………………………….49 圖3-3. DNA 100 bp marker分析物循環伏安法測試圖……………...……………..50 圖3-4. 微電泳晶片結合間接電化學偵測電活性試劑Ru(bpy)32+在不同濃度的電泳圖…………………………………………………………………………………..51 圖3-5. Ru(bpy)32+循環伏安圖………………………………………………….……52 圖3-6. Ru(bpy)32+不同掃描速率循環伏安圖………………………..……..….……52 圖3-7. DNA平板膠體電泳圖…..……………………………………………….…..53 圖3-8. 微電泳晶片結合間接電化學偵測DNA，在不同分離電壓的電泳圖……..54 圖3-9. 多巴胺和兒茶酚電流訊號隨時間改變的趨勢(系統變數)………………..57 圖3-10. 多巴胺對兒茶酚電流訊號比值隨時間改變的趨勢(系統變數)..………..57 圖3-11. 多巴胺和兒茶酚電流訊號隨時間改變的趨勢(環境變數)………...….….57 圖3-12. 多巴胺和兒茶酚電流訊號比值隨時間改變的趨勢(環境變數)……..…..57 圖3-13. 多巴胺和兒茶酚層析峰半高寬隨HPC濃度變化的趨勢……………..…60 圖3-14. 多巴胺和兒茶酚解析度隨HPC濃度變化的趨勢…..………...............….60 圖3-15. 多巴胺和兒茶酚層析峰半高寬隨實驗時間的變化…………………...…61 圖3-16. 多巴胺和兒茶酚解析度隨添加試劑變化的趨勢………………………...62 圖3-17. 多巴胺和兒茶酚隨不同管柱修飾劑添加變化之層析圖………...............63 圖3-18. 訊號峰不對稱因子計算示意圖…………………………………………...64 圖3-19. 不同清洗方法對管壁修飾影響的電泳圖………………………………...66 圖3-20. 不同pH溶液修飾管壁對樣品滯留時間的影響………………………….67 圖3-21. 不同pH溶液修飾管壁對EOF的影響…………………………………….67 圖3-22. 0.1 M NaOH不同修飾時間對樣品滯留時間……………………………...68 圖3-23. 0.1 M NaOH不同修飾時間與EOF關係…………………………………..68 圖3-24. 多巴胺和兒茶酚滯留時間隨實驗長時間進行下的變化，探討pH改質的穩定性………………………………………………………………………………..69 表目錄 表1-1. HPCE的各種方法與其分離依據…………………………………….…...11 表1-2. 毛細管電泳之檢測方法…………………………………………………..14 表1-3. 永久性的管壁改良修劑……………..…………………………………....21 表1-4. 動力學去活化方法………………………………………………………..22 表2-1. 外接式電極晶片製作流程圖…………………………………………..…32 表2-2. 短流道微電泳晶片其分離電壓對神經傳導物質的影響…………….….47 表3-1. 微電泳晶片間接電化學偵測法分析DNA鹼基片段之操作條件……....55 表3-2. HPC濃度改變對多巴胺的影響。(HPC未經過濾)…………………….…59 表3-3. 多巴胺和兒茶酚在各種修飾劑改質下，其HPW、N、Rs的變化，使用不同分離電壓1 kV、2 kV來做比較………………………………..……………..63 表3-4. 晶片修飾前後訊號峰不對稱因子的改變………………………………..64 表3-5. 不同pH溶液修飾下對三種離子陽離子、中性離子、陰離子的影響…….67
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