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研究生： 王蘭玉
Wang, Lan-Yu
論文名稱： 蛋白質分析之微全程分析生醫晶片系統之研發
Development of Micro Total Analysis System(μ-TAS) for Protein Analysis
指導教授： 陳淑慧
Chen, Shu-Hui
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2002
畢業學年度： 90
語文別： 中文
論文頁數： 132
中文關鍵詞： 固相萃取 、免疫分析 、蛋白質分析 、生醫晶片
外文關鍵詞： protein analysis, bio-chip, immunoassay, solid-phase extraction
相關次數： 點閱：102  下載：6 
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    • 本研究發展兩種類型之微全程分析生醫晶片系統用於蛋白質分析，這兩種微流體晶片的流體驅動原理都建立在本實驗室所研發的連續壓力流與電泳偶合微流體進樣技術上。

    第一類型之晶片系統整合了固相萃取與電泳分離模式。此整合型晶片系統可用於蛋白質分析前之預濃縮。將固相萃取之碳十八之卡匣連接微流體晶片。此整合型晶片將清洗/沖堤步驟與電泳分離和線上（on-line）偵測結合在一起。於研究結果顯示，其Cy5-BSA之校正曲線斜率為0.99以上，且濃縮程度可達一個級數以上。

    第二類型之晶片系統整合具有免疫反應之小珠與電泳分離模式，用於免疫分析前之蛋白質濃縮。此類型之整合裝置，具有一固定化抗體（第一抗體）卡匣連接微流體晶片。於研究結果顯示，沖堤出之抗原仍具有活性。利用此裝置，由第一抗體濃縮之抗原經由分離管道時可被第二抗體辨識進而鍵結。利用磷酸化酪胺酸-牛的血清蛋白（P-BSA）當測試模型，P-BSA經第一抗體（固定化anti-BSA）濃縮後，沖堤出之P-BSA被第二抗體（磷酸化酪胺酸抗體，anti-P）快速辨識。以上的結果可利用BSA當控制組（negative control）來比對驗證。傳統方法中利用免疫沈澱法，膠電泳分離並合併點漬法來偵測磷酸化蛋白質，其時間冗長且步驟繁雜。對於偵測磷酸化蛋白質利用此整合型微流體晶片為相當有力快速的偵測方式而且試劑耗損量少。

    In this study, we developed two kinds of integrated devices for protein analysis. These devices are based on the flow-through design of the microchip for the coupling of hydrodynamic and electrokinetic flows.

    The first device was designed to integrate the solid phase extraction and electrophoretic separation and was applied for protein pre-concentration prior to separation. A C18 micro-cartridge was connected to the hydrodynamic inlet of the microchip. Using this device, the wash/elution step can be integrated on-line with electrophoretic separation and detection on the microchip. Results indicated that the calibration curve of Cy5-BSA obtained from this integrated device has a R2 value greater than 0.99 and a concentration enhancement about one order of magnitude.

    The second device was designed to integrate the immunoreaction bed and electrophoretic separation and was applied for protein purification prior to immunoseparation. A first-antibody immobilized micro-cartridge was connected to the hydrodynamic inlet of the microchip. Results indicated that the eluted antigen still remained its activity. Using this device, the concentrated antigen from the first antibody bed was recognized by the second antibody in the separation channel via immunoseparation. Using BSA conjugated phosphotyrosine (P-BSA) as the test model; P-BSA was concentrated by the first antibody (anti-BSA) and then recognized by the second antibody, anti-phosphotyrosine antibody. The results were further confirmed for the comparing with that obtained using BSA as the negative control. The traditional method for detecting phosphoproteins is using immunoprecipitation followed by gel electrophoresis and southern blotting. This process is slow and time-consuming. This device has a great potential to be applied for rapid detection of specific phosphoproteins with a great reduction in the analysis time and sample consumption.

    致謝I 中文摘要II 英文摘要IV 目錄VI 表目錄XI 圖目錄XII 第一章 序論 1-1生醫晶片1 1-2微全程分析生醫晶片系統 (Micro Total System，m-TAS)2 1-3晶片上電場驅動流體之原理7 1-3-1電泳原理7 1-3-2晶片式電泳9 第二章 連續壓力流偶合微流體電泳晶片系統之架設 2-1研究動機10 2-2晶片的設計11 2-3卡匣(Cartridge) 12 2-4進樣模式…13 2-5電壓操控與壓力流模式之優點14 2-6連續壓力流偶合晶片式電泳系統模組15 2-7其它實驗所需使用之儀器與設備18 第三章 螢光染料之標示 3-1研究背景21 3-2螢光染料標示形式…21 3-2-1非共價性標示之螢光染料22 3-2-2共價性標示之螢光染料23 3-3實驗部分24 3-3-1藥品…24 3-3-2實驗方法24 3-4結果與討論27 3-4-1 Sypro Orange標示BSA之探討27 3-4-2 Cy5標示胜、蛋白質之探討28 3-4-3 定量分析之探討 31 3-4-4 蛋白質分析之CE方法與傳統膠電泳之比較 32 3-5結論32 第四章 具固相萃取與電泳分離之整合型晶片系統 4-1研究背景34 4-2實驗部分35 4-2-1藥品36 4-2-2實驗方法36 4-3結果與討論40 4-3-1卡匣與微流體晶片離線（Off-line）之探討40 4-3-2卡匣與微流體晶片線上（On-line）之探討 41 4-3-3卡匣與微流體晶片線上濃縮（On-line concentration） 之探討42 4-3-4校正曲線之探討42 4-4結論43 第五章免疫分析整合微流體晶片系統 5-1研究背景45 5-2研究目的46 5-3實驗部分47 5-3-1藥品47 5-3-2實驗方法48 5-4結果與討論56 5-4-1抗原與抗體之免疫反應56 5-4-2固相免疫分析法之探討（抗原與抗體之親和力）58 5-4-3一次抗體免疫分析之探討60 5-4-4一次抗體於晶片上免疫分析之探討61 5-4-5二次抗體於晶片上免疫分析之探討61 5-4-5-1 Cy5-BSA經anti-BSA agarose bead（第一抗體）後， 與第二抗體（anti-P）反應之探討63 5-4-5-2 Cy5-P-BSA經anti-BSA agarose bead（第一抗體）後， 與第二抗體（anti-P）反應之探討65 5-5結論67 第六章 結論與未來展望 6-1結論69 6-1-1共價性染料標示之胜與蛋白質69 6-1-2固相萃取之整合型微流體晶片70 6-1-3免疫分析之整合型微流體晶片71 6-2未來展望72 參考文獻74

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