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研究生： 許弼凱
Hsu, Bi-kei
論文名稱： 微流體電泳晶片之線上濃縮與蛋白質分離
Microchip Electrophoresis System with On Chip Protein Concentration and Separation Functions
指導教授： 陳淑慧
Chen, Shu-hui
學位類別： 碩士
Master
系所名稱： 工學院 - 奈米科技暨微系統工程研究所
Institute of Nanotechnology and Microsystems Engineering
論文出版年： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 93
中文關鍵詞： 微流體電泳晶片
外文關鍵詞： Microchip Electrophoresis System
相關次數： 點閱：103  下載：1 
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    • 中文摘要
    從生物組織中所分離出的蛋白質種類相當的多且複雜度高，但是蛋白質卻具有相當的潛力成為生物或疾病標誌物。但目前常用的蛋白質分離工具普遍存在著樣品消耗量大、分析時間長、不易與其他分析儀器結合等問題。這樣的平台將很難用來發展成檢驗用分析方法，而利用微機電製程發展的微流體晶片(Microfluidic Chip) 擁有著快速分析、微型化、易操作、高整合性及降低樣品與試劑的損耗等優點，因此近年來成為熱門的分析工具。
    本研究中我們利用十字形PMMA 6 cm 晶片，以電壓進樣的方式在分離管道六公分處做偵測，並使用膠電泳(Gel Electrophoresis)的模式，在電泳緩衝液中添加高分子聚合物HPMC(Hydroxypropyl methyl cellulose , Mw 12,000)，溶液中所形成的孔洞充當分子篩用來分離具有FITC染料標示的蛋白質標準品FITC-protein makers (Tripsin 20 kDa 、Carbonic anhydrase 29 kDa、Alcohol dehydrogenase 39 kDa、 BSA 66 kDa、β-Galactosidase 116 kDa 、Myosin 205 kDa)。
    另外，由於微流體晶片可容許的樣品體積少及蝕刻管道的光徑短，使得晶片式電泳以光學偵測法分析樣品時的偵測靈敏度通常無法達到實際要求。因此本研究中以等速電泳(isotachophoresis，ITP) 結合膠電泳(Gel Electrophoresis，GE)的模式，發展蛋白質線上濃縮晶片用來提升偵測靈敏度，經過蛋白質線上濃縮晶片所得到的蛋白質樣品訊號有著20~40倍的提升。

    Abstract
    The separation of specific proteins from complicated protein samples has become a major topic because the proteins isolated from biological tissues are in complex mixtures.However, proteins hold great potentials to become disease markers.Current analytical tools, nevertheless have drawbacks including large sample consumption, long analysis time, and are difficult to integrate with other instruments. Microchip electrophoresis has become one of the most powerful analytical tools in recent years due to its high throughput, miniaturization, ease of handling, ease of integration and low consumption of samples and reagents.
    In this study, we used a cross channel PMMA (polymethylmethacry- late) microfluidic chip with an effective separation length of 6 cm to separate FITC-labeled protein markers (Tripsin 20 kDa 、Carbonic anhydrase 29 kDa、Alcohol dehydrogenase 39 kDa、 BSA 66 kDa、β-Galactosidase 116 kDa 、Myosin 205 kDa). Samples were injected electrokinetically and polymers hydroxypropyl methyl cellulose , Mw 12,000 (HPMC) was added in buffers as molecular sieving matrix. Detection was performed at a distance of 6 cm from the cross. Gel electrophoresis on a microchip is gaining popularity and other polymers like HPMC are also being used instead of polyacrylamide or polyethylene glycol separation buffers. We use the sieving mode to separate FITC-protein makers ( Tripsin 20 kDa 、Carbonic anhydrase 29 kDa、Alcohol dehydrogenase 39 kDa、 BSA 66 kDa、β-Galactosidase 116 kDa 、Myosin 205 kDa)。
    Due to the low sample loadability and shallow depth of microchannels, the sensitivity of microchip electrophoresis is limited, especially when optical detector is used for detection. In the second study, we integrated isotachophoresis (ITP) and gel electrophoresis (GE) and developed an on-line concentration procedure for protein separation. The sensitivity was enhanced about 20-40-fold with the on line concentration procedure.

    目 錄 中文摘要------------------------------------------------------I 英文摘要----------------------------------------------------III 誌謝---------------------------------------------------------V 目錄--------------------------------------------------------VI 圖目錄------------------------------------------------------IX 第一章 序論 1.1 微小化生化分析系統---------------------------------------1 1.2 蛋白質分離-----------------------------------------------2 1.3 研究目的-------------------------------------------------3 第二章 微流體晶片 2.1 前言-----------------------------------------------------4 2.2 微流體晶片的優點-----------------------------------------5 2.3 微流體晶片的材質與修飾技術-------------------------------6 2.4 微流體晶片的材料選擇與設計-------------------------------7 2.5 PMMA微流體晶片的製造-------------------------------------8 第三章 利用微流體晶片分離蛋白質 3.1 前言----------------------------------------------------18 3.2 毛細管電泳原理------------------------------------------19 3.2.1 電泳------------------------------------------------19 3.2.2 Zeta電位及電滲透流-----------------------------------21 3.3 膠電泳(Gel Electrophoresis)------------------------------22 3.4 晶片式電泳儀的架設--------------------------------------24 3.5 樣品與緩衝液的配置--------------------------------------25 3.6 實驗部分------------------------------------------------27 3.6.1 偵測蛋白質樣品的進樣--------------------------------27 3.6.2 FITC-BSA 與FITC-CAS 的分離---------------------------28 3.6.3 FITC-Makers的分離------------------------------------31 3.6.4 FITC-protein makers訊號的鑑定------------------------33 3.7 螢光染料的標示------------------------------------------37 3.7.1 Nano Orange標示蛋白質樣品----------------------------39 3.7.2 5-IAF標示蛋白質樣品----------------------------------40 3.7.3 結論------------------------------------------------42 第四章 晶片式電泳的線上濃縮 4.1 前言----------------------------------------------------61 4.2 等速電泳(ITP)結合膠電泳(GE)------------------------62 4.3相對訊號強度的降低(wide dynamic range)--------------------63 4.4 線上濃縮晶片的設計與操作--------------------------------65 4.5 線上濃縮晶片的電路設計----------------------------------66 4.6 樣品與緩衝液的配置--------------------------------------66 4.7 實驗部分------------------------------------------------68 4.7.1 FITC-BSA與FITC-CAS的分離---------------------------68 4.7.2 FITC-protein makers的分離---------------------------70 第五章 結論與未來展望 5.1 結論----------------------------------------------------86 5.2 未來展望------------------------------------------------87 參考文獻----------------------------------------------------89 圖目錄 第二章 微流體晶片 圖2-1 微流體晶片自動化的概念--------------------------------15 圖 2-2 適合用來製造微流體晶片的高分子結構-------------------15 圖 2-3 晶片的設計-------------------------------------------16 圖 2-4 光罩的設計-------------------------------------------10 圖 2-5 熱壓機與玻璃母模-------------------------------------17 圖2-6 PE膜與PMMA晶片結合------------------------------------16 第三章 利用微流體晶片分離蛋白質 圖3-1毛細管內壁解離、電雙層、界面電位之示意圖-----------------43 圖3-2毛細管電泳中電滲透流( EOF )的示意----------------------44 圖3-3 晶片電泳儀--------------------------------------------45 圖3-4 樣品進樣與分離----------------------------------------46 圖3-5 CCD觀察蛋白質樣品的進樣--------------------------------46 圖3-6 CCD捕捉到樣品進樣的影像--------------------------------47 圖3-7 光電倍增管(PMT)在分離管道中偵測FITC-CAS---------------48 圖3-8 FITC-BSA 與FITC-CAS 的分離-----------------------------49 圖3-9 蛋白質混合物波峰鑑定圖--------------------------------50 圖3-10a CAS (23 kDa)------------------------------------------51 圖3-10b BSA (66 kDa)------------------------------------------51 圖3-10c CAS (23 kDa)；BSA (66 kDa)------------------------------51 圖3-11不同濃度的電泳緩衝液分離FITC-protein makers------------52 圖3-12 50kDa的microcorn分離FITC-protein makers---------------53 圖3-13 FITC-protein makers加入FITC-BSA-----------------------54 圖3-14 FITC-protein makers加入FITC-CAS-----------------------55 圖3-15 非共價性螢光染料的發光機制---------------------------56 圖3-16 5-IAF的反應示意圖-------------------------------------57 圖3-17 Nano-orange標示CAS與BSA-------------------------------58 圖3-18 利用5-IAF標示CAS、OVA、BSA電泳圖-----------------------59 圖3-19 混合5-IAF標示CAS、OVA、BSA電泳圖-----------------------60 第四章 晶片式電泳的線上濃縮 圖 4-1同濃度的FITC-BSA與FITC-CAS分別在十字晶片與蛋白質線上濃縮晶片上做偵測---------------------------------------76 圖4-2 DNA線上濃縮PMMA晶片---------------------------------77 圖4-3 蛋白質線上濃縮PMMA晶片-------------------------------77 圖 4-4 晶片操作的電路設計-----------------------------------78 圖 4-5 FITC-BSA 與FITC-CAS在十字晶片上的分離-----------------79 圖 4-6 FITC-BSA 與FITC-CAS在線上濃縮晶片上的分離------------80 圖 4-7 FITC-protein makers在線上濃縮晶片上的分離-------------81 圖 4-8 BSA 66 kDa、β-Galactosidase 116 kDa 、Myosin 205 kDa在線上濃縮晶片上的分離---------------------------------82 圖 4-9 Tripsin 20 kDa 、Carbonic anhydrase 29 kDa、Alcohol dehydrogenase 39 kDa在線上濃縮晶片上的分離------------83 圖 4-10 FITC-protein makers在十字晶片上的分離----------------84 圖 4-11 FITC-protein makers在線上濃縮晶片上的分離------------85

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