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研究生：	劉晉維 Liu, Chin-Wei
論文名稱：	可促進親和性感測進行的介電濕潤驅動微流體晶片開發 An Affinity Sensor Improved by EWOD Actuator-based Microfluidic Chip
指導教授：	張憲彰 Chang, Hsien-Chang
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：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	75
中文關鍵詞：	電阻抗分析、蛋白質A 、交流電滲流、介電濕潤
外文關鍵詞：	Electro-chemical impedance spectroscopy, Protein A, AC electroosmosis, Electro-wetting on dielectric (EWOD)
相關次數：	點閱：85 下載：2
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某些特定蛋白質常是臨床上檢驗與疾病判定的指標，而藉由抗原抗體間高度的專一性結合反應，再搭配標的性或非標的性的第二抗體，所形成的親和性免疫分析法，遂得以將待測物濃度定量。但這些以精密光學儀器為基礎的檢驗設備，本身雖已有價格昂貴、操作繁瑣耗時的問題，卻也常出現有偽陰性等之缺點。在這檢測過程中，各反應步驟後對未接合上的物質之沖洗，其沖洗勁道被視為造成此問題的核心。基於此，本研究設計以微機電技術，製作一種由修飾有(1)介電層之串聯式電極組成的電濕潤(Electrowetting; EWOD)操控，與(2) 抗體(IgG)的金電極之兩區塊所構成的微流體免疫晶片。前者藉由電極逐次的極化-非極化之操控，而迅速呈親、疏水性相鄰的兩區，終使含有檢體的水溶液被帶往感測區移動，而運送至後者的感測區之電極上。此區乃以單端含硫醇基之11-MUA的自組性單層膜(SAM)修飾於金電極後，以EDC/NHS活化其另一端之羧基之後，再導入IgG與其鍵結，沖洗去為鍵結上的IgG後，以小牛血清蛋白(BSA)填埋空位後，便可作為protein A的感測探針。此外，在(1)與(2)之交會處，特以氧電漿來製作一超親水流道，形成兩個親疏水性差異極大的表面，致使液滴被運送至此，即能自發性的移動到感測電極上，這也可運用成為親和反應後的沖洗電極之目的。另外，電極也於感測前被施予8 Vpp、500 Hz之電訊號處理，亦即藉由交流電滲流(ACEOF)的施加，液珠內產生渦流，而更促使了親和反應時間，從原本的需1小時縮減為50秒，即能達檢測之功效。經電化學阻抗分析結果可得知，電極表面上的阻抗變化量(ΔRet)對於含protein A溶液的濃度，在1-50 ng/ml具有良好線性相關。此微流體晶片系統未來可繼續探討更低極限之操作外，也將可朝數位系統化之多工性免疫檢測晶片目標邁進。

Some specific proteins existing and correlating with disease in the blood or the food, its concentration changes or structural change, is considered as the symbol of disease development. On clinic, immunoassay is applied to detect these substances and measure the antibody or antigen concentrations owing to their high bio-specific recognition interaction with their complementary target. In fact, the drawback of immune analytical instrument which based on optical method not only is high price and complicated operation, but false negative detection is often occurred. Among this, the fail in eluting processes for cleaning away the unbonding substances to be the main reason can be considered. To promote this, a microfluidic immuno-chip which is made by micro electro-mechanical technology and combining two zones that are modified (1) a series of insulator-coated electrodes as electro-wetting on dielectric (EWOD) construction and (2) a antibody (IgG) - modified gold electrode. The former is designed for creating a droplet containing target sample and transporting it in chip by EWOD. By stepwise operating the electrodes rapidly to be hydrophilic and hydrophobic, the sample was moved to the sensing zone. The later is for detecting the concentration of target sample based on measuring the extent of impedance change. The self-assembly monolayer, 11-MUA, possessing a thiol group in one side will spontaneously bind onto gold electrode and a carboxylic group in the other side was activated by the agents of EDC/NHS that may promote the bind with antibody through its amino group. After the blocking treatment with bovine albumin serum, this zone will be used as for detecting Protein A. we also treated the intersection of zone 1 and 2 by oxygen plasma to allow the sensing zone to be more hydrophilic that will spontaneously achieve movement and promote the feasibility in sample transportation and electrode elution. Moreover, AC eletroosmosis flow (ACEOF) was introduced by setting the sensing electrode at 8 Vpp with 500 Hz before detection that will reduce the time for affinity reaction dawn to be 50 sec from 1 hr. As a result, the resistance change (ΔRet) by electro-chemical impedance spectroscopy for detecting protein A showed a linear correlation in the range of 1-50 ng/ml. The microfluidic system can be systemized for multiplex immuno-detection chip in the future.

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 前言.............................................1
1-2 微機電系統技術...................................2
1-3 數位式微流體晶片.................................2
1-4 介電濕潤操控技術.................................5
1-4-1 表面張力.......................................5
1-4-2 介電濕潤現象...................................7
1-4-3 介電材料與驅動電壓............................11
1-4-4 介電濕潤現象之四種微流體操控模式..............12
1-4-5 交流電滲流原理................................14
1-5 酵素免疫分析法..................................16
1-5-1 免疫分析法基本理論............................16
1-5-2 抗原與抗體之定義..............................16
1-5-3 抗原與抗體的結合力............................17
1-5-4 酵素免疫分析法................................17
1-6 電化學檢測......................................18
1-7 研究動機........................................27
1-8 文獻回顧........................................28
1-9 研究架構........................................32

第二章 實驗與製程...................................33
2-1 實驗架設與系統架構..............................33
2-1-1 儀器設備......................................33
2-1-2 實驗試劑......................................34
2-1-3 系統架構......................................35
2-2三維晶片結構.....................................38
2-2-1 電極設計......................................38
2-2-2 兩階段親疏水自發性驅動設計....................40
2-2-3 整體晶片路徑規劃與設計........................41
2-2-4 一次抗體於金電極上的修飾過程..................43
2-3 元件製程........................................44
2-3-1 玻璃基材表面清洗..............................44
2-3-2 金屬電極製程..................................45
2-3-3 介電層與疏水層塗佈............................46

第三章  結果與討論..................................47
3-1 數位式微流體免疫分析系統........................47
3-1-1 晶片設計參數..................................48
3-1-2 介電濕潤於液滴的產生與輸送操作................48
3-1-3 自發性親疏水驅動..............................53
3-1-4 交流電滲流作用力..............................55
3-2 免疫反應於電化學檢測............................57
3-2-1 模擬電極之等效電路的建立......................58
3-2-2 在不同修飾步驟之CV行為與EIS量測...............60
3-2-3 固定修飾時間下Protein A的電化學阻抗檢測.......65
3-2-4 以交流電滲流不同作用時間的緻密度變化..........68
3-2-5 晶片沖洗效率探討..............................70

第四章 結論與未來展望...............................71
4-1 結論............................................71
4-2 未來展望........................................72
參考文獻............................................73
                                    

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校內：2010-08-03公開
校外：2010-08-03公開

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