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研究生:楊東潔
研究生(外文):Dong-Jie Yang
論文名稱:整合交流電滲流之阻抗式核酸生物感測晶片的研發
論文名稱(外文):To Develop Impedimetric Nucleic Acid Biosensors Chip Integrated with AC Electroosmosis Flow
指導教授:吳靖宙
指導教授(外文):Ching-Chou Wu
口試委員:胡仲祺洪敏勝
口試日期:2011-07-19
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物產業機電工程學系所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:121
中文關鍵詞:交流電滲流電化學阻抗分析頻譜核酸生物感測晶片雜合時間
外文關鍵詞:Alternating current-electroosmosis flowElectrochemical impedence spectroscopyNucleic acid bio-sensorHybridization time
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專一性去氧核醣核酸(deoxyribonucleic acid, DNA)片段皆數奈米等級的小分子,在溶液中僅藉布朗運動的與探針DNA(probe DNA, pDNA)進行雜合,此雜合方式限制了檢測極限及增加反應時間,此問題是目前親和性感測器的發展瓶頸之一。本研究採用交流電滲流(alternate current-electroosmosis flow, AC-EOF)控制含有DNA液體的流動,並整合至沙門氏菌(salmonella)核酸感測晶片。實驗中對最佳電控條件與溶液配方進行探討,以期能降低檢測極限與縮短雜合反應時間。
在AC-EOF最佳操作條件與電極設計的探討中發現,使用electric triple layer模型可對電動力學古典平均理論值進行校正,在低導電度液中,可使理論值與實際直有很高的吻合度(如D.I.water與1 mM Tris中最高流速的操作頻率分別約為75與200 Hz)。在1 mM Tris中最佳頻率下,半徑為200 μm盤電極粒子收集量分別為半徑300 μm與400 μm盤電極的1.73與2.1倍,因此選用半徑200 μm的盤電極作為後續的DNA雜合實驗評估。比較在有無1 mM Tris中AC-EOF驅動對雜合效率的影響。結果顯示AC-EOF電驅動技術使飽和雜合時間能縮短至3 min,為靜置雜合反應時間的0.033倍,其雜和後電子轉移電阻△Ret-tDNA可達21.44±1.36 kΩ,且為靜置雜合△Ret-tDNA的1.43倍。在TES(NaOH)內進行量測,其檢測線性範圍為10-11~10-17 M,最低檢測極限可達10-17 M。此晶片的開發大幅提升微流體控制與DNA檢測的整合,並利於病原菌特定基因片段的快速檢測。

The specific deoxyribonucleic acid (DNA) fragments are in the order of sveral nanometers. The hybridization between probe DNA (pDNA) and the specific DNA in suspension liquid only is carried out by the Brownian motion. The hybridization approach limits the detection limit and increases the response time, which is one of the problems of the bioaffinity sensors. In this study, we integrated the alternating current–electroosmosis flow (AC-EOF) control into the Salmonella nucleic acid affinity chips to promote the rate and amount of DNA hybridization. In the experiments, the optimal conditions of AC-EOF control and solution recipe were explored so as to lower detection limit and shorten the response time of hybridization.
The correction of electric-triple layer model to the classical formula can obtain a better fitting of applied frequency and average flow velocity between the theoretical values and experimental data in a low conductivity solution. For example, the operation frequency of largest flow velocity for double disttlled water (1.2 μS/cm) and 1 mM Tris (6.1 μS/cm) buffer was 75 Hz and 200 Hz, respectively.
In the optimal frequency, the disk electrode of 200-μm wide radius had the most collected particles dissolved in 1 mM Tris, which was 1.73 and 2.1 times larger than that collected by the electrodes of 300-μm and 400-μm wide radius, respectively. Therefore, the 200 μm radius electrode was used for the DNA hybridization experiments.In comparison with the stationary hybridization in 1 mM Tris, the AC-EOF driving could reduce the hybridization time to 3 min, which was 0.033 times smaller than that in stationary condition. Moreover, the electron-transfer resistance (ΔRet-tDNA) measured by electrochemical impedence spectroscopy reached 21.44±1.36 kΩ, which is 1.43 times as large as that obtained in stationary hybridization. The linear range and detection limit detected in 10 mM TES (pH 7.0) was 10–11–10–17 M and 10–17 M, respectively. The chip design can greatly facilitate the integration of microfluidic control and DNA measurement for the detection of specific gene fragments of microbes.

目錄
摘要................................................................I
Abstract............................................................ II
目錄.............................................................. IV
圖目錄...........................................................VIII
表目錄........................................................... XVI
符號表........................................................XVII
緒論.............................................................................................................1
1.1前言....................................................................................................................1
1.2核酸生物感測器之討論....................................................................................2
1.2.1生物感測器的原理與發展.......................................................................2
1.2.2 DNA自組裝與帶電之特性........................,,,,.........................................5
1.2.3循環伏安法的原理與DNA檢測之應用...............................,,,,.............8
1.2.4電化學阻抗頻譜分析之原理與對DNA檢測的應用...……,,,,………11
1.3電動力現象與理論計算………………………………………………….….19
1.3.1電極溶液介面之電雙層機制……………………………………….…19
1.3.2 AC-EOF理論……………………………..…………………………...23
1.3.2.1 介面電容之Gouy-Chapman 與 Stren修飾理論..........................23
1.3.2.2 AC-EOF驅動方式……….…………...………….………...…... 26
1.3.2.3 AC-EOF最佳流速之頻率於古典平均流速與RC model理論27
1.3.3 DEP之理論………………………………………..…………………..30
1.3.3.1誘導偶極…………………………………………………………..30
1.3.3.2 DEP效應……………………………………………….…………32
1.3.4電熱理論………………………………………………………...….…34
1.3.5交流電動力學之特性探討……………………………………………36
1.4電動力學技術於親和性感測器之應用文獻探討………………………….38
1.5研究目的架構……………………………………………………………….40
第二章 材料與實驗方法……………………………………………………………44
2.1實驗試劑與設備……………………………………………………………44
2.1.1實驗試劑………………………………………………...……………44
2.1.2 實驗設備……………………………………………………………..47
2.2 ITO玻璃微電極製作.....................................................................................50
2.3薄膜金電極微製程製作................................................................................54
2.4螢光粒子配置與AC-EOF量化流程............................................................57
2.5電控後膠體粒子收集效果之螢光量化........................................................59
2.6沙門氏桿菌核酸檢測流程............................................................................60
2.7電化學量測....................................................................................................63
2.7.1檢測溶液配方.......................................................................................63
2.7.2 CV與EIS檢測條件與分析................................................................63
2.7.3 EIS等效電路設計................................................................................64
2.8 原子力顯微鏡分析.......................................................................................66
第三章 結果與討論....................................................................................................67
3.1古典平均流速數值模擬與RC model計算之最佳AC-EOF的操作頻率..67
3.1.1螢光膠體粒子流速量化.......................................................................71
3.1.2 D.I. water中最佳驅動頻率探討...........................................................73
3.1.3 1 mM Tris中最佳驅動頻率探討..........................................................75
3.1.4 1 mM KCl中最佳驅動頻率探討.........................................................77
3.1.5 Electric triple layer 模型對古典平均流速公式之修正......................79
3.1.6表面粗糙度對middle layer與黏滯度補償基數之探討........................83
3.1.7 ETL數值分析、古典平均流速與實驗比較……….….......................85
3.2 電控後膠體粒子收集效果之螢光量化……….…......................................91
3.3 核酸感測器特性之探討……….…..............................................................93
3.3.1於不同液體中探針修飾程度的探討...................................................93
3.3.2不同修飾步驟之電化學特性探討.......................................................95
3.4以EIS評估不同配方之雜合飽和時間.........................................................98
3.4.1 液體導電度對雜交反應的影響..........................................................99
3.5 EIS對交流電滲流於沙門氏DNA chip之評估...........................................102
3.5.1探討不同驅動電壓對電極MCH/pDNA複合層之評估.....................102
3.5.2 AC-EOF驅動方式雜合量對反應時間的影響..................................105
3.5.3比較遠離AC-EOF最佳操作頻率下雜合量對反應時間的影響.....107
3.5.4 pDNA專一性探討..............................................................................109
3.5.5檢測極限以其靈敏度.........................................................................111
第四張 結論..............................................................................................................112
參考文獻....................................................................................................................113
附錄............................................................................................................................121

圖目錄
圖1.1 電極/溶液介面之EDL結構示意圖....................................................................4
圖1.2單股DNA結構..................................................................................................6
圖1.3相鄰鹼基對形成適當的堆疊,使pz軌域可能產生重疊(π-stack) ...................7
圖1.4 (a)循環伏安法給予的三角波電位 (b)循環伏安圖..........................................9
圖1.5電極與溶液所產生的等效電路圖。其中電解液阻抗以Rs表示、溶液中氧
化還原物質擴散到電極表面所遭受的阻抗阻抗以Zw表示、電極與溶液間
的EDL電容以Cdl表示以及電子轉電阻抗以Ret表示..................................12
圖1.6電子轉移與擴散所引起不同的奈氏圖:(a)為擴散與電子轉移阻抗共同貢獻
之曲線;(b)為擴散行為所支配之曲線;(c)僅為電子轉移所支配之曲線....17
圖1.7極/溶液介面之EDL結構示意圖。φd表外漢姆茲層之電位、介達電位(Zeta,
ζ)表擴散層起始之電位(c點表示)、x軸之a點為內漢姆茲層之邊介,a-b
之區域為oHP與,b表為擴散層之起始位置,電位於b點後由指數下降分
佈......................................................................................................................22
圖1.8 電極/溶液介面物理特性組成之電路元件。Lgap表示電極間距,L與ΔL分
別表示離二電極中心的電極距離與其單位長度,ΔCdl表示在ΔL距離下之
電雙層電容,ΔRb表示在L位置下單位長度ΔL所相對應之溶液阻抗....26圖1.9 AC-EOF驅動方式之理論示意圖。虛線所指為電場方向;綠球表示陽離子;
紅球表示陰離子;藍色鍵頭表示流體渦流方向;紅鍵頭表示庫倫力方向...27
圖1.10物質較外在環境極化能力高(a)或低(b)關係圖。1.10(a)表示極化能力比外
在環境高時,介面內部的電荷累積會多於介面外部,此時物質的偶極距
方向與電場的方向相同;反之,圖1.10(b)當物體的極化能力比外在環境
低時,大部分的電荷存在於物體外部對抗電場,而物體偶極距方向與電
場方向相反……………………………………………………………………………………………….31
圖1.11受介電泳力作用之物體在非均勻電場中移動情形。紫色粒子正受正介電
泳(pDEP)效應所驅動往強電場區移動;綠色粒子正受負介電泳(nDEP)效
應所驅動往弱電場區移動…………………………………………………………….………….33
圖1.12電熱(焦耳熱效應)之流體驅動示意圖。π(ω)為ETL作用力與方向性之重
要的指標(a),當π(ω)項即為負值,此刻的ETL驅動渦流產生方向示意圖
………………………………………………………………………………………………….…………..….35
圖2.1檢測電極製作流程………………………………………………………………….……………….….50
圖2.2盤-環電極光罩設計與其尺寸………………………………………………….………………….51
圖2.3 (左)乳膠片之絕緣層光罩;(右)顯影後完整電極區域示意…….………………….53
圖2.4 (左)圖為良好完整之電極晶片(右)圖為過蝕刻失敗晶片…….…………………….53
圖2.5 薄膜金電極製作流程…………………………………………..……………………..…..54
圖2.6 完整盤環金電極製作。盤直徑400 μm、環寬100 μm與電極間距50 μm工作
電極總面積為2 μm2,實驗中透過環電極設計可使用交流電控技術,探討工
作電極修飾pDNA後與目標物進行雜合反應之後的阻抗變化量………...…...56
圖2.7 螢光粒子之交流電動力檢測平台…………………………..….…………………..……...57
圖2.8 真實流速取樣方式之示意圖。左圖表示於1 mM KCl 電控條件為3 Vp-p,1 K
Hz,2 min的收集圖像。右圖表單一顆粒子進入盤(Disk)電極開始(0秒)至10秒所移動之距離…………………………..….…………………..……………………...…...58
圖2.9 電控條件為3 Vp-p、200 Hz於1 mM Tris 溶液中施作2 min前(a)後(b)其螢光
收集量化取樣與分析。上圖為螢光影像,虛線圈選區域為ImageJ軟體分析螢光強度的區域;下圖為所相對應的強度曲線…………..…………………….…...59
圖2.10 沙門氏桿菌特異性片斷檢測流程示意圖。1為不同配方修飾於帶負電性金
電極表面之評估;2為使用MCH進行blockibg與轉置貼附之ssDNA後之評
估;3為傳統方是雜合飽和時間之評估;3’為使用AC-EOF電控之雜合反
應之評估…………………………..….…………………..……………………………………......62
圖2.11 (a)阻抗實部與虛部所組成之奈氏圖。(b)上圖為不同掃描頻率下總阻抗值
(包含實部與虛部,其單位為歐姆),右下為不同掃描頻率所對應阻抗原件
之相角圖。(矩形線表示有擴散效應影響之介面現象,三角則否) ……......64
圖2.12 電極介面有擴散現象(a)與無擴散效應時(b)所使用等效電路。Rs為溶液等
效阻抗,CPE常相位元件所表之電雙層等效電容,Ret為電子轉移阻抗,
Zw為擴散引起的阻抗..….…………………..…………………………………...………......65
圖2.13 AFM分析原理示意圖..….…………………..………………..…………………...………......66
圖3.1 依平均流速公式計算在盤電極間內緣處(L:25μm)的頻率對流速的關係
圖。施加電壓為3 Vp-p.…………………..………………..…………………...……….…......69
圖3.2 實驗流速取樣方式之示意圖。左圖表示於1 mM Tris 電控條件為3 Vp-p,
500 Hz經AC-EOF驅動,單一顆粒子進入盤(Disk)電極開始(0秒)至10秒所移
動之圖像取樣……………..…………………………………..…………………...……….…......71
圖3.3 溶液為1 mM Tris於施加3Vp-p於頻率500 Hz下距離與流速的特性曲線圖..72
圖3.4 溶液為1 mM Tris於施加3 Vp-p頻率500 Hz經AC-EOF驅動2 min後所得到之
停滯點……………..…………………………………..…………………...……….……………......72
圖3.5 在D.I. water中施加3 Vp-p於不同頻率(100、150、200、300、500與700 Hz)
下所得粒子的位置與移動流速的關係圖,Distance表示在盤電極的邊緣往
中心計算的距離圖3.6 1 mM KCl不同頻率流動距離與流速的特性曲線..74
圖3.6 施加3 Vp-p電位於D.I. water中以100 Hz(a)、150 Hz (b)、200 Hz(c)與300
Hz(d)驅動粒子分佈2 min後螢光影像圖…………………...……….……………......74
圖3.7 在1 mM Tris中施加3 Vp-p於不同頻率(100、150、200、300、500與700 Hz)
下所得粒子的位置與移動流速的關係圖,Distance表示在盤電極的邊緣往
中心計算的距離………………...……….……...……….………...……….……………….......76
圖3.8 施加3 Vp-p電位於1 mM Tris中以100 Hz (d)、150 Hz (c)、200 Hz (c)與300
Hz(d) 驅動粒子分佈2 min後螢光影像圖圖3.9 電極介面等效電路之(a)固
液介面等效電路模型(b)ETL之等…………………...……….…………………............76
圖3.9 在1 mM KCl中施加3 Vp-p於不同頻率(100、150、200、300、500與700 Hz)
所得粒子的位置與移動流速的關係圖,Distance表在盤電極邊緣往中心的
距離………………...……….……...……….………...……….…………………….………….......78
圖3.10 施加3 Vp-p電位於1 mM KCl中以100 Hz (d)、500 Hz (c)、700 Hz (c)與900
Hz(d) 驅動粒子分佈2 min後螢光影像圖…….…………………….…………...........78
圖3.11 於大電壓稀釋溶液中電極/溶液介面之ETL模型;λs、λm與λd分別表示stern
layer、middle layer與diffuse layer之厚度圖3.13 原子力顯微鏡檢測ITO表
面粗糙度示意圖….…………………….…………......... ….…………………….…………....80
圖3.12 電極介面等效電路之(a)固液介面等效電路模型,(b)ETL之等效電路與(c)
簡化後ETL之等效電路。Ro、Ctl、Rb分別表示電極之氧化層電阻、ETL電
容與溶液電阻,其中Ctl又可細分成stern layer(Cs)、middle layer (Cm)與
diffuse layer (Cd) 之電容所構成,其中電子於各層間傳遞時所受之阻力又
可細分為Rs 、Rm 與Rd………….…………......... ….…………………….………………..81
圖3.13 原子力顯微鏡檢測ITO表面粗糙度示意圖,Ra≒4~5nm,最大起伏為
7 nm……….………….................................... ….…………………….…………………………..88
圖3.14 於盤環電極(Rdisk:500 μm、Dgap:50 μm 與Wring:100 μm)。 驅動電壓3 Vp-p
下,比較評估D.I. water於實驗(藍虛線)、古典平均流速(紅虛線)公式與
ETL模擬公式(綠虛線)之平均流速-頻率之特性曲線…………………………….89
圖3.15 於盤環電極(Rdisk:500 μm、Dgap:50 μm 與Wring:100 μm)。驅動電壓3 Vp-p
下,比較評估1mM Tris於實驗(藍虛線)、古典平均流速(紅虛線)公式與ETL
模擬公式(綠虛線)之平均流速-頻率之特性曲線………………………………….89
圖3.16 於盤環電極(Rdisk:500 μm、Dgap:50 μm 與Wring:100 μm)。驅動電壓3 Vp-p
下,比較評估1 mM KCl於實驗(藍虛線)、古典平均流速(紅虛線)公式與
ETL模擬公式(綠虛線)之平均流速-頻率之特性曲線…………………………….90
圖3.17 於盤-環ITO電極(固定Dgap:50 μm 與Wring:100 μm。Rdisk分別為:200 μm(a)
、300 μm(b)與400 μm (c)),電控條件為3Vp-p,150 Hz於1 mM Tris 溶液2
min之螢光收集量化分析。信號值取之部分為電控後盤電極所對應之單一
電極(Single)與總面積(total area)的螢光強度。(n=3) ………………………….92
圖3.18 經piranha /aqua regia清潔之後裸金電極(bare),於含有1 μMp DNA的
Tris(EDTA) buffer與Tris(NaCl) buffer分別修飾2小時之奈氏圖。其檢測溶
液為TES(NaOH) ........ ….…………………….…………………………….………………..94
圖3.19 使用Rdisk為200 μm之薄膜金電極經piranha /aqua regia處理後,依序修飾
pDNA(1 μM)、MCH(1 mM)、tDNA(1 nM)。雜合配方為Tris(NaCl),量
測溶液為TES(NaOH),所得之CV圖(a)與奈氏圖(b) …….………………97
圖3.20 Tris之結構式。不帶電荷之Tris分子(a)與離子吸附H+離子使末端形成
NH3+的官能基帶正電之Tris分子(b) …………………………….……………....…..100
圖3.21 MCH/pDNA修飾後的電極隨在Tris(NaCl) buffer(a)與1mM Tris (pH9.3)於
tDNA雜合 (30、60、90、120與150 min)後量測所得奈氏圖。量測溶液
TES(NaOH) ……………………….................................................…….……………....…..100
圖3.22 MCH/pDNA修飾後的電極隨在Tris(NaCl) buffer與1 mM Tris於tDNA雜合
(30、60、90、120與150 min)後 EIS電阻變化量(△Ret = Ret-dsDNA –
Ret-MCH)……………………………………………………………………………………………101
圖3.23 MCH/ssDNA電極隨不同電控振幅條件(1 Vp-p、1.5 Vp-p、2 Vp-p、2.5 Vp-p、
3 Vp-p) 於頻率為200 Hz之1 mM Tris溶液中,電控時間為每30 s停止10 s
共180 s之 EIS電阻變化量(△Ret = Ret-afterMCH - Ret-MCH)。(n=3) ……….…..104
圖3.24 MCH/pDNA經由電控條件於振幅1.5 Vp-p頻率為200 Hz之1mM Tris中,
tDNA電控雜合 (30、60、90、120、150與180秒鐘) 量測所得奈氏圖。
量測溶液為含 5 mM Fe(CN)63-/4-的10 mM TES (pH 7.0) ……………..........106
圖3.25 MCH/pDNA經由電控條件於振幅1.5 Vp-p頻率為200 Hz之1mM Tris ( pH
9.3)中,AC-EOF驅動後tDNA雜合時間(30、60、90、120、150與180秒鐘)
EIS電阻變化量(△Ret = Ret-pDNA - Ret-MCH)。(n=3) ……………......…......106
圖3.26 MCH/pDNA經由電控條件於振幅1.5 Vp-p頻率為400 Hz之1mM Tris
中,tDNA電控雜合 (120、180、240、300、360與420秒鐘) 量測所得奈氏圖。量測溶液為含 5 mM Fe(CN)63-/4-的10 mM TES (pH 7.0)。
圖3.27 MCH/pDNA經由電控條件於振幅1.5 Vp-p頻率為200 Hz與400 Hz之
1mM Tris(pH9.3)中,AC-EOF驅動後tDNA雜合時間EIS電阻變化量(△Ret = Ret-pDNA - Ret-MCH)。(n=3) ….........................……......…................107
圖3.28 MCH/ssDNA修飾後的電極以靜置方式(Tris(NaCl))時間為30、60、90、
120與150 min(藍線)和AC-EOF驅動(1.5 Vp-p頻率為200 Hz於Tris) 時間為30、60、90、120與150 s(紅線)後電阻之便化△Ret- mDNA(Ret-mDNA - Ret-MCH)。...............................…......…..........................................................109
圖3.29 探針專一性反應之檢測。.........…………......…..........................................110
圖3.30 MCH/pDNA經由電控條件於振幅1.5 Vp-p頻率為200 Hz於1 mM Tris(pH
9.3)中,依序雜交上不同濃度的tDNA (10-18 M ~ 10-9 M)後,AC-EOF驅動後tDNA雜合時間EIS電阻變化量,每次雜合時間為120 s。△Ret-tDNA = (Ret-AC-EOF - Ret-MCH)。(n=3) .....................................................................111
附錄圖一 MCH/ssDNA修飾後的電極隨在40±1℃的Tris(NaCl) buffer與1 mM Tris (pH 9.3)於tDNA雜合 (30、60、90、120與150 min)後 EIS阻抗變化量(△Ret-tDNA = Ret-AC-EOF - Ret-MCH)。(n=3) .........................................................................121
表目錄
表1-1 以循環伏安法檢測DNA的範例文獻列表.........……......….........................10
表1-2交流阻抗(EIS)於核酸檢測分析之文獻........…….......…................................18
表1-3上述 AC-EOF、 DEP與ETL之特性說明…….......…................................37
表1-4 應用交流電動力技術提升親和性感測器之方法...…...................................39
表2-1 實驗中所使用探針與目標基因序列........…….......…...................................61
表3-1 不同溶液組成相對應之EDL內厚度(λd)、導電度(σ)、擴散係數(D)與以RC
model與古典平均流速公式數值計算所得之最佳AC-EOF的操作頻率.....69
表3-2 ETL模型之數值分析之三層電容厚度(λs、λm與λd)、介電常數、頻率修正、
粗糙度與黏滯度之補償基數(γ、δ與Λ) .…….......…...................................88
表3-3經piranha /aqua regia清潔後之薄膜金電極(bare),於含有1 μM pDNA的
Tris(EDTA) buffer與Tris(NaCl) buffer分別修飾2小時之奈氏圖。其檢測
溶液為TES(NaOH)。(n=3) ........…….......….................................................94
表3-4 使用Rdisk為200 μm之金電極經piranha /aqua regia處理後於傳統靜置雜合,
依序形成pDNA(1 μM)、MCH(1 mM)、tDNA(1 nM),其雜合配方為Tris
(NaCl),量測溶液為TES(NaOH),經由電路模型模擬電極介面現象得到相
對應元件值。(n=3) .......…….......…...............................................................97


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