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研究生:李佳言
研究生(外文):Chia-Yen Lee
論文名稱:整合型微流體系統及其在核酸分析上之應用
論文名稱(外文):Integrated Microfluidic Systems for DNA Analysis
指導教授:李國賓李國賓引用關係
指導教授(外文):Gwo-Bin Lee
學位類別:博士
校院名稱:國立成功大學
系所名稱:工程科學系碩博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:93
中文關鍵詞:核酸分析微流體系統微機電
外文關鍵詞:DNA analysismicrofluidic systemMEMS
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隨著微機電系統與微製程技術的發展,利用此技術所製造的各種整合型微流體元件,已逐漸應用於生醫樣品的處理與分析之自動化 ◦ 使用於電泳分離之微型裝置及檢測機制已陸續開發成功,並可提供較少樣品消耗量、較高解析度與較短的處理時間等優點 ◦ 然而樣品前處理仍需要耗費大量的時間與人工,因此一種可進行樣品前處理(如: 細胞培養、細胞裂解、DNA萃取、DNA與試劑的混合及複製DNA等步驟)的整合型微流體系統是迫切需要的◦
本研究開發出使用於細胞培養環境監控之溫溼度感測器、進行細胞裂解與DNA複製的微型溫度控制系統及電力驅動之主動式微型流體混合器 ◦ 並透過對上述裝置的適當組合,設計並製造出可執行相關DNA複製功能的整合型微流體系統 ◦
本研究成功地發展出一種附有整合型溫度感測器以進行溫度信號飄移補償的溼度感測器之嶄新技術, 此微型感測器係建立於以白金薄膜當作溫度感測元件,並利用另一懸浮於距玻璃底材表面一段極小距離之可移式電極形成電容電極 ◦ 當相對溼度改變時,此裝置之電容量測值即會因為此懸浮結構與玻璃底材距離之改變而產生變化 ◦ 此微型溼度感測器之基本元件乃氮化矽/矽製成之懸臂樑, 該懸臂樑表面並塗佈一層具濕氣吸附性質之聚合物薄膜(聚醯亞胺),當環境相對溼度變化時即會引起此溼度感測層之膨脹並引起懸臂樑之彎曲,進而引起此懸臂樑與底材間電極距離之改變而導致電容值之變化 ◦ 實驗數據證實了本研究設計之微型懸臂樑之低勁度與前端之大面積電極可產生高靈敏度(2.0 nF/%R.H.) ◦ 為了補償因溫度變化造成之電容量測值飄移,本研究將一微型白金薄膜電阻溫度感測器整合於其中 ◦ 實驗數據顯示此感測器在高溼度(>65% R.H.)下具有極低之信號遲滯值(hysteresis) ◦ 在本研究中,電阻與電容之量測值與相對溼度之關係進行了詳盡的探討,且數值與實驗結果都顯示了本研究之溼度感測器具有極高之線性度 (R2= 0.9989)、極高之穩定度(±0.76%)與極快之響應時間(1.10秒) ◦
本研究並提出一種建立在玻璃晶片上之微型溫度控制系統之新式製程技術,其包含了微型溫度感測器與微型加熱器,可進行細胞裂解(cell lysis, 95oC)與DNA複製(Polymerase Chain Reaction (PCR), 95oC - 57oC - 72oC)等需要特定溫度或溫度循環之生醫反應◦ 本研究係利用微機電技術製造白金薄膜電阻形成溫度感測與加熱元件,使用載玻片作為絕緣層,並將以上元件裝置於一微型槽內,以對槽中物質進行溫度控制,此微型槽係以PDMS模造法製成之 ◦ 置於微型槽中之微型溫度感測器與微型加熱器可極為準確地量測槽中物質溫度,並有效地控制其溫度◦ 實驗數據顯示因為此微型系統極低的熱慣性(thermal inertia),可產生急速的加熱(20oC/sec)與冷卻速度(10oC/sec),並可在72分鐘內完成30次PCR溫度循環◦ 利用此微型溫度控制系統進行酵素消化作用與DNA 複製所需的特定溫度與溫度循環皆在本研究中成功地完成 ◦
本研究亦提出一種利用電動力驅動造成場效應以改變玻璃管道壁面上之介面電位(zeta potential)之微型流體混合器,此混合器係蝕刻玻璃底材形成主流體管道及側面埋入式電極管道,並將金/鉻沉積於側面管道之上以形成電容電極,其電容效應可改變在電滲透流(electroosmotic flow, EOF)中壁面上局部區域電雙層(electrical double layer, EDL)內之介面電位 ◦ 此介面電位之局部改變對於單一流體可進行速度之控制,並可對異質的流體產生快速之混合 ◦ 實驗數據證實微型管道中之流體速度確可藉由側面電極造成之電容效應引起管壁介面電位變化,進而對微流體進行主動式控制 ◦ 與其它使用橫跨微型管道之平面電極相較,此埋入式側面電極可避免因不良之晶片接合造成之流體洩漏及提供直接光學偵測之可行性 ◦ 本研究之微流體混合器除了顯現出極為有效的混合效果,也可對流體行為進行控制◦
最後,本研究將前述之微型流體混合器與微型溫度控制系統整合於同一晶片之上, 以進行DNA複製及其前處理◦ 在此微流體系統當中,細胞首先於微型細胞裂解反應器中進行細胞裂解以釋出DNA,萃取之DNA、PCR引子及試劑皆被電滲透流驅動通過一段附有電動力驅動之微型流體混合器之管道以進行混合,該混合均勻之檢體隨即進入PCR反應器進行DNA複製 ◦ 實驗結果證明本研究之整合型微流體系統除了可自動地完成檢體前處理步驟,並可大幅地節省了操作時間與人工 ◦ 相信此研究對於生醫檢測系統之微小化, 必可帶來具前瞻與突破性之貢獻 ◦
MEMS (Micro-Electro-Mechanical-Systems) technologies and micromachining techniques have been increasingly employed in the integration of microfluidic devices designed to automate the generation and analysis of biomedical samples. Miniaturized devices for electrophoresis separation and sensing mechanisms have been successfully developed and provide such benefits as lower sample consumption, higher resolution, and improved detection speeds. However, the sample pretreatment process still requires significant time and effort. Therefore, an emerging requirement exists to develop integrated microfluidic systems for sample pretreatment operations such as cell culture monitoring, cell lysis, DNA extraction, DNA/reagent mixing and DNA amplification.
This study develops temperature / relative humidity sensors for applications such as cell culture monitoring, micro temperature control systems for cell lysis / DNA amplification, electroosmotic flow fluid controllers, and active electrokinetically-driven micro-mixers for the homogeneous mixing of samples. An appropriate combination of such devices permits the design and fabrication of integrated microfluidic systems capable of performing a diverse range of functions for DNA amplification.
This study successfully develops a novel technique for the fabrication of micro humidity sensing devices with integrated temperature sensors for signal drift compensation. A MEMS-based device is developed in which thin-film platinum resistors serve as temperature sensing elements and a microstructure suspended at a small distance above the surface of a glass substrate acts as the movable electrode of a capacitor. A mechanism is proposed to measure the capacitance between the suspended wafer structure and the glass substrate at different values of relative humidity. The fundamental component of the MEMS-based humidity sensor is a nitride/silicon cantilever coated with a water-absorbent polymer film (polyimide). A variation in humidity induces moisture-dependent bending of this microcantilever, which subsequently changes the measured capacitance between the cantilever and the substrate. The current experimental data demonstrate that the low stiffness of the microcantilever and the large electrode area on the microcantilever tip yield a high degree of sensitivity, i.e. 2.0 nF / % RH. To compensate for the temperature drift of the capacitance signals, the humidity sensor is integrated with a micro resistance-type temperature detector comprised of platinum resistors. The experimental data indicate a low hysteresis value at high relative humidity (>65% RH). The relationship between the measured resistance/capacitance and the relative humidity is fully investigated and documented. The numerical and experimental results reveal a high degree of linearity (R2 = 0.9989), a high stability (±0.76%) and an acceptable response time (1.10 s).
This study proposes a novel technique for the fabrication of an on-chip temperature control system comprising micro temperature sensors and micro heaters located on a glass substrate designed for cell lysis (95oC) and DNA amplification (95oC – 57oC – 72oC) applications. A MEMS technique is developed in which sensing/heating elements formed of thin-film platinum resistors, and an electrical isolation layer formed by a cover slip, are located within a micromachined chamber fabricated by a PDMS casting process. Significantly, the MEMS-based on-chip temperature control system is located within the micro chamber itself. This arrangement permits the temperature to be precisely measured and effectively controlled. The experimental data reveal that the resulting low thermal inertia of the developed micro system yields high heating and cooling rates, i.e. 20oC/sec and 10oC/sec, respectively. It is shown that 30 PCR (polymerase chain reaction) thermal cycles can be achieved in less than 72 minutes. Furthermore, this study demonstrates that the proposed temperature system is fully capable of supporting the enzyme digestion and DNA sample amplification process.
This study also presents a novel active electrokinetically-driven micro-mixer which uses localized capacitance effects to induce zeta potential variations along the surface of silica-based microchannels. The proposed mixer is fabricated by etching bulk flow and electrode shielding channels into glass substrates and then depositing Au/Cr thin films within the latter channels to form capacitor electrodes, which establish localized zeta potential variations near the Electrical Double Layer (EDL) region of the electroosmotic flow (EOF) within the microchannels. The potential variations induce flow velocity changes within homogeneous fluids and a rapid mixing effect in inhomogeneous fluids. The current experimental data confirm that the fluid velocity can be actively controlled by using the capacitance effect of the shielding electrodes to vary the zeta potential along the channel walls. Compared with the common use of planar electrodes across the microchannels, the use of buried shielding electrodes prevents current leakage caused by poor bonding and facilitates optical observation during operation. The present experimental results show that the developed microfluidic device permits a high degree of control over the fluid flow and yields an efficient mixing performance.
At last, this study integrates the developed micro-mixer with the on-chip temperature control system, and applies the integrated chip to the DNA amplification process. In this amplification process, the cell lysis is initially performed in a micro lysis reactor. Extracted DNA, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the electrokinetically-driven micro mixer. The homogeneous mixture is then thermally cycled in a PCR chamber to perform DNA amplification. The results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The developed microfluidic systems provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems.
English Abstract I
Chinese Abstract IV
Acknowledgements VII
Table of Contents VIII
List of Tables XI
List of Figures XII
Nomenclature XIX

Chapter 1 :
INTRODUCTION 1
1.1 Impact of MEMS technologies on bio-applications 1
1.2 Development of micro DNA chips 2
1.3 Development of Lab-on-a-Chip (LOC) 2
1.4 Motivation and objectives 3
1.5 Thesis organization 4

Chapter 2 :
MICROMACHINE-BASED HUMIDITY SENSORS WITH INTEGRATED TEMPERATURE SENSORS FOR SIGNAL DRIFT COMPENSATION 8
2.1 Introduction 8
2.2 Design 11
2.3 Fabrication 12
2.4 Results and discussion 13

Chapter 3 :
MICROMACHINE-BASED TEMPERATURE CONTROL SYSTEMS FOR BIOMEDICAL APPLICATIONS 19
3.1 Introduction 19
3.2 Design 22
3.3 Fabrication 24
3.4 Results and discussion 25

Chapter 4 :
ACTIVE ELECTROKINETICALLY-DRIVEN MICRO-MIXERS UTILIZING ZETA POTENTIAL VARIATION INDUCED BY FIELD EFFECT 28
4.1 Introduction 28
4.2 Design 31
4.3 Fabrication 33
4.4 Results and discussion 34

Chapter 5 :
INTEGRATED MICRO-PCR SYSTEMS FOR DNA AMPLIFICATION 38
5.1 Introduction 38
5.2 Design 38
5.3 Fabrication 39
5.4 Results and discussion 40

Chapter 6 :
CONCLUSIONS 43
6.1 Overview of the dissertation 43
6.2 Future studies 46

REFERENCES 47

TABLES 55

FIGURES 57
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