成功大學博碩士論文系統

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研究生：	黃富駿 Huang, Fu-Chun
論文名稱：	整合型微流體即時檢測系統應用於核酸增幅與電泳分離及偵測 An Integrated Microfluidic System for Nucleic Acid Amplification, Electrophoresis Separation and On-line Optical Detection
指導教授：	李國賓 Lee, Gwo-Bin
學位類別：	博士 Doctor
系所名稱：	工學院 - 工程科學系 Department of Engineering Science
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	61
中文關鍵詞：	聚合酶連鎖反應、毛細管電泳、微型加熱器、微型溫度感測器、晶片實驗
外文關鍵詞：	PCR, CE, micro heater, micro temperature sensor, lab-on-a-chip, μ-TAS
相關次數：	點閱：81 下載：1
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近年來，“晶片實驗室”一直是醫學工程發展之趨勢，目的是將傳統實驗室系統化分析流程整合於單一微型晶片系統中。本論文所研發之整合型微流體晶片系統，乃是整合分子生物、半導體製程、以及微流體三大技術所發展出來; 此整合型晶片關鍵系統包括微型聚合酶連鎖反應晶片系統、毛細管電泳晶片系統及即時螢光偵測系統，整合為一晶片實驗室。該系統可將DNA基因檢測之傳統生化分析程序，包含細胞裂解、去氧核醣核酸(deoxyribonucleic acid, DNA)複製與分離，整合於單一晶片，同時將光纖嵌入於晶片內，即時偵測DNA分離後的螢光訊號。
首先，聚合酶連鎖反應晶片係利用金屬「鉑」，製作電阻式微型溫度感測器及微型加熱器，此溫度控制系統置於反應槽下方，則可即時地對反應槽控制溫度。此外，本論文更提出新式聚合酶連鎖反應晶片之設計，將片狀式微型電阻加熱器改為柵狀，以電路中並聯之原理來補償反應槽邊界的溫度，使反應區的溫度更為均勻。而毛細管電泳晶片係使用壓克力(polymethyl methacrylate，PMMA)或聚碳酸脂(polycarbonate，PC)為底材，並以3D模具射出成型，同時製作出微管道與儲液槽，接著在常溫常壓下以PE/TPE膜接合於管道面，形成完整的微型毛細管電泳晶片。此新型的晶片製作方法，克服了傳統上以高溫高壓結合法所產生製成整合與生物修飾的缺點。此製程由於製作成本及困難度大幅降低，使得晶片具有可拋棄式的優點。
為了將生物樣品從聚合酶連鎖反應晶片流暢地輸送到毛細管電泳晶片，本研究利用微型幫浦與微型閥門將兩種功能性晶片連結形成一整合型晶片。最後，並在毛細管電泳晶片管道末端伸入兩光纖，光纖的一端導入入射光，以激發有標定螢光分子之待測DNA，另一端光纖則用來偵測其激發之DNA螢光訊號。從細胞裂解、增殖遺傳分子特殊片段、微流體的傳輸、電泳分析片段大小及螢光訊號即時偵測等流程，皆自動化地整合在此晶片實驗室中。
本研究利用晶片實驗中微型聚合酶連鎖反應晶片成功地增殖肺炎雙球菌的基因，並利用後端的毛細管電泳晶片將基因片段分離，最後再由光纖傳導螢光訊號至光電轉換器，並準確地判讀出增殖片段之大小。此外，晶片系統亦針對登革熱病毒(RNA)病毒，進行反轉錄聚合酶連鎖反應。
本論文以微機電製程技術，製作出聚合酶連鎖反應晶片、毛細管電泳晶片、微型幫浦及微閥門，並成功地將上述功能整合於單一晶片實驗室上。此晶片系統之優點具有：快速基因判讀、可攜性高、可拋棄式晶片、避免交叉汙染。對於生物體特定基因的研究上，將可代替傳統費時的大型儀器，應用於生物醫學、環境工程與食品工業等領域上。

This dissertation presents an integrated microfluidic chip capable of performing DNA/RNA (Deoxyribonucleic acid/Ribonucleic acid) amplification, electrokinetic sample injection and separation, and on-line optical detection of polymerase chain reaction (PCR) products in an automatic mode. In the device introduced here, DNA/RNA samples are first replicated using a micromachine-based PCR module or reverse transcription polymerase chain reaction (RT-PCR) module and then transported by a pneumatic micropump to a sample reservoir. The samples are subsequently driven electrokinetically into a microchannel, where they are separated electrophoretically and then detected optically using an optical fiber integrated into the device. The various modules of the integrated microfluidic chip are fabricated from cheap biocompatible materials, i.e. polydimethylsiloxane, polymethylmethacrylate and soda-lime glass. The functionality of the device is demonstrated through its successful application to the DNA-based bacterial detection of Streptococcus pneumoniae and the RNA-based detection of Dengue-2 virus. It is shown that the low thermal inertia of the PCR/RT-PCR modules reduces the sample and reagent consumption and shortens the reaction time.
In order to further decrease the sample volume and to enhance disposability, a uniform temperature chip for PCR and capillary electrophoresis (CE) chip sealed with a polyethylene/thermoplastic elastomer (PE/TPE) film are developed.
The uniform-temperature PCR chip was fabricated on soda-lime glass using MEMS technologies. A fence-type design of micro-heaters was used to improve the thermal uniformity of the reaction chamber. Experimental data show that the temperature distribution of the reaction chamber (diameter is 3 mm) is less than 1.0℃ in the PCR chamber at 52.0℃.
The PE/TPE film packaged CE chip is performed at atmospheric pressure and room temperature, which is a fast, easy and reliable bonding method to form a sealed CE chip. The fabrication of polymethylmethacrylate (PMMA) and polycarbonate (PC) microfluidic channels is accomplished by using an injection-molding process, which could be mass-produced for commercial applications. In addition to microfluidic CE channels, three-dimensional reservoirs for storing bio-samples, and CE buffers are also formed during this injection-molding process. The functionality of the mass-produced CE chip is demonstrated through its successful separation of x-174/HaeⅢ DNA markers within 2 minutes.
The integrated microfluidic device proposed in this study represents an important contribution to the fields of molecular biology, genetic analysis, infectious disease detection, and other biomedical applications.

Abstract.................................................Ⅰ
中文摘要.................................................Ⅲ
致謝.....................................................Ⅴ
Table of Contents........................................Ⅶ
List of Tables...........................................Ⅹ
List of Figures..........................................XI
Nomenclature............................................XVI

Chapter 1： Introduction
  1-1 MEMS technology and biotechnology...................1
  1-2 Bio-MEMS and lab-on-a-chip..........................1
  1-3 Literature survey...................................2
    1-3-1 Micro PCR chips.................................2
    1-3-2 Micro CE chips..................................3
    1-3-3 Integrated PCR/CE chips.........................6
  1-4 Motivation and objectives...........................8

Chapter 2：Materials and methods
  2-1 DNA replication....................................10
2-1-1 Polymerase chain reaction..........................10
2-1-2 Reverse transcription polymerase chain reaction....11
2-1-3 Micro PCR chips with edge temperature compensation.11
  2-2 Electrophoresis....................................13
2-2-1 Gel electrophoresis................................13
2-2-2 Capillary electrophoresis..........................14
2-2-3 Mciro CE chips packaged with PE/TPE films..........14
2-3 Integrated PCR/CE system.............................21
  2-3-1 Design...........................................22
2-3-2 Fabrication........................................26
2-3-3 Experimental setup.................................29
  2-4 Sample preparation.................................30

Chapter 3：Results and discussion
  3-1 Micro PCR chips with edge temperature compensation.32
    3-1-1 Thermal cycle..................................32
    3-1-2 Temperature uniformity.........................34
    3-1-3 Nucleic acid amplification.....................36
  3-2 Micro CE chips.....................................38
    3-2-1 DNA marker separation and on-line detection....40
    3-2-2 Characterization of micro CE separation........44
  3-3 Integrated PCR/CE system...........................44
    3-3-1 Characterization of integrated PCR/CE chips....45
    3-3-2 Detection of DNA-based bacteria................47
    3-3-3 Detection of RNA-based virus...................49

Chapter 4： Conclusions and future work
  4.1 Overview of dissertation...........................50
  4.2 Future work........................................51

References...............................................52
Biography................................................57
Publication List.........................................58
                                    

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校內：2008-12-10公開
校外：2008-12-10公開

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