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系統識別號 U0026-0812200911364074
論文名稱(中文) 微型自動化核酸增幅系統快速偵測感染性疾病之病原菌
論文名稱(英文) Automatic Nucleic Acid Amplification Microsystems for Rapid Pathogen Diagnosis of Infectious Disease
校院名稱 成功大學
系所名稱(中) 微機電系統工程研究所
系所名稱(英) Institute of Micro-Electro-Mechancial-System Engineering
學年度 93
學期 2
出版年 94
研究生(中文) 廖家陞
研究生(英文) Chia-Sheng Liao
電子信箱 Q2692403@ccmail.ncku.edu.tw
學號 q2692403
學位類別 碩士
語文別 英文
論文頁數 90頁
口試委員 口試委員-張志欽
指導教授-李國賓
口試委員-劉校生
口試委員-吳俊忠
中文關鍵字 反轉錄聚合酶連鎖反應  多樣式聚合酶連鎖反應  聚合酶連鎖反應  生物晶片  微機電系統  微型全分析系統  氣動式微閥門  氣動式微幫浦  微流體晶片  複合式聚合酶連鎖反應  疾病偵測 
英文關鍵字 Pneumatic microvalves  Pneumatic micropumps  Microfluidic chip  Diagnosis  Multiple  μ-TAS  PCR  MEMS  Biochip  RT PCR  Multiplex PCR 
學科別分類
中文摘要   隨著生物化學起飛、分子遺傳學崛起,新的生物技術以分子為單位重新銓釋生物的現象。然而,微機電技術則實為一項極重要的路徑,使人類能操控小至數微米的細胞世界。作者冀期,科技的進步不只使生活更為方便,且能將自身的努力更進一步回饋於生命的本身。
  本研究提出一個新的微型自動化系統,基於核酸放大的原理而應用於快速疾病偵測。微系統採用微機電製成技術,整合一微流體控制模組及一微型溫度控制模組。其中微型溫度控制模組是由一電阻式微型加熱器及溫度感測器所組成。本研究利用同一種金屬-鉑,製成的微型加熱器及溫度感測器置於反應槽之內,可以即時地偵測溫度,以獲得一個快速、均勻、穩定的溫度循環控制。而微流體控制模組則包含儲液槽、微幫浦、微閥門及微流道。藉由微流體控制模組的整合,可使整個檢測的流程自動化進行,以減少人為造成的失誤。
  本文中,首先以五種上呼吸道感染性細菌之去氧核醣核酸(DNA),大量複製出特徵片段及抗藥性基因片段。接著,更進一步以兩階段式的反轉錄聚合酶連鎖反應複製偵測出兩種病毒之核醣核酸(RNA)。另外,本研究更研發複合式(multiple)及多樣式(multiplex)聚合酶連鎖反應晶片。複合式聚合酶連鎖反應可在數個反應槽中,平行地處理多種不同或相同之檢測樣本。多樣式聚合酶連鎖反應則可在單一反應槽中,複製同一檢體之特徵片段及抗藥性基因片段。最後,整個實驗的流程可統整為:細胞裂解、樣品傳輸、反轉錄反應、聚合酶連鎖反應,使用者皆可十分方便的進行自動化快速疾病偵測。
  實驗資料顯示,藉由微型溫度控制模組的快速升降溫,可使以去氧核醣核酸為基因體的微生物可以在15分鐘內成功地複製其特徵片段,以作為疾病的偵測診斷。而以核醣核酸為基因體的病毒也只需約一個鐘頭。本研究之微系統,可使用市售9伏特的鎳氫電池作為其操作的電源,在封裝之後為一可攜帶式的檢測儀器。相信無論在基因分析、分子生物學、感染性疾病檢測及其他許多生物的應用上,本研究可提供了一個極為關鍵的檢測平台,對於快速自動化的放大核酸片段有極大的助益。
英文摘要  Rapid advances in biochemistry and bio-genetics lead to molecular biology, which is revolutionizing scientific thought. A micro-electro-mechanical-system (MEMS) allows human manipulation of micrometer-scale molecules. Advancement in biotechnology will hopefully improve the quality of human life.
 This investigation presents an automatic rapid diagnosis microsystem based on nucleic acid amplification. The miniature system is fabricated using MEMS techniques, and consists of a micro temperature control module and a microfluidic control module. The heating and temperature sensing elements of micro temperature control module are both fabricated using platinum, and are located within the reaction chambers to generate rapid and uniform thermal cycling. The microfluidic control module, including the reservoir, micropumps, microvalves and microchannel, enables automatic testing with minimal human intervention.
 This study proposes a microsystem to detect several genes associated with the DNA-based upper respiratory tract infection microorganisms, their corresponding antibiotic-resistant gene and RNA-based virus. An efficiency multiplex amplification of many targets of interests in one reaction is feasible using the proposed micro device. Additionally, multiple PCR, a novel approach of processing of various samples with different thermal cycle conditions in parallel, is developed for fast diagnosis. Multiple PCR is much faster than the traditional bench-top PCR machine system. The proposed microsystem also realizes a two-step reverse transcription polymerase chain reaction. The complete detection process, including cell lysis, sample/reagents transportation, cDNA synthesis and amplification, can be automatically performed in a user-friendly manner.
 The experimental data demonstrate that the high heating/cooling rates of the microsystem (20℃/sec and 10℃/sec, respectively) permit successful amplification of DNA microorganisms within 15 minutes and of RNA virus within one hour. The portable microsystem is packaged and operated by a 9-volt battery. The developed microsystem provides a crucial tool for many applications such as genetic analysis, molecular biology and infectious disease detection.

論文目次 Abstract I
中文摘要 III
致謝 V
Table of Contents VII
List of Tables XI
List of Figures XII
Nomenclature XXI


Chapter 1: Introduction 1
1.1 MEMS and Bio-MEMS technology 1
1.2 Nucleic Acids Amplification for Diagnosis 1
1.3 Literature Survey 2
1.4 Motivation and Objectives 3


Chapter 2: Theory 6
2.1 DNA and RNA 6
2.2 Cell Lysis 7
2.3 Polymerase Chain Reaction 8
2.4 Reverse Transcription Polymerase Chain Reaction 8
2.5 Slab-gel Electrophoresis 9
2.6 Micro Temperature Control Module 10
2.6.1 Micro Heaters 11
2.6.2 Micro Temperature Sensors 11
2.7 Pneumatic Micropumps and Microvalves 13
2.7.1 Design 14
2.7.2 Membrane Activation Theory 14
2.8 ASIC Control system 15


Chapter 3: Fabrication 22
3.1 Overview of Fabrication 22
3.2 On-chip Temperature Control Module 24
3.2.1 Glass Substrate Cleaning 24
3.2.2 Patterning 25
3.2.3 Metal deposition 29
3.2.4 Lift-off fabrication 29
3.3 Micro Fluidic Control Module 30
3.3.1 Substrate cleaning 31
3.3.2 Su-8 Mold Fabrication 31
3.3.3 PDMS Casting 32


Chapter 4: Materials and Methods 41
4.1 Materials 41
4.1.1 DNA-based Bacteria in Upper Respiratory Tract Infectious Disease 41
4.1.2 RNA-based Dengue Virus and Enterovirus 42
4.1.3 RT and PCR Reagents 43
4.2 Methods 43
4.2.1 On-chip Cell Lysis 43
4.2.2 Micro PCR chip 44
4.2.3 Multiplex and Multiple PCR 45
4.2.4 Reverse Transcription PCR 46
4.2.5 Microsystem Packaging 48


Chapter 5: Results and Discussion 57
5.1 Micro Temperature Control Module 57
5.1.1 Calibration of Temperature Sensors 57
5.1.2 Temperature Uniformity 58
5.1.3 Thermal Cycling Test 58

5.2 Microfluidic Control Module 59
5.2.1 Micro-Pneumatic Valves 60
5.2.2 Micro-Pneumatic Pumps 60

5.3 Nucleic Acid Amplification Results 61
5.3.1 Detection of DNA-based Microorganism Genes and Antibiotic-resistant Genes 61
5.3.2 Detection of RNA-based Virus 62
5.3.3 Detection Limits and Specificity 63
5.3.4 Multiplex and Multiple PCR Amplification 63


Chapter 6: Conclusions and Future Work 80


References 82

Biography 91

Publication 92
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