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系統識別號 U0026-0812200912035996
論文名稱(中文) 微型聚合酶連鎖反應晶片之性能測試
論文名稱(英文) Characterization of Micromachine-based Polymerase Chain Reaction Microchips
校院名稱 成功大學
系所名稱(中) 工程科學系碩博士班
系所名稱(英) Department of Engineering Science
學年度 94
學期 2
出版年 95
研究生(中文) 陳奕諭
研究生(英文) Yi-Yu Chen
電子信箱 n9693137@mail.ncku.edu.tw
學號 n9693137
學位類別 碩士
語文別 中文
論文頁數 89頁
口試委員 口試委員-楊瑞珍
指導教授-李輝煌
口試委員-李國賓
中文關鍵字 微型溫度感測器  微幫浦  聚合酶連鎖反應  微機電系統  微閥門  微型加熱器 
英文關鍵字 Micro heater  Micro Temperature Sensor  PCR  Microvalve  MEMS  Micropump 
學科別分類
中文摘要 本研究針對一個新式的微型流動式核酸增幅晶片進行性能測試,本晶片成功利用微機電系統製程技術製作,根據核酸放大原理可應用於病原菌的偵測,此晶片包含了微型溫度控制模組以及微流體傳輸模組,皆採用微機電製程技術製作完成,其中微型溫度控制模組利用同一種金屬-鉑(Platinum, Pt)製作而成,利用其穩定的金屬特性所製成的微型加熱器(Micro heater)以及微型溫度感測器(Micro temperature sensor)做為核酸增幅反應區並可即時地偵測與監控反應區內之實際溫度,本研究的特點在於配合微流體傳輸模組設計了三個環繞式的反應區,每個反應區分別提供聚合酶連鎖反應所需要的三個特定溫度,利用微氣動幫浦及微閥門來傳輸反應區內的檢體及試劑,因此在一般桌上型的熱循環機(Thermal cycling machine)中常見的因聚合酶連鎖反應的溫度循環所造成的升降溫時間消耗問題已不復存在;再者微流體傳輸模組乃採用生物相容性極高的高分子材料(polydimethylsiloxane, PDMS)製作而成的微型氣動式幫浦來自動化傳輸檢體,並可隨檢測需要由微幫浦控制電路彈性的調整反應時間,藉此達成聚合酶連鎖反應需要的溫度循環而完成核酸放大的目的。
本研究利用人類常感染的上呼吸道傳染疾病之病原菌檢體做為檢測樣本,包含肺炎鏈球菌(Streptococcus pneumoniae)檢體以及A型鏈球菌檢體(Group A Streptococcus, GAS),實驗結果顯示在微型晶片上均可以成功達成核酸增幅的目的,此外相較於一般桌上型機器此微型晶片更可節省超過70% 的檢測時間,自動化操作的微型晶片可讓使用者更方便且更快速地進行核酸增幅反應。
關鍵字:微機電系統、聚合酶連鎖反應、微型加熱器、 微型溫度感測器、微幫浦、微閥門。



英文摘要 This study investigates the performance of a new micro flow-through polymerase chain reaction (PCR) chip fabricated by using micro-electro-mechanical-system (MEMS) technology. The micro PCR chip was used for fast nucleic acid amplification. The PCR chip comprised a micro thermal control module and a microfluidic transport module. The micro thermal control module was composed of a micro heater and a temperature sensor to heat the sample and monitor the actual temperature in the reaction chambers. The platinum was used to fabricate the micro heater and the micro temperature sensor due to its stable property. The microfluidic transport module has a triangular layout. Three reaction sections individually provide a specific temperature for denaturation, annealing and extension process, respectively, and the microfluidic transport module transport the sample in the reaction chamber smoothly. Therefore, heating and cooling process can be performed efficiently. Besides, the microfluidic transport module can change the reaction time easily by using the microfluidic control system and the reaction temperature can be controlled precisely by the micro temperature control system.
The study employed the developed system for detection of upper respiratory tract infectious bacteria, including the Streptococcus pneumoniae and the Group A Streptococcus. Experimental data show that the micro PCR chip can successfully amplify the target DNA sequence. Besides, the micro chip can save more than 70% detection time when compared with the traditional PCR machine.
Keywords:MEMS, PCR, Micro heater, Micro Temperature Sensor, Micropump, Microvalve



論文目次 摘 要 i
Abstract ……………………………………………………………………iii
誌 謝 v
目 錄 vii
縮寫及符號說明 xi
表 目 錄 xiii
圖 目 錄 xiv

第一章 緒論 1
1-1 生醫微機電系統簡介 1
1-2 研究動機及目的 2
1-3 研究方法 4
1-4 文獻回顧 5
1-4.1 微型聚合酶連鎖反應晶片 5
1-4.2 氣動式微幫浦 8
1-5 論文架構論述 10
第二章 理論及設計 17
2-1 細胞裂解 17
2-2 聚合酶連鎖反應 18
2-3 平版膠電泳 20
2-4 流動式聚合酶連鎖反應晶片 21
2-4.1 微型溫度控制模組 21
2-4.2 微流體傳輸模組 25
2-5 晶片控制系統 27
第三章 晶片製作與實驗材料 38
3-1 製程規劃 38
3-2 光罩設計與製作 39
3-3 晶片製程技術 40
3-3.1 晶片清潔 40
3-3.2 黃光微影製程 43
3-3.3 金屬薄膜沉積 46
3-3.4 蝕刻與剝離製程 47
3-3.5 微注模製程 48
3-3.6 封裝與接合技術 49
3-4 晶片製作流程 50
3-4.1 微型加熱器 50
3-4.2 微型環繞式氣動幫浦 52
3-5 實驗材料 54
第四章 結果與討論 61
4-1 固定式PCR晶片 61
4-1.1 複合式PCR與多樣式PCR 61
4-1.2 純化檢體之檢測 63
4-2 流動式PCR晶片 65
4-2.1 溫度感測器校正 65
4-2.2 溫度穩定度測試 66
4-2.3 微流體傳輸晶片測試 67
4-2.4 流動式PCR晶片檢測 68
4-3 固定式PCR晶片核酸增幅結果 69
4-3.1 專一性與成功率統計分析 69
4-3.2 複合式PCR與多樣式PCR偵測 71
4-4 流動式PCR晶片之偵測極限與專一性 72
第五章 結論及未來展望 82
5-1 結論 82
5-2 未來展望 83

參考文獻 84
自 述 89

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