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研究生:黃崇哲
研究生(外文):Chung-Che Huang
論文名稱:微型玻片式PCR加熱模組之研究
論文名稱(外文):The Study of Slide-based PCR Thermal Cycler
指導教授:陳林祈
口試委員:黃振康洪敏勝盧彥文
口試日期:2013-07-09
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物產業機電工程學研究所
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:2013
畢業學年度:102
語文別:中文
論文頁數:77
中文關鍵詞:即時定量聚合&;#37238;鏈鎖反應矽膠加熱片PCR晶片H1N1
外文關鍵詞:quantitative polymerase chain reactionflexible heaterPCR chipH1N1
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即時定量聚合&;#37238;鏈鎖反應(real-time quantitative polymerase chain reaction, real-time qPCR)是結合DNA擴增與螢光感測來對病原體檢測方法。在實驗室的高靈敏度、低偵測極限儀器體積通常較龐大,因此想發展可攜式的即時定量PCR感測器。本研究針對PCR加熱模組微小化,以輕薄的矽膠加熱片作為加熱單元,並利用PDMS做成PCR晶片。PCR加熱模組以壓克力為基材,利用CNC銑床進行加工為所需形狀,將加熱單元、風扇、散熱鋁塊以及熱電偶等元件安置其中。其升降溫速率分別為1.1與0.7°C/s,溫度振幅可小於0.1°C。PCR晶片為配合暗場式螢光模組的訊號量測,利用鋁翻模製作反應槽高度為1mm,反應槽所容納反應試劑體積為10μl。為避免聚合&;#37238;吸附在PDMS表面上,添加2.5% PVP於反應試劑中也不會對聚合&;#37238;有抑制現象。在自製加熱模組驗證中,使用H1N1的互補DNA序列作為標的,當黏合溫度為50°C 時有較高的產率。分別以自製加熱模組與市售儀器進行核酸擴增,而後利用洋菜膠電泳分析。自製加熱模組PCR擴增曲線以sigmoid model進行曲線配適,可得到擴增效率為0.97(市售)與0.70(自製)。日後若能整合暗場式螢光感測模組,成為可攜式即時定量PCR儀器,對防疫現場實測或是居家醫療的概念實現,會有很大幫助。

Real-time quantitative polymerases chain reaction (real-time qPCR) is combined DNA amplification and fluorescent sensing for pathogen detection. Because laboratory instruments with high sensitivity and low detection are usually bulky, the concept of portable real-time qPCR is developed. This research is focused on the miniaturization of PCR thermal module. We select thin silicone film as heating unit and used PDMS as PCR chip. This PMMA-based device is constructed out by CNC machining, and it is modularized compactly in order to accommodate silicone film, fan, heat sink and thermocouple. Heating and cooling rate of the heating module is 1.1 and 0.7°C/s separately and the temperature amplitude is smaller than 0.1°C. In order to suit the dark-field fluorescent sensing module, the height of reaction chamber is 1mm and the chamber contains 10μl per reaction. Taq polymerase is apt to absorb on the surface of PDMS, 2.5% PVP is added in PCR mixture to avoid absorption and it doesn’t inhibit polymerase activity. We use the cDNA sequence of H1N1 as target template. In order to improve PCR efficiency, we choose 50 °C as annealing temperature. In home-made heating module authentication, commercial PCR instrument is a control group. The fluorescent signal is analyzed by sigmoid model fitting to the amplified efficiency, and the efficiency were found to be 0.97 (commercial instrument) and 0.70 (homemade). If we can integrate dark-field fluorescent sensing module, a portable real-time qPCR instrument will achieve the concepts of on-site detection or point-of-care.

誌謝 i
中文摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 xi
第一章、前言 1
1.1 研究背景 1
1.2 研究動機 2
1.3 研究目的 3
1.4 研究架構 4
第二章、文獻探討 5
2.1 聚合&;#37238;鏈鎖反應 5
2.1.1 PCR機制與理論分析 5
2.1.2 real-time qPCR偵測機制 7
2.1.3 螢光光學偵測模組設計 10
2.2 微流道晶片及溫度循環模組 13
2.2.1 靜態式微PCR系統及其加熱模組 13
2.2.2 連續流(flow-through)式微PCR系統 16
2.2.3 PCR晶片蒸發以及氣泡問題 20
2.3 流感病毒之介紹與偵測方法 22
2.3.1 流感病毒 22
第三章、研究方法 24
3.1 實驗儀器與設備 24
3.2 實驗藥品 25
3.3 加熱模組控制與設計 26
3.3.1 熱電偶(thermocouple)性能測試 26
3.3.2 撓性超薄電熱片性能測試 27
3.3.3 自製加熱模組設計與製作 27
3.4 PCR晶片製作與測試 30
3.4.1 SU8負光阻PCR晶片製程 30
3.4.2 CNC鋁模PCR晶片製程 32
3.4.3 熱學測試-封裝及添加物對PCR之影響 33
3.4.4 光學測試-螢光訊號擷取 34
3.5 加熱模組整合測試 35
3.5.1 模擬病原體製作 35
3.5.2 自製加熱模組參數調整 36
3.5.3 PCR效率曲線分析 36
3.5.4 PCR偵測極限分析 37
3.6定量PCR系統驗證 38
第四章、結果與討論 39
4.1 加熱模組控制與設計 39
4.1.1 熱電偶訊號量測與訊號處理 39
4.1.2 加熱器設計組裝升溫測試 41
4.1.3 flow simulation熱流評估模擬 47
4.2 PCR晶片製作與測試 49
4.2.1 PCR晶片封裝測試 49
4.2.2 添加物對PCR產率之影響 51
4.2.3 螢光訊號擷取測試 53
4.3 加熱模組整合測試 56
4.3.1 PCR溫度循環測試 56
4.3.2 PCR反應時間調整 58
4.3.3 PCR晶片的選擇 61
4.4 加熱模組效能分析 62
4.4.1 PCR效率curve fitting 62
4.2.2 PCR偵測極限curve fitting 64
第五章、結論與建議 66
5.1 結論 66
5.2 未來展望與建議 69
參考文獻 70


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