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研究生:鄭凱懋
研究生(外文):Kai-Mao Cheng
論文名稱:利用數位微流體平台進行聚合酶鏈鎖反應及數位聚合酶鏈鎖反應
論文名稱(外文):PCR and Digital PCR on Digital Microfluidic Platform
指導教授:范士岡
指導教授(外文):Shih-Kang Fan
口試委員:張鈺盧彥文
口試委員(外文):Yu ChangYen-Wen Lu
口試日期:2019-03-16
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:機械工程學研究所
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:2019
畢業學年度:107
語文別:中文
論文頁數:91
中文關鍵詞:數位微流體晶片B型肝炎非小細胞肺癌介電濕潤PCRdPCR微孔洞陣列
DOI:10.6342/NTU201900726
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本研究希望可以利用數位微流體平台 (Digital microfluidic platform, DMF)操控含有B型肝炎病毒DNA (HBV DNA)的奈升等級試劑,進行聚合酶鏈鎖反應 (Polymerase chain reaction, PCR)。PCR,是一種利用人工的方式進行指定序列的DNA倍增技術。在實驗中,含有HBV DNA的PCR試劑將會被利用移液器加入到數位微流體晶片上。接著利用介電濕潤力 (Electrowetting on dielectric, EWOD)進行試劑的移動並且藉由黏附在晶片背部的加熱片形成晶片上不同的溫度區間。使試劑可以在不同的溫度區間內運動,並達成DNA指定序列放大之結果。其濃度會以螢光測量的方式進行實驗數據的驗證。在本次的實驗當中,PCR每圈所需時間為50秒並且僅需要100 nL的試劑體積即可完成,不論是在速度上及所需體積上都優於傳統PCR的表現。另外,本研究也期望可以操作另一項PCR的延伸應用,數位聚合酶鏈鎖反應 (Digital polymerase chain reaction, dPCR)。其可以利用大量分散的微小液滴進行DNA絕對濃度的估算,是一項相當具有潛力的技術。因此本研究期望可以藉由dPCR檢測因服用抗肺癌藥物所產生之突變基因T790M。本文是利用油水介面的表面張力進行大量且快速的液體分散,快速的在4秒內進行22500個微孔洞的液體分散後,利用加熱系統進行升降溫使每個獨立的分散液體進行dPCR,並在濃度為1818 copies/μL的DNA濃度下成功的進行PCR反應。但在未來,期望這項技術在未來可以與數位微流體平台進行結合發展出含有DNA萃取技術的dPCR的Point of care (POC)技術。
In this research, we applied PCR on Digital microfluidic platform (DMF) by manipulating the droplet with HBV virus DNA in different temperature region. PCR is an artificial way to increase the special sequence in DNA structure. In this experiment the PCR reagent with HBV virus DNA was put on DMF platform by the pipetteman. Then the EWOD force moved the reagent in different temperature region which was generated from the heating film adhered on the back side of the chip and performed the PCR on EMF platform. The experimental results were confirmed by the fluorescence of the droplet. when the concentraction of the DNA solution got higher, the fluorescent of the solution became stronger. The reagent cost in this experiment was about 100 nL, and the cycle time was about 50 seconds. Both two parameters are better than traditional PCR.
There were also another experiments in this research, Digital PCR (dPCR). It is an advanced version of the PCR. dPCR had ability to get the absolute concentraction of the DNA solution by analyzing lots of nano-droplets, therefore, it is a powerful technique. In this research, we applied the silicon chip with the microwell array on non-small cell lung cancer diagnosis. PCR solution was loaded in the reservoir then dispensed by injection method and dispensed in 4 seconds. After droplet dispensing, the chip will be put into the heating system to carry out the PCR process. Then the DNA concentration can be calculated by Poisson distribution. In this experiment DNA concentration detection limit can approach to 1818 copies/μL.
致謝......................................i
中文摘要 ..................................ii
ABSTRACT..................................iii
目錄.......................................iv
圖目錄.....................................viii
表目錄.....................................xii
第一章 緒論...............................1
1.1 研究背景...............................1
1.2 文獻回顧...............................5
1.2.1 數位微流體平台........................5
1.2.2 聚合酶鏈鎖反應........................6
1.2.3 數位聚合酶鏈鎖反應....................10
1.2.4 利用矽晶圓微孔洞晶片進行聚合酶鏈鎖反應..18
1.2.5 利用PCR技術進行HBV檢測................20
1.2.6 利用dPCR技術進行NSCLC檢測.............21
1.2.7 技術比較..............................22
1.3 研究動機及目標..........................25
第二章 理論介紹............................26
2.1 介電濕潤................................26
2.2聚合酶鏈鎖反應............................30
2.3 加熱片原理...............................34
2.4 數位聚合酶鏈鎖反應........................36
第三章 實驗架設與材料介紹....................42
3.1 晶片製程.................................42
3.1.1數位微流體晶片..........................42
3.1.2 dPCR晶片..............................46
3.2 實驗架設.................................49
3.2.1 PCR實驗架設............................49
3.2.2數位聚合酶鏈鎖反應實驗架設................51
3.3 實驗材料介紹..............................56
第四章 實驗結果與討論........................60
4.1 PCR......................................60
4.1.1 PCR試劑校正.............................60
4.1.2 PCR溫度控制.............................60
4.1.3 PCR油相環境選擇.........................63
4.1.4 PCR實驗結果.............................65
4.1.5利用核殼液滴結構進行PCR...................66
4.2 dPCR.....................................68
4.2.1 矽晶圓微孔洞陣列晶片.....................68
4.2.2數位聚合酶鏈鎖反應液滴分散.................69
4.2.2.1利用微流體幫浦進行液體分散...............69
4.2.2.2利用介電濕潤力在微孔洞陣列中進行液體分散..74
4.2.2.3利用介電濕潤力在SU-8微孔洞陣列中進行液體分散.76
4.2.3 dPCR測試結果............................77
4.2.3.1 溫度量測..............................77
4.2.3.2 孔洞表面積體積比過大...................78
4.2.3.3 dPCR試劑校正..........................79
4.2.3.4 dPCR測定DNA濃度.......................81
第五章 結論與未來展望.........................83
5.1結論.......................................83
5.2未來計畫...................................83
參考文獻......................................85
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