楊燿州臺灣大學:機械工程學研究所楊欣穎Yang, Xin-YingXin-YingYang2010-06-302018-06-282010-06-302018-06-282009U0001-1908200922035800http://ntur.lib.ntu.edu.tw//handle/246246/187089本研究開發一種新型的電磁驅動式核酸萃取聚合酶連鎖反應微晶片系統，其整個系統體積為72mm × 52.5mm × 11mm，僅手機般的大小，包括所有的控制電路、微流道、微幫浦、閥、線圈、壓電片、微加熱器以及微溫度感測器，可以說是令人耳目一新的生醫晶片(biochip)元件系統。本系統採用微機電(MEMS)製程技術製作以玻璃為基材的加熱器，以及利用SU-8製程技術製作矽基材的氣腔母模。另外，藉由MDX-40R加工機，製作出適合的壓克力流道母模，再透過PDMS翻模技術與氧氣電將接合製作出本實驗的拋棄式微流道晶片。利用印刷電路板及積體電路組成控制系統，系統的外殼是使用藉由雷射加工的製作壓克力為材料。本研究利用磁珠在特定pH值以下的環境，會吸引DNA的原理，再藉由線圈吸引磁珠，使磁珠穿越不同的腔體，達到萃取DNA的目的，再藉由熱氣動式微幫浦推動液體進入聚合酶連鎖反應槽，透過溫度轉換，達成聚合酶連鎖反應結果。由於本系統的微流道晶片為可拋棄式，所以在每次使用上僅需更換微流道部分，達成低成本、可拋棄式的耗材。本實驗利用微加熱器以及微溫度感測器，並藉由比例微分積分(PID)程式寫入微處理器中做溫度，控制PCR腔體的溫度。本研究所開發的核酸萃取聚合酶連鎖反應晶片所需的前處理時間，從用手操作的30分鐘縮減成6分鐘，而本晶片的PCR反應時間，從商用機台150分鐘縮減到74分鐘，所需DNA樣本溶液也從商用機台的25μL減少到只需10μL。由實驗結果顯示，本晶片前處理萃取DNA濃度為2.5ng/μL,由PCR跑膠結果圖可知道，可達成PCR反應，並有效縮減商用機器所需之時間與DNA樣本溶液的使用量。In this work, we develop a DNA extraction and polymerase chain reaction (PCR) microchip utilizing magnetic beads driven by electromagnetic force. The size of the self-contained system, which comprises a microfluidic chip, micro-heaters, micro-temperature sensors, coils, a piezoelectric-plate and a control module, is 72mm × 53.5mm × 11mm. The parameters such as cycle times, cycle numbers and reaction temperatures can be adjusted and controlled by a microcontroller. The device can fully operates with a 12V DC voltage, and does not require any external pumping equipment or bulky power supply.he microfluidic chip is composed of three layers, a PDMS microchannel layer, a PDMS air chamber membrane, and a glass cover. A sample loading chamber, a wash buffer chamber, an elution buffer chamber and a PCR chamber are fabricated on the microfluidic chip. A linear array of coils, which is located underneath the microfluidic chip, is also implemented in the system. The coil array is in charge of manipulating magnetic beads, on which DNA are attached, through the microchannel and and chambers. Since all solutions for DNA extraction and PCR are loaded in different chambers prior to the operation, thermo-pneumatic microvalves, which are integrated in the microfluidic chip, are used to isolate those solutions. Besides, an integrated thermo-pneumatic micropump is used to actuate DNA solution into the PCR chamber. Both microvalves and micropump in the system perform well with a very mild temperature elevation. In the PCR chamber, heaters and temperature sensors fabricated by MEMS techniques were implemented, and the PID scheme are employed to control the temperature to desired level with desired durations. ransient temperature of the PCR chamber is measured by an infrared thermometer. An infrared thermal imager is used to observed temperature distribution in the PCR chamber and near the thermo-pneumatic microvalves. Concentration of the DNA extracted from blood is detected by a UV-visible spectrophotometer. Slab gel electrophoresis experiments are carried out to demonstrate the PCR results. The proposed system only needs 10μL PCR liquid sample and takes less than 74 minutes for a whole PCR process.致謝 III要 IVbstract V錄 VII目錄 X目錄 XIV一章 緒論 1.1前言 1.3.1樣品預處理晶片 3.3.2聚合酶連鎖反應晶片 6.3.2.1腔體式 8.3.2.2連續流式 11.3.2.3熱對流式 13.3.2.4其它類型 14.3.3整合式樣本前處理及聚合酶連鎖反應晶片 16.4論文架構 18二章 理論與操作原理 19.1 DNA樣品前處理原理 19.2聚合酶連鎖反應原理 20.3磁珠在腔體內移動原理 23.4熱氣動式微幫浦與閥之原理 24.5電阻式熱感測元件電路原理 27三章 元件設計 32.1微流道晶片設計 32.1.1腔體設計 33.1.2熱氣動式微閥設計 36.2外殼設計 38.3生物晶片組裝設計 39四章 製程方法與步驟 41.1製程規劃 41.2光罩設計 42.3製程技術及原理 45.3.1基材清潔 45.3.2微影製程 46.3.3金屬蒸鍍 47.4微流道製程 48.4.1熱氣動式微閥之SU-8母模製程 48.4.2流道之壓克力母模 51.4.3 PDMS轉印製程 52.4.4 PDMS與玻璃接合 54.5微加熱器與微溫度感測器製程 57.5.1 黃光微影製程 57.5.2 E-beam鍍膜製程 58.6電路及系統整合 60.6.1控制及感測電路設計 60.6.2線圈製作 64.6.3微樣品前處理聚合酶連鎖反應晶片組裝 65五章 實驗量測與討論 67.1控制系統 67.2實驗環境 70.2.1 DNA萃取及試劑調配 70.2.2商用分光光度計 72.2.3商用PCR機台 72.2.4電泳設備 73.2.5螢光照相系統 75.2.6紅外線測溫槍 76.2.7紅外線溫度熱像儀 77.3流道上溫度表現與討論 78.4熱氣動式微閥的表現與討論 80.5 DNA extraction結果與討論 81.6 PCR結果與討論 82六章 結論與未來展望 84.1結論 84.2未來展望 85考文獻 864641209 bytesapplication/pdfen-US聚合酶連鎖反應生醫晶片微機電技術SU-8PDMSDNA extractionPCRbiochipMEMSthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187089/1/ntu-98-R96522725-1.pdf