- 學位論文
電磁驅動式核酸萃取聚合酶連鎖反應微晶片系統之開發
Development of a DNA-extraction and Polymerase-chain-reaction Microchip by using Magnetic Beads and Thermo-pneumatic Valves
摘要
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。
並列摘要
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. The 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. Transient 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.