聚合脢連鎖反應高分子元件之設計

PDMS (polydimethylsiloxane) Based Polymerase Chain Reaction Component Design

林旻翰(Min-Han Lin)

指導教授 ： 胡文聰

國立臺灣大學/工學院/應用力學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02521

摘要

本論文利用高分子材料--聚二甲基矽氧烷製作出可拋棄式的聚合脢連鎖反應元件，並利用重力驅動的方式設計一個往覆式的反應平台，驅動流體到94℃、55℃和72℃等三種不同的溫度區域進行聚合脢連鎖反應。聚二甲基矽氧烷是一個有彈性、製備簡單、便宜且透明的高分子材料；利用雷射雕刻機製作出聚二甲基矽氧烷翻模用的母模，如此可以快速的製造出所需要的流道尺寸而省去花在黃光製程的時間。由於聚二甲基矽氧烷的透氣性，容易在實驗過程中產生氣泡而阻斷實驗的進行，沈積上一層厚度為4 μm 的聚對二甲苯在聚二甲基矽氧烷上可以改善這問題的產生。同時，在溫度的模擬上，利用ANSYS來幫助整個實驗溫度系統的設計。
在熱傳方面，利用熱電阻所量測出來的試劑溫度結果顯示，試劑可在30秒的時間內從94℃降到55℃、或在15秒內升溫到72℃或94℃等進行反應所需的溫度。溫度量測和模擬的結果相比較是互相吻合的。吾人利用一長度為773 bps的DNA片段進行聚合脢連鎖反應的實驗；實驗中，試劑分別在DNA分離的溫度（94℃）停留30秒、引子鍊合的溫度（55℃）和引子延伸的溫度各停留一分鐘來完成一次聚合脢連鎖反應複製過程。結果顯示利用第一代的平台和流道進行實驗，可以有將DNA片段進行複製放大的成果顯現，但其效率尚不及市售的反應平台所呈現的結果。吾人推測流道中的液體蒸發問題是一個影響效率的重要因素，因此需要設計改善這問題的發生。

關鍵字

聚合脢連鎖反應 ； 聚二甲基矽氧烷 ； 往覆式

並列摘要

This thesis presents a new device to perform polymerase chain reaction (PCR) using polydimethylsiloxane (PDMS) polymer as disposable PCR channel. Particular feature in the device include gravity-fed, circularly reciprocating action to ensure the PCR reagent is in contact with a specific temperature region during the PCR cycle of three temperatures – 94oC, 55oC, 72oC. The PCR channel (3.5 mm width by 1 mm height) is fabricated by molding PDMS against a laser scriber engraved PMMA master, with a 4 µm thick parylene film coated on the PCR channel inner and outer surfaces to alleviate bubble formation at high temperature. Simulation, using ANSYS, of heat transfer of the device is also undertaken to aid the design of the device.

Results from temperature measurement, using resistive temperature detectors (RTD) sensors, show that the reagent can be cooled down to 55oC (from 94oC ) in 30 sec, or heated up to 72oC and 94oC in 15 sec, in agreement with ANSYS predictions. PCR is demonstrated by a 773 bps DNA template, which is incubated at 94oC for 30 sec, 55oC for 1 min., and 72oC for 1 min. to perform DNA denaturation, primer annealing, and extension of DNA template, respectively. Using the present first-generation device, DNA amplification is clearly proven, however, the efficiency is not as good as commercial PCR machine. Evaporation in the reagent is believed to be the key problem that needs to be improved.

並列關鍵字

PCR ； PDMS ； reciprocating

參考文獻

[12] 楊龍杰, “微小尺寸下的液體量測與驅動,” 物理雙月刊, 25卷3期, 2003年6月

[1] Phillip Belgrader, Steve Young, Bob Yuan, Michael Primeau, Lee A. Christel, Farzad Pourahmadi, and M. Allen Northrup, “A Battery-Powered Notebook Thermal Cycler for Rapid Multiplex Real-Time PCR Analysis,” Anal. Chem., Vol. 73, pp. 286-289, 2001

[2] Martin U.Kopp, Andrew J. de Mello, Andreas Manz, “Chemical Amplication: Continuous-Flow PCR on a Chip,” Science, Vol. 280, 15 May 1998

[3] Madhavi Krishnan, Victor M. Ugaz, Mark A. Burns, “PCR in a Rayleigh-Benard Convection Cell,” Science, Vol. 298, 25 October 2002

[5] Jong Wook Hong, Teruo Fujii, Minoru Seki, Takatoki Yamamoto, Isao Endo, “Integration of amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip,” Electrophoresis, Vol. 22, pp. 328-333, 2001