微流道表面鈍化效應對聚合酶連鎖反應之影響

The effect of microchannel surface passivation on polymerase chain reaction

邱顯丞(Xian-Cheng Qiu)

指導教授 ： 陳志堅

國立屏東科技大學/工學院/生物機電工程系所/碩士(2014年)

https://doi.org/10.6346/NPUST.2014.00306

摘要

聚合酶連鎖反應（PCR）是可以複製特定的DNA片段的技術，然而此技術需使用一台體積龐大的熱循環儀才可進行，因此為了改善這個問題，本文研製了一個連續流體方式PCR晶片。首先以商用模擬軟體CFD-ACE+TM模擬本文所配置的三個溫度區域對於晶片之影響，確認溫度分布後，便可加入微流道進行模擬，探討裝置的可行性與相關參數。實驗方面，首先以微機電製程技術與聚二甲基矽氧烷(PDMS)翻模技術，製作出具有微流道之晶片，而溫度的部分是在晶片下方兩側，配置兩個不同溫度的加熱模組，而中間設置冷卻區域，將熱能傳導至散熱區，使得晶片中央溫度下降，而溫度控制是使用8051單晶片與測溫元件所組成，透過上述之條件，實際檢測晶片之溫度發現到晶片內與外具有7~8K的溫度差，因此運用此結果搭配紅外線熱像儀，便可達成三個穩定溫區，成功建立出PCR所需之環境後，接著對於晶片的微流道表面進行優化，本文採用三種表面鈍化試劑進行比較，吐溫 20（Tween 20）濃度為20%時，能明顯減少PDMS表面的甲基（CH3）光譜，而表面的平均粗糙度約61nm，明顯優於其他兩種，接著運用試劑均可溶於水的特性，以去離子水進行觀察，發現Tween 20可使PDMS改變為親水性，因此可了解到Tween 20鈍化效果最為良好。接著實際運用於生物實驗，結果顯示本文裝置成功複製了DNA片段，擴增時間為50min，並且透過鈍化處理能有效的幫助DNA複製，接著對於裝置進行探討，發現流量在1μl/min與樣品量在10μl以上時，雖然無法媲美市售PCR熱循環儀，但是無論實驗時間或實驗結果，均能達成一個最佳之平衡點。
關鍵詞：連續流體式PCR、微流體晶片、聚合酶鏈鎖反應、鈍化反應、聚二甲基矽氧烷。

關鍵字

連續流體式PCR ； 微流體晶片 ； 聚合酶鏈鎖反應 ； 鈍化反應 ； 聚二甲基矽氧烷

並列摘要

The polymerase chain reaction (PCR) is a technique that can duplicate specific DNA fragments, however, conducting the technique requires the use of a very large-scale thermal cycler. In order to accommodate for this problem, this study has developed a continuous flow PCR chip. Initially, CFD-ACE+TM commercial simulation software is used to simulate the impact that the three temperature zones configured in this paper have on the chip. After confirming the temperature distribution, microfuidic channels can be added to the simulations in order to investigate the feasibility and related parameters of the apparatus. In the experimentation, initially, a microelectromechanical processing technique and polydimethylsiloxane (PDMS) formwork technique were used to create a chip with microfuidic channels. For the temperature, on the two sides of the bottom of the chip, two heating modules were configured with different temperatures and a cooling zone was set up in the center to allow the thermal energy to transfer to the heat dissipation zone and allow the temperature of the chip’s center to decrease. The temperature control was composed of an 8051 single chip and temperature measurement device, which, the actual detection of wafer temperature was found to have the chip temperature difference between the inside and outside within 7-8K, Thus the use of this result with infrared thermal imager, by using the abovementioned conditions, could create three stable temperature zones.
After successfully establishing the environment necessary for the PCR, optimization was conducted on the surface of the chip’s microfluidic channels. This paper used three types of surface passivation reagents to conduct comparisons and discovered that when the polysorbate 20 (Tween 20) has a concentration of 20%, it can significantly reduce the methyl (CH3) spectroscopy on the PDMS surface, resulting in an average surface roughness which is approximately 61nm. This outcome was significantly more favorable than the other two types. Also, as all the reagents used are water soluble, deionized water was used to conduct observations, and it was discovered that the Tween 20 can cause the PDMS to become hydrophilic; thus it can be seen that the Tween 20 has the optimal passivation effect. Furthermore, when applied to actual biological experiments, the results indicated that the apparatus successfully duplicated the DNA fragments and that use of passivation treatment can effectively help the response of the DNA copies. The experimental time was 50 min.Then for the device to explore, discover traffic 1μl/min and the sample volume at the time of 10μl or more, although not comparable to commercially available PCR thermal cycler, but no matter the time or the experimental results, the best able to reach a balance.
Keywords：Continuous fluid PCR、Microfluidic chip、Polymerase chain reaction、Passivation、PDMS

並列關鍵字

Continuous fluid PCR ； Microfluidic chip ； Polymerase chain reaction ； Passivation ； PDMS

參考文獻

[28] 梁文憲，2006，兩段式熱循環微流道PCR晶片，大同大學生物工程學系碩士論文，臺北。

[27] 謝雅玉，2005，用於微晶片之聚雙甲基矽氧烷基材表面穩定化學修飾，國立成功大學化學系碩士論文，臺南。

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