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Title: | 於圓柱陣列微流道中以脈衝式電場分離DNA之研究 Research of DNA Separation in Microchannels with Post Array under Pulsed Electric Field |
Authors: | Jui-Ting Huang 黃睿亭 |
Advisor: | 謝之真(Chih-Chen Hsieh) |
Keyword: | DNA電泳,脈衝式電場,圓柱陣列微流道,DNA分離, DNA electrophoresis,DNA separation,pulsed electric field,post array,microfluidics, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 本研究測試在圓柱陣列微流道中以間歇性的電場進行大DNA的分離。在直流電場下,DNA分子有一定機率與圓柱進行碰撞、上鉤、而後脫鉤,不同大小的DNA經歷此過程所需的時間不同,因此能達到分離。但在高電場下,DNA常以脫鉤時之直線型態於圓柱之間前進,與圓柱之碰撞機率大幅降低。我們認為施以將電場適時關閉再打開之間歇性電場可改善此不利碰撞的分子型態、提高高電場下的分離效率。
我們利用黃光微影及電漿蝕刻在熔融石英基板上製作出圓柱陣列,以繼電器及電腦迴路控制電場的開關,由單分子實驗影像可觀測到DNA在電場為關時確實會由脫鉤時之直線狀回縮,且在50 V/cm之間歇性電場下經過2 mm之圓柱陣列即可將T4及λ-DNA分離。 更進一步,我們預期對λ-及T4 DNA的分離應存在一最佳化電場開、關時間(ton、toff),因此參考初步模擬結果(from陳致安)及實驗量測之T4 DNA特徵鬆弛時間與λ-及T4 DNA之特徵脫鉤時間,選取間歇性電場之ton、toff參數分別為ton= 100、350、1500毫秒及toff= 1500、3000、6000、12000毫秒,初步檢視ton、toff兩參數之影響。 由模擬結果,我們預期當toff大於T4之特徵回縮時間(對應參數toff≧3000毫秒)及ton介於T4及λ-DNA之特徵脫鉤時間之間(ton= 350毫秒)時會具有較好的解析度。在固定通道長度下隨toff由1500毫秒上升至12000毫秒,分離效果增加;而比較ton為100、350及1500毫秒之結果,ton= 100毫秒下有較好的分離結果,與模擬之趨勢不同。 考慮分離所需時間,ton= 100毫秒下所需分離時間較長、經歷的ton、toff循環數較多。為使各組參數下DNA所經歷之循環數相同,我們依ton選取不同偵測終點。初步結果顯示,ton為350毫秒之分離優於100毫秒之結果,但當ton大於350毫秒後於不同通道的分離結果趨勢不盡相同。因此,對於ton之於分離解析度之效應仍需更多實驗結果以與模擬進行驗證。 Separating long DNA in microchannels with post array is a promising alternative to the conventional gel as less time is required. The hooking process alters the mobility of DNA with different degree according to their molecular weight. However, the occurrence of channeling phenomenon limits the operating conditions to low Peclet number (Pe) and further restricts the separation efficiency of a post array with fixed length. We use an intermitant electric field in replace of the continuous one to separate λ-(48.5 kbp) and T4(165.6 kbp) DNA in a fused silica microchannel with a hexagonal post array of 1 micron diameter and 3 micron pitch. Baseline resolution is achieved in our initial experiments under 50 V/cm. The channeling phenomenon is found being suppressed due to the relaxation of stretched DNA during the “off” period of the electric field. We also correlate ton with the first trapping time of DNA. Separations with varying duration of the time intervals ton and toff are conducted to examine the effect of ton and toff on DNA separation efficiency. The results indicate that toff longer than three times of the relaxation time of T4 with ton just enough for λ-DNA to unhook leads to the best resolution of separation. Compared to the results of continuous electric field, an intermitant one enables the separation to be conducted under higher Pe. With properly tuned ton and toff, the resolving power of a channel with fixed length increases only at the expense of time. The ability of this electric field scheme to be easily incorporated with existing devices proofs again its great flexibility. More elaborate relation between the time intervals and the DNA to be separated needs to be constructed for real-world applications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55057 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 化學工程學系 |
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ntu-104-1.pdf Restricted Access | 11.55 MB | Adobe PDF |
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