於圓柱陣列微流道中以間歇式電場分離DNA－電場強度及開關頻率之影響

Abstract

本研究探討在圓柱陣列微流道中以不同間歇性電場之參數分離大片段DNA。DNA在直流電場驅動下，於微流道中有機率與圓柱發生碰撞，而有上鉤、脫鉤之行為，不同大小的DNA經歷此過程所花費的時間不同而有分離效果。但在連續性的電場下，DNA會以脫鉤後的拉伸狀態前進而不易經歷碰撞，故分離效率降低。我們認為將電場以固定頻率進行開、關，可以改善此狀況，並提高DNA在通道中的碰撞機率以及分離效果。 我們由單分子實驗影像可觀測到DNA在電場關閉時會由脫鉤時之直線狀回縮，利用此現象，我們可使用較高強度(Pe>1)之間歇性電場，如50 (Pe =3.27)或90 V/cm(Pe =5.88)，使DNA在經過5 mm之圓柱陣列即可將T4及λ-DNA分離。 間歇式電場提供多個可控變數：電場開、關時間(ton、toff)及電場強度(E)。本研究將固定toff以供我們研究ton及E對分離的影響。我們以T4及λ-DNA的特徵脫鉤時間劃分出三個區間：(1)較λ-DNA之脫鉤時間短之't' _'on' ^'short' 、(2)介於兩DNA脫鉤時間之't' _'on' ^'middle' 與(3)較T4 DNA之脫鉤時間長之't' _'on' ^'long' 。在一固定電場下，討論ton之選擇對於DNA分離效果及時間的影響。若固定電場開、關次數，則介於ttrap,λ及ttrap,T4中間的ton有較高解析度；若固定偵測終點，則't' _'on' ^'middle' 中較靠近ttrap,λ的ton使得DNA經歷更多碰撞而有較高的分離效果。而't' _'on' ^'short' 與't' _'on' ^'long' 之分離效果皆較't' _'on' ^'middle' 差。 電場大小對分離的影響，也和ton之選擇有關：若選擇't' _'on' ^'middle' ，則低電場有較好的分離效率；若選擇't' _'on' ^'long' ，則應使用高電場。而相同距離下，'t' _'on' ^'middle' 之結果普遍比't' _'on' ^'long' 者佳。雖然低電場之分離效果較高電場之分離好，但經由間歇式電場，實驗結果也證明了可以用高電場進行快速的大DNA之分離。此外，經由調整ton及toff，此裝置具有分離各種不同大小DNA之潛力。

Unlike the time consuming conventional gel electrophoresis, microchannels and nanochannels act as alternatives to separate large DNA in a shorter period of time. Though some of existing DNA separating devices are time-saving, such as channels with post array, those devices can only resolve length around the order of λ-DNA (48.5kbp). Some devices are able to resolve large DNA like T4 DNA (165.6kbp), like nano-pillar array. However, those take a long period of time. We apply an intermittent electric field to replace the continuous one to separate λ-(48.5kbp) and T4(165.6kbp) DNA in a fused silica microchannel with a hexagonal post array of 1 micron diameter and 3 micron pitch. We can successfully resolve large DNA under low electric field (20 V/cm), and even under higher ones (50 and 90 V/cm). Baseline resolution is achieved in our experiments around 15 minutes. Inside the post array, the mobility of DNA can be reduced with different degrees according to their molecular weight by the hooking process. However, the occurrence of channeling phenomenon under continuous electric field restricts not only the operating field strength but also the efficiency of a fixed length of channel with post array.The channeling phenomenon is found to be suppressed by the relaxation of stretched DNA during the “off” period of the electric field. We also correlate ton with the trapping time of DNA under different electric field. Separations with varying duration of the time interval ton and electric field E are conducted to examine the effect of ton and E on DNA separation efficiency with toff fixed at 3 times of the relaxation time of T4. The results indicate that under low electric field, ton in the middle of the trapping time of T4 and λ-DNA has the best resolution of separation for a fixed period of time, and ton close to the trapping time of λ-DNA has the best resolution of separation for a fixed channel length. Compared to continuous electric field, an intermittent one enables the separation to be conducted under higher Pe. With properly tuned ton and E, 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 proves again its great flexibility. In addition, we also compare our simulation results to our experimental results, and the simulation results show high consistency with experimental ones, which proves the credibility of simulation. With simulation, we can predict the result before conducting an experiment, or do further research on tough conditions.