於微流道中結合介電泳與正向應力分離DNA之研究

依大小分離DNA在生物學及病理學上都有廣泛的應用，而近期有許多研究是以微流道電泳進行DNA之分離，因其有裝置體積小、樣品體積需求低及分離速度快等優點。文獻中的微流體電泳裝置，主要是於流道中設置障礙物，利用較長的DNA更易被障礙物阻擋的原理來達到平行於電場方向上的分離。我們在先前的研究中設計出利用正向應力(normal stress)來分離DNA的微流體裝置，其是利用DNA沿著彎曲的電力線移動時，若受到拉伸則會產生往曲率中心的正向應力，因越長的DNA會受到越大的正向應力，所以能使DNA產生垂直於電場方向之分離。為了更進一步提升以正向應力分離DNA的效率，在本研究中我們嘗試以交流電引發DNA的負介電泳(negative dielectrophoresis，nDEP)效應來與正向應力拮抗，使不同長短的DNA達到更好的分離。我們以λ-DNA(48.5 kbp)和T4 DNA(165.6 kbp)做為分離之對象，以改變緩衝溶液組成及交流電頻率的方式尋找能引發DNA負介電泳的實驗條件。我們於2.2X TBE(Tris/ Borate/ EDTA)緩衝液、0.5X TBE緩衝液及5 mM 磷酸緩衝液中都只觀測到明顯的正介電泳(positive dielectrophoresis，pDEP)現象。而於電導率為187 μS/cm磷酸緩衝液和含5 mM MgCl2之磷酸緩衝液中，則無法觀察到明顯的介電泳現象。雖然已嘗試文獻中所提到能夠引發DNA負介電泳的環境條件，但在實驗中都只能引發正介電泳或沒有明顯介電泳現象。有鑑於引發DNA負介電泳的困難，我們未來將改利用DNA正介電泳，並嘗試改變微流道設計來改變正向應力的方向，使較長的DNA向微流道中心移動，再利用DNA正介電泳與正向應力拮抗，達到更好的DNA分離。

關鍵字

微流道； DNA分離；電泳；正向應力；介電泳

並列摘要

Separating DNA according to size has a wide range of applications in biology and pathology. Many recent studies have conducted DNA separation in microfluidic devices due to their advantages such as small size, low sample volume requirements and fast separation. The microfluidic DNA separation devices in literature mainly used the principle that longer DNA is more easily blocked by obstacles to achieve separation in the direction parallel to the applied field. Different from the typical studies, however, we have designed a microfluidic device that uses normal stress to separate DNA. The normal stress only becomes significant on a curved and stretched DNA, and always points toward the center of curvature of the DNA contour. Since longer DNA experiences larger normal stress, DNA can be separated in the direction perpendicular to the applied field. In this study, we tried to introduce negative dielectrophoresis (nDEP) effect in order to improve the efficiency of our normal stress-based DNA separation device. We took λ-DNA (48.5 kbp) and T4 DNA (165.6 kbp) as the model DNA for separation and looked for the experimental conditions that can trigger nDEP of DNA by changing the composition of the buffer and the frequency of the AC field. Although we have tried the experimental conditions from several studies reporting DNA nDEP, we only observed positive dielectrophoresis (pDEP) in 2.2X TBE (Tris/ Borate/ EDTA) buffer, 0.5X TBE buffer and 5 mM phosphate buffer (PB). Moreover, no obvious DEP was observed in PB with a conductivity of 187 μS/cm and PB containing 5 mM MgCl2. Due to the difficulty to trigger nDEP of DNA, we would suggest that the future design of such a device uses pDEP instead. This could be done by redesigning the microchannel and reversing the direction of normal stress so that longer DNA moves to the center of the device while the shorter DNA moves to the sidewall of the channel due to pDEP effect.