利用絕緣式介電泳於微流道中分離DNA之研究

Abstract

近年來以微流道分離DNA的研究因其相較傳統技術更加快速且便利的優勢而興盛，其中以介電泳力來分離的方法逐漸受到重視，在相關研究中又多以絕緣式介電泳來分離，因為相較於電極式介電泳，絕緣式介電泳的成本較為低廉，而且因為其電極離實際上分離的區域相當遠的原因，能夠克服電極式介電泳在電極處積垢以及因為化學反應而產生氣泡進而影響實驗的問題，因此本研究希望能設計出有別於以往的絕緣式介電泳分離方法的新穎裝置，我們以T4-DNA(165.6 kbp) 和λ-DNA (48.5 kbp)來做為DNA的分離代表對象。 我們提出的裝置設計是以電泳和介電泳的結合應用作為核心，並以不同的DNA在實驗上電泳速度相同但在相同介電泳條件下會有不同程度的介電泳力來做為分離的原理，讓介電泳力作用於DNA電泳的垂直方向，並且不斷重複此一過程，以此達成垂直於電泳方向的分離。通道的設計則是考量如何利用通道構型產生不均勻電場和適當的電力線分布以達到分離之目的。此裝置雖是以分離DNA而設計，但依靠的原理是電力線在流道內的分布以及其與介電泳的結合應用，因此理論上此設計並不局限於分離DNA，若設計得宜此裝置應可分離各式物質。 我們根據上述的原則設計了一種類似於三稜鏡的裝置，也就是混合的DNA一起進入通道後會因為介電泳性質的不同而產生不同的電泳方向。將分離單元設計為水滴的形狀並將其重複排列於微流道中，稱其為分離區，再利用微流道本身的構型控制電力線的分布來分離DNA，裝置本身可以分為入口、分離區及出口。DNA進入分離區之後會受介電泳力而偏離原先的電泳路徑，並且受力會因DNA的大小而不同，以此來分離DNA。在原始的裝置設計中我們確認了上述效應的影響，但因為通道構型的設計會使部分分離區無法發揮功效，並且還會增加DNA的擴散，因此我們在第二種設計中改良出口部分，以此讓分離區全都能發揮功效，同時把分離區擴大並改善電力線的分布，也成功讓DNA偏移更多。其中較小之DNA偏移的比較大之DNA更多，雖然證實此裝置的確具有分離效果但與我們預期較大之DNA應偏移較多相反，我們推測原因為分離區結構和DNA間的體積排斥效應所導致，因此在最後的相關驗證中以λ-DNA和λ-DNA的碎片作為分離對象並成功將其分離。 在我們的研究中，此種裝置設計仍有優化之空間，若能有效設計通道幾何結構以及分離單元之幾何形狀，有望能夠在成功分離DNA的同時更進一步增加分離效率。

In recent years, research on DNA separation using microfluidic channels has become popular due to its advantages of being faster and more convenient compared to traditional techniques. Among these methods, the use of dielectrophoresis (DEP) for DNA separation has become increasingly important. Among different application modes of dielectrophoresis, insulator-based dielectrophoresis (iDEP) has become more popular compared to electrode-based dielectrophoresis (eDEP) because iDEP avoids issues associated with electrode fouling and bubble formation. In current study, we focus on utilizing the dielectrophoretic force field generated using iDEP to design a novel device for DNA separation. We employ T4-DNA (165.6 kbp) and λ-DNA (48.5 kbp) as representative DNA targets for separation. The designed principle of our device integrates electrophoresis and dielectrophoresis as the core mechanism. The separation principle relies on the fact that different DNA exhibiting the same electrophoretic velocity while experiencing varying degrees of dielectrophoretic forces under identical conditions. By arranging the electrophoresis and dielectrophoresis in the orthogonal directions, DNA separation by size can be achieved. Based on the principles mentioned above, we designed a device resembling a prism where DNA with different sizes, entering into the device at the same inlet, would leave the device at different position. The device consists of three major parts: the inlet, the separation zone and the outlet. The separation zone is an assembly of repeating separation units with a shape of a droplet and dielectrophoresis is generated between the separation units due to the asymmetric electric field. When DNA enters the separation zone, it deviates from the original electrophoretic path due to dielectrophoretic forces, which vary based on DNA size, enabling DNA separation. Using the primitive device, we confirmed the proposed separation principle. However, due to the flaws in the device design, some part of the separation zone was unused, resulting a deteriorated separation. Therefore, in the modified design, we adjusted the the exit to ensure all separation zone were effective and also enlarged the separation zones to further enhance the separation efficiency. Although DNA with different sizes were shown to leave the separation zone in different path, smaller DNA exhibited more significant deviation than larger DNA, contrary to our expectations. We found this is caused by the excluded volume effects between the separation units and DNA. In the final validation, λ-DNA and its fragments were successfully separated using the modified device. To conclude, we have proposed a novel combination of dielectrophoresis and electrophoresis for DNA separation and a prototype device was demonstrated. Based on the finding of this study, we can further improve the device for more effective and continuous separation of DNA. While the device is designed for DNA separation, it should also be capable of separating various substances such as particles, cells and proteins.