可交換式模組化微流元件運用於細胞核酸之萃取技術

Abstract

本研究接續實驗室前人所開發的模組化微流元件，將其運用於細胞、病患檢體的微量DNA 萃取。傳統的 DNA 萃取方法必須使用定量滴管以及離心機搭配不同劑量的化學藥劑逐次將細胞裂解、DNA 聚合、細胞殘骸排除、蛋白質分解等等不同的功能，在化學環境下逐步清洗，最後再去除雜質後留下DNA。 然而，較近期的研究及產品則轉向於使用DNA 載具(二氧化矽薄膜或磁珠)來提高DNA 的萃取量。傳統的萃取方法中，因為DNA 分子本身具有帶電荷極性結構，在酒精的非極性環境中，DNA 分子會相互黏合在一起形成一個大型、肉眼可見的團塊。在沖洗細胞殘骸的過程中容易有化學試劑殘留或是DNA 片段被沖洗脫落造成損失等問題。然而，運用DNA 載具的優點是，將DNA 分子上帶有負電荷的磷酸鹽結構與二氧化矽分子在強離子環境下透過離子橋連接，形成一個強力的離子鍵結，這種方法能夠減少DNA 分子的遺漏，也可以透過載具清洗的方式把化學試劑的殘留量減少。 本研究將使用 PDMS(聚二甲基矽氧烷)做為模組的成型材料，在經過特定功能設計的鋁製模具中製作、組裝。模組運用微米磁珠做為DNA 載具，具有清洗DNA、分離液體預防交叉汙染、傳遞載具等功能。在模組化元件中達成微量DNA的萃取。相較於複雜且昂貴的MEMS 製程以及商用的大型儀器，使用模具可以快速生產具有競爭力的功能元件，其簡單且有效的操作方式有利於將此DNA 萃取方法快速推廣，而且模組化元件萃取結果的效率並不遜於精密的MEMS 微流晶片或已經商用的萃取方法。 未來，運用此優勢生產操作簡便且便宜的DNA 萃取模組，推廣至居家照護 用途(Point Of Care, POC)，或是貧窮國家針對傳染性疾病的快速篩檢將相當具有潛力。

The thesis will introduce the swappable fluidic module which was developed by the LAB senior, and it will be implemented on DNA extraction from the HBV Hepatitis B virus or human cells in this research. The traditional DNA extraction method must use pipet and centrifuge for different chemical reagent preparation, then lysis cells, bind DNA, eliminate the cell debris and finally decompose the protein or other PCR inhibitor. After all the process above, we clean the samples step by step, then DNA fragment will finally remain in the solution. In standard DNA extraction method, since the DNA structure have the phosphate backbone on it, when the solution is in neutral condition, for example high concentration of ethanol, the DNA fragment will bind with each other, forming a big, visible aggregation. We utilize this property to extract the DNA from the cells, but it has some drawbacks because of the DNA fragment will be washed out or contaminating substance remained in the aggregation, it will lead to bad influence for the upcoming process. However, the recent research and commercial approach turn to use the DNA carrier such as Silicon dioxide membrane or magnetic beads to enhance the DNA extraction efficiency. To resolve the problems mentioned above, scientist developed the DNA carrier method to bind DNA stronger than before. The DNA carriers are coated with silicon dioxide on it, when the carrier and DNA are in the strongly ionized environment, the ion will bind the phosphate backbone and SiO2 together, forming a strong ion-bind between them. By this method, it can reduce the quantity of washed out DNA and enhance the purity of solution after washing steps. We will use PDMS(poly-dimethylsiloxane) as the material to manufacture the specially designed modules. These modules have the functions such as washing DNA, separating the reagents and transporting the beads. By the development of DNA carrier magnetic beads, we can perform the DNA extraction on our newly released method. Comparing to the complex and expensive MEMS chip or commercial machine, our modules can be manufactured at fast speed and low cost. The easy-handled and functional modules can be promoted to the public easily. Besides, the DNA extracted by module have the great efficiency compared to the commercial methods. In the future, it can promote to the point-of-care POC platform or even use it to diagnose the infectious illness in remote area or bad medical condition place.