本論文主要是透過介電濕潤(Electrowetting on dielectric, EWOD)晶片應用於生殖醫學。而介電濕潤晶片的優點在於製程簡單方便且成本低廉、容易操作微流體的生成、傳輸、分離、結合等。介電濕潤晶片應用於生殖醫學上的應用是將數位微流體晶片作為微量DNA萃取的平台，將傳統利用磁珠去萃取DNA的方法重現於晶片上，希望藉由數位微流體晶片的特性，利用更少量的體積去抓取DNA，並提高其抓取率。
體外受精（In vitro fertilization , IVF）是一項令人興奮的科學成就，對人類生活產生了巨大影響。提高受精率和生育健康寶寶將是重要的研究目標。在過去，醫生選擇胚胎來定植卵巢取決於胚胎的外觀，而不是胚胎的基因型。這是因為傳統的臨床基因檢測需要至少為ng /μl的DNA濃度。然而，胚胎培養基中DNA的濃度僅為pg /μl的量級或甚至低於fg /μl。如果我們可以通過EWOD系統從胚胎培養基中提取cell-free DNA(cfDNA)，它將對臨床生殖醫學產生重大影響。
本實驗分為三個步驟:首先，我們先去測量所需應用到生物性緩衝液的接觸角，並且實際將buffer操作在晶片上於油浴及空氣的環境下，希望藉此了解到哪一種環境較適合進行DNA的萃取;接著，我們利用從工研院取得的設計好的哺乳類動物DNA(106 copies/µl)，去確認是否可以成功在晶片上去萃取DNA，並且與傳統法做比較，並且發現晶片上萃取DNA可以有效提高DNA的抓取率。再來，我們藉由改變一些萃取的步驟及介電層的材質去提高抓取的效率;最後，我們將檢體改成2.5天及3.5天臨床的鼠胚培養置換液去進行DNA的萃取，其濃度低於pg/µl甚至是fg/µl，以傳統方法去進行萃取是極為困難去取得這麼低含量的DNA且最低體積需要8µl，但我們利用EWOD晶片成功僅用50nl的體積即可成功抓取到DNA。利用EWOD晶片可以從50nl的2.5天臨床鼠胚培養置換液萃取到25~200fg 的DNA量; 利用EWOD晶片可以從50nl的3.5天臨床鼠胚培養置換液萃取到0~3fg 的DNA量。
接著，我們利用設計好的Sry引子去將在EWOD晶片上對鼠胚培養置換液進行的DNA萃取結果進行基因判定，企圖利用此方法使我們在體外受精的過程中且在未傷害胚胎本體的情況下，對鼠胚進行性別的判定。我們目前已經成功地利用鼠胚培養置換液分辨出鼠胚的性別且也藉由正負控制組以及內部校正組去驗證實驗的可靠性。這代表了通過EWOD系統從胚胎培養基中提取cfDNA，再對提取的DNA進行基因檢測是可行的，這將對臨床生殖醫學提供一嶄新的發展。
關鍵字：DNA萃取、電潤濕、數位微流體、磁珠

This study presents the application of electrowetting on dielectric(EWOD) chip on clinical reproductive medicine. The advantage of EWOD chip is simple fabrication, low cost, and easily to operate microfluidic generating, transporting, separating, merging.In this study, we have developed a platform to extract DNA in clinical reproductive medicine with EWOD system.We follow the traditional MBs-based extraction kit and perform on our EWOD chip. Electrowetting-on-dielectric (EWOD) with magnetic beads (MBs) would be a good way to improve the DNA extraction due to its high flexibility and low reagent consumption.
In vitro fertilization (IVF) was an exciting scientific achievement that maintains a great impact on human lives. Improve the fertilization rate and give birth to healthy baby would be the important research goal. In the past, doctors chose embryos to colonize ovary depend on embryos' appearance, but not the fetus genotype. That is because traditional clinical genetic testing requires a concentration of DNA at least of ng/μl. However, the concentration of DNA in embryo medium culture would only be the order of pg/μl or even low at fg/μl. If we can extract cfDNA from the embryo culture medium by EWOD system, it will give a big impact of the clinical reproductive medicine.
We divide our experiment to three parts: First, a preliminary study of all reagents was conducted prior to adapting the on-chip DNA extraction protocol, the droplet manipulation by EWOD for all reagents, on both air or oil environments; Second, we take the designed mammal DNA as sample, which is provided from The Industrial Technology Research Institute (ITRI, Taiwan), and the concentration of sample is order of 106 copies/µl. We used designed mammal DNA to test the reliability of our platform, compared our result with the result of traditional method. Besides, we change the material of dielectric layer and some DNA extraction protocol in order to add our result of recovery rate; Third, we change the sample from designed mammal DNA to the clinical 2.5 days and 3.5days embryo culture. The clinical embryo buffer of mouse has low DNA concentration less than 1pg/μl or even 1fg/μl. It is very difficult to extract DNA from the embryo buffer with traditional method due to its low concentration and traditional method require 8µl at least. However, we not only extract DNA successfully from the embryo buffer but also just use 50nl sample to extract DNA. We have extracted 25~200fg DNA from 50 nl 2.5 days mouse medium culture with EWOD system; We have extracted 0~3fg DNA from 50 nl 3.5 days mouse medium culture with EWOD system.
We use designed Sry primer to do determine the DNA extraction results of the mouse embryo medium culture which do with EWOD chip.This method to allow us to determine the sex of the mouse embryo during the in vitro fertilization without harming the embryo itself. We have distinguished the sex of the mouse embryo with DNA extraction result which do from medium culture with EWOD system. It is feasible to extract cfDNA from embryo culture medium by EWOD system, and then perform genetic testing on the extracted DNA. It is an amazing result for the clinical reproductive medicine.

Keywords: DNA extraction; electrowetting; digital microfluidics; magnetic beads

目錄
中文摘要 I
ABSTRACT III
目錄 VI
第一章 緒論 1
1.1 前言 1
1.2 基因檢測 1
1.3 微流體系統 2
1.4 研究目標 3
第二章 文獻回顧 5
2.1介電濕潤現象與微液珠操控 5
2.2介電層材料之探討 10
2.3 介電濕潤系統於生醫領域與DNA之相關文獻 12
第三章 研究方法及系統架設 18
3.1 介電濕潤原理 18
3.2 共平面式介電濕潤系統 21
3.3 介電濕潤晶片製程 22
3.4 介電濕潤晶片之NI PXI-6512系統架設及建立 27
3.5哺乳類DNA樣品與臨床老鼠胚胎置換液樣品來源與用途 30
3.6 DNA萃取所需之藥品及其功用 31
3.7 引子的選擇 32
3.8即時聚合酶連鎖反應原理 33
3.9 膠體電泳原理 36
第四章 DNA萃取之實驗結果 37
4.1 EWOD晶片之微液滴操控 37
4.2 生醫緩衝液在晶片操作的可行性 37
4.3傳統的DNA萃取實驗 39
4.4 以哺乳類DNA樣品在晶片上進行DNA萃取 40
4.5 如何提高DNA萃取的回收率 43
4.5.1 系統化規劃優化DNA萃取方法 43
4.5.2 改變緩衝液的體積比例需求 44
4.5.3 改變加熱溫度 46
4.5.4 改變加熱時間 47
4.5.5 增加洗脫DNA次數 48
4.5.6 優化方式的選擇與操作 49
4.6 EWOD晶片的重複利用可行性 51
4.7 以臨床老鼠胚胎培養置換液樣品進行DNA萃取 52
第五章 以胚胎培養置換液樣進行胚胎性別判別 73
5.1 基因檢測在人工生殖方面面臨的挑戰 73
5.2 正負控制組(POSITIVE AND NEGATIVE CONTROL)以及內部校正組(INTERNAL CONTROL)的選擇 74
5.3 對控制組進行QPCR及膠體電詠 75
5.4以臨床老鼠胚胎培養置換液樣品去判斷鼠胚性別 78
第六章 結論 83
第七章 未來工作 85
6.1將樣品從鼠胚培養置換液換成人類胚胎置換液 85
6.2後續DNA序列判定的方法 86
文獻參考 88

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