隨著科技知識的發展，生物醫學微機電系統 (Bio-MEMS) 研究正朝著芯片實驗室 (LOC) 設備的方向發展。數字微流體 (DMF) 是一種液體處理技術，允許在開放的電極陣列上進行單獨的液滴控制。對於這種即時護理系統，針對電潤濕 (EWOD) 機制優化的 DMF 系統是一種潛在的技術。在芯片實驗室領域，EWOD微流控生化分析設備有著廣泛的應用。
數字微流體 EWOD 平台適用於基於磁珠 (MB) 從小鼠胚胎培養基中提取無細胞 DNA (cf-DNA)。基於 EWOD 的提取協議僅使用微量提取生物試劑。
需要對傳統和基於 EWOD 的 cf-DNA 的提取性能進行比較分析研究。從台灣林口長庚紀念醫院獲得的小鼠基因組 DNA (gDNA) 用於比較分析研究以常規和 EWOD 方法提取 cf-DNA 的性能。代替 cf-DNA，加載一微升已知濃度的小鼠 gDNA，並以常規和 EWOD 方式執行基於 MB 的提取方案。定量聚合酶鏈反應 (qPCR) 計算常規和 EWOD 提取的回收率。已知濃度的 gDNA 用作陽性對照。根據 q-PCR 結果，常規技術產生 14.8% 的 gDNA，而 EWOD 產生 36.74% 的 gDNA。因此，EWOD 平台上基於 MB 的 cf 提取提供了更好的性能。
鉀補充 SOM (KSOM) 用作小鼠胚胎培養基。小鼠胚胎髮育在4.5天內主要分為四個階段。在這些階段中，選擇小鼠胚胎髮育2.5天（E2.5）和3.5天（E3.5）的胚胎培養基進行cf-DNA提取。 EWOD 提取使用 100V 的電壓和 2kHz 的頻率。從 KSOM 小鼠胚胎培養基的 E2.5 和 E3.5 天提取無細胞 DNA。 E 2.5 樣本中 cf-DNA 的平均數量為 91.47 fg，E3.5 天為 3.28 fg。因此，可以在 DMF EWOD 平台中提取飛克數量的 cf-DNA。
對於任何進一步的研究，例如測序，飛克數量的 cf-DNA 處於亞臨界/亞閾值濃度。據我們所知，我們需要最少皮克/納克 DNA 量進行進一步分析。我們展示了一種突破性的基於 PCR 的機制，稱為“改進的嵌套 PCR”，可將這種亞臨界濃度的 cf-DNA 擴增放大至納克範圍並進行 DNA 測序。基本局部比對搜索工具 (BLAST) 用作序列相似性搜索軟件，以確認查詢和主題之間的識別百分比。測序結果顯示查詢序列和主題序列之間的核苷酸同一性超過 97%。因此，我們可以確認基於 EWOD 的 cf-DNA 提取和基於 PCR 的納克級擴增（改進的嵌套 PCR）方法的真實性。
要了解完整的基因組信息，我們無法進行基於 PCR 的擴增。基於 PCR 的擴增僅擴增特定基因類型。因此，可以通過全基因組測序（WGS）獲得完整的基因組信息。 WGS 需要納克範圍的 DNA。因此，需要進行全基因組擴增 (WGA) 以將 DNA 擴增至納克範圍。但是，執行 WGA 必須需要最小皮克量的 cf-DNA。 E2.5 和 E3.5 胚胎培養基只有飛克範圍的 cf-DNA。因此，我們不能將此提取用於 WGA（以保持納克範圍）和 WGS。為了獲得完整的基因組信息，我們在整個小鼠胚胎生長過程中保持培養基不變。因此，培養基可以收集每個胚胎髮育階段釋放的 cf-DNA。在最後階段，胚胎培養基用於基於 MB 的 EWOD cf-DNA 提取和全基因組擴增 (WGA)。 cf-DNA 的放大納克範圍經過 WGS 併計算了帶有插入缺失（插入和缺失）的單核苷酸多態性 (SNP)。用於基於 EWOD 的 cf-DNA 提取的胚胎培養基用作對照樣品。對照樣品（胚胎培養基）和 EWOD 提取的 cf-DNA 的 SNP 和插入缺失進行了比較，併計算了每條染色體中 SNP 和插入缺失的百分比。 WGS分析也可以確定胎兒性別。
結果表明，基於 DMF EWOD 技術的微尺度 cf-DNA 提取可用於獲取全基因組信息。這些發現表明，使用 EWOD 提取 DNA 是一種可行的選擇。所有這些發現將為著名的芯片實驗室概念鋪平道路。

Biomedical micro-electromechanical systems (Bio-MEMS) research is progressing towards lab-on-a-chip (LOC) devices as scientific and technological knowledge develops. Digital microfluidics (DMF) is a liquid-handling technology that allows for individual droplet control on an open array of electrodes. For such point-of-care systems, a DMF system optimized for an electro-wetting-on-dielectric (EWOD) mechanism is a potential technology. In the lab-on-a-chip field, EWOD microfluidic biochemical analysis devices have a wide range of applications.
The digital microfluidic EWOD platform is adapted for a magnetic bead (MB) based extraction of cell-free DNA (cf-DNA) from mouse embryo culture medium. Only microscale quantity of extraction bio-reagents are utilized for the EWOD based extraction protocol.
A comparative analytical study on extraction performance between conventional and EWOD-based cf-DNA needs to be carried out. Mouse genomic DNA (gDNA) obtained from Chang Gung Memorial Hospital, Linkou, Taiwan, is utilized for the comparative analytical study on the extraction performance of cf-DNA in both conventional and EWOD ways. In place of cf-DNA, one microliter of mouse gDNA with known concentration is loaded and performed the MB-based extraction protocol in conventional and EWOD ways. Quantitative polymerase chain reaction (qPCR) calculates the recovery rate on both conventional and EWOD extraction. The known concentration of gDNA is used as the positive control. According to q-PCR results, the conventional technique yielded 14.8%, while the EWOD yielded 36.74% of gDNA. Hence the MB-based cf-extraction on the EWOD platform gives better performance.
Potassium-supplemented SOM (KSOM) is used as the mouse embryo culture medium. Mouse embryo development can majorly be classified into four stages within 4.5 days. Among these stages, the embryo culture medium at 2.5 days (E2.5) and 3.5 days (E3.5) of mouse embryo development is selected for cf-DNA extraction. EWOD extraction used a voltage of 100V and a frequency of 2kHz. Cell free-DNA was extracted from E2.5 & E3.5 days of KSOM mouse embryo-culture medium. The average quantity of cf-DNA in samples of E 2.5 is 91.47 fg and for E3.5 days is 3.28 fg. Hence, the extraction of cf-DNA in femtogram quantity can be possible in the DMF EWOD platform.
For any further study, such as sequencing, the cf-DNA in femtogram quantity is in subcritical/subthreshold concentration. To the best of our knowledge, we need a minimum of picogram/nanogram DNA amounts for further analysis. We demonstrated a ground-breaking PCR-based mechanism named ‘modified nested PCR’ to amplify this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. The Basic Local Alignment Search Tool (BLAST) is employed as a sequence similarity search software to confirm the identification percentage between query and topic. The sequencing result revealed more than 97 percent nucleotide identity between the query and subject sequences. Hence we can confirm the authenticity of the EWOD-based cf-DNA extraction and the PCR-based nanogram level amplification (modified nested PCR) method.
To understand the complete genome information, we cannot be able to perform PCR-based amplification. PCR-based amplification only amplifies the specific gene type. Hence, the complete genome information can be obtained by whole genome sequencing (WGS). WGS needs a nanogram range of DNA. Hence, whole genome amplification (WGA) needs to be performed to amplify DNA to the nanogram range. However, a minimum picogram quantity of cf-DNA must be required for performing WGA. E2.5 and E3.5 embryo culture medium have only a femtogram range of cf-DNA. Hence we cannot use this extraction for WGA (to maintain nanogram range) and WGS. To get the complete genome information, we maintain the culture medium as such throughout the mouse embryo growth. As a result, the culture medium can collect the released cf-DNA from each embryo's developmental stage. In the final stage, the embryo culture medium is utilized for the MB-based EWOD cf-DNA extraction and whole genome amplification (WGA). The amplified nanogram range of cf-DNA has undergone WGS and calculated the single nucleotide polymorphism (SNP) with the indel (insertion and deletion). The embryo culture medium used for EWOD based cf-DNA extraction is used as the control sample. The SNP and indel of both control sample (embryo culture medium) and EWOD extracted cf-DNA has compared and percentage of SNP and indel in each chromosome has calculated. The fetal gender determination is also possible from the WGS analysis.
The results demonstrate that microscale cf-DNA extraction on the DMF EWOD technology can be used to obtain whole-genome information. These findings suggest that using EWOD to extract DNA is a viable option. All of these findings will pave the way for a well-known lab-on-a-chip concept.

Abstract i
摘要 iv
Acknowledgment vii
Contents viii
List of Figures xi
List of Tables xv
Chapter 1 Introduction - 1 -
1.1 Motivation - 3 -
Chapter 2 Literature Review - 5 -
2.1 Lab-on-a-Chip - 5 -
2.2 Electrowetting-On-Dielectric - 5 -
2.2.1. Droplet Cutting in EWOD - 9 -
2.3 Genetic Testing - 11 -
2.4 In-Vitro Fertilization (IVF) - 11 -
2.5 Deoxyribonucleic Acid (DNA) - 12 -
2.5.1 Magnetic Property of DNA - 13 -
2.5.2 Utility of Magnetic Property of DNA for extraction - 14 -
2.6 Magnetic Beads - 14 -
2.6.1 MBs in DMF - 15 -
2.6.2 MBs in Nucleic Acid Extraction on DMF Platform - 16 -
2.7 Significance of Wash Buffer - 17 -
2.7.1 On-Chip MB Washing Protocol - 18 -
2.8 Concept of DNA Extraction - 21 -
2.8.1 DNA Extraction Methods - 22 -
2.9 Polymerase Chain Reaction (PCR) - 25 -
2.9.1 Quantitative PCR - 26 -
2.10 Agarose Gel Electrophoresis - 26 -
2.11 Mouse Embryo Developmental Stages - 28 -
2.12 Cell-Free DNA (cf-DNA) - 29 -
2.13 DNA Sequencing - 33 -
2.14 Next Generation Sequencing - 34 -
2.14.1 Whole Genome Sequencing - 35 -
2.15 Background - 37 -
Chapter 3 Materials, Equipment & Methodology - 38 -
3.1 Materials & Equipment - 38 -
3.1.1 Dropbot - 39 -
3.1.2 Electro-Wetting-On-Dielectric Chip - 43 -
3.1.2.1 Electrodes in EWOD Chip - 44 -
3.1.2.2 Droplet Generation on EWOD Chip - 45 -
3.1.2.3 Droplet Mixing on EWOD Chip - 46 -
3.2 Methodology - 47 -
3.2.1 Embryo Culture Medium - Potassium Simplex Optimized Medium (KSOM) - 48 -
3.2.2 Bio-Reagents requirement for cf-DNA extraction - 49 -
3.2.3 Typical way of cf-DNA Extraction - 50 -
3.2.3.1 cf-DNA extraction steps in detail - 50 -
3.2.4 Modification in typical way of cf-DNA extraction - 52 -
3.2.5 Adaptation of extraction procedure towards EWOD Platform - 54 -
3.2.6 EWOD Chip Cleaning Procedure - 59 -
3.2.7 qPCR Analysis - 60 -
3.2.8 Modified Nested PCR - 61 -
3.2.9 Agarose Gel Electrophoresis Procedure - 64 -
Chapter 4 Results & Discussion - 67 -
4.1 Fixation of Bio-Buffer Quantity - 67 -
4.2 q-PCR Analysis - 69 -
4.2.1 Standard Curve - 70 -
4.2.1.1 Procedure - Standard Curve - 70 -
4.3 A Comparison – Conventional & EWOD Extraction - 72 -
4.3.1 Aid of EWOD in cf-DNA Extraction - 73 -
4.4 Quantitative analysis on extracted cf-DNA on EWOD platform - 74 -
4.5 Efficiency of Chip Cleaning Process - 75 -
4.6 Improvement in cf-DNA Extraction Protocol - 76 -
4.7 Modified Nested PCR Analysis - 79 -
4.8 DNA Sequencing Analysis - 79 -
4.9 Modification in Embryo Culture Medium - 82 -
4.10 Whole Genome Sequence Analysis - 83 -
Chapter 5 Conclusion & Future Scope - 88 -
Reference - 90 -

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