整合型微流體平台運用於檢測BRCA1基因突變及卵巢癌風險評估

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研究生：	鄭宇宏 Cheng, Yu-Hung
論文名稱：	整合型微流體平台運用於檢測BRCA1基因突變及卵巢癌風險評估 An Integrated Microfluidic Platform for Detecting BRCA1 Gene Mutation and Risk Assessment of Ovarian Cancer
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	許耿福 Hsu, Keng-Fu 張晃猷 Chang, Hwan-You
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	122
中文關鍵詞：	卵巢癌、早期風險評估、BRAC1基因變異、液態檢體、微流體
外文關鍵詞：	Ovarian cancer, cancer risk assessment, BRCA1 mutation, liquid biopsy, microfluidics
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卵巢癌，又稱為“婦女的隱形殺手”。台灣每年約會有1300個新案例發生，而其死亡率在女性癌症中排名第七位，更是在婦科癌症中排名第一。主要原因為卵巢癌難以在早期被篩檢出。早期卵巢癌的五年存率有92%，而到了晚期，五年存活率掉到僅僅17%，因此如何有效且準確的在早期就發現卵巢癌是非常重要的。近期BRAC1基因變異已經被證實與卵巢癌的發生有很大的關連，有此基因變異的群眾往往有更高的機率會罹患卵巢癌。液態檢體是一門新興的技術，提供了需多關於腫瘤資訊的生物標記物如: 游離DNA，許多篇研究也在游離DNA中發現BRAC1基因變異，可提供卵巢癌的檢測及評估新的工具。因此本研究欲利用微流體系統來自動化萃取病人血漿檢體中的游離DNA並檢測BRAC1的基因變異來提供臨床醫生風險評估。此系統整合了兩個模組: 第一模組為”游離DNA萃取模組”利用渦流型微混和器均勻混和游離DNA及類矽磁珠，能在45分鐘內從200微升的血漿檢體萃取游離DNA並達到76%抓取率；第二模組為”游離DNA定量模組”，運用連動式微幫浦平均分配4.5微升的純化檢體進入反應區並執行90分鐘的晶片上即時單核苷酸多態性聚合酶連鎖反應，目前檢測極限達到50拷貝數。藉由自動化游離DNA萃取及定量過程，此發展系統在”晶片實驗室”的應用有很好的潛力。

Ovarian cancer is known as "a silent killer of women". In Taiwan, there are 1,300 new cases each year, and its mortality rate ranks the seventh among female cancers and the first among gynecological cancers. It is due to the fact that ovarian cancer is difficult to be diagnosed at early stages. The five-year survival rate of early-stage ovarian cancer is 92%, but at the late stages, drops to only 17%. Thus, early diagnosis of ovarian cancer is critical. Recently, BRCA1 gene mutations have been confirmed to be highly related to the occurrence of ovarian cancer. It is evident that patients with this gene mutation usually have a higher chance of developing ovarian cancer in their lifetime. Liquid biopsy such as cell-free DNA (cfDNA) is an emerging technology that brings more information about tumors from the blood. Many studies have found BRAC1 gene mutations in cfDNA could serve a potential tool for diagnosis and risk assessment of ovarian cancer. Therefore, an integrated microfluidic system was developed in this work to automatically extract cfDNA from patient plasma samples and detect BRAC1 germline and somatic mutations to provide clinicians a risk assessment in an automatic format within 120 min. Two modules were integrated in this system; the first module was called “cfDNA extraction module” equipped with a vortex-type micromixer that can mix cfDNA and silica-like magnetic beads to achieve an extraction rate of 76% from 200 µL of plasma sample in 45 min. The second module was a “cfDNA quantification module” which activated a consecutive micropump to distribute 4.5 µL of purified cfDNA sample evenly into reaction zones to perform on-chip real-time single nucleotide polymorphism polymerase chain reaction (SNP-qPCR) within 90 min. The limit of detection was found to be as low as 50 copies. By automating the cfDNA extraction and cfDNA quantification process, the developed system presents a great potential for lab-on-a-chip (LOC) applications.

Abstract    2
摘要    4
致謝    5
Table of Contents    7
List of figures    10
List of tables    19
Nomenclature and abbreviations    21
Chapter 1 Introduction    24
1 Ovarian cancer    24
2 BRCA1 gene mutation    26
3 Liquid biopsy and cell-free DNA    28
3.1 Cell-free DNA and ovarian cancer    28
3.2 Microfluidic applications on cell-free DNA    29
4 Single nucleotide polymorphism polymerase chain reaction (SNP-PCR)    30
5 Motivation and novelty    32
Chapter 2 Materials and methods    35
1 Preparation of samples and reagents    35
1.1 BRCA1 germline/somatic mutation specific primers    35
1.2 DNA samples and PCR reagents preparation    41
1.3 Clinical samples preparation    42
1.5 cfDNA quantification by arthrobacter luteus (ALU) primers    48
2 Chip design and microfabrication    50
3 Working principle and characterization of microfluidic chip    56
3.1 Pneumatic microvalves and micropumps    56
3.2 Vortex-typed micromixer    58
4 Experimental setup and procedure    61
Chapter 3 Results and discussion    65
1 Characterization of the integrated microfluidic chip    65
1.1 Pumping volume and uniformity of micropump    65
1.2 Mixing index of vortex-typed micromixer    70
2 Optimization of cfDNA extraction    72
2.1 Calibration curve and recovery rate calculation    72
2.2 Capture rate of cfDNA    72
3 SNP-PCR for detecting BRAC1 mutation    75
3.1 Optimization of on-bench SNP-PCR condition for germline mutation    75
3.2 Optimization of on-bench SNP-PCR condition for somatic mutation    77
3.3 Spiked experiments of BRAC1 mutation detection    77
4 SNP-qPCR for detecting BRAC1 mutation    80
4.1 Optimization of on-bench SNP-qPCR condition for germline mutation    80
4.2 On-chip SNP-qPCR for germline mutation    80
4.3 Optimization of on-bench SNP-qPCR operating condition for somatic mutation    85
4.4 On-chip SNP-qPCR for somatic mutation    85
5 Sensitivity tests of BRCA1 mutation detection on the microfluidic platform    90
5.1 Limit of detection of SNP-PCR    90
5.2 Limit of detection of SNP-qPCR    90
6 Specificity tests of BRCA1 mutation detection on the microfluidic platform    95
7 Cell-line tests by SNP-PCR    97
8 Clinical tests by SNP-PCR    100
8.1 Clinical tests by SNP-PCR    100
8.1 Spiked experiments of clinical samples    105
Chapter 4 Conclusions and future perspectives    109
1 Conclusions    109
2 Future perspectives    111
2.1 Clinical tests    111
2.2 LOD of the microfluidic system    111
2.3 Increase detection numbers on the microfluidic chip    111
2.4 Specific primers for real-time PCR    112
2.5 Ovarian cancer prognosis    112
References    113
Supplementary information    122

                                

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