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研究生:鄭宇宏
研究生(外文):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
論文名稱(外文):An Integrated Microfluidic Platform for Detecting BRCA1 Gene Mutation and Risk Assessment of Ovarian Cancer
指導教授:李國賓李國賓引用關係
指導教授(外文):Lee, Gwo-Bin
口試委員:許耿福張晃猷
口試委員(外文):Hsu, Keng-FuChang, Hwan-You
口試日期:2021-09-09
學位類別:碩士
校院名稱:國立清華大學
系所名稱:動力機械工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
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 cancercancer risk assessmentBRCA1 mutationliquid biopsymicrofluidics
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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
1.1 Ovarian cancer 24
1.2 BRCA1 gene mutation 26
1.3 Liquid biopsy and cell-free DNA 28
1.3.1 Cell-free DNA and ovarian cancer 28
1.3.2 Microfluidic applications on cell-free DNA 29
1.4 Single nucleotide polymorphism polymerase chain reaction (SNP-PCR) 30
1.5 Motivation and novelty 32
Chapter 2 Materials and methods 35
2.1 Preparation of samples and reagents 35
2.1.1 BRCA1 germline/somatic mutation specific primers 35
2.1.2 DNA samples and PCR reagents preparation 41
2.1.3 Clinical samples preparation 42
2.1.5 cfDNA quantification by arthrobacter luteus (ALU) primers 48
2.2 Chip design and microfabrication 50
2.3 Working principle and characterization of microfluidic chip 56
2.3.1 Pneumatic microvalves and micropumps 56
2.3.2 Vortex-typed micromixer 58
2.4 Experimental setup and procedure 61
Chapter 3 Results and discussion 65
3.1 Characterization of the integrated microfluidic chip 65
3.1.1 Pumping volume and uniformity of micropump 65
3.1.2 Mixing index of vortex-typed micromixer 70
3.2 Optimization of cfDNA extraction 72
3.2.1 Calibration curve and recovery rate calculation 72
3.2.2 Capture rate of cfDNA 72
3.3 SNP-PCR for detecting BRAC1 mutation 75
3.3.1 Optimization of on-bench SNP-PCR condition for germline mutation 75
3.3.2 Optimization of on-bench SNP-PCR condition for somatic mutation 77
3.3.3 Spiked experiments of BRAC1 mutation detection 77
3.4 SNP-qPCR for detecting BRAC1 mutation 80
3.4.1 Optimization of on-bench SNP-qPCR condition for germline mutation 80
3.4.2 On-chip SNP-qPCR for germline mutation 80
3.4.3 Optimization of on-bench SNP-qPCR operating condition for somatic mutation 85
3.4.4 On-chip SNP-qPCR for somatic mutation 85
3.5 Sensitivity tests of BRCA1 mutation detection on the microfluidic platform 90
3.5.1 Limit of detection of SNP-PCR 90
3.5.2 Limit of detection of SNP-qPCR 90
3.6 Specificity tests of BRCA1 mutation detection on the microfluidic platform 95
3.7 Cell-line tests by SNP-PCR 97
3.8 Clinical tests by SNP-PCR 100
3.8.1 Clinical tests by SNP-PCR 100
3.8.1 Spiked experiments of clinical samples 105
Chapter 4 Conclusions and future perspectives 109
4.1 Conclusions 109
4.2 Future perspectives 111
4.2.1 Clinical tests 111
4.2.2 LOD of the microfluidic system 111
4.2.3 Increase detection numbers on the microfluidic chip 111
4.2.4 Specific primers for real-time PCR 112
4.2.5 Ovarian cancer prognosis 112
References 113
Supplementary information 122
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