針對心血管疾病使用微流體系統偵測存在於胞外囊泡的小分子核糖核酸

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研究生：	鄭鴻琳 Cheng, Hong-Lin
論文名稱：	針對心血管疾病使用微流體系統偵測存在於胞外囊泡的小分子核糖核酸 Detecting MicroRNA from Extracellular Vesicles for Cardiovascular Diseases by Using a Microfluidic System
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	王玉麟 Wang, Yu-Lin 林彥亨 Lin, yen-heng
學位類別：	碩士 Master
系所名稱：	工學院 - 奈米工程與微系統研究所 Institute of NanoEngineering and MicroSystems
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	69
中文關鍵詞：	心血管疾病、胞外囊泡、微型核醣核酸、微流體
外文關鍵詞：	Cardiovascular diseases, Extracellular vesicles, MicroRNAs, Microfluidics
相關次數：	點閱：1 下載：0
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根據世界衛生組織的報告，在全球死亡人數裡，因為心血管疾病而死亡的人數佔了非常大的比例。在2015年超過1770萬人的死亡死於心血管疾病，所以心血管疾病被認定為是全球主要死因之一。在台灣，心血管疾病在十大死因中排名第二，造成的醫療支出對於全民健保系統也造成重大的負擔。然而傳統的檢測心血管疾病的方法包括斷層掃描、心電圖和心肌灌注成像掃描。雖然這些檢測的方法已被醫院廣泛應用，但是這些方法相對昂貴且不能在早期階段診斷出心血管疾病。另外，創新的分子生物標誌物如血液中的肌鈣蛋白I，C-反應蛋白，N端前腦利鈉肽以相對簡單，快速和非侵入式的方式對早期的心血管疾病可以檢測出徵兆。近幾年來，從細胞外囊泡釋放的微型核醣核酸已被認為是重要的心血管疾病生物標誌物用於心血管疾病的早期偵測及預後監控。在這項研究中，我們開發了一個整合型微流體系統搭載了高靈敏的場效應晶體管，且這微流體系統整合下列幾項功能:利用磁珠抓取檢體中的胞外囊泡、裂解胞外囊泡、純化出與心血管疾病相關的微型核醣核酸、以及場效電晶體檢測釋放的微型核醣核酸的濃度。整個偵測的流程可自動化地操作單一晶片在五小時內完成且對目標微型核醣核酸21和126的偵測濃度極限達到飛莫爾濃度範圍可符合人體生理濃度。在未來，這整合型微流體晶片將有非常大的潛力成為可以用於心血管疾病的早期診斷的工具。

According to World Health Organization’s reports, cardiovascular diseases (CVDs) are one of the major causes of death globally, responsible for over 17.7 million deaths in 2015. It is not only one of the leading causes of death worldwide but also second amongst the top ten leading causes of deaths in Taiwan. Traditional detecting methods include cardiac computerized tomography scan, electrocardiography and myocardial perfusion imaging scan. Although diagnosis of CVDs through bio-imaging techniques is common in hospitals, those methods are relatively costly and cannot diagnose CVDs at early stages. In contrast, the level of novel molecular biomarkers in blood, such as troponin I, C-reactive protein, or N-terminal pro-brain natriuretic peptide, provide an indication for early detection and prognosis monitoring of CVDs in a relatively simple, rapid and non-invasive manner. Recently, micro ribonucleic acids (miRNAs) extracted from extracellular vesicles (EVs) have been recognized as promising biomarkers for early detection of CVDs and prognosis monitoring. However, detection and quantification of miRNAs by using the existing methods are relatively labor-intensive and time-consuming. In this study, an integrated microfluidic system equipped with highly sensitive field-effect transistors which was capable of performing EVs extraction, EVs lysis, target miRNAs extraction and miRNAs detection has been reported. The entire detection process could be automated within five hours on a single chip and the detection limit of miRNAs was observed to be in a femtomolar range for two targeted miRNA, including miR-21 and miR-126, which meets the requirement of physiological concentrations. This integrated microfluidic system has great potential to be used as a tool for early detection of CVDs.

Table of contents
Abstract    I
摘要    III
Table of contents    IV
List of figures    IX
List of tables    XVI
Abbreviations and nomenclature    XVII
Chapter 1 Introduction    1
1-1 Cardiovascular Diseases    1
1-2 Extracellular vesicles (EVs) and micro ribonucleic acids (miRNAs)    3
1-3 Literature survey    5
1-3-1 Microfluidics    5
1-3-2 Field-effect transistor    7
1-3-3 EVs detection by using microfluidic systems    8
1-4 Novelty and motivation    9
Chapter 2 Materials and methods    12
2-1 Cell culture and isolation of sample model of EVs    12
2-1-1 Quantification of EVs yields    13
2-1-2 Identification of EVs    14
2-1-3 EVs extraction on bench    16
2-2 Quantification methods about capture rate of EVs extraction    16
2-3 Quantification methods about capture rate of miRNAs extraction    17
2-4 Design and fabrication process of the microfluidic chip    20
2-4-1 Design of integrated microfluidic chip    21
2-4-2 Fabrication of integrated microfluidic chip    25
2-4-3 Working principle of microfluidic chip    26
2-5 Characterization of chip performances    28
2-5-1 Measurement of pumping rate    28
2-5-2 Measurement of mixing index    29
2-5-3 Measurement of wall shear force    30
2-6 Experimental setup    32
2-7 Experimental procedure    34
Chapter 3 Results and discussion    39
3-1 Performance of the integrated microfluidic system    39
3-1-1 Pumping rate of micropump    39
3-1-2 Mixing index of micromixer    41
3-1-3 Shear force of the micromixer    43
3-2 Image data of capturing EV by using CD63-coated magnetic beads    45
3-2-1 SEM images of captured EV on CD63-coated magnetic beads    47
3-2-2 Fluorescent image of captured EV on CD63-coated magnetic beads    49
3-3 Capture rate of EVs extraction    50
3-3-1 EVs extraction at different operation applied gauge pressure    51
3-3-2 EVs extraction at different time    52
3-4 Capture rate of miRNAs extraction    53
3-4-1 miRNAs extraction at different operation frequencies    53
3-4-2 miRNAs extraction at different time    55
3-5 FET detection    56
3-6 miRNAs detection by using the integrated microfluidic system    58
Chapter 4 Conclusions and future perspectives    61
4-1 Conclusions    61
4-2 Future perspectives    62
References    64


                                

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