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研究生： 戴健軒
Tai, Chien-Hsuan
論文名稱： 整合型微流體系統應用於流感傳染病及癌症細胞致癌基因之快速檢測
Integrated Microfluidic Systems for Rapid Detection of Influenza Infections and Diagnosis of Oncogenes in Cancer Cells
指導教授： 李國賓
Lee, Gwo-Bin
學位類別： 博士
Doctor
系所名稱： 工學院 - 工程科學系
Department of Engineering Science
論文出版年： 2013
畢業學年度： 101
語文別： 英文
論文頁數： 113
中文關鍵詞： 流感病毒 、DNA 微陣列 、螢光原位雜交 、微機電系統 、微流體
外文關鍵詞： Influenza virus, DNA microarray, FISH, MEMS, microfluidics
相關次數： 點閱：150  下載：3 
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    • 最近，每年很多人死於流感。流感是由流感病毒引起的急性呼吸道感染疾病，是一種快速傳播的病毒。迄今為止，全球已有超過10000例死亡人數，而此死亡人數在流感高峰期預估增加至百萬人。流感大流行受到全球各國廣泛關注，成為當前公共衛生重要的議題。分子檢測可以提供一個高靈敏度及高專一性應用於流感檢測，傳統的分子診斷方法是利用反轉錄聚合酶鏈反應 （RT-PCR）和平板跑膠法鑑定來檢測流感傳染病。然而，整個程序需要約4個多小時之久。因此，急需發展一個有效的平台可提供快速篩選季節和新型流感病毒。除了傳染性疾病，對人類健康嚴重構成威脅的是遺傳性疾病和癌症，DNA微陣列和螢光原位雜交（FISH）兩大技術已廣泛被用於檢測相關的遺傳性疾病和癌症。對於傳統的這二種技術，它們通常有著繁雜的實驗流程、耗時的雜交過程及昂貴的試劑。另外，他們還需要數位訓練有素的專業人員來執行整個實驗過程。
    為了解決上述這些問題，本論文提出了三種新型的微流體整合平台應用於流感及癌症相關疾病檢測。此三種微流體平台整合微幫浦，微混合器和溫度控制模組。這些微流體整合平台能夠自動化處理臨床生物樣品，遺傳基因的鑑定和相關疾病的診斷。首先提出是流感整合型平台，該系統能快速診斷和分類不同的亞型流感病毒。此一整合型平台能進行樣品的前處理，核酸擴增和光學檢測流感病毒。整個流程，包括病毒裂解，萃取核糖核酸（RNA），反轉錄（RT），聚合酶鏈反應（PCR），以及光學檢測，成功地整合再一起。從光學檢測模組獲得的信號，能夠準確區分甲型流感A/H1N1（infA/H1N1），甲型流感A/H3N2（infA/H3N2），乙型流感B（INFB），陽性和陰性對照樣本。實驗結果表明，該開發的微流體系統可以成功地在60分鐘內區分同的亞型流感病毒。此外，本系統也成功地應用在92名患者喉頭檢體測試。且有著超過90%之靈敏度及100%之專一性。

    接下來針對DNA微陣列技術本論文提出第二型整合型平台，此整合型平台能夠自動地進行樣品的前處理和微陣列的雜交過程。整個步驟包括細胞裂解、信使核糖核酸（mRNA）的反轉錄（RT）及互補脫氧核糖核酸（cDNA）的雜交純化，都能在這個整合型平台自動執行。相比較於傳統方式整個實驗流程須達十個小時，藉由這個整合型微流體晶片平台，可以在六小時內自動執行整個實驗，且可以避免因為人為操作不當而產生汙染及可以減少生物樣品和試劑的消耗。因此，本晶片系統期待在不久的將來可以提供一個有用的平台在遺傳分析和診斷應用。最後，本論文所提出之第三型整合型平台為應用於螢光原位雜交技術應用於偵測細胞染色體異常。此整合型平台能夠完整自動化執行繁雜螢光原位雜交技術且比傳統方式有許多優勢，包括減少生物樣品和試劑的消耗，自動化減少人工汙染且比較傳統方法需要10小時，此平台只需3小時內即可達成。我們希望此開發系統能在不久的將來應用於檢測傳染病和遺傳性疾病。

    Recently, many people die from influenza every year. The influenza virus is a type of rapidly transmitted virus, infecting the upper respiratory tract and sometimes causing acute viral respiratory illness. To date, more than 10,000 cases worldwide have died of the disease and this number increases to millions in some pandemic years. This virus has raised serious concerns in the public health worldwide. Molecular detection may provide a more sensitive and selective diagnosis. Traditional reverse transcription polymerase chain reaction (RT-PCR) and slab-gel electrophoresis can be used for detection of an influenza infection. However, it usually takes more than 4 hours to perform the entire assay. Therefore, methods and tools for the rapid screening and diagnosis of seasonal and novel influenza infections are in great demand. In addition to infectious diseases, genetic diseases and cancer present serious threats to human health. DNA microarray and the fluorescence in situ hybridization (FISH) techniques have been widely used to detect related genetic diseases and cancers. Though effective, these two methods usually involve labor-intensive and time-consuming hybridization processes that require costly reagents and well-trained personnel to perform the entire assay.
    In order to address these problems, the current study presents three new microfluidic-based platforms for the detection of influenza and cancer. Fully integrated with micropumps, micromixers, and temperature control module, these microfluidic systems are capable of pre-treatment of clinical bio-samples, identification of specific genes and diagnosis of associated diseases. The first of such systems is an integrated microfluidic system that performs sample pre-treatment, nucleic acid amplification, and optical detection for the molecular diagnosis of influenza viruses. The entire analysis protocol – including virus lysis, ribonucleic acid (RNA) extraction, reverse transcription (RT), polymerase chain reaction (PCR), and optical detection – was successfully performed within an automated microfluidic system, which enabled rapid diagnosis and subtyping of multiple strains of the influenza virus. Signals obtained from the integrated optical detection module could accurately differentiate influenza A/H1N1 (infA/H1N1), influenza A/H3N2 (infA/H3N2), influenza B (infB), and positive and negative control samples. The experimental results demonstrated that this developed microfluidic system can successfully distinguish between infA/H1N1, infA/H3N2 and infB within 60 min. As a demonstration for the effectiveness of this platform, oral swabs from 92 patients were tested using the developed microfluidic system, which demonstrated over 90% sensitivity and 100% specificity. The second developed platform, which combines DNA microarray technology with microfluidics, is capable of automatically performing the sample pretreatment and hybridization processes that are required for microarray-based detection – including cell lysis, messenger ribonucleic acid (mRNA) extraction, reverse transcription (RT), complementary deoxyribonucleic acid (cDNA) purification, and hybridization. The employment of this microfluidic system dramatically shortens the analysis time from over 10 hours to 6 hours. This developed system may provide a useful platform for applications in genetic analysis and diagnosis. The third platform entails a novel integrated microfluidic chip capable of detecting cancer by performing a complete FISH recipe and probing the chromosomal abnormality of cells in an automated fashion. In this study, several functional components were integrated on a single chip to perform automatic FISH on the microfluidic platform. The novel microfluidic system carried out the entire process automatically within 3 hours, where the conventional manual method required over 10 hours for performing the entire protocol. We expect pre-clinical testing of the developed system for detecting infectious and genetic diseases may lead to a promising technique.

    Abstract I 摘要 III 致謝 V Table of Contents VI List of Tables X List of Figures XI Abbreviation XXI Nomenclature XXIII Chapter 1 Introduction 1 1.1 MEMS and Microfluidic Technology 1 1.2 Infectious and Genetic Diseases 1 1.2.1 Infectious Diseases 2 1.2.2 Genetic Diseases 2 1.2.3 Influenza Viruses 3 1.2.4 Oligonucleoyide Microarrays 4 1.2.5 Fluorescence In Situ Hybridization (FISH) 5 1.3 Motivation and Objectives 5 Chapter 2 Theory and Chip Design 12 2.1 Microfluidic Control Systems 12 2.1.1 Chip Manufacturing Process 12 2.1.2 Circular-type Micropump 12 2.1.3 Suction-type Micropump 13 2.1.4 Mixing Efficient 13 2.1.5 Membrane Deformation Theory 14 2.2 Nucleic Acid Amplification Techniques 15 2.2.1 Polymerase Chain Reaction (PCR) and Reverse-Transcription PCR (RT-PCR) 15 2.3 Design of the Temperature Control Module 16 Chapter 3 An integrated microfluidic platform for rapid detection and subtyping of influenza viruses from clinical samples 21 3.1 Introduction 21 3.2 Materials and methods 25 3.2.1 Experimental process 25 3.2.2 Chip design 25 3.2.3 Fabrication process 27 3.2.4 Virus preparation 27 3.2.5 One-step RT-PCR protocol 28 3.3 Results and discussion 30 3.3.1 Characterization of the micropump 30 3.3.2 Selectivity test 30 3.3.3 Limit of detection of the integrated microfluidic system 32 3.3.4 Diagnosis of influenza infections using clinical specimens 35 3.4 Conclusion 35 Chapter 4 An integrated microfluidic system capable of sample pretreatment and hybridization for microarrays 49 4.1 Introduction 49 4.2 Materials and methods 52 4.2.1 Chip design 52 4.2.2 Fabrication process 53 4.2.3 Experimental process 54 4.2.3.1 Reverse-transcription (RT) process 56 4.2.3.2 Preparation of the oligonucleotide array membrane 57 4.2.3.3 Preparation of digoxigenin-labeled cDNA targets and hybridization reagents 57 4.2.4. Polymerase chain reaction (PCR) process 58 4.3 Results and discussion 59 4.3.1 Characterization of the microfluidic control module 59 4.3.2 Verification of extracted mRNA 61 4.3.3 Verification of hybridization 62 4.4 Conclusion 63 Chapter 5 A Novel Integrated Microfluidic Platform to Perform Fluorescence in situ Hybridization for Chromosomal Analysis 76 5.1 Introduction 76 5.2 Materials and methods 78 5.2.1 Chip design 78 5.2.2 Fabrication process 80 5.2.3 Experimental process 80 5.2.4 Sample preparation 81 5.3. Results and discussion 82 5.3.1 Characterization of the micropump 82 5.3.2 FISH analysis 84 5.4 Conclusion 85 Chapter 6 Conclusions and Future Work 93 6.1 Review of the Dissertation 93 6.2 Future Works 94 References 96 Publication List 112

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