臺灣博碩士論文加值系統

生物醫學檢測與生物科技應用於人類疾病之診斷與分析於二十一世紀以來，已逐漸扮演著不可或缺的重要腳色。近年來，更由於預防醫學觀念的發達以及臨床醫學的突破，快速醫學篩檢與疾病之診斷應用於基因分析與臨床檢測更是獲得高度之重視與發展。因此，傳統上除了整體檢測過程中所需的分子診斷以及基因偵測技術外，臨床檢體之前處理與濃縮萃取的程序於檢驗程序中亦是不可或缺的一環。然而於傳統臨床醫學之疾病檢測中，生物檢體之前處理諸如細胞分離、去氧核糖核酸(DNA)以及核糖核酸(RNA)的萃取與純化尤其複雜，往往耗費許多的處理時間與人力成本，且其繁複的人為操作與檢測步驟，往往增加檢體之損耗與疾病診斷之準確性。因此，隨著近年來微機電系統 (micro-electro-mechanical-systems，MEMS)與微製程技術之成熟，在許多不同的應用領域中有顯著的發展與整合價值。其中，又以微流體技術為基礎所建構之生物醫學分析晶片，更是具快速疾病診斷之發展潛力與市場價值。藉由微機電製程技術所生產之微流體生醫檢測晶片，其具有高檢測靈敏度、可拋棄式、可攜帶性、低樣品及檢體消耗量、低耗能、體積小以及成本低等優點，相較傳統基因分析與疾病檢測技術下，有著突破性的發展價值與潛力。
因此，於臨床傳醫學檢測與疾病診斷中，從生物檢體中純化、分離且萃取目標細胞、病原菌甚至是DNA，是分子生物檢測的第一步。而有效的目標檢體純化的方法，除了可以從臨床體液中(諸如全血、痰唾液等)直接萃取出高品質以及高純度的目標檢體外，其操作步驟與程序更要簡單、快速、準確以及低成本。因此，本論文提出四型以微型免疫磁珠為實驗架構之微流體晶片系統，自動化地進行快速檢體純化與疾病之檢測。本論文所提出之平台主要是將微型磁性顆粒進行表面修飾，並將免疫學原理中抗體針對目標抗原之專一性極高之特性，以萃取臨床體液中之特定目標癌細胞、病毒或DNA，並將以濃縮、純化、分離後，於晶片上自動地完成聚合?連鎖反應 (PCR)。此外並藉由光學訊號之分析，進行快速傳染性與遺傳性性疾病之相關基因判讀。本論文所發展之平台除了可以減少人為操作的不穩定性，更可大幅縮短操作時間與步驟，以達自動化快速疾病檢測之目的。此四組微流體晶片系統分別用人類遺傳基因之多型性(polymorphism)、地中海貧血、卵巢癌及肺癌，以及流行性感冒病毒進行快速篩檢與診斷。第一型晶片乃藉由一微型磁場所產生之磁力，將表面修飾專一性抗體之磁珠與人體之白血球細胞 (HWBC)結合後由全血中純化萃取而出，進而再將細胞裂解並萃取基因體DNA後並進行基因多型性之診斷與分析。第二型晶片為第一型晶片之改良，於本晶片上已大幅改善上述平台中較為繁複的檢體萃取步驟，並改良以往於臨床樣品取得時所需的侵入式萃取步驟(如抽血)。此平台應用表面修飾之微型磁珠於較易取得之唾液檢體中，藉由緩衝液濃度之調整直接進行基因體DNA之純化，並利用光學系統於基因分析後進行地中海貧血之基因螢光訊號判讀。而第三型平台為建構一三維(3D)立體結構之微流體晶片平台，目的是用於解決一般微型化系統中難以處理體積較多之臨床檢體之問題，尤其是當處理相對濃度極低之檢體如循環的腫瘤細胞 (CTCs)等，可以快速且專一的將腫瘤細胞從體液中分離出，並進行癌症基因組(oncogenes panel)之鑑定。而本論文所提出之第四型微流體晶片平台乃一吸取式微流體晶片應用快速流行性感冒病毒之快速篩檢。於系統中係利用類似三明治免疫分析法(sandwich-like immunoassay)於微型磁珠上進行目標病毒之螢光檢測分析。此整合型系統藉由主動式微幫浦與微閥門的控制下，能自動化地進行檢測操作流程，並大幅的縮短檢測之時間及提高其效率。本論文所提出之四型晶片系統經過驗證可發現其疾病檢測之DNA、腫瘤細胞基因以及流行感冒病毒之偵測極限濃度約莫為12 pg/μL，~10 cells以及0.128 HAU，且利用微型晶片高升降溫速率之優點，相對於傳統大型檢測儀器，可以節省近50%的檢測時間。相信於不久的將來，本晶片系統所提供之快速自動化檢測平台，將於基因分析、分子生物以及快速疾病之偵測將有極大之助益。

Rapid and accurate diagnosis of various diseases has been of a great need for biomedical applications. Due to advances in preventive medicine and clinical diagnosis, tools for the rapid analysis of genetic mutation associated with hereditary diseases and infectious diseases have attracted significant interests in recent years. The entire diagnostic process usually involves several critical steps such as sample pre-treatment, genetic identification and data analysis. The sample pre-treatment processes such as extraction and purification of the target nucleic acids prior to genetic analysis are essential in molecular diagnostics. The genetic identification process may require delicate and complicated apparatus for nucleic acid amplification, sequencing and detection. Traditionally, pre-treatment of clinical biological samples (e.g. the extraction of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and target cells isolation) and the analysis of genetic genes associated with genetic/infectious diseases are typically a lengthy and costly process. These labor-intensive and time-consuming processes usually result in a high-cost per diagnosis and may hinder their practical applications. As well, the accuracy of the diagnosis may be affected owing to potential contamination from manual processing. Alternatively, due to significant advances in micro-electro-mechanical-systems (MEMS) and microfluidic technology, there are numerous miniature systems employed in biomedical applications, especially for the rapid diagnosis of genetic/infectious diseases. A number of advantages including automation, compactness, disposability, portability, lower cost, less diagnosis time, lower consumption of samples and reagents, and lower power consumption can be realized by using these microfluidic-based platforms. As a result, microfluidic-based systems are becoming promising platforms for genetic analysis, molecular biology and for the rapid detection of diseases.
Consequently, the current study proposes four new microfluidic-based platforms capable of pre-treatment of clinical bio-samples, identification of genetic genes and diagnosis of associated diseases. The combination of magnetic beads and microfluidic technology enables the realization of these micro-systems. For the first system, human white blood cells (HWBCs) can be purified and isolated from the clinical whole blood, followed by extracting the genomic DNA (gDNA) with the incorporation of surface-modified magnetic beads. The single nucleotide polymorphism (SNP) genotyping can be then identified by using the built nucleic acid amplification module. In the second system, the microfluidic system integrated with three functional devices including a sample purification module for gDNA extraction from saliva samples, a self-compensated polymerase chain reaction (PCR) module for the detection of genetic mutation and an external optical detection module for end-point analysis of a gene assay has been used for the detection of ?-thalassemia-1 deletion. The third microfluidic system is also proposed for the rapid isolation and detection of target cancer cells in an automatic manner. A new 3-dimensional (3D) microfluidic system has been developed to solve the fundamental and challenging problems in handling the clinical bio-samples with relatively large volume in the microfluidic system, especially to identify the rare cancer cells such as circulating tumor cells (CTCs) in bodily fluids during the early stage. For the fourth micro system, a suction-based microfluidic system for rapid detection and optical analysis of influenza viral particles has been demonstrated. As a whole, the developed systems may provide promising point-of-care platforms for rapid diagnosis of diseases.

Abstract I
中文摘要 III
致謝 V
Table of Contents VII
List of Tables XV
List of Figures XVI
Abbreviation XXVII
Nomenclature XXXI
Chapter 1: Introduction 1
1.1 MEMS-based microfluidic technologies 1
1.1.1. Introduction to microfluidics 1
1.1.2. Microfluidics for lab-on-a-chip and point-of-care applications 2
1.2 Immunological and molecular diagnosis 4
1.2.1 Purification of bio-samples 4
1.2.2 Immunological diagnosis 4
1.2.3 Molecular diagnosis 6
1.2.4 Reverse transcription and polymerase chain reaction 7
1.3 Motivation and Objectives 8
1.4 Scope and structure of the dissertation 9
Chapter 2: Theory and Design 15
2.1 Overview of the designed magnetic-bead-based micro systems 15
2.2 Microfluidic control system 16
2.2.1 Pneumatic-driven microfluidic control system 16
2.2.2 Suction-type microfluidic control system 16
2.2.3 Membrane activation theory 17
2.2.4 Working principle of microfluidic control module 17
2.2.4.1 Microfluidic control module driven by compressed air 18
2.2.4.2 Microfluidic control module driven by negative air pressure 19
2.3 Active micro-mixer 19
2.3.1 Concept of membrane-type micro-mixer 20
2.3.2 Design of vortex-type micro-mixer 20
2.3.3 Mixing efficiency 21
2.4 Magnetic bio-separator module 21
2.4.1 Model of magnetic bio-separator 22
2.4.2 Design of the bio-separator 24
2.5 Optical detection module 24
2.6 Nucleic acid amplification technique 25
2.6.1 Molecular biology 25
2.6.2 Cell lysis 26
2.6.3 Nucleic acid amplification 27
2.6.4 Optimization of the nucleic acid amplification module 28
2.7 Slab-gel electrophoresis 31
Chapter 3: Extraction of gDNA and detection of SNP genotyping utilizing an integrated magnetic bead-based microfluidic platform 36
3.1 Introduction 36
3.2 Materials and methods 39
3.2.1 Experimental process 39
3.2.2 Design 42
3.2.2.1 Design of membrane-type micro-mixer 44
3.2.2.2 Working principle of two-way circular micropump 45
3.2.3 Fabrication 46
3.2.3.1 Assembly of the integrated microfluidic system 46
3.2.3.2 Fabrication of the micro bio-separator module and the nucleic acid amplification module 47
3.2.3.3 Fabrication of the microfluidic control module 48
3.2.4 Genetic analysis of methylenetetra-hydrofolate reductase (MTHFR) C677T region and the design of primers 48
3.2.5 PCR reagents 50
3.3 Results and discussion 50
3.3.1 Characterization of the microfluidic control module 51
3.3.2 Performance of biological applications 52
3.3.3 Mixing efficiency 53
3.3.4 Binding effect 54
3.3.5 Trapping effect of magnetic beads 56
3.3.6 Analysis of genetic genes 57
3.4 Summary 59
Chapter 4: Microfluidic System for the Rapid Detection of ?-Thalassemia-1 Deletion Using Saliva Samples 69
4.1 Introduction 69
4.2 Materials and methods 71
4.2.1 Operational procedure 71
4.2.2 Design and fabrication of the integrated system 72
4.2.2.1 gDNA extraction module 73
4.2.2.2 Self-compensated PCR module 75
4.2.2.3 Optical detection module 76
4.2.3 Sample preparation and nucleic acid amplification 76
4.2.3.1 Standard procedure for saliva samples collection 77
4.2.3.2 gDNA extraction materials and process 77
4.2.3.3 PCR materials and process 78
4.3 Results and discussion 79
4.3.1 Characterization of the integrated platform 79
4.3.1.1 Microfluidic devices 79
4.3.1.2 Circular microcoils array 80
4.3.1.3 PCR module 80
4.3.2 Performance of the gDNA extraction 81
4.3.2.1 gDNA extraction from saliva with different storage conditions 82
4.3.2.2 Elution effect of extracted gDNA 83
4.3.3 Detection of ?-thalassemia-1 deletion 84
4.3.3.1 Optical analysis of ?-thalassemia-1 deletion 84
4.3.3.2 Limitation of detection 85
4.4 Summary 86
Chapter 5: Rapid Isolation and Detection of Cancer Cells by Utilizing Integrated Microfluidic Systems 97
5.1 Introduction 97
5.2 Materials and methods 100
5.2.1 Experimental procedures 100
5.2.2 Chip design 102
5.2.3 Experimental setup 106
5.2.4 Fabrication process of the integrated microfluidic system 107
5.2.5 Cell lines 108
5.2.6 Antibodies and immunomagnetic beads 109
5.2.7 Isolation of cancer cells 109
5.2.8 Thermal lysis and reverse transcription 110
5.2.9 PCR panels 110
5.2.10 Primer sets 112
5.3 Results and discussion 112
5.3.1 Characterization of the 3D microfluidic incubator 112
5.3.2 Characterization of the microfluidic control module and the nucleic acid amplification module 115
5.3.3 Binding between the cancer cells and beads 116
5.3.4 Mixing efficiency 117
5.3.5 Volumetric effects 118
5.3.6 Detection of expressed genes 118
5.3.7 Limitation of detection 119
5.3.8 Verification of clinical ascites 120
5.3.9 End-point optical analysis of ovarian and lung cancer 121
5.4 Summary 122
Chapter 6: A Magnetic-bead-based Immunoassay for the Rapid Purification and Detection of Influenza Viruses utilizing Suction-type Microfluidic Systems 137
6.1 Introduction 137
6.2 Materials and methods 139
6.2.1 Working principle 139
6.2.2 Chip design and experimental setup 142
6.2.3 Fabrication 144
6.2.4 Virus strain 144
6.2.5 Immunoassay reagents 145
6.3 Results and discussion 146
6.3.1 Characterization of the microfluidic system 146
6.3.2 Detection of immunoassay 148
6.4 Summary 150
Chapter 7: Conclusions and Future Work 159
References 161
Biography 175
Publication list 176

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