基於核酸的檢測是一項十分流行應用於DNA、RNA 等核酸分子偵測的技術。這項技術可以用於癌症的早期診斷，遺傳性疾病的確認。通常檢測樣本中核酸的數量太低以至於難以偵測到，因此，核酸檢測的第一步通常是將核酸增幅放大到可以偵測的水準。聚合酶連鎖反應（PCR）是一個最常使用於核酸增幅的方法。在PCR過程中，反應樣本會在兩到三個不同的操作溫度之間循環。反應過程中的熱循環要求精確穩定的控制。為了簡化相對複雜的熱循環過程，一些恆溫式的核酸增幅方法得到了發展。恆溫式的核酸增幅方法例如恆溫式環形核酸增幅法（LAMP）可以在固定的溫度下進行，因此也不需要進行熱循環的過程，這極大的減少了溫度控制模組的複雜性以及能量的消耗，使得該方法更適於基於微流體技術的應用。和PCR相比，LAMP有更好的靈敏性和專一性，反應時間也更短，以在一個小時內完成反應。LAMP的這些優點使得它成為了一個具有前景的基於核酸的檢測技術。在生物醫學應用中，確定原始樣本中核酸的量是十分重要的。樣本中核酸的原始濃度可以使用即時PCR的方式得到。但是這個定量的過程需要一個參照物或者是標準曲線。而且，在這種大體積的反應時無法區分目標物濃度上細微的差異。為了克服這些缺點，發展出了數位化的核酸增幅方法。在本研究中，我們提出了一個用於進行數位化LAMP分析的新方法，通過整合基於微流體的液滴乳化技術和基於流體力學的捕獲技術來形成一個大小均勻的液滴陣列，並將該陣列用於數位化的LAMP實驗。該方法是將LAMP 反應溶液通過微流體乳化液滴技術形成單分散的乳化液滴，每顆液滴都可以充當一個反應容器。基於流體力學的捕獲技術則被用於固定這些液滴從而形成一個液滴陣列。我們設計的整合型微流體晶片可以產生大小均勻的液滴，大小差異是小於3%，同時這些液滴可以在流體力學的作用下被依序固定在設計好的陷阱微結構中。除此之外，我們的LAMP反應也是可以成功的在油包水的乳化液滴中完成。

Nucleic acid technology (NAT) based detection is a popular technique to detect nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be used for early diagnosis of cancer, confirmation of genetic disease, and many others. Usually, the copy number of the nucleic acid molecules in a sample is too less to be detected. As a result, the first step of nucleic acid detection is to amplify the nucleic acid molecules to a detectable level. Polymerase chain reaction (PCR) is one of the most commonly used methods for DNA or RNA amplification, in which nucleic acid samples go through thermal cycling between two or three different operating temperatures. The thermal cycling requires accurate and robust control of the operating temperature. To simplify the complicated processes of thermal cycling, isothermal amplification methods have been developed. Isothermal amplification reactions such as loop-mediated amplification (LAMP) can be implemented at a fixed temperature with no need for thermal cycling, which reduces the complexity of the temperature control module and consumes less energy, making them more suitable for microfluidics-based applications. Moreover, when compared with PCR, LAMP is more sensitive and specific. Furthermore, it takes less than one hour to finish the amplification process. These advantages make LAMP become a promising method for NAT based detection. In biomedical applications, it is often of great significance to quantify the accurate nucleic acid molecule copies in original samples. The original DNA copies can be measured by real-time PCR. However, the quantification relays on a reference or a standard curve. Besides, this bulk reaction fails to distinguish subtle difference of the target copy number. In an effort to overcome the disadvantages, digital DNA amplification has been developed. In this study, we reported a new method to implement array-based digital LAMP analysis, in which we integrated the emulsion droplet microfluidic device and hydrodynamic trapping techniques to form a droplet array which could be further applied digital LAMP assay. By generating monodisperse water-in-oil droplets which contain LAMP reaction mixture, the bulk LAMP reaction mixture could be partitioned into many separate compartments. Each droplet could function as a reaction chamber. Hydrodynamic trapping technique was further used for immobilizing the droplets to form a droplet array, which is good for later analysis of the result. In this work, we used our integrated microfluidic chip to demonstrate digital LAMP assay. The microfluidic chip could be able to generate uniform droplet with a size variation less than 3% and the droplet could be hydrodynamically immobilized to form a droplet array. Besides, the LAMP assay could be successfully implemented in water-in-oil droplet.

Table of Contents
Abstract……. I
摘要………… III
List of Figures VII
List of Tables XIII
Abbreviations and Nomenclature XIV
Chapter 1 Introduction 1
1.1 MEMS and Microfluidic Technology 1
1.2 Nucleic Acid Detection 2
1.3 Formation of Emulsion Droplet Using Microfluidic Devices 4
1.4 Hydrodynamic Trapping of Micro-particle Using Microfluidic Devices 6
1.5 Motivation and Objectives 8
Chapter 2 Theory 19
2.1 Quantification Method of Digital Nucleic Acid Detection 19
2.2 Emulsion Droplet Formation and Trapping 20
2.2.1 Definition and Characteristics 20
2.2.2 Emulsifying Agents 21
2.2.3 Concept of Hydrophilic and Lipophilic Balance (HLB) 22
2.2.4 Hydrodynamic Trapping 23
Chapter 3 Materials and Methods 29
3.1 Design of the Emulsion Droplet Formation and Trapping Chip 29
3.2 Fabrication of the Emulsion Droplet and Formation Trapping Chip 29
3.2.1 Lithography Process 29
3.2.2 PDMS Casting and Chip Assembly 31
3.3 Sample Preparation 32
3.4 Experimental Procedure 34
3.5 Experimental Setup 34
3.6 Numerical Simulation 35
Chapter 4 Results and Discussion 47
4.1 Droplet Formation and Trapping Using Integrated Microfluidic Chip 47
4.1.1 Numerical Simulation of the Trapping Process 47
4.1.2 The Process of Droplet Formation 47
4.1.3 Size Distribution of Emulsion Droplets 48
4.1.4 Hydrodynamic Trapping of Droplets 48
4.2 Droplet Array-based LAMP Reaction 49
4.2.1 Optimization of the Fluorescence Dye 49
4.2.2 Result of LAMP reaction 50
Chapter 5 Conclusions and Future Perspectives 61
5.1 Conclusions 61
5.2 Future Perspectives 62
REFERENCES 64

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