近幾十年來，傳染病對我們的全球公共健康造成了嚴重威脅，每年都造成全世界數百萬人死亡，特別是在開發中國家。儘管許多體外診斷（in vitro diagnostic, IVD）設備已在醫療院所中廣泛使用，但仍存在一些問題待克服，例如檢驗時間長、靈敏度低、成本過高以及儀器體積大，這些因素都會阻礙其在資源較匱乏的環境使用。因此，建立快速精準、高靈敏、價格適中，且無需複雜的操作或是大型儀器，進而實現真正的自動化“從樣品到答案” 的即時護理（point-of-care, POC）的體外診斷設備是迫切需要的。本論文提出三種整合式自微流體裝置在分子診斷上的應用。這些自驅動微流體裝置，皆使用了新開發的聚二甲基矽氧烷 (PDMS) 表面處理技術，使晶片具液流自傳輸功能。首先是應用於數位式環形核酸恆溫增幅法 (loop-mediated isothermal amplification, LAMP) 的整合型自驅動微流體晶片，整個檢驗流程可利用毛細力造成的流體自驅動完成，以在此自驅動微流控設備實現核酸的絕對定量。結果顯示，此晶片可在30分鐘內成功定量抗萬古黴素腸球菌的基因，其最低定量濃度為11條核酸。此外，我們也開發了另一款自驅動的微流體晶片，結合疏水性微型閥門的設計，可以在晶片上快速鑑定A型流感（H1N1）病毒。整個檢驗流程僅需透過毛細作用力便能進行，所得到的檢驗結果也能利用肉眼直接判讀，每反應檢測極限僅為3×10-4 HAU，與目前用於臨床上的靈敏度相當。最後，為了將上述的檢驗流程自動化，我們設計了一台自動化的可攜式體外檢測裝置，用於病毒和細菌等病原體的快速檢測。該系統能夠在40分鐘內自動執行分子診斷的所有必要步驟，包括病原體純化，核酸擴增和光學檢測。分析過程可以藉由智能手機操作和監視，其針對A型流感病毒與多重抗藥性金黃色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)的檢驗極限分別為每反應3×10-3 HAU和30個菌落。可以預見，這種由智慧型手機控制的設備可以用作臨床病原體檢測的平台，期望在不久的將來可成為及時醫療照護應用的有用工具，特別是在資源有限的開發中國家。

Infectious diseases pose a serious threat to global public health in recent decades, accounting to millions of deaths annually worldwide, particularly in developing countries. Although numerous in vitro diagnostic (IVD) devices have been widely employed in clinical settings, several challenges such as long turn-around time, low sensitivity, high cost as well as bulky instrument requirement, still need to be addressed, which may hinder their application in resource-limited settings. Therefore, it is essential to establish a rapid, accurate, sensitive and affordable IVD without complicated manipulations/instruments to realize truly automated “sample-to-answer” point-of-care (POC) product. In this dissertation, three integrated self-driven microfluidic devices which implement a novel polydimethylsiloxane (PDMS) surface treatment technology have been presented and used for molecular diagnosis of infectious diseases. First, an integrated microfluidic chip was developed to carry out digital loop-mediated isothermal amplification (LAMP) for absolute nucleic acid quantification. The liquid-handling process could be automatically performed on this chip via capillary forces as a result of the PDMS surface treatment which in turn could carry out digital LAMP on a self-driven microfluidic device. As a proof of concept, amplification of a gene specific to a vancomycin-resistant enterococcus strain was performed on the developed microfluidic chip within 30 min at a limit of detection (LOD) of only 11 copies. Furthermore, the developed hydrophilic treatment technique was then adopted to establish an autonomous microfluidic device for rapidly identifying influenza A (H1N1) virus by using reverse transcription LAMP (RT-LAMP) assay. The entire diagnostic process could be performed via capillary forces and the liquid flow could be regulated by hydrophobic soft microvalves. The LOD was measured to be only 3×10−4 hemagglutinin units (HAU)/reaction, which is sensitive enough for clinical applications. Finally, an automated IVD system has been developed for rapid detection of pathogens, including virus and bacteria. The system was capable of automatically carrying out all necessary steps for molecular diagnosis, including pathogen purification, nucleic acid amplification and optical detection within 40 min and the LODs were experimentally found to be 3.2 × 10−3 HAU/reaction for influenza A (H1N1) virus and 30 colony-forming units (CFU)/ reaction for methicillin-resistant Staphylococcus aureus (MRSA), respectively. The analytical process could be controlled and monitored by a smartphone. Thus, it is envisioned that this smartphone-controlled apparatus may serve as a platform for clinical, POC pathogen detection, particularly in resource-limited settings in the near future.

Abstract I
中文摘要 III
誌謝 V
Table of Contents VI
List of Tables IX
List of Figures X
Abbreviations and Nomenclature XVI
Symbols XIX
Chapter 1 Introduction 1
1.1 Molecular diagnostics and infectious diseases 1
1.1.1 Nucleic acid technologies 1
1.2 Microfluidic technologies 5
1.2.1 NAT in microfluidics 5
1.3 In vitro diagnostics 7
1.3.1 Autonomous microfluidics 8
1.3.2 Material selection in capillary-driven microfluidics 9
1.4 Motivation and novelty 13
1.4.1 Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated PDMS 13
1.4.2 An integrated self-driven microfluidic device for rapid detection of the influenza A (H1N1) virus by reverse transcription loop-mediated isothermal amplification 15
1.4.3 A sample-to-answer, portable platform for rapid detection of pathogens with a smartphone interface 19
1.5 Scope and structure of the dissertation 22
Chapter 2: Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated PDMS 24
2.1 Introduction 24
2.2 Materials and methods 25
2.2.1 VRE genomic DNA and LAMP reagents 25
2.2.2 Chip design, fabrication, and surface treatment 27
2.2.3 Hydrophilicity of the UV-cured glue film 29
2.2.4 LAMP biocompatibility of hydrophilic PDMS 29
2.2.5 Digital LAMP 30
2.2.6 Cell biocompatibility of the hydrophilic PDMS layer 31
2.3 Results and discussion 32
2.3.1 Characterization of the microfluidic chip 32
2.3.2 Characterization of the hydrophilic, UV-cured glue film 34
2.3.3 Absolute quantification of DNA using digital LAMP 35
2.4 Summary 37
Chapter 3: An integrated self-driven microfluidic device for rapid detection of the influenza A (H1N1) virus by reverse transcription loop-mediated isothermal amplification 47
3.1 Introduction 47
3.2 Materials and methods 48
3.2.1 Preparation of aptamer-conjugated magnetic beads 48
3.2.2 Influenza virus and bacterial strains and RT-LAMP reagents 48
3.2.3 Chip fabrication and design 50
3.2.4 Experimental procedure 53
3.2.5 Mixing efficiency 55
3.3 Results and discussion 55
3.3.1 Characterization of the microfluidic chip 55
3.3.2 Characterization of the mixing efficiency 57
3.3.3 Specificity and sensitivity of the RT-LAMP reaction 58
3.3.4 LOD of the integrated microfluidic device 59
3.4 Summary 61
Chapter 4: A sample-to-answer, portable platform for rapid detection of pathogens with a smartphone interface 75
4.1 Introduction 75
4.2 Materials and methods 76
4.2.1 Overview of the approach 76
4.2.2 Virus and bacteria strains 77
4.2.3 Preparation of affinity molecule-conjugated magnetic beads 78
4.2.3 Colorimetric LAMP assay 78
4.2.4 Chip design and fabrication 80
4.2.5 Experimental procedures 83
4.2.6 Custom-made portable pathogen diagnostic system 84
4.2.7 Mixing efficiency 85
4.3 Results and discussion 86
4.3.1 Characterization of the custom-made portable system 86
4.3.2 Optimization of the colorimetric LAMP assay 88
4.3.3 Sensitivity and specificity of the colorimetric LAMP assay 89
4.3.3 The limit of detection of the custom-made portable system 92
4.4 Summary 92
Chapter 5: Conclusions and future perspectives 109
References 113
Publication list 136

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