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研究生： 林穎
Lin, Ying
論文名稱： 結合旋轉擴散技術和環介導等溫核酸增幅晶片進行呼吸道疾病之多功檢測
Combining Rotational Diffusometry with LAMP-on-chip for Multiplex Detection of Respiratory Diseases
指導教授： 莊漢聲
Chung, Han-Sheng
學位類別： 碩士
Master
系所名稱： 工學院 - 生物醫學工程學系
Department of BioMedical Engineering
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 73
中文關鍵詞： 呼吸道疾病 、環介導等溫擴增 、引子設計 、多功檢測 、微晶片 、快速檢測 、低檢體體積 、雙性粒子 、旋轉布朗運動 、影像處理
外文關鍵詞： Respiratory diseases, loop-mediated isothermal amplification (LAMP), primer design, multiplex detection, microchip, rapid testing, low sample volume, Janus particles (JPs), rotational Brownian motion, image processing
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    • 呼吸道疾病（如流感和新冠肺炎）對全球健康構成了重大挑戰。由於這些疾病具有高傳染性和潛在致命性，它們已成為公共衛生的首要關注點。目前，傳統診斷方法如快速檢測套件和聚合酶鏈鎖反應核酸檢測（PCR test）各有優缺點。快速檢測套件操作簡便，可在短時間內提供結果，但其準確性相對較低，尤其在敏感性和特異性方面尚有改進空間；而聚合酶鏈鎖反應核酸檢測雖具備較高的準確性和靈敏度，但檢測過程耗時、成本高昂，且需要專業設備與技術支持。
    為解決上述挑戰，我們結合旋轉擴散測量法與環介導等溫擴增技術(LAMP)，將反應環境引入微流道中，開發出一種新型多重檢測方法。該技術能同時檢測多種呼吸道疾病，旨在提升診斷速度與準確性，同時降低檢測成本。我們設計了具備多重檢測功能的微流道環介導等溫擴增技術（LAMP-on-chip），並使用雙性粒子實時監測核酸擴增過程。在我們的研究當中，該裝置實現 20 分鐘內達到 772.4 fg/μL 的檢測極限，且能同時檢測多達3種呼吸道病原體，包含新冠病毒和A型流感(H1、H5)病毒，適合臨床診斷症狀相似但病原體不同的患者。
    透過分析雙性粒子的旋轉布朗運動相關時間（Cross-correlation time），可即時判斷核酸擴增是否成功，大幅提升診斷效率。儘管微流道環介導等溫擴增技術的最低檢測限略高於傳統聚合酶鏈鎖反應核酸檢測，其在快速、多重檢測及即時現場診斷方面的優勢足以彌補這一不足。
    雙性粒子的微流道環介導等溫擴增技術在本論文中展現了在早期且準確檢測傳染病中的強大潛力。此技術不僅顯著提升了診斷效率，還能減少重複檢測需求，節省時間與資源；在傳染病管理中也有助於改善患者減輕醫療系統負擔。我們預期本論文中所發展的這種多功能檢測平台可為未來新興疾病的快速診斷提供了廣泛應用前景，並在公共衛生管理與傳染病控制中發揮關鍵作用，有望為全球健康挑戰做出積極貢獻。

    Respiratory diseases, such as influenza and COVID-19, pose significant challenges to global health. Due to their high transmissibility and potential lethality, these diseases have become a top priority in public health. Currently, traditional diagnostic methods, such as rapid diagnostic test kits and polymerase chain reaction (PCR) tests, have their respective advantages and limitations. Rapid test kits are easy to operate and provide results within a short time, but their accuracy is relatively low, particularly in terms of sensitivity and specificity. On the other hand, PCR tests offer high accuracy and sensitivity but are time-consuming, costly, and require specialized equipment and technical expertise.
    To address these challenges, we combined rotational diffusion measurement and loop-mediated isothermal amplification (LAMP) techniques, integrating the reaction environment into a microfluidic chip to develop a novel multiplex diagnostic method. This technology enables simultaneous detection of multiple respiratory diseases, aiming to improve diagnostic speed and accuracy while reducing costs. We designed a microfluidic LAMP platform (LAMP-on-chip) with multiplex detection capabilities, employing Janus particles (JPs) to monitor nucleic acid amplification in real-time. This device achieves a detection limit of 772.4 fg/μL within 20 minutes and can detect multiple pathogens simultaneously, making it suitable for diagnosing patients with similar symptoms but different pathogens.
    By analyzing the cross-correlation time of the rotational Brownian motion of JPs, nucleic acid amplification success can be determined in real-time, significantly enhancing diagnostic efficiency. Although the limit of detection (LOD) of LAMP-on-chip is slightly higher than that of traditional PCR tests, its advantages in rapid, multiplex detection and point-of-care testing (POCT) compensate for this limitation.
    The Janus particle-based on LAMP-on-chip platform demonstrates strong potential for early and accurate detection of infectious diseases. This platform not only significantly improves diagnostic efficiency but also reduces the need for repeated testing, saving time and resources. In infectious disease management, it helps improve patient outcomes and alleviates the burden on healthcare systems. The development of this multifunctional diagnostic platform offers extensive applications for rapid diagnosis of emerging diseases and plays a critical role in public health management and infectious disease control, making a meaningful contribution to addressing global health challenges.

    摘要 i ABSTRACT iii ACKNOWLEDGEMENT v CONTENTS vi LIST OF TABLES viii LIST OF FIGURES ix CHAPTER 1 INTRODUCTION 1 1.1 Motivation and Overview 1 1.2 Viral Respiratory Diseases 2 1.2.1 Influenza Virus 3 1.2.2 Coronavirus and SARS-CoV-2 4 1.3 State-of-the-art Diagnostics for Viral Respiratory Diseases 5 1.4 Loop-Mediated Isothermal Amplification (LAMP) on Chip 7 1.4.1 Integration of LAMP with Microfluidic Chip Technology 9 1.4.2 Applications of LAMP-on-chip Device 9 1.5 Aims and Contributions of this Thesis 11 CHAPTER 2 MATERIAL AND METHODS 13 2.1 Construction, Growth and Extraction of Plasmids 13 2.2 Multiplexing Chip 14 2.2.1 Chip Fabrication 14 2.2.2 Chip Performance 16 2.3 JPs Fabrication 17 2.4 Experimental Flow 21 2.5 Primer Design 22 2.6 LAMP with JPs 25 2.6.1 On-chip Reaction 26 2.6.2 Quantification of JP-Enabled Rotational Diffusivity 26 2.6.3 Rotational Diffusometry with Cross-correlation Analysis 30 CHAPTER 3 RESULTS AND DISCUSSION 32 3.1 Characterizations of Transparent Heating Plate 32 3.2 Effect of Lyophilized JPs on Chip 36 3.3 Evaluations of Designed Primers for Influenza-H1-cDNA and Influenza-H5- cDNA 37 3.3.1 Specificity of Influenza-H1-cDNA and Influenza-H5- cDNA 37 3.3.2 LODs of Influenza-H1-cDNA, Influenza-H5-cDNA, and SARS-CoV-2-nsp2-cDNA in Tubes 38 3.4 Effects of LAMP-on-chip 40 3.4.1 LODs of Influenza-H1-cDNA, Influenza-H5-cDNA, and SARS-CoV-2-nsp2-cDNA on LAMP-on-chip 41 3.4.2 LAMP-on-chip with JPs 42 3.4.2.1 Influenza-H1-cDNA Effect: Time-Based Analysis 43 3.4.2.2 LOD and LOQ of Influenza-H1-cDNA 44 3.4.2.3 Influenza-H5-cDNA Effect: Time-Based Analysis 45 3.4.2.4 LOD and LOQ of Influenza-H5-cDNA 46 3.5 Multiplexing Test Results 48 CHAPTER 4 CONCLUSIONS 49 CHAPTER 5 FUTURE WORK 52 REFERENCES 54 APPENDIX I 57 APPENDIX II 58

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