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研究生： 李文斌
Lee, Wen-Bin
論文名稱： 整合型微流體系統於個人化醫療抗生素自動化快速篩選之應用
Applications of Integrated Microfluidic Devices for Automatic Rapid Drug Screening of Precision Medicine
指導教授： 李國賓
Lee, Gwo-Bin
李炫昇
Lee, Mel S.
口試委員: 張晃猷
Chang, Hwan-You
陳致真
Chen, Chih-Chen
郭峯志
Kuo, Feng-Chih
學位類別： 博士
Doctor
系所名稱： 工學院 - 動力機械工程學系
Department of Power Mechanical Engineering
論文出版年： 2024
畢業學年度： 112
語文別： 英文
論文頁數： 147
中文關鍵詞： 精準藥物 、抗菌藥敏試驗 、細菌抗藥性 、最小抑菌濃度 、藥物篩選 、微流體
外文關鍵詞： precision medicine, antimicrobial susceptibility testing, antimicrobial resistance, minimum inhibitory concentration, drug screening, microfluidics
相關次數： 點閱：2  下載：0 
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    • 抗生素的氾濫及不當使用所衍生出的細菌多重抗藥性問題，一直是重要的全球議題，多重抗藥性細菌在醫院引起的院內感染，使得有效治療的抗生素減少，造成治療上的困難；隨之而來的對抗細菌感染的難度增加將導致病人住院時間更長，預後更差，進而造成醫療費用的顯著增加。為了有效使用抗生素預防多重抗藥性問題持續惡化，抗生素敏感性試驗是臨床上最常用來評估抗生素對細菌療效的方法。此研究計畫書提出了三種整合型微流體系統平台，分別可對臨床多重抗藥性菌株自動化進行單一種，特定兩種以及數種抗生素感受性試驗。首先整合氣動式微幫浦與常閉式微閥門等微流體元件，可將當前需手動操作的細菌感受性試驗，改用以自動化完成以及顏色變化來判讀結果，提供快速準確的抗生素最低抑菌濃度。接著透過改良之晶片設計模組，便可執行合併抗生素抑制濃度試驗，分析特定兩種抗生素之合併使用後是否具有協同作用，作為臨床醫師在投藥上的診斷依據。此外，透過新型的薄膜式微流體元件及細菌生長指示劑的導入，此整合型微流體系統可於4.5小時內經由比色結果獲得準確的抗生素最小抑菌濃度以及不同藥物組合之效果。本研究中介紹的基於微流體系統的抗生素藥敏試驗檢測方法相較於傳統方法具有多項優勢，包括減少試劑消耗、縮短周轉時間和自動化複雜過程。這些創新解決了對快速、準確且易用的抗生素藥敏試驗系統的需求，這對抗擊細菌多重抗藥性的崛起和改善患者護理至關重要，並顯示了其更廣泛診斷應用的潛力，最終為全球對抗抗菌素抗藥性的努力作出貢獻。

    The overuse and misuse of antibiotics have led to the significant global issue of multi-drug resistance (MDR) in bacterial strains. Hospital-acquired infections caused by MDR bacteria reduce the effectiveness of available antibiotics, increasing treatment difficulties and associated healthcare costs. To effectively use antibiotics and prevent the worsening of MDR, antimicrobial susceptibility testing (AST) is crucial for assessing the efficacy of antibiotics. This dissertation explores the development and integration of microfluidic systems for AST, focusing on three innovative devices: The first designed microfluidic systems, as a proof of concept, demonstrated the feasibility of determining the Minimum Inhibitory Concentration (MIC) using pneumatic micropumps paired with normally-closed microvalves. Based on the broth microdilution method provided by CLSI, it enabled automated sample injection, transport, and mixing within nine minutes. Tests conducted on a standard strain of Enterococcus and four VRE genotypes (14 samples in total) showed that the MIC values obtained on-chip were consistent with those derived from the current clinical method, Etest®.
    Additionally, by using a duplicated chip design and a new model that applied two antibiotics against a single pathogen, the second approach was adapted for parallel AST with combinational antibiotics. The new method accurately and automatically performs on-chip dispensation, dilution, and mixing, enabling the determination of the Fractional Inhibitory Concentration Index (FICI) of two antibiotics, similar to the checkerboard assay.
    Moreover, the third advanced microfluidic device, combining normally-closed microvalves and a modified new membrane-type microfluidic component, enabled the entire on-chip AST process—including sample dispensation, 2-fold serial dilutions of three antibiotics, and different antibiotic combinations—to be completed within five minutes. The final colorimetric result was obtained after just 4.5 hours of incubation. This automated microfluidic platform significantly enhances the efficiency and accuracy of rapid AST, representing a major advancement in precision medicine. By tailoring antibiotic treatments to the specific needs of individual patients, this platform has the potential to improve patient outcomes and address the challenge of antimicrobial resistance.

    Acknowledgements I Abstract II 摘要 IV Table of Contents VI List of Figures IX List of Tables XII Nomenclature and Abbreviations XIII Chapter 1 Introduction 1 1-1 Bio-Micro-Electro-Mechanical-System (Bio-MEMS) and Microfluidic System 1 1-2 Antimicrobial Resistance (AMR) 4 1-3 Antimicrobial Susceptibility Testing (AST) 5 1-4 Precision Medicine in Infectious Diseases 9 1-5 Motivation and Objectives 12 1-5-1 A Microfluidic Device for Minimum Inhibitory Concentration Determination of Antimicrobial Susceptibility Testing by Using Liquid Broth Dilutions 12 1-5-2 A Microfluidic Device for Antimicrobial Susceptibility Testing of Combined Antibiotics by Using Broth Dilution Method 15 1-5-3 Automatic and Rapid Antimicrobial Susceptibility Test on an Integrated Microfluidic Device 18 1-6 Scope and Structure of the Dissertation 21 Chapter 2 A Microfluidic Device for Minimum Inhibitory Concentration determination of Antimicrobial Susceptibility Testing by Using Liquid Broth Dilutions 23 2-1 Introduction 23 2-2 Materials and Methods 24 2-2-1 Antibiotic and Biological Material Preparation 24 2-2-2 Experimental Procedures of on-chip AST 26 2-2-3 Chip Design and Fabrication 29 2-2-4 Operation of the Microfluidic Chip 34 2-2-5 Use of a Vital Stain Kit to Detect Live/dead Bacteria 38 2-2-6 Comparison of On-Chip AST and Etest® 38 2-3 Results and Discussion 39 2-3-1 Characterization of the Microfluidic Chip 39 2-3-2 Performance of On-Chip AST via a Fluorescence Vital Staining 42 2-3-3 Visual Assessment of On-Chip AST 44 2-3-4 On-Chip AST with Clinical Strains of Vancomycin-Resistant Enterococcus (VRE) 46 2-4 Summary 51 Chapter 3 A Microfluidic Device for Antimicrobial Susceptibility Testing of Combined Antibiotics by Using Broth Dilution Method 53 3-1 Introduction 53 3-2 Materials and Methods 54 3-2-1 Preparation of Antibiotics and Microorganism 54 3-2-2 Manual AST for Single and Combination Antibiotics 55 3-2-3 On-Chip AST Assays with Antibiotic Combination 55 3-2-4 Chip Design and Fabrication 58 3-2-5 Verification of On-Chip AST for the Function of Antibiotic Combination 64 3-3 Results and Discussion 65 3-3-1 Characterization of the Pneumatically-Driven Microfluidic Components 65 3-3-2 Fluorescent Results for Verification of Antibiotic Combinations 71 3-3-3 AST Results with Two Antibiotics for Dual MIC Analysis 75 3-3-4 AST Results of Pairwise Antibiotic Combination 78 3-4 Summary 82 Chapter 4 Personalized Antibiotic Screening on an Integrated Microfluidic Device 84 4-1 Introduction to Precision Medicine in AST 84 4-2 Materials and Methods 87 4-2-1 Reagents and Antibiotics 87 4-2-2 Principle of the Integrated Microfluidic Device 88 4-2-3 Chip Design and Fabrication 92 4-2-4 Operation of the Microfluidic Components 96 4-2-5 Performance Assessment of Membrane-Type Micropump/Micromixer 96 4-2-6 Manual AST Assay Using Broth Microdilution 98 4-3 Results and Discussion 99 4-3-1 Characterization of the Microfluidic Chip 99 4-3-2 Verification of the Automated Dilution On-Chip 106 4-3-3 MIC Determination On-Chip with Individual Antibiotics 107 4-3-4 Investigation of Combination Effects for Personalized Antibiotic Treatment 116 4-4 Summary 121 Chapter 5 Implications for Clinical Practice and Public Health 122 5.1 Impact on Clinical Practice 122 5.2 Public Health Implications 123 5.3 Economic Considerations 124 5.4 Regulatory Issues 125 5.5 Future Directions for Research and Development 125 5.6 Conclusion 126 Chapter 6 Conclusions 127 6-1 Summary of Key Findings 127 6.2 Implications for Clinical Practice 128 6.3 Recommendations for Future Research 129 6.4 Conclusion 131 References 132 Publication list 140

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