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研究生： 房彥伶
Fang, Yen-Ling
論文名稱： 快速檢測敗血症細菌之整合型微流體系統研發
An Integrated Microfluidic System for Earlier Pathogen Identification in Bacteria Septicemia
指導教授： 李國賓
Lee, Gwo-Bin
口試委員: 陳致真
Chen, Chih-Chen
李炫昇
Lee, Mel S.
學位類別： 碩士
Master
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： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 86
中文關鍵詞： 細菌分離 、微流體 、薄膜過濾 、磁珠 、甘露聚糖結合凝集素 、敗血症 、聚合酶連鎖反應
外文關鍵詞： bacteria isolation, microfluidics, membrane-based filtration, magnetic beads, mannose binding lectin, sepsis, polymerase chain reaction
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    • 敗血症是指病菌侵入人體後造成的全身性嚴重發炎反應，隨病程發展會造成器官功能衰竭，臨床上皆是先給予經驗性抗生素，等待抗生素敏感性報告出爐，再調整最適合的抗生素。但是現行以抽血培養再塗盤的敗血症檢測從確認病菌到找出正確抗生素，約需3天。因此，敗血症的快速診斷有助於臨床醫生及時進行抗生素治療，從而降低細菌感染的死亡率。本篇研究目標是提出一個可以快速從人類全血中分離、檢測格蘭氏陰性菌及陽性菌的整合型微流體晶片，此晶片是一個整合三個功能性模組的薄膜過濾裝置；(一)攪拌增強過濾模組，只有小於濾膜孔徑的粒子可以在後端被收集，血球細胞皆被帶走。此模組可以每分鐘處理20.74 μL全血，並移除99.47%的血球; (二)以磁珠為基底的細菌抓取模組，用修飾過的奈米磁珠抓取細菌，磁珠-細菌複合物會被下方放置磁鐵收集。在晶片上的優化後，可以在20分鐘得到56%至85%的細菌收集率; (三)細菌辨識模組，利用聚合酶鏈鎖反應放大目標細菌基因以鑑定細菌，在晶片上最高的偵測極限為每反應5 CFU，最低為1 CFU。值得注意的是，從全血中進行細菌分離、抓取、辨識的整個過程都可以在同一晶片上自動化完成。因此，這種整合型的微流體系統將來可以臨床醫生更多診斷線索。此外，也可以為敗血病患者提供適當的抗生素治療組合。

    Rapid diagnosis of sepsis may assist clinicians to initiate a timely and effective antibiotic therapy and therefore reduce mortality of bacterial infections. In this work, an integrated microfluidic system for fast isolation of bacteria for both gram-positive and gram-negative bacteria directly from human whole blood was presented. Since enrichment and detection of bacteria in whole blood have proven difficult, we developed an integrated microfluidic device comprising a membrane-based filtration module, a micromixer containing magnetic beads surface-coated with flexible neck regions of mannose-binding lectin (FcMBL) such that they could be used for bacteria capture, and a polymerase chain reaction (PCR) module for bacteria identification. First, the stirring-enhanced filtration module was used for sample pretreatment, which can separate white blood cells (WBCs), red blood cells (RBCs) and bacteria continuously. With this approach, most of the blood cells including WBCs and RBCs were removed while bacteria passed the filter. The filtration rate was measured to be around 20.74 μL-min−1 and removal efficiency was as high as 99.47%. The second module, the magnetic bead-based bacterium isolating device using the micromixer, was characterized by capture rates ranging from 56% to 85%. The final module performed on-chip PCR. With TaqMan probes, bacterium identification can be performed by detecting the fluorescence signals. The limit of detection was measured to be as low as 5 CFU/reaction. The entire process including sample pretreatment (90 minutes), bacteria isolation (20 minutes) and identification (90 minutes) could be completed on a single device within 4 hours, which is far less than the time needed for the conventional culture-based approach. This is the first time that an integrated microfluidic device with the ability to capture and differentiate bacterial species by using protein A-FcMBL magnetic beads was reported. This may be a promising way for healthcare professionals to provide timely treatments and improve time for adequate therapies.

    Abstract I 中文摘要 III Acknowledgements V Table of contents VII List of Figures X Abbreviations and nomenclature XIII Chapter 1: Introduction 15 1-1 Bio-MEMS and microfluidic System 15 1-2 Sepsis 17 1-3 Microfluidic techniques for bacteria detection 20 1-4 FcMBL 25 1-5 Motivation and novelty 26 1-6 The structure of thesis 29 Chapter 2 Materials and methods 31 2-1 Fabrication of the microfluidic chip 31 2-2 Design of the integrated microfluidic chip 35 2-3 Design and working principle 40 2-4 Experimental Setup 44 2-5 Experimental procedure 46 2-6 Sample Preparation 50 2-6-1 Preparation of the bacterial and blood samples 50 2-6-2 Preparation of the FcMBL-coated magnetic beads 50 2-6-3 PCR reagents and the designed primers 53 Chapter 3 Results and Discussion 55 3-1 Stirring-enhanced filtration 55 3-2 Capture ability of the FcMBL‑coated magnetic beads 62 3-3 Optimization of PCR conditions 65 3-4 Specificity test for detection of the targeted bacteria 68 3-5 Limits of detection of the bacteria 71 3-6 On-chip PCR and fluorescence detection 73 Chapter 4 Conclusions and future perspectives 78 References 81 Appendix 85

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