臺灣博碩士論文加值系統

細菌的鑑定與分類對我們的環境十分重要，其影響範圍包括生態鏈、農業、公共衛生與臨床診斷。從過去至現在，已有相當多種細菌檢測方式，而細菌藥敏試驗是其中一個臨床上常見的細菌檢驗，在可以用來判斷菌株的抗藥性與否、評估最適當的抗生素劑量並避免藥物的過度使用。然而，因臨床樣本的細菌數量相當低，必須經由一至兩天的血液培養並達一定的檢測濃度才可進行抗藥性的測試，並且，抗生素的篩檢上需要再額外消耗一天的時間。然而，在長時間的檢測中，容易增加嚴重的感染性疾病對患者的死亡率，為解決此問題，我們設計一個快速細菌抗藥性篩檢平台，透過微流井技術，將細菌濃度低的臨床樣本進行濃縮，同時透過微流道技術來進行微環境的控制，以達到縮短細菌培養時間的效果。此外，我們結合表面增強拉曼散射 (surface-enhanced Raman scattering, SERS) 光學檢測技術，利用其免標定、快速檢測的特性，在短時間內完成抗藥性篩檢。希望能透過此平台的設計，建構出一個快速、免標定的細菌檢測和藥敏試驗，並提供高度細菌感染的患者準確的用藥與治療。

Bacterial identification and characterization are important to our environment, ecosystem, agriculture, healthcare and clinical diagnosis. Antibiotic susceptibility test (AST) is a common bacteria detecting method for selecting proper antibiotic treatment and preventing any overuse or misuse of antibiotics. However, due to the low bacterial concentration of the clinical sample, conventional AST usually requires a prolonged bacterial culturing process, which increases the mortality of seriously infected patients. To address this problem, we propose a rapid AST platform for bacteria detection. We use surface-enhanced Raman scattering (SERS) technology to rapidly analyze the antibiotic susceptibility and combine a microwell device in our platform for initial sample concentration enrichment. In addition, our platform can also manipulate microenvironment by using a microfluidic channel. With the SERS technology integrated with microwell and microfluidic device, we hope this platform could achieve highly sensitive, label-free bacteria detection and rapid AST to enable a timely and accurate sepsis treatment.

口試委員會審定書 #
誌謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS iv
LIST OF FIGURES vii
Chapter 1 Introduction 1
1.1 Research Background 1
1.1.1 Bacteria detection and AST 1
1.1.2 SERS and rapid AST 2
1.1.3 Challenges of SERS-based rapid AST 4
1.2 Literature Review 5
1.2.1 Comparison of rapid AST method 5
1.2.2 Microfluidic-based concentrating method 8
1.3 Microwell for bacteria investigation 10
1.4 Research Motivation 11
1.5 Thesis structure 12
Chapter 2 Experimental Design 13
2.1 SERS detection on two-dimensional SERS substrate 13
2.1.1 Raman scattering 13
2.1.2 Surface-enhanced Raman scattering 14
2.1.3 Two-dimensional SERS-active substrate 15
2.2 Microwell-based concentration 16
2.2.1 Centrifugation for microwell-based bacteria trapping 16
2.2.2 Microwell rehydration and isolation 18
2.3 Microwell and microfluidic device design 18
2.3.1 Microwell size and geometry 18
2.3.2 Microfluidic device design 20
Chapter 3 Materials and Methods 21
3.1 Materials 21
3.2 Bacteria preparation 21
3.3 Device fabrication 21
3.3.1 Microwell and microfluidic channel fabrication 21
3.3.2 Two-dimensional SERS-active substrate fabrication 24
3.4 Microwell-SERS detecting procedures 25
3.5 SERS measurement and spectral processing 26
3.6 Conventional-SERS detecting procedures 27
Chapter 4 Results and Discussion 28
4.1 Results of microwell and microfluidic device 28
4.1.1 Centrifugation time optimization 28
4.1.2 Bacteria trapping of different sample concentration 30
4.1.3 Rehydration efficiency 33
4.2 Microwell cross-contamination tests 34
4.3 SERS detection of different bacteria concentration 35
4.4 Microwell SERS-AST detection 37
Chapter 5 Conclusion 40
Chapter 6 Future Work 41
Reference 44

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