整合型微流體系統平台於分子診斷技術之應用

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研究生：	張文馨 Chang, Wen-Hsin
論文名稱：	整合型微流體系統平台於分子診斷技術之應用 Applications of integrated microfluidic systems for molecular diagnosis
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	李國賓李炫昇楊瑞珍吳旻憲王玉麟曾繁根陳宗嶽
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：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	134
中文關鍵詞：	微流體、恆溫環狀擴增法、聚合酶鏈鎖反應、奈米金探針、水產養殖病原菌、蝴蝶蘭、關節周邊組織感染、微機電系統、微型全分析系統
外文關鍵詞：	microfluidics, loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), nanogold probe, aquaculture pathogens, Phalaenopsis orchid, periprosthetic joint infection, Micro-Electro-Mechanical-Systems (MEMS), micro-total-analysis-system (μTAS)
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查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

隨著社會的進步，個體間的距離越來越近，許多快速傳播的感染病因而出現。這些感染病不僅威脅人類健康甚鉅，亦造成嚴重經濟損失。早期偵測及診斷是遏止傳染疾病擴散的關鍵因素。因此許多研究學者投身於開發既正確又快速的偵測及診斷工具。在所有偵測工具中，分子診斷已被廣泛地應用於感染病偵測因為其具有高靈敏性和專一性。分子偵測可提供正確且靈敏的偵測結果，這些結果可作為處置方法之參考。然而，複雜的程序及操作者所造成的誤差和汙染使得分子診斷尚無法成為一良好的田間試驗或即時醫療工具。
為了解決以上問題，本論文提出四種整合式微流體系統，分別可以直
由魚類，蘭花及人類關節液樣本中對細菌及病毒等病原體以分子診斷的方式進行偵測。這些整合式微流體系統借助生物微機電系統的知識，將數個微流體元件整合至單一生物晶片上而達成晶片實驗室的想法。此外，整合式微流體系統可自動化執行傳統生醫實驗中所有手動步驟，不需操作者介入而省下時間金錢，因此可避免操作者所造成的誤差和汙染。
本論文針對這些整合式微流體系統的效能進行確效，結果顯示其具有高度專一性，且不同系統之靈敏度分別為20條DNA拷貝數(恆溫環狀擴增法及螢光偵測)，35皮克質體去氧核糖核酸(恆溫環狀擴增法及濁度偵測)，200菌落形成單位(聚合酶鏈鎖反應及螢光偵測)及100個菌落形成單位(奈米金探針偵測)。此外，本研究以具有溫度控制模組，液體傳輸模組及光學偵測模組的整合式控制系統自動化地於短時間內完成包含分離去氧核糖核酸/核糖核酸/細菌，利用聚合酶鏈鎖反應或恆溫環狀擴
增進行核酸增幅，奈米金探針偵測或光學偵測之所有實驗流程。實驗結果顯示這些微型全分析系統可於不遠的將來作為田間試驗或即時醫療之有用工具。

As the society progresses, many rapidly-spread infectious diseases emerge because the distance between individuals is getting closer and closer. Not only do these infectious diseases threaten people health significantly but they also cause huge economic loss. The key factor to cease the spread of infectious diseases is early detection and diagnosis.Researchers have devoted to develop detection and diagnostic tools which are accurate and rapid. Among all detection tools, molecular diagnosis has been extensively employed in infectious disease detection because of its high sensitivity and high
specificity. It may provide accurate and sensitive detection results which could be references for making proper treatment decisions. However, complicated processes,human errors and contamination prevent its potential to be an in-filed or point-of-care
tool.
To overcome the above-mentioned disadvantages, four different integrated microfluidic systems were developed to detect pathogens including bacteria and viruses
from aquaculture, orchids and human joint fluidic sample directly are presented in this dissertation. By means of the knowledge from bio-Micro-Electro-Mechanical-System
(Bio-MEMS), the integrated microfluidic systems integrated several microfluidic components into one single biochip to realize to concept of Lab-on-a-Chip (LOC).Furthermore, the integrated control systems can be used to perform all manual steps in a traditional biomedical experiment automatically with less human intervention to save labor and cost. With Bio-MEMS, human error and contamination can also be reduced. The performances of the integrated microfluidic systems were validated in this dissertation and the results showed that the purposed microfluidic systems were highly specific with sensitivities as low as 20 copies of plasmid deoxyribonucleic acid (DNA) for loop-mediated isothermal amplification (LAMP) with fluorescent detection, 35 pg of plasmid DNA for LAMP with turbidity detection, 200 colony formation units (CFU) for polymerase chain reaction (PCR) with fluorescent detection and 100 CFU for nanogold probe detection, respectively. Moreover, in this study, the whole
experimental procedures including DNA/RNA/bacteria isolation, nucleic acid amplification by PCR or LAMP, nanogold probes detection or optical detection can
be performed on an integrated control system which consisted of a temperature control module, a liquid transportation module and an optical detection module
automatically within a short period of time. Based on the experiment data, these micro-total-analysis-systems (μTAS) are promising tools for in-field diagnosis or
point-of-care in the near future.

Abstract…………………………………………………………………………………I
中文摘要………………………………………………………………………………III
致謝……………………………………………………………………………………V
Table of Contents……………………………………………VI
List of Figures………………………………………………………………………VIII
List of Tables……………………………………………XV
Abbreviation and Nomenclature………………VIII
Chapter 1: Introduction………………………………………1
1 Molecular diagnosis………………………………………………………………1
1.1 PCR…………………………………………………………………………3
1.2 LAMP………………………………………………………………………4
2 MEMS-based nucleic acid amplification using microfluidic technologies……4
2.1 Performing PCR in microfluidic systems……………………………………..9
2.2 Performing LAMP in microfluidic
systems…………………………………10
3 Motivation and objectives………………………………………………………13
3.1 Rapid purification and detection of pathogens in agricultural species using LAMP on an integrated microfluidic system……………………………… 13
3.2 Direct purification and detection of viruses directly from the fresh leaves of Phalaenopsis orchid using an integrated microfluidic system ……...……...13
3.3 Rapid isolation and diagnosis of live bacteria from human joint fluids by using an integrated microfluidic system ……………………………………14
3.4 An integrated microfluidic system for rapid detection and typing of live bacteria from human joint fluidic samples …………………………………..15
4 Scope and structure of the dissertation…………………………………………..15
Chapter 2: Rapid purification and detection of pathogens in agricultural species using LAMP on an integrated microfluidic system ………………………17
1 Introduction………………………………………………..……………………17
2 Materials and methods………………………………………………………….21
2.1 Experimental procedure……………………………………….……………..21
2.2 Chip design…………………………………………………………………..23
2.3 Fabrication process…………………………………………………………25
2.4 Working principle of the pneumatic micro-pump …………………………26
2.5 Custom-made control system ………………………………………………27
2.6 Primer and nucleotide probe design ………………………………………29
2.7 Preparation of nucleotide-probe-conjugated magnetic beads……………….30
2.8 Positive control construction………………………………………………30
2.9 Preparation of infected fish samples………………………………………30
2.10 PCR ………………………………………………………………………31
2.11 Electrophoresis…………………………………………………………… 31
3 Results and discussion…………………………………………………………31
3.1. Performance of the pneumatic micropump…………………………………31
3.2. Performance of the custom-made control system…………………………32
3.3. Sensitivity…………………………………………………………………34
3.4. Detection of pathogen from fish samples…………………………………36
4 Summary…………………………………………………………39
Chapter 3: Direct purification and detection of viruses directly from the fresh leaves of a Phalaenopsis orchid using an integrated microfluidic system……………………………………………………………………40
1 Introduction…………………………………………… ………………………40
2 Materials and methods…………………………………………………………44
2.1 Experimental procedure……………………………………………………44
2.2 Chip design…………………………………………………………………46
2.3 Fabrication process…………………………………………………………49
2.4 The microfluidic system……………………………………………………50
2.5 Primer and nucleotide probe
design………………………………………51
2.6 Preparation of nucleotide-probe-conjugated magnetic beads…………….….52
2.7 RNA extraction………………………………………………………………52
3 Results and Discussion………………………………………… ………………53
3.1 Performance of the integrated microfluidic chip…………………………….53
3.2 Performance of the optical detection unit……………………………………54
3.3 Optimum hybridization temperature of the magnetic beads coated with virus
specific probes………………………………………………………………55
3.4 Sensitivity of the integrated microfluidic system……………………………57
3.5 Virus detection using fresh orchid leaves and the integrated microfluidic
system……………………………………………………………………….59
4 Summary………………………………………………………63
Chapter 4: Rapid isolation and diagnosis of live bacteria from human joint fluids
by using an integrated microfluidic system……………………………66
1 Introduction……………………………………………………………………66
2 Materials and methods……………………………………69
2.1 Experimental procedure……………………………………………69
2.2 Chip design and fabrication………………………………………………….72
2.3 Preparation of EMA………………………………………………………77
2.4 Preparation of vancomycin-conjugated magnetic beads……………………77
2.5 PCR primers and PCR reaction……………………………………………78
2.6 Preparation of dead bacteria…………………………………………………78
2.7 Positive control construction………………………………………………78
2.8 Clinical specimen……………………………………………………………79
2.9 Statistical analysis……………………………………………………………79
3 Results and discussion………………………………………80
3.1 The pumping rate of the transportation units…………………………80
3.2 Optimization of EMA pre-treatment of live bacteria diagnostic assay……81
3.3 LOD of the proposed system………………………………………………84
3.4 Clinical specimen tests……………………85
4 Summary………………………………………………………………………87
Chapter 5 An integrated microfluidic system for rapid detection and typing of live bacteria from human joint fluidic samples………………………………89
1 Introduction……………………………………………………………………89
2 Materials and methods…………………………………………………………92
2.1 Experimental procedures……………………………………………………92
2.2 Chip design…………………………………………………………………94
2.3 Preparation of nanogold-conjugated 16S probes……………………………97
2.4 Bacterial genomic DNA extraction…………………………………………98
2.5 PCR primers and PCR reaction……………………………………………98
3 Results and discussion…………………………………………………………100
3.1 Characterization of the integrated microfluidic chip………………………100
3.2 Detection of live bacteria using nanogold-conjugated 16S probes……….100
3.3 Detection of live bacteria on the proposed integrated microfluidic system..101
3.4 Typing of live bacteria using PCR reaction with specific primer sets……104
4 Summary………………………………………………………………………107
Chapter 6: Conclusions and future perspectives…………………………………108
References……………………………………………………………………………113
Publication lists………………………………………………………………………130
                                

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