利用雙層適體快速檢測A型流行性感冒病毒之整合型微流體系統

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研究生：	曾宜婷 Tseng, Yi Ting
論文名稱：	利用雙層適體快速檢測A型流行性感冒病毒之整合型微流體系統 Integrated Microfluidic System for Rapid Detection of the Influenza H1N1 Virus Using a Sandwich-based Aptamer Assay
指導教授：	李國賓 Lee, Gwo Bin
口試委員:	楊瑞珍陳致真
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：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	54
中文關鍵詞：	螢光、甲型H1N1流行性感冒病毒、甲型流行性感冒病毒、微流體、三明治型適體測定
外文關鍵詞：	Fluorescence, H1N1, Influenza A virus, Microfluidics, Sandwich-based aptamer assay
相關次數：	點閱：3 下載：0
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A型H1N1 流感病毒的迅速蔓延最近幾年受到密切的關注，因此非常迫切需要研發一種能定點照護的自動化快速檢測病毒的儀器。傳統上的流感篩檢有許多關鍵的缺點，它們操作耗時、勞力密集，而且專一性很低。在本研究，我們提出一種新方法，使用螢光檢測法在整合型微流體系統中自動化操作，將新流感病毒利用三明治型的雙層適體執行。透過微流體晶片讓整體的檢測時間縮短至30分鐘。需要使用的試劑量也能降低五倍。由於適體與病毒間的高親和力和高專一性讓檢測靈敏度顯著提升到0.032 HAU。實驗數據顯示我們開發的微流體系統可以成功的檢測出A型H1N1流感病毒。

The rapid spread of influenza-associated H1N1 viruses has caused serious concern in recent years. Therefore, there is an urgent need for the development of automatic, point-of-care devices for rapid diagnosis of the influenza virus. Conventional approaches suffer from several critical issues; notably, they are time-consuming, labor-intensive, and are characterized by low specificity. In this work, we present a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system. The entire detection process was shortened to 30 minutes using this chip-based system, and reagent consumption was decreased 5-fold in comparison to traditional methods. The limit of detection was significantly improved to 0.032 HAU due to the high affinity and high specificity of the H1N1-specific aptamers, suggesting that this microfluidic system could be useful in the detection of the H1N1 virus in situ.

Table of Contents
Abstract    1
摘要    2
誌謝    3
Table of Contents    4
List of Tables    6
List of Figures    7
Abbreviations and nomenclature    11
Chapter 1 Introduction    13
1 Influenza A H1N1    13
2 Methods for the diagnosis of influenza infections    13
2.1 Virus culture    14
2.2 Polymerase chain reaction techniques    14
2.3 Rapid influenza diagnostic tests    14
3 Comparison of H1N1 specific aptamer and antibody    15
4 Diagnosis using sandwich-based assay    15
5 MEMS-based microfluidic technology    20
6 Motivation and objectives    20
Chapter 2 Materials and methods    22
1 Design of the magnetic bead-based microfluidic chip    22
1.1 Experimental procedure    22
1.2 Design of detection chip    24
2 Fabrication of the microfluidic system    27
2.1 Microfluidic control module    27
2.2 Master mold casting    28
2.3 PDMS casting and chip bonding    29
3 Sample preparation    31
3.1 Virus strains    31
3.2 H1N1-specific aptamer    31
3.3 Bead-conjugated aptamers    32
3.4 Fluorescence-modified aptamer    33
4 Polymerase chain reaction and gel electrophoresis    34
5 Experimental setup and optical detection    35
Chapter 3 Results and discussion    37
1 Characterization of microfluidic chip    37
1.1 Chip structure    37
1.2 Pumping rate of the micro-pump    38
2 Confirmation of sandwich binding assay    39
2.1 Virus capturing by aptamer-conjugated magnetic beads    39
2.2 FAM-modified aptamer    41
3 Virus capturing by aptamer-conjugated magnetic beads and FAM-coated aptamer    43
3.1 Limit of detection of the integrated microfluidic system    43
3.2 Specificity tests of the integrated microfluidic system    46
3.3 Methodological comparison    50
Chapter 4 Conclusions and future perspectives    51
1 Conclusions    51
2 Future perspectives    51
References    52

                                

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