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
1.1 Influenza A H1N1 13
1.2 Methods for the diagnosis of influenza infections 13
1.2.1 Virus culture 14
1.2.2 Polymerase chain reaction techniques 14
1.2.3 Rapid influenza diagnostic tests 14
1.3 Comparison of H1N1 specific aptamer and antibody 15
1.4 Diagnosis using sandwich-based assay 15
1.5 MEMS-based microfluidic technology 20
1.6 Motivation and objectives 20
Chapter 2 Materials and methods 22
2.1 Design of the magnetic bead-based microfluidic chip 22
2.1.1 Experimental procedure 22
2.1.2 Design of detection chip 24
2.2 Fabrication of the microfluidic system 27
2.2.1 Microfluidic control module 27
2.2.2 Master mold casting 28
2.2.3 PDMS casting and chip bonding 29
2.3 Sample preparation 31
2.3.1 Virus strains 31
2.3.2 H1N1-specific aptamer 31
2.3.3 Bead-conjugated aptamers 32
2.3.4 Fluorescence-modified aptamer 33
2.4 Polymerase chain reaction and gel electrophoresis 34
2.5 Experimental setup and optical detection 35
Chapter 3 Results and discussion 37
3.1 Characterization of microfluidic chip 37
3.1.1 Chip structure 37
3.1.2 Pumping rate of the micro-pump 38
3.2 Confirmation of sandwich binding assay 39
3.2.1 Virus capturing by aptamer-conjugated magnetic beads 39
3.2.2 FAM-modified aptamer 41
3.3 Virus capturing by aptamer-conjugated magnetic beads and FAM-coated aptamer 43
3.3.1 Limit of detection of the integrated microfluidic system 43
3.3.2 Specificity tests of the integrated microfluidic system 46
3.3.3 Methodological comparison 50
Chapter 4 Conclusions and future perspectives 51
4.1 Conclusions 51
4.2 Future perspectives 51
References 52

References
[1]. R. J. Garten, C. T. Davis, C. A. Russell, et al., Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans, Science, 2009, 325, 197-201.
[2]. S. S. Shrestha, D. L. Swerdlow, R. H. Borse, et al., Estimating the burden of 2009 pandemic influenza A (H1N1) in the United States (April 2009-April 2010), Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2011, 52 Suppl 1, S75-82.
[3]. K. Tharakaraman and R. Sasisekharan, Influenza Surveillance: 2014–2015 H1N1 “Swine”-Derived Influenza Viruses from India, Cell host & microbe, 2015, 17, 279-282.
[4]. D. S. Hui, N. Lee and P. K. Chan, Clinical management of pandemic 2009 influenza A(H1N1) infection, Chest, 2010, 137, 916-925.
[5]. Itolikar., Shobha., M. Y., et al., H1N1 Revisited After Six Years: Then and Now., Journal of The Association of Physicians of India, 2015, 63.
[6]. D. S. Leland and C. C. Ginocchio, Role of cell culture for virus detection in the age of technology, Clinical microbiology reviews, 2007, 20, 49-78.
[7]. R. L. Atmar, B. D. Baxter, E. A. Dominguez, et al., Comparison of reverse transcription-PCR with tissue culture and other rapid diagnostic assays for detection of type A influenza virus, Journal of clinical microbiology, 1996, 34, 2604-2606.
[8]. L. L. Poon, K. H. Chan, G. J. Smith, et al., Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays, Clinical chemistry, 2009, 55, 1555-1558.
[9]. C. Steininger, M. Redlberger, W. Graninger, et al., Near-patient assays for diagnosis of influenza virus infection in adult patients, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases, 2009, 15, 267-273.
[10]. D. J. Faix, S. S. Sherman and S. H. Waterman, Rapid-test sensitivity for novel swine-origin influenza A (H1N1) virus in humans, The New England journal of medicine, 2009, 361, 728-729.
[11]. J. F. Drexler, A. Helmer, H. Kirberg, et al., Poor clinical sensitivity of rapid antigen test for influenza A pandemic (H1N1) 2009 virus, Emerging infectious diseases, 2009, 15, 1662-1664.
[12]. X. Fang and W. Tan, Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach, Accounts of chemical research, 2010, 43, 48-57.
[13]. L. Y. Hung, T. B. Huang, Y. C. Tsai, et al., A microfluidic immunomagnetic bead-based system for the rapid detection of influenza infections: from purified virus particles to clinical specimens, Biomedical microdevices, 2013, 15, 539-551.
[14]. T. Hermann and D. J. Patel, Adaptive recognition by nucleic acid aptamers, Science, 2000, 287, 820-825.
[15]. Y. Lin, R. James, Heil., et al., Aptamer based two-site binding assay., U.S. Pat., No. 20,020,037,506. 28, 2002.
[16]. K. Ikebukuro, C. Kiyohara and K. Sode, Novel electrochemical sensor system for protein using the aptamers in sandwich manner, Biosensors & bioelectronics, 2005, 20, 2168-2172.
[17]. Y. H. Tennico, D. Hutanu, M. T. Koesdjojo, et al., On-chip aptamer-based sandwich assay for thrombin detection employing magnetic beads and quantum dots, Analytical chemistry, 2010, 82, 5591-5597.
[18]. S. J. Lee, Y. S. Kwon, J. E. Lee, et al., Detection of VR-2332 strain of porcine reproductive and respiratory syndrome virus type II using an aptamer-based sandwich-type assay, Analytical chemistry, 2013, 85, 66-74.
[19]. J. W. Park, S. Jin Lee, E. J. Choi, et al., An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening, Biosensors & bioelectronics, 2014, 51, 324-329.
[20]. I. Shiratori, J. Akitomi, D. A. Boltz, et al., Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity, Biochemical and biophysical research communications, 2014, 443, 37-41.
[21]. H. C. Lai, C. H. Wang, T. M. Liou, et al., Influenza A virus-specific aptamers screened by using an integrated microfluidic system, Lab on a chip, 2014, 14, 2002-2013.
[22]. N. Maluf, An introduction to microelectromechanical systems engineering., Artech House, 2000.
[23]. C. H. Weng, T. B. Huang, C. C. Huang, et al., A suction-type microfluidic immunosensing chip for rapid detection of the dengue virus, Biomedical microdevices, 2011, 13, 585-595.
[24]. A. C. Hurt, R. Alexander, J. Hibbert, et al., Performance of six influenza rapid tests in detecting human influenza in clinical specimens, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 2007, 39, 132-135.