臺灣博碩士論文加值系統

在診斷領域中，免疫測定法是一種常見的利用抗體-抗原相互作用之敏感性和特異性來檢測分析物的方法，傳統的免疫分析法是利用擴散與沉降的方式使抗原抗體行專一性的結合，然通常需花費冗長的時間(數小時)、所費不貲，且所能呈現的檢測靈敏度及檢測極限也是一大考驗。此對於一些急性感染病症或初期癌症的病患非但有緩不濟的窘迫，且可能也因此錯過了有效的治療黃金時間。有鑑於此，本研究發展了一種三維介電泳微流體晶片之免疫檢測平台，其結合微流體、電動力學，搭配生物微粒之操控以達快速檢測之目的。執行時，先以Protein A修飾於次微米顆粒，在微管道連續注入下將產生剪切應力，此時若施加適當的交流電電場，將對該微粒誘發負介電泳力，繼而匯諸於管道的末端之上下層對稱的V型電極組之前。由於在該處被聚集的絕緣性微粒夾縫間會自然形成極高電場，若能設定約在0.8 ul/mL的流速，且擇以恰當交流電的電壓與頻率，則會使分子量相對極小的螢光抗體(Anti-protein A-FITC IgG)呈現正介電泳，遂被吸引至該微粒夾縫間，且與微粒上的Protein A產生抗原-抗體反應而結合。若輔以光學系統的進行螢光度分析，即能在數分鐘之內獲得其螢光強度變化，進而定量抗體濃度之濃度。其此，本檢測系統亦嘗試了模擬登革熱病毒感染進行重組非結構性蛋白(recombinated nonstructure protein, rNS1)的檢測，遂將上述的Protein A 更換為登革熱病毒單株抗體(DN5C6)化學修飾於次微米顆粒上，待測抗原更換為螢光抗原-抗體(rNS1/2E8-Streptavidin 488)，完成其後相同步驟，便能以螢光強度變化定量rNS1之濃度，迄今已可在10分鐘內即可測得明顯的螢光亮度。

Immunoassay is a common biochemical platform to measure the sensitivity and specificity of protein-protein or antigen (Ag)-antibody (Ab) interactions. Diffusion and settlement are general mode of actions introduced in the assay to monitor the dynamics of the interactions. However, complicated procedures and prolonged processing time may generate false or ambiguous signals to interfere with research hypotheses or even clinical diagnostic outcomes. In this study, we have developed a three-dimentional immune dielectrophoresis (3D-IDE) microfluidic chip to advance the use of traditional immunoassays for fast and accurate diagnosis. The binding of protein A and anti-protein A Ab was used as a model here to demonstrate the procedures and the effectiveness of 3D-IDE microfluidic chip in measuring their specific interaction. First, protein A was immobilized on micron particles as a binding target. Second, with the shear-force of the continuous fluid injection of the microchannel, appropriate AC signal was then applied in the solution to induce negative DEP force to the particles that can be trapped at the tip of the V shaped electrode. Due to the high electric field between the aggregated particles, the detection anti-protein A IgG, which was conjugated with fluorochrome FITC, on particles can be found to bind with protein A within minutes. As the IgGs on the micron particles were conjugated with FITC, the fluorescent signals trapped at the electrode can be observed by optical system and fluorescent intensity, which reflects the amount of interacting Ag-Ab complex, can be quantified. We have applied the concept of this 3D-IDE microfluidic chip to detect the infection of Dengue virus by studying the interaction of virus non-structural protein NS1 and its specific monoclonal Ab DN5C6, In this testing, DN5C6 was substitute for protein A for the immobilization on the micron particles, yet the rNS1 protein obtained as a streptavidin conjugate was used an antigen for subsequent sandwich immune reaction using secondary antibodies conjugated with a flurochrome Alexa 488 (rNS1/ 2E8-Streptavidin 488). Our 3D-IDE result indicates that the NS1 antigen and DN5C6 specific interacting complex can be effectively quantified within 10 min. This pilot study indicates that the 3D-IDE microfluidic chip can be effectively used to study Ag-Ab interaction for fast diagnostic purposes of viral infection and may have wide applications to other types of protein-protein interaction.

Abstract I
中文摘要 III
致謝 IV
Table of Content V
List of Figures VII
List of Tables X
Chapter 1 Introduction 1
1.1 Background 1
1.2 Conventional Detection Method 2
1.2.1 Immunoassay Techniques 2
1.2.2 Molecular Biology Techniques 5
1.3 Novel Techniques 6
1.3.1 Micro-Electro-Mechanical System Technology 7
1.3.2 Microfluidic Techniques 9
1.3.3 Electrical Techniques 11
1.3.4 Other Techniques 12
1.4 Theory 13
1.4.1 Dielectrophoresis (DEP) 13
1.4.2 Force of Particles in Micro-channel 17
1.4.3 Application of Dielectrophoresis for Biological Analysis 19
1.5 Motivation and Objectives 20
Chapter 2 Material and Methods 22
2.1 The Working Principle of Immuno-Chip 22
2.2 Chip Fabrication 24
2.2.1 Chip design 25
2.2.2 Metal Electrode Fabrication 26
2.2.3 Micro Channel Fabrication 28
2.3 Instruments of Experiment 30
2.4 Sample preparation 31
2.5 Beads Immobilization 32
2.6 Experimental Setup 33
Chapter 3 Result and Discussion 35
3.1 Beads Packing 35
3.2 Detection of Anti-protein A 41
3.2.1 Flow Cytometry Responds of Immobilization 42
3.2.2 The Optimal Flow Rate of Ag-Ab Interaction 44
3.2.3 Detection Specificity of Anti-Protein A 45
3.2.4 Detection Sensitivity of Anti-protein A 47
3.3 Detection of Dengue Virus Nonstructural Protein 49
3.3.1 Flow Cytometry Responds of Immobilization 50
3.3.2 Detection Specificity and Sensitivity of rNS1 Protein 52
Chapter 4 Conclusion 59
Chapter 5 Prospect 60
References 61
Personal Information 66

[1] H. C. Ng Alphonsus, U. Uvaraj, and A. R. Wheeler (2010). Immunoassays in microfluidic systems. (review), Analytical and Bioanalytical Chemistry 397: 991-1007.
[2] N. Chiem and D. Jed Harrison (1997). Microchip-Based Capillary Electrophoresis for Immunoassays: Analysis of Monoclonal Antibodies and Theophylline., Analytical Chemistry 69: 373-378.
[3] V. K. Yadavalli and M. V. Pishko (2004). Biosensing in microfluidic channels using fluorescence polarization., Analytica Chimica Acta 507: 123-128.
[4] E. Engvall and P. Perlman (1971). Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 9: 871-874.
[5] B. K. Van Weemen and A. H. W. M. Schuurs (1971). Immunoassay using antigen-enzyme conjugates. FEBS Letters 15: 232-236.
[6] Centers for Disease Control and Prevention (CDC) dengue: Laboratory Guidance and Dignostic Testing.
[7] B. D. Lindenbach and C. M. Rice (2003). Molecular biology of flaviviruses. Advances in Virus Research 59: 23-61
[8] P. Dussart, B. Labeau, G. Lagathu, P. Louis, M. R. T. Nunes, S. G. Rodrigues, C. Storck-Herrmann, R. Cesaire, J. Morvan, M. Flamand, and L. Baril (2006). Evaluation of an Enzyme Immunoassay for Detection of Dengue Virus NS1 Antigen in Human Serum. Clinical and Vaccine Immunology 13: 1185-1189
[9] R. W. Peeling, H. Artsob, J. L. Pelegrino, P. Buchy, M. J. Cardosa, S. Devi, D. A. Enria, J. Farrar, D. J. Gubler, M. G. Guzman, S. B. Halstead, E. Hunsperger, S. Kliks, H. S. Margolis, C. M. Nathanson, V. C. Nguyen, N. Rizzo, S. Vázquez, and S. Yoksan (2010). Evaluation of diagnostic tests: dengue. Nature Reviews Microbiology 8: 2459
[10] E. Huhtamo, E. Hasu, N. Y. Uzcátegui, E. Erra, S. Nikkari, A. Kantele, O. Vapalahti, and H. Piiparinen (2010). Early diagnosis of dengue in travelers: Comparison of a novel real-time RT-PCR, NS1 antigen detection and serology. Journal of Clinical Virology 47:49-53.
[11] V. Tricou, H. T. Vu, N. V. Quynh, C. V. Nguyen, H. T. Tran, J. Farrar, B. Wills, and C. P. Simmons (2010). Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses BMC Infectious Diseases 10:142-149.
[12] J. Mairhofer, K. Roppert, and P. Ertl (2009). Microfluidic Systems for Pathogen Sensing: A Review. Sensors 9: 4804-4823.
[13] J. Heo and S. Z. Hua, (2009). An Overview of Recent Strategies in Pathogen Sensing. (review) Sensors 9: 4483-4502.
[14] Y. H. Liu, C. H. Wang, J. J. Wu, and G. B. Lee (2012). Rapid detection of live methicillin-resistantStaphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis. Biomicrofluidics 6: 034119.
[15] C. Valat, B. Limoges, D. Huet, and J. L. Romette (2000). A disposable Protein A-based immunosensor for flow-injection assay with electrochemical detection. Analytica Chimica Acta 404: 187-194.
[16] M. Bart, E. C. A. Stigter, H. R. Stapertb, G. J. de Jonga, and W. P. van Bennekoma (2005). On the response of a label-free interferon-immunosensor utilizing electrochemical impedance spectroscopy. Biosensors and Bioelectronics 21: 49-59.
[17] R. Elshafeya, C. Tlili, A. Abulrob, A. C. Tavares, and M. Zourob (2013). Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain tissue. Biosensors and Bioelectronics 39: 220-225.
[18] K. M. Hansen and T. Thundat (2005). Microcantilever biosensors. Methods 37: 57-64.
[19] A. J. C. Eun, L. Huang, F. T. Chew, S. F. Y. Li, and S. M. Wong (2002). Detection of two orchid viruses using quartz crystal microbalance (QCM) immunosensors. Journal of Virological Methods 99: 71-79.
[20] B. Johnsson, S. Löfås, and G. Lindquist (1991). Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Analytical Biochemistry 198: 268-277.
[21] M. Mujika, S. Arana, E. Castaño, M. Tijero, R. Vilares, J. M. Ruano-López, A. Cruz, L. Sainz, and J. Berganza (2009). Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network Biosensors and Bioelectronics 24: 1253–1258.
[22] C. M. Ho and Y. C. Tai (1998). Micro-electro-mechanical-systems (MEMS) and fluid flows. Annual Review of Fluid Mechanics 30: 579-612.
[23] Y. Huang , E. L. Mather, J. L. Bell, and M. Madou (2002). MEMS-based sample preparation for molecular diagnostics Analytical and Bioanalytical Chemistry 372: 49-65.
[24] J. M. Yang, J. Bell, Y. Huang, M. Tirado, D. Thomas, A. H. Forster, R. W. Haigis, P. D. Swanson, R. B. Wallace, B. Martinsons, and M. Krihak (2002). An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays. Biosensors and Bioelectronics 17: 605-618.
[25] L. Gervais and E. Delamarche (2009). Toward one-step point-of-care immunodiagnostics using capillary-driven microfluidics and PDMS substrates. Lab on a Chip 9: 3330-3337.
[26] J. H. Lee, B. K. Oh, B. Choi, S. Jeong, and J. W. Choi (2010). Electrical detection-based analytic biodevice technology. BioChip Journal 4: 1-8
[27] W. Liang, W. Yi, S. Li, R. Yuan, A. Chen, S. Chen, G. Xiang, and C. Hu (2009). A novel, label-free immunosensor for the detection of α-fetoprotein using functionalised gold nanoparticles. Clinical Biochemistry 42: 1524-1530.
[28] J. W. Choi, B. K. Oh, Y. H. Jang, and D. Y. Kang (2008). Ultrasensitive immunoassay for prostate specific antigen using scanning tunneling microscopy-based electrical detection. Applied Physics Letter 93: 033110
[29] H. A. Pohl (1978). Dielectrophoresis(book).Cambridge University Press.
[30] R. Pethig (2010). Review Article—Dielectrophoresis: Status of the theory, technology, and applications. Biomicrofluidics 4: 022811.
[31] L. Yang (2009). Dielectrophoresis assisted immuno-capture and detection of foodborne pathogenic bacteria in biochips. Talanta 80: 551-558.
[32] O. K. Koo, Y. Liu, S. Shuaib, S. Bhattacharya, M. R. Ladisch, R. Bashir, and A. K. Bhunia (2009). Targeted Capture of Pathogenic Bacteria Using a Mammalian Cell Receptor Coupled with Dielectrophoresis on a Biochip. Analytical Chemistry 81: 3094-3101
[33] C. F. Chou and F. Zenhausern (2003). Electrodeless dielectrophoresis for micro total analysis systems. IEEE Engineering in Medicine and Biology Magazine 22: 62-67.
[34] Z. Gagnon, S. Senapati, and H. C. Chang (2010). Optimized DNA hybridization detection on nanocolloidal particles by dielectrophoresis. Electrophoresis 31: 666-671.
[35] I. F. Cheng, H. W. Han, and H. C. Chang (2012). Dielectrophoresis and shear-enhanced sensitivity and selectivity of DNA hybridization for the rapid discrimination of Candida species. Biosensors and Bioelectronics 33: 36-43.
[36] M. Javanmard, A. H. Talasaz, M. Nemat-Gorgani, D. E. Huber, F. Pease, M. Ronaghi, and R. W. Davis (2009). A Microfluidic Platform for Characterization of Protein—Protein Interactions. IEEE Sensors Journal 9: 883-891.
[37] S. Tuukkanen, J. J. Toppari, V. P. Hytonen, A. Kuzyk, M. S. Kulomaa, and P. Torma (2005). Dielectrophoresis as a tool for nanoscale DNA manipulation. International Journal of Nanotechnology 2:280-291.
[38] L. Zheng, S. Li, P. J. Burke, and J. P. Brody (2003). Towards single molecule manipulation with dielectrophoresis using nanoelectrodes. Proceedings of the Third IEEE Conference on Nanotechnology 1: 437.
[39] C. A. Marquette and L. J. Blum (2006). Beads Arraying and Beads Used in DNA Chips. Topics in Current Chemistry 261: 113-129.
[40] L. Zheng, J. P. Brody, and P. J. Burke (2004). Electronic manipulation of DNA, proteins, and nanoparticles for potential circuit assembly. Biosensors and Bioelectronics 20: 606-619
[41] I. F. Cheng, H. C. Chang, D. Hou, and H. C. Chang (2007). An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting. Biomicrofluidics 1: 021503.
[42] M. I. Gómez, A. Lee, B. Reddy, A. Muir, G. Soong, A. Pitt, A. Cheung, and A. Prince (2004). Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nature Medicine 10: 842-848.
[43] C. C. Lin, Y. M. Yang, Y. F. Chen, T. S. Yang, and H. C. Chang (2008). A new protein A assay based on Raman reporter labeled immunogold nanoparticles. Biosensors and Bioelectronics 24: 178-183.
[44] T. Takeuchi, O. Hosono, J. Koide, K. Amano, H. Sekine, and T. Abe (1991). The presence of anti-protein A antibodies in patients with rheumatoid arthritis. The presence of anti-protein A antibodies in patients with rheumatoid arthritis. Scandinavian Journal of Immunology 33: 585-592.
[45] N. T. Thanh and Z. Rosenzweig (2002). Development of an aggregation-based immunoassay for anti-protein A using gold nanoparticles. Analytical Chemistry 74: 1624-1628.
[46] P. R. Young, P. A. Hilditch, C. Bletchly, and W. Halloran (2000). An antigen capture Enzyme-Linked Immunosorbent Assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. Journal of Clinical Microbiology 38: 1053-1057.
[47] A. L. Sophie, M. T. Drouet, P. Roux, M. P. Frenkiel, M. Arborio, A. M. Durand-Schneider, M. Maurice, I. L. Blanc, J. Gruenberg, and M. Flamand (2005). The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes: implications for viral infectivity. Journal of Virology 79: 11403-11411.
[48] S. Alcon, A. Talarmin, M. Debruyne, A. Falconar, V. Deubel, and M. Flamand (2002). Enzyme-Linked immunosorbent assay specific to dengue virus Type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. Journal of Clinical Microbiology 40: 376-381.