鮑氏不動桿菌之快速檢測在院內極其重要以預防醫療照護相關感染。尤其針對細菌檢測的早期診斷可以有效增加治療功效。在本研究中，我們提出了一種整合型定點照護設備，可在30分鐘內以雙重適體技術檢測細菌。此設備整合光二極體激發螢光之模組，其接收到的訊號可被光二極體激發，並以光電二極管量化數據，由此可實行一具有高靈敏性及高專一性之細菌量化診斷。整個過程可以在由永久性磁鐵及電磁鐵之組合之電磁控平台上操作。在施10伏特之電壓時，微型幫浦可以達到270 µL/min的傳輸速率，並適合用於定點照護上之應用。本研究使用磁珠系統，表面接上AB菌專一適體來抓取樣品中的細菌，並用第二段適體接上量子點以定量細菌。檢測極限為每反應103顆鮑氏不動桿菌，與本文所提及之一篇文獻比較後有相當的競爭力。此新的生物技術不僅縮小了設備的尺寸，且具有低消耗及低成本的特性。此研究是首次顯示一可以執行雙重適體技術之細菌檢測的全自動化定點照護設備並使用電磁驅動之微型幫浦及微型混合器。在不遠的將來，此設備在臨床上快速診斷AB菌具有不容小覷之潛力。

Rapid detection of Acinetobacter baumannii (AB) is important in hospitals to prevent healthcare-associated-infections. The earlier the diagnosis could be performed, the better the efficacy of treatment that could be achieved. In this study, an integrated microfluidic device for bacterial diagnosis within 30 minutes utilizing a new dual-aptamer assay for point-of-care (POC) applications has been developed. The microfluidic device was further integrated with a light-emitting diode (LED) induced fluorescence module, such that the signals could be induced by a LED and quantified with a photodiode, for the quantitative diagnosis of bacteria with high sensitivity and specificity for POC applications. The entire procedure could be performed on an electromagnetic-driven platform equipped with micropumps, microvalves and micromixers with a combination of permanent magnets and electromagnets. The pumping rate of the micropump was measured to be 270 µL/min with an applied voltage of 10 V, which is amenable for POC applications. Magnetic beads surface-coated with AB-specific aptamers were used to capture bacteria in samples. Then a second aptamer and quantum dots were used to quantify the amount of bacteria. The limit of detection (LOD) for AB diagnosis was experimentally found to be 102 colony-forming unit (CFU)/reaction, which is comparable with the ones reported in literature. Not only could the new method of the biological assay minimize the dimensions of the equipment, but also reduces the power consumption and cost. This is the first time that an automatic POC device was demonstrated which could perform a dual-aptamer assay to diagnose AB by using electromagnetic-driven microfluidic system. It has great potential for fast diagnosis of AB in clinics in the near future.

Abstract I
摘要 III
致謝 IV
List of figures VII
Nomenclature and abbreviations XV
Chapter 1 Introduction 1
1.1 Acinetobacter baumannii 1
1.2 Aptamers in biological applications 4
1.3 Fluorescent detection system 7
1.4 Point-of-care diagnostics 9
1.5 MEMS and bio-MEMS based microfluidic technology 10
1.6 Motivation and novelty 13
Chapter2 Materials and methods 15
2.1 Design and fabrication of the electromagnetic microfluidic chip 15
2.1.1 Chip design-micropump, microvalves and micromixer 15
2.1.3 Chip fabrication procedure 19
2.1.4 Operating principle of the electromagnetic-driven microfluidic platform 23
2.1.5 Fabrication of the electromagnetic driven platform 25
2.2 Sample preparation 27
2.2.1 Preparation of bacterial samples and aptamers 27
2.2.2 Procedure of the magnetic beads surface-coated with specific aptamer 29
2.2.3 Procedure of the quantum dots coated with specific aptamer 30
2.3 Experimental procedure 31
2.3.1 Benchtop procedure 31
2.3.2 Microfluidic chip procedure 31
2.4 Experimental setup 34
2.5 Circuit design 36
2.6 Methods of characterizing the electromagnetic-driven micropump, microvalves and micromixer 38
2.7 Methods of optimal the conditions of the dual aptamer assay 39
Chapter3 Results and discussion 41
3.1 Characterization of the developed microfluidic chip 41
3.1.1 Pumping volume & pumping rate of the microfluidic chip 41
3.1.2 Mixing efficiency of the microfluidic chip 45
3.2 Performance of electromagnet 47
3.3 Stability of LED-induced fluorescence module 49
3.4 Optimization of the dual aptamer assay 51
3.5 Fluorescent signal of the benchtop dual aptamer assay 56
3.6 Performance of the dual aptamer assay on the microfluidic chip 59
Chapter 4 Conclusions and future perspectives 65
4.1 Conclusions 65
4.2 Future perspectives 67
References 68
Publication list 73

[1] C. Berrettoni, S. Berneschi, R. Bernini, A. Giannetti, I. A. Grimaldi, G. Persichetti, G. Testa, S. Tombelli, C. Trono, and F. Baldini, "Optical Monitoring of Therapeutic Drugs with a Novel Fluorescence- Based POCT Device," Procedia Engineering, vol. 87, pp. 392-395, 2014.
[2] L. Gao, Y. Lyu, and Y. Li, "Trends in Drug Resistance of Acinetobacter baumannii over a 10-year Period: Nationwide Data from the China Surveillance of Antimicrobial Resistance Program," Chinese Medical Journal, vol. 130, no. 6, pp. 659-664, 2017.
[3] E. Wenzler, D. A. Goff, J. E. Mangino, E. E. Reed, A. Wehr, and K. A. Bauer, "Impact of rapid identification of Acinetobacter Baumannii via matrix-assisted laser desorption ionization time-of-flight mass spectrometry combined with antimicrobial stewardship in patients with pneumonia and/or bacteremia," Diagn Microbiol Infect Dis, vol. 84, no. 1, pp. 63-68, 2016.
[4] L. Dijkshoorn, A. Nemec, and H. Seifert, "An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii," Nature Reviews Microbiology, Review Article vol. 5, p. 939, 2007.
[5] J. C. Lagier, S. Edouard, I. Pagnier, O. Mediannikov, M. Drancourt, and D. Raoult, "Current and past strategies for bacterial culture in clinical microbiology," Clin Microbiol Rev, vol. 28, no. 1, pp. 208-36, 2015.
[6] K. Kim, J. H. Guo, Z. X. Liang, and D. L. Fan, "Artificial Micro/Nanomachines for Bioapplications: Biochemical Delivery and Diagnostic Sensing," (in English), Advanced Functional Materials, Article vol. 28, no. 25, p. 19, Jun 2018, Art. no. 1705867.
[7] R. Coico, "Gram Staining," Current Protocols in Microbiology, vol. 00, no. 1, pp. A.3C.1-A.3C.2, 2006.
[8] R. Patel, "New Developments in Clinical Bacteriology Laboratories," Mayo Clinic Proceedings, vol. 91, no. 10, pp. 1448-1459, 2016/10/01/ 2016.
[9] S. Bashiri, F. N. Mansoor, and Z. Valadkhani, "Expansion of a highly sensitive and specific ELISA test for diagnosis of hydatidosis using recombinant EgB8/2 protein," (in English), Iranian Journal of Basic Medical Sciences, Article vol. 22, no. 2, pp. 134-139, Feb 2019.
[10] N. Alizadeh, M. Y. Memar, B. Mehramuz, S. S. Abibiglou, F. Hemmati, and H. Samadi Kafil, "Current advances in aptamer-assisted technologies for detecting bacterial and fungal toxins," (in eng), J Appl Microbiol, vol. 124, no. 3, pp. 644-651, Mar 2018.
[11] A. R. Oliphant, C. J. Brandl, and K. Struhl, "Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein," Mol Cell Biol, vol. 9, no. 7, pp. 2944-9, 1989.
[12] A. D. Ellington and J. W. Szostak, "In vitro selection of RNA molecules that bind specific ligands," nature, vol. 346, no. 6287, p. 818, 1990.
[13] C. Tuerk and L. Gold, "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase," Science, vol. 249, no. 4968, pp. 505-510, 1990.
[14] J. Voskuil, "Commercial antibodies and their validation," (in eng), F1000Research, vol. 3, pp. 232-232, 2014.
[15] Z. Cao, R. Tong, A. Mishra, W. Xu, G. C. Wong, and Y. Lu, "Reversible cell‐specific drug delivery with aptamer‐functionalized liposomes," Angewandte Chemie International Edition, vol. 48, no. 35, pp. 6494-6498, 2009.
[16] J. G. Bruno and J. L. Kiel, "In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection," Biosensors and Bioelectronics, vol. 14, no. 5, pp. 457-464, 1999.
[17] J. Wang, X. Cai, G. Rivas, H. Shiraishi, and N. Dontha, "Nucleic-acid immobilization, recognition and detection at chronopotentiometric DNA chips," Biosensors and Bioelectronics, vol. 12, no. 7, pp. 587-599, 1997.
[18] M. S. Song, S. S. Sekhon, W. R. Shin, H. C. Kim, J. Min, J. Y. Ahn, and Y. H. Kim, "Detecting and Discriminating Shigella sonnei Using an Aptamer-Based Fluorescent Biosensor Platform," (in English), Molecules, Article vol. 22, no. 5, p. 12, 2017, Art. no. 825.
[19] J. G. Bruno, T. Phillips, T. Montez, A. Garcia, J. C. Sivils, M. W. Mayo, A. Greis, and L. Metrix, "Development of a Fluorescent Enzyme-Linked DNA Aptamer-Magnetic Bead Sandwich Assay and Portable Fluorometer for Sensitive and Rapid Listeria Detection," (in English), Journal of Fluorescence, Article vol. 25, no. 1, pp. 173-183, Jan 2015.
[20] X. Wang and Q. Zhao, "A fluorescent sandwich assay for thrombin using aptamer modified magnetic beads and quantum dots," Microchimica Acta, vol. 178, no. 3-4, pp. 349-355, 2012.
[21] Y. H. Tennico, D. Hutanu, M. T. Koesdjojo, C. M. Bartel, and V. T. Remcho, "On-chip aptamer-based sandwich assay for thrombin detection employing magnetic beads and quantum dots," Analytical chemistry, vol. 82, no. 13, pp. 5591-5597, 2010.
[22] S. J. Xiao, P. P. Hu, X. D. Wu, Y. L. Zou, L. Q. Chen, L. Peng, J. Ling, S. J. Zhen, L. Zhan, and Y. F. Li, "Sensitive discrimination and detection of prion disease-associated isoform with a dual-aptamer strategy by developing a sandwich structure of magnetic microparticles and quantum dots," Analytical chemistry, vol. 82, no. 23, pp. 9736-9742, 2010.
[23] S. Rasoulinejad and S. L. M. Gargari, "Aptamer-nanobody based ELASA for specific detection of Acinetobacter baumannii isolates," J Biotechnol, vol. 231, pp. 46-54, 2016.
[24] R. Stoltenburg, C. Reinemann, and B. Strehlitz, "SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands," Biomol Eng, vol. 24, no. 4, pp. 381-403, 2007.
[25] M. Berner, U. Hilbig, M. B. Schubert, and G. Gauglitz, "Laser-induced fluorescence detection platform for point-of-care testing," Measurement Science and Technology, vol. 28, no. 8, p. 085701, 2017.
[26] L. Novak, P. Neuzil, J. Pipper, Y. Zhang, and S. Lee, "An integrated fluorescence detection system for lab-on-a-chip applications," Lab Chip, vol. 7, no. 1, pp. 27-9, Jan 2007.
[27] T. Kamei and T. Wada, "Contact-lens type of micromachined hydrogenated amorphous Si fluorescence detector coupled with microfluidic electrophoresis devices," Applied physics letters, vol. 89, no. 11, p. 114101, 2006.
[28] J. L. Fu, Q. Fang, T. Zhang, X. H. Jin, and Z. L. Fang, "Laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement," Analytical chemistry, vol. 78, no. 11, pp. 3827-3834, 2006.
[29] J. Webster, M. Burns, D. Burke, and C. Mastrangelo, "Monolithic capillary electrophoresis device with integrated fluorescence detector," Analytical chemistry, vol. 73, no. 7, pp. 1622-1626, 2001.
[30] A. Klotz, A. Brecht, C. Barzen, G. Gauglitz, R.D. Harris, G.R. Quigley, J.S. Wilkinson, and R. A. Abuknesha, "Immunofluorescence sensor for water analysis," 1998.
[31] P. Yeh, N. Yeh, C.-H. Lee, and T.-J. Ding, "Applications of LEDs in optical sensors and chemical sensing device for detection of biochemicals, heavy metals, and environmental nutrients," Renewable and Sustainable Energy Reviews, vol. 75, pp. 461-468, 2017.
[32] R. Bilan, I. Nabiev, and A. Sukhanova, "Quantum Dot-Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery," (in English), Chembiochem, Review vol. 17, no. 22, pp. 2103-2114, Nov 2016.
[33] A. J. Tudos, G. J. Besselink, and R. B. Schasfoort, "Trends in miniaturized total analysis systems for point-of-care testing in clinical chemistry," Lab Chip, vol. 1, no. 2, pp. 83-95,
2001.
[34] S. K. Vashist, "Point-of-Care Diagnostics: Recent Advances and Trends," Biosensors, vol. 7, no. 4, p. 62, 2017.
[35] C.-M. Ho and Y.-C. Tai, "MICRO-ELECTRO-MECHANICAL-SYSTEMS (MEMS) AND FLUID FLOWS," Annual Review of Fluid Mechanics, vol. 30, no. 1, pp. 579-612, 1998.
[36] T. Vilkner, D. Janasek, and A. Manz, "Micro total analysis systems. Recent developments," Analytical chemistry, vol. 76, no. 12, pp. 3373-3386, 2004.
[37] D. R. Reyes, D. Iossifidis, P.-A. Auroux, and A. Manz, "Micro total analysis systems. 1. Introduction, theory, and technology," Analytical chemistry, vol. 74, no. 12, pp. 2623-2636, 2002.
[38] P. A. Auroux, D. Iossifidis, D. R. Reyes, and A. Manz, "Micro total analysis systems. 2. Analytical standard operations and applications," Analytical chemistry, vol. 74, no. 12, pp. 2637-2652, 2002.
[39] S. Shoji and M. Esashi, "Microflow devices and systems," Journal of Micromechanics and Microengineering, vol. 4, no. 4, p. 157, 1994.
[40] M. H. Wu, S. B. Huang, Z. Cui, Z. Cui, and G. B. Lee, "A high throughput perfusion-based microbioreactor platform integrated with pneumatic micropumps for three-dimensional cell culture," (in eng), Biomed Microdevices, vol. 10, no. 2, pp. 309-19, 2008.
[41] M. M. Aeinehvand, L. Weber, M. Jimenez, A. Palermo, M. Bauer, F. F. Loeffler, F. Ibrahim, F. Breitling, J. Korvink, M. Madou, D. Mager, and S. O. Martinez-Chapa, "Elastic reversible valves on centrifugal microfluidic platforms," (in English), Lab on a Chip, Article vol. 19, no. 6, pp. 1090-1100, Mar 2019.
[42] R. Tang, H. Yang, Y. Gong, M. You, Z. Liu, J. R. Choi, T. Wen, Z. Qu, Q. Mei, and F. Xu, "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection," (in eng), Lab Chip, vol. 17, no. 7, pp. 1270-1279, Mar 29 2017.
[43] J. H. Wu, C. H. Wang, Y. D. Ma, and G. B. Lee, "A nitrocellulose membrane-based integrated microfluidic system for bacterial detection utilizing magnetic-composite membrane microdevices and bacteria-specific aptamers," (in eng), Lab Chip, vol. 18, no. 11, pp. 1633-1640, May 29 2018.
[44] J. Li, K. W. Chang, C. H. Wang, C. H. Yang, S. C. Shiesh, and G. B. Lee, "On-chip, aptamer-based sandwich assay for detection of glycated hemoglobins via magnetic beads," Biosens Bioelectron, vol. 79, pp. 887-93, May 15 2016.
[45] J. H. Wu, C. H. Wang, Y. D. Ma, and G. B. Lee, "A nitrocellulose membrane-based integrated microfluidic system for bacterial detection utilizing magnetic-composite membrane microdevices and bacteria-specific aptamers," Lab on a Chip, vol. 18, no. 11, pp. 1633-1640, 2018.
[46] S. Park, H. Kim, S.-H. Paek, J. W. Hong, and Y.-K. Kim, "Enzyme-linked immuno-strip biosensor to detect Escherichia coli O157:H7," Ultramicroscopy, vol. 108, no. 10, pp. 1348-1351, 2008/09/01/ 2008.
[47] C.-H. Wang, J.-J. Wu, and G.-B. Lee, "Screening of highly-specific aptamers and their applications in paper-based microfluidic chips for rapid diagnosis of multiple bacteria," Sensors and Actuators B: Chemical, vol. 284, pp. 395-402, 2019/04/01/ 2019.