本研究所提出一整合型自動化微流體晶片系統，此微流體晶片包含微型幫浦、渦旋式微混合器、微閥門、微流道以及微型加熱器，進行以磁珠抓取目標後做聚合酶連鎖反應，並以此裝置進行微生物之偵測。金黃色葡萄球菌是一種最常見引起傳染性疾病的微生物，常發生在醫院和醫療設施。其最為嚴重的是一種抵抗所有可用青黴素和β-內醯胺類抗生素 (β-lactam)，就是抗甲氧西林類藥物的金黃色葡萄球菌 (methicillin-resistant Staphylococcus aureus, MRSA)，成為一種在醫院最普遍的抗藥性的細菌；抗藥性金黃色葡萄球菌會在人體內快速生長，易導致感染者會有重症情況甚至是死亡風險，因此偵測此微生物在臨床上具有需求性，為了改善傳統微生物偵測的缺失本研究提出在生物檢體前處理時，加入溴化乙碇 (ethidium monoazide;以下簡稱 EMA)藥品。偵測檢體內呈現有存活之細菌。
其中微幫浦在操作條件頻率為35 Hz通入氣壓為20 psi時，驅動液體流速為每分鐘267 l。另外，渦旋式微混合器在操作條件頻率為9 Hz通入氣壓為15 psi時，可達到最高混合效率96.96 ％。本研究成功地在微流體晶片上對數種細菌檢體進行聚合酶連鎖反應，除了抗藥性金黃色葡萄球菌之外皆不會有非預期的產物出現，代表本晶片對於特定檢體具有相當高的專一性，另外在偵測極限部份，磁珠修飾抗體或去氧核醣核酸探針所得到的極限分別為106 CFU/ml及104 CFU/ml，皆比傳統醫院檢測所需的108 CFU/ml低，且成功結合使用EMA藥物來輔助判斷抗藥性金黃色葡萄球菌之存活狀態，檢測時間也由3天縮短至150分鐘。本研究成功的實驗證明微流體晶片系統在生醫檢測上可簡化偵測流程、縮短檢測時間並且提高了靈敏度。

The study presents a microfluidic system integrated with several crucial components including a vortex-type micro-mixer, micro-pumps, micro-valves and a micro temperature control module for fast detection of bacteria. The microfluidic chip can automatically perform magnetic beads-based extraction of sample and the subsequent polymerase chain reaction (PCR) for nucleic acid amplification. Staphylococcus aureus (S. aureus, SA) infection is one of the most common infectious diseases that occur in hospitals and healthcare facilities. More seriously, methicillin-resistance Staphylococcus aureus (MRSA), which is bacterium resistant to all available penicillin and β-lactam antimicrobial drugs, has become one of the most prevalent antibiotic resistant pathogens in hospitals. Live MRSA can grow rapidly inside the human body and can be vital for patients. In this study, ethidium monoazide (EMA) was first used to verify if the captured bacteria is alive. Then automatic isolation and detection of MRSA were performed on the microfluidic system.
The micropump and micromixer were first characterized. The maximum pumping rate of the micropump was measured to be 267 l/min at a driving frequency of 35 Hz and an air pressure of 20 psi. The vortex-type micro-mixer could achieve a mixing efficiency as high as 96.96% within 10 seconds under an applied pressure of 15 psi at a driving frequency of 9 Hz. Experimental data showed that the micro PCR module can successfully amplify the target DNA sequence of SA and MRSA with a high specificity. The detection limit of this microsystem using magnetic beads coated with antibody or DNA probes were experimentally found to be106 CFU / ml and 104 CFU / ml, respectively, while the conventional method was 108 CFU / ml. The total reaction time of this developed method was about 150 minutes, which was much faster than the conventional method. The developed microfluidic system may provide a powerful tool for fast disease diagnosis

1. R.J. Wilfinger, P. H. Bardell and D. S. Chhabra, “The resonistor a frequency selective device utilizing the mechanical resonance of a substrate,” IBM J. 12, 113-118 ,1968.
2. T. Vilkner , D. Janasek and A. Manz , “Micro total analysis systems: Recent developments,” Analytical Chemistry. 76, 3373-3386, 2004.
3. K. K. Jaln, “Biotechnological application of lab-chips and microarrays,” Trends in Biotechnology.18, 278-280, 2000.
4. R. Raiteri , M. Grattarola and R. Berger, “Micromechanics senses biomolecules,” Materials Today. 5, 22-29, 2002.
5. D. R. Reyes, D. Iossifidis, P. A. Auroux, and A. Manz, “Micro Total Analysis Systems. 1. Introduction, Theory, and Technology,” Analytical Chemistry. 74, 2623-2636,2002.
6. J. S. Czachor and T. E. Herchline,“Bacteremic nonmenstrual staphylococcal toxic shock syndrome associated with enterotoxins A and C,” Clin. 32, E53-6,2001.
7. M. Finland , “ Emergence of antibiotic resistance in hospitals,1935-1975.” Reviews of Infectious Diseases. 1, 4-22,1979.
8. M. Barber, “Methicillin-resistant staphylococci,” J. Clin. Pathol. 14,385-93,1961.
9. Y. Katayama, T. Ito, and K. Hiramatsu, “A new class of genetic element, Staphylococcal cassette chromosome mec, encodes methiciln resistance in Staphylococcus aureus,” Antimicrob. Agents Chemother. 44,1549-1555,2000.
10. B. Guignard, J.M. Entenza, and P. Moreillon,"Beta-lactams against methicillin-resistant Staphylococcus aureus." Curr Opin Pharmacol .5(5),479-89,2005.
11. M.L. Chen, S.C. Chang, H.J. Pan, P.R. Hsueh, L.S. Yang, S.W. Ho, K.T. Luh, “Longitudinal analysis of methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan.” J. Formos. Med. Assoc. 98,426-32,1999.
12. CDC “Staphylococcus aureus resistant to vancomycin--United States, 2002.” MMWR Morb. Morta.l Wkly Rep. 51,565-7,2002.
13. M.A. Northrup, M.T. Ching, R.M. White, and R.T. Wltson, “DNA amplification with a microfabricated reaction chamber,” Transducers, Chicago, USA, 924-926, 1993.
14. A.T. Woolley, D. Hadley, P. Landre, A.J. deMello, R.A. Mathies, and M.A. Northrup, “Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device,” Analytical Chemistry.68(23),4081-4086, 1996.
15. E.T. Lagally and R.A. Mathies, ”Integrated PCR-CE system for DNA analysis to the single molecule limit,” Micro Total Analysis Systems,117-118, 2001.
16. 廖家陞，“微型自動化核酸增幅系統快速偵測感染性疾病之病原菌”，國立成功大學微機電系統工程研究所碩士論文，2005
17. M.U. Kopp, A.J.d. Mello and A. Manz, “Chemical amplification: continuous-flow PCR on a chip,” Science.280(5366), 1046-1048,1998.
18. J. Liu, M. Enzelberger, S. Quake, “A nanoliter rotary device for polymerase chain reaction,” Electrophoresis.23(10),1531-1536, 2002.
19. P.J. Obeid, T.K. Christopoulos, H.J. Crabtree, and C.J. Backhouse, “Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection,” Analytical Chemistry. 75, 288-295, 2003.
20. J.H. Wang, L.J. Chien, T.M. Hsieh, C.H. Luo, W.P. Chou, P.H. Chen, P.J. Chen, D.S. Lee and G.B. Lee, “Micromachined Flow-through Polymerase Chain Reaction Chips Utilizing Multiple Membrane Activations,” Journal of Micromechanics and Microengineering, 17, 367-375, 2007.
21. E. Makino, T. Mitsuya and T. Shibata , “Fabrication of TiNi shape memory micropump,” Sensors and Actuators A.88, 256-262, 2001.
22. A. Olsson, P. Enoksson, G. Stemme, E. Stemme, “ A vlaveless planar pump isotropically etched in silicon,” Micromechanics Europe,120-123, 1995.
23. M.A. Unger, H.P. Chou, T. Thorsen, A. Scherer and S.R. Quake, “Monolithic microfabricated valves and pumps by multilayer soft lithography,” Science.288, 113-116, 2000.
24. A.D. Stroock, S.K.W. Dertinger, A. Ajdari, I. Mezić, H.A. Stone and G.M. Whitesides, “Chaotic mixer for microchannels,”Science.295,647-651,20021.
25. Z. Yang, H. Goto, M. Matsumoto and R. Maeda, “Active micromixer for microfluidic systems using lead-zirconate-titanate (PZT)-generated ultrasonic vibration”,Electrophoresis. 21, 116–9,2000.
26. H.Y. Tseng, C.H. Wang, W.Y. Lin and G.B. Lee “Membrane-activated microfluidic rotary devices for pumping and mixing,” Biomed. Microdevices. 9, 545–54,2007.
27. K.Y. Lien, C.J. Liu, Y.C. Lin, P.L. Kuo, and G.B. Lee, “Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform” Microfluidics and Nanofluidics, 6, 539-555 1, 2009
28. S.Y. Yang, J.L. Lin and G.B. Lee, “A Vortex-type Micromixer Utilizing Pneumatic-driven Membranes,” Journal of Micromechanics and Microengineering, Vol. 19, 2009
29. H.K. Nogva , S.M. Dromtorp , H. Nissen , K. Rudi, “Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease.” PCR BioTechniques.34, 810 812–813,2003.
30. K. Rudi, B. Moen, S.M. Drømtorp, and A.L. Holck, “Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples.” Appl. Environ. Microbiol. 71, 1018–1024,2005.
31. S. Wang and R.E. Levin, “Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR.” J. Microbiol.Methods, 64, 1–8,2006.
32. G.L. Coffman, J.W. Gaubatz, K.L. Yielding, L.W. Yielding, ”Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with ethidium analog.” J. Biol. Chem. 257, 13205–13297,1982.
33. A. Nocker and A.K. Camper, “Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.” Appl. Environ. Microbiol. 72, 1997–2004,2006.
34. C.M. Bitar, C.G. Mayhall, V.A. Lamb, T.J. Bradshaw, A.C. Spadora, H.P. Dalton,”Outbreak due to methicillin－and rifampin－resistant Staphylococcus aureus ： epidemiology and eradication of the resistant strain from the hospital,” Infect Control .8 ,15 －23,1987.
35. N.A. Campbell, J.B. Reece, “Biology,” 6th edition, Benjamin Cummings,2002.
36. K.B. Mullis, F. Ferre and R.A. Gibbs, “The polymerase chain reaction,” Boston, Birkhauser, USA. 1994.
37. 蘇慧慈， “原位分子生物學技術” 徐氏基金會， 1996.
38. I. Brodie, J.J. Muray, "The physics of microfabrication," Plenum Press, New York, 1982.
39. H. Esch, G. Huyberechts, R. Mertens, G. Maes, J. Manca, W. De Ceuninck, and L. De Schepper, "The stability of Pt heater and temperature sensing elements for silicon integrated tin oxide gas sensors," Sensors and Actuators B-Chemical. 65, 190-192, 2000.
40. C.H. Wang and G.B. Lee, “Automatic bio-sampling chips integrated with micro-pumps and micro-valves for disease detection.” Biosensors and Bioelectronics. 21, 419-425, 2005
41. C.H. Wang and G.B. Lee, "Pneumatic-driven peristaltic micropumps utilizing serpentine-shape channels," Journal of Micromechanics and Microengineering. 16, 341-348, 2006.
42. 林家澍編譯，“分子生物學速成＂ 合記圖書，2002.
43. 陳奕諭，“微型聚合酶連鎖反應晶片之性能測試” ，國立成功大學工程科學研究所碩士論文，2006
44. 簡良如，“新型吸取式三區微流體反應晶片應用於聚合酶連鎖反應” ，國立成功大學工程科學研究所碩士論文，2008
45. B.E. Slentz, N.A. Penner and F.E. Regnier, "Capillary Electrochromatography of Peptides on Microfabricated Poly(dimethylsiloxane) Chips Modified by Cerium(IV)-catalyzed Polymerization," Journal of Chromatography A. 948, 225–233, 2002.
46. Y.H. Liu, K.Y. Lien, C.H. Wang, J.J. Wu and G.B. Lee,“ Rapid Isolation and Detection of Methicillin-Resistant Staphylococcus Aureus on a microfluidic system,” IEEE NEMS 2011, Kaohsiung, Taiwan, February, 2011.