臺灣博碩士論文加值系統

本研究利用雷射光鉗操弄微米級微粒子之特性，進行單細胞之捕捉與拖曳操弄，探討其應用在臨床研究與檢測的可行性，實驗標的物分別為直徑約20 µm和1 µm的羊水細胞及金黃色葡萄球菌，內容包括：(1)利用光鉗揀選置於微流道晶片之羊水細胞，因應實際流道尺寸與細胞拖曳路徑，探討較佳的光鉗操弄參數，包括雷射功率和拖曳速度，進行細胞分離； (2) 針對金黃色葡萄球菌進行光鉗作用力量測，探討其不同等級抗藥性所獲致的光鉗作用力之關係，試著區分萬古黴素敏感性金黃色葡萄球菌 (VSSA) 菌株及萬古黴素中度抗性金黃色葡萄球菌(VISA)菌株。
以上實驗成果預期將有助於單細胞培養及單細胞全基因體定量分析，以及提供臨床醫師較快速的檢測方法，以便即時正確用藥。

In this study, the manipulation characteristics of laser tweezers exerted at micro-sized particles are applied for investigating the feasibilities of clinical studies and examinations of single cells by trapping and dragging the experimental subjects: amniotic fluid cells (20 µm) and staphylococcus (1 µm). The experiments include (1) to collect the amniotic fluid cells which are put in the microfluidic chip for the cells separation: to investigate the practical operation parameters, i.e. laser power and dragging speed, in order to harmony with the real dragging route in the micro-channels; (2) to measure the exerted forces of laser tweezers which are at the different resistances of staphylococcus aurous in order to distinguish the strains of VSSA and VISA.
The above experimental achievements are expected to contribute on the development of the culture and the quantitative analysis of whole genome of single cell, and a fast examination of staphylococcus aurous for medical doctor to prescribe for treatment.

摘要 I
Abstract V
誌謝 VI
目錄 VIII
圖目錄 XI
表目錄 XV
第一章 緒論 1
1-1前言 1
1-2目的 3
1-3方法 3
第二章 理論與文獻回顧 8
2-1 雷射光鉗 10
2-1-1光鉗原理 10
2-1-2光鉗在生醫研究應用的發展 21
2-2 生物細胞 34
2-2-1羊水細胞 34
2-2-2金黃色葡萄球菌 37
第三章 實驗方法 46
3-1 實驗架設 46
3-2 樣本製作與實驗操作 50
3-2-1微流道設計概念 53
3-2-2微型裝置的製造 54
3-2-3實驗操作 54
第四章 結果與討論 62
4-1細胞微流道 62
4-2羊水細胞操弄與檢測 68
4-2-1操弄之光學參數 68
4-2-2細胞培養與細胞濃度之關係 71
4-2-3 微珠模擬拖曳 72
4-2-4羊水細胞拖曳及細胞培養 73
4-3金黃色葡萄球菌抗藥性檢測 80
第五章 結論 86
附錄 87
參考文獻 93
簡歷 98

參考文獻
[1]2008 Fluxion Biosciences, Inc http://www.iul-instruments.de/pdf/168_BioFlux_Viscosity_TechNote-1038-01.pdf
[2]2011國健局年報 P14
[3]A. Ashkin ,“Acceleration and trapping of particles by radiation pressure,” Phys Rev Lett, Vol. 24, No. 4, 1970, pp.156-9.
[4]A. Ashkin, Dziedzic J, Bjorkholm J, Chu S,“Observation of a single-beam gradient force optical trap for dielectric particles” Opt Lett, Vol. 11, No. 5, 1986, pp.288.
[5]A. Ashkin, J.Dziedzic, T. Yamane,“Optical trapping and manipulation of single cells using infrared laser beams,” Nature, Vol. 330, No. 6105, 1987, pp. 769–771.
[6]A. Ashkin,“Forces of a single-beam gradient laser trap on a dielectric sphere in the ray optics regime,” Biophys J, Vol. 61, No. 2, 1992, pp. 569-82.
[7]A. H. Handyside, R.J.A. Penketha, R.M.L. Winstona, J.K. Pattinsonb, J.D.A. Delhantyc, E.G.D. Tuddenhamb” Biopsy of human preimplatation embryos and sexing by DNA amplification” The Lancet , Vol. 333, 1989, pp. 347-349.
[8]A. Prusa and M. Hengstschläger, “Amniotic fluid cells and human stem cell research-A new connection,” Medical Science Monitor, Vol. 8, 2002, pp.253-257.
[9]A. Schulman, “The search for alternative sources of human pluripotent stem cells,” Stem Cell Reviews, Vol. 1, 2005, pp.291-292.
[10] A. Wood, “Ethics and embryonic stem cell research,” Stem Cell Reviews, Vol. 1, 2005, pp. 317–324.
[11] Anonymous: Breakpoints in in-vitro antibiotic sensitivity testing. Report by aworking party of the British Society for Antimicrobial Chemotherapy. J Antimicrob Chemother 1988;21:701-10.
[12] B. S. Cho, T. G. Schuster, X. Zhu, D. Chang, G. D. Smith, and S. Takayama, "A microfluidic device for separating motile sperm from nonmotilesperm via inter-streamline crossings," in 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine & Biology, 2002, pp.156-159.
[13] C. L. Lin,“Opto-Mechanical Application of Microstructured Materials : 2) Laser-driven polymer micro-sensors,” 2004, p. 56.
[14] C. Luo, H. Li, C. Xiong, X. Peng, Q. Kou, Y. Chen, H. Ji and Q. Ouyang, “The combination of optical tweezers and microwell array for cells physical manipulation and localization in microfluidic device.” Biomedical Microdevices, Vol. 9, Number 4, 2007, pp.573-578.
[15] C. T. Lim , E. H. Zhou, A. Li, S.R.K. Vedula, H. X. Fu “Experimental techniques for single cell and single molecule biomechanics” Materials Science and Engineering C 26 2006, pp. 1278 – 1288.
[16] D. D. Carlo, L. Y. Wu and L. P. Lee, “Dynamic single cell culture array.” Lab Chip, 2006, pp. 1445-1449.
[17] D. Fauza, “Amniotic fluid and placental stem cells,” Best Practical Research in Clinical Obstetrics and Gynecology, Vol. 18, 2004, pp. 877-891.
[18] D. M. Sievert, M. L. Boulton, G. Stoltman, “Staphylococcus aureus resistant to vancomycin - United States” 2002. MMWR 2002;51:565-7.
[19] D. Miller, V. Urdaneta, ”Vancomycin-resistant Staphylococcus aureus – Pennsylvania”, 2002 (Reprinted from MMWR, vol 51,2002, pg 902,)." Jama-Journal of the American Medical Association 288(17): 2116-2116.
[20] E. Leclerc, Y. Sakai, and T. Fujii, “Cell culture in 3D microfluidic structure of PDMS.” Biomedical Microdevices, 2003, pp. 109-114.
[21] F. M. Kamm, “Ethical issues in using and not using embryonic stem cells,” Stem Cell Reviews, Vol. 1, 2005, pp.325-330.
[22] G. Gouesbet, B. Maheu, G. Gr''ehan,“Light scattering from a sphere arbitrarily located in a gaussian beam, using a bromwich formulation,” Journal of the Optical Society of America A, Vol. 5, No.9, 1988, pp. 1427-43.
[23] G. M. Hale, and M. R. Querry, “Optical constants of water in the 200-nrn to 200-mm wavelength region,” Appl. OpL, vol. 12, 1973, pp. 555-563.
[24] H. Hanaki, A. K. Kuwahara, V. S. Boyle, R. S. Daum, H. Labischinski, K. Hiramatsu, “Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50.” J Antimicrob Chemother. Aug. 1998;42(2):199-209.
[25] H. Hoehn. And D. Salk, “Morphological and biochemical heterogeneity of amniotic fluid cells in culture,” Methods in Cell Biology, Vol. 26, 1982, pp.11-34.
[26] H. Liang, K. T. Vu, Priya Krishnan, T. C. Trang, David Shin, S. Kimel, and M. W. Berns “Wavelength Dependence of Cell Cloning Efficiency after Optical Trapping” Biophysical Journal, Vol. 70 March 1996, pp. 1529-1533.
[27] H. Van de Hulst,“Light scattering by small particles,” New York: Dover, 1981.
[28] H. Zhang and K.K. Liu” Optical tweezers for single cells” J. R. Soc. Interface, 2008, pp. 671-690.
[29] Hiramatsu K, Aritaka N, Hanaki H, et al: Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 1997;350:1670-3.
[30] I. A. Vorobjev, H. Liang, W. H. Wright, and M. W. Berns “Optical trapping for chromosome manipulation: a wavelength dependence of induced chromosome bridges” Biophysical Society, Vol. 64, February 1993, pp. 533-538.
[31] Invitrogen com http://www.docstoc.com/docs/37060852/Useful-Numbers-for-Cell-Culture.
[32] J. W. Schmidt, A. Greenough, M. Burns, A. E. Luteran and D. G. McCafferty, “Generation of ramoplanin-resistant staphylococcus aureus,” FEMS Microbiol. Lett. 2010, pp.104-111.
[33] John A. Ryan, Ph.D. Corning Incorporated Life Sciences 900 Chelmsford St Lowell, MA 01851; http://catalog2.corning.com/Lifesciences/media/pdf/Subculturing_protocol.pdf
[34] K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of Photo Damage to Escherichia Coli in Optical Traps,”Biophysical Journal, Vol. 77, 1999, pp.2856–2863.
[35] K. Hiramatsu, H. Hanaki, T. Ino,” Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility,” J Antimicrob Chemother,1997, Vol. 40, pp.135-6.
[36] K. Hiramatsu, N. Aritaka, H. Hanaki, “Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin.” Lancet, 1997;350:1670-3.
[37] L. Cui, H. Murakami, K. Kuwahara-Arai,”Contribution of a thickened cell wall and its glutamine nonamidated component to the vancomycin resistance expressed by Staphylococcus aureus Mu50.” Antimicrob Agents Chemother, 2000, Vol. 44,pp. 2276-85.
[38] L. Cui, X. Ma, K. Sato, K. Okuma, F. C. Tenover, E. M. Mamizuka, C. G. Gemmell, Mi-Na Kim, Marie-Cecile Ploy, N. E. Solh, V. Ferraz, and K. Hiramatsu, “Cell Wall Thickening Is a Common Feature of Vancomycin Resistance in Staphylococcus aureus” J Clin Microbiol. Jan. 2003; 41(1): 5–14.
[39] L. Weissman, “Stem cells: Units of development, units of regeneration, and units in evolution,” Cell, Vol. 100, 2000, pp.157-168.
[40] M. B. Avison, P. M. Bennett, R. A. Howe,” Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50.” J Antimicrob Chemother, 2002, Vol. 49, pp. 255-60.
[41] M. Gosden, “Amniotic fluid cell types and culture,” British Medical Bulletin, Vol. 39, 1983, pp.348-354.
[42] M. Ichikawa and K. Yoshikawa, “Optical transport of a single cell-sized liposome” Appl. Phys. Let., Vol. 79, 2001, pp. 4598-4600.
[43] M. Mitka, “Amniotic cell show promise for fetal tissue engineering,” The Journal of the American Medical Association, Vol. 286, 2001, pp. 2083.
[44] N. Rosenthal, “Prometheus’s vulture and the stem-cell promise,” New England Journal of Medicine, Vol. 349, 2003, pp. 267-274.
[45] P. V. Guillot, K. O’Donoghue, H. Kurata, and N. M. Fisk, “Fetal stem cells: Betwixt and between,” Seminars in Reproductive Medicine, Vol. 24, 2006, pp.340–347.
[46] Q. You, L. Cai , J. Zheng , X. Tong , D. Zhang , Y. Zhang ” Isolation of human mesenchymal stem cells from third-trimester amniotic fluid” International Journal of Gynecology and Obstetrics, (2008) 103, pp. 149–152.
[47] R. Burger, I. K. Dimov and J. Ducrée, “Highly efficient single cell capturing under stagnant flow conditions on a centrifugal microfluidic platform.” Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences, Nov.-1-5, 2009, Jeju, Korea.
[48] S. N. Bhatia, U. J. Balis, et al. (1998). "Microfabrication of hepatocyte/fibroblast co-cultures: Role of homotypic cell interactions." Biotechnology Progress, 1998, pp. 378-387.
[49] T. Fujii, “PDMS-based microfluidic devices for biomedical applications.” Microelectronic Engineering, 2002, pp. 907-914.
[50] T. Turetsky, E. Aizenman, Y. Gil, N. Weinberg, Y. Shufaro, A. Revel, N. Laufer, A. Simon, D. Abeliovich and B.E. Reubinoff, “Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.” Hum. Reprod. 2008, 23 (1): pp. 46-53.
[51] W. H. Wright,G. J. Sonek and M.W. Berns,“Parametric study of the forces on microspheres held by optical tweezers,” Appl Opt, Vol. 33, 1994, pp. 1735.
[52] Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Trombergt ”Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers” Biophysical Journal Vol. 68 May 1995, pp. 137-2144.
[53] Y. Tadir,W. H. Wright,M.W. Berns,“Cell micromanipulation with laser beams,” In G.I.F.T., From Basics to Clinics, GL Capitanio, RH Asch, L De Cecco, S Croce (eds). New York, Raven Press, 1989, pp. 359-368.
[54] 台大醫院基因醫學部 分子遺傳實驗室, http://www.dna.org.tw/Default.asp
[55]行政院衛生署國民健康局『臨床細胞遺傳學實驗室操作指引』序 P7
[56]吳佳全, “細胞分裂影響因素及與癌變關係” 中國浙江大學課程論文 www.zjubiolab.zju.edu.cn/lesson/news.php?&news_catalogId=11&page=2
[57] 吳崇安,黃鈞正,邱爾德,“光學嵌住之理論探討,” 物理雙月刊, Vol. 22, No. 5, 2000, pp. 485-488.
[58]金黃色葡萄球菌,維基百科, http://zh.wikipedia.org/wiki/%E9%87%91%E9%BB%83%E8%89%B2%E8%91%A1%E8%90%84%E7%90%83%E8%8F%8C
[59] 徐玄修、林永昇、殷宏林、謝哲偉 “生醫微流體晶片之製程平台” 國家實驗研究院 儀器科技研究中心, 2006
[60]曾彥方， ”光鉗作用力在生物細胞力學感測上的應用開發” ，中臺科技大學醫學工程暨材料研究所碩士論文，民國99年7月(2010)
[61]衛生署疾病管制局, http://www.cdc.gov.tw/info.aspx?treeid=45da8e73a81d495d&nowtreeid=1BD193ED6DABAEE6&tid=D5AD02736F6C8AB0