Please use this identifier to cite or link to this item:
標題: 細胞裂解微流體模組之開發
Development of microfluidic module for cell lysis
作者: 李孟鴻
Lee, Mong-Hong
關鍵字: cell lysis;細胞裂解;microfluidic module;erythrocyte;yeast;Bacillus;微流體模組;紅血球;酵母菌;枯草桿菌
出版社: 食品暨應用生物科技學系所
引用: [1] R.C. Anderson, X. Su, G.J. Bogdan, and J. Fenton, “A miniature integrated device for automated multistep genetic assays,” Nucl. Acids Res., vol. 28, Jun. 2000, p. e60. [2] M. Schena, D. Shalon, R.W. Davis, and P.O. Brown, “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science (New York, N.Y.), vol. 270, Oct. 1995, pp. 467-470. [3] J.J.W. Chen, R. Wu, P. Yang, J. Huang, Y. Sher, M. Han, W. Kao, P. Lee, T.F. Chiu, F. Chang, Y. Chu, C. Wu, and K. Peck, “Profiling Expression Patterns and Isolating Differentially Expressed Genes by cDNA Microarray System with Colorimetry Detection,” Genomics, vol. 51, Aug. 1998, pp. 313-324. [4] D. Erickson and D. Li, “Integrated microfluidic devices,” Analytica Chimica Acta, vol. 507, Apr. 2004, pp. 11-26. [5] 許育群, “微流體電泳晶片之建構及其在毛細電泳檢測上的應用,” 國立中興大學碩士論文, 2003. [6] D.C. Duffy, J.C. McDonald, O.J.A. Schueller, and G.M. Whitesides, “Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane),” Analytical Chemistry, vol. 70, Dec. 1998, pp. 4974-4984. [7] D.T. Ross, U. Scherf, M.B. Eisen, C.M. Perou, C. Rees, P. Spellman, V. Iyer, S.S. Jeffrey, M. Van de Rijn, M. Waltham, A. Pergamenschikov, J.C. Lee, D. Lashkari, D. Shalon, T.G. Myers, J.N. Weinstein, D. Botstein, and P.O. Brown, “Systematic variation in gene expression patterns in human cancer cell lines,” Nat Genet, vol. 24, Mar. 2000, pp. 227-235. [8] C. Debouck and P.N. Goodfellow, “DNA microarrays in drug discovery and development,” Nat Genet. [9] X. Wen, S. Fuhrman, G.S. Michaels, D.B. Carr, S. Smith, J.L. Barker, and R. Somogyi, “Large-scale temporal gene expression mapping of central nervous system development,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, Jan. 1998, pp. 334 -339. [10] J.L. STROMINGER, J.T. PARK, and R.E. THOMPSON, “Composition of the cell wall of Staphylococcus aureus: its relation to the mechanism of action of penicillin,” The Journal of Biological Chemistry, vol. 234, Dec. 1959, pp. 3263-3268. [11] N.H. Georgopapadakou and J.S. Tkacz, “The fungal cell wall as a drug target,” Trends in Microbiology, vol. 3, Mar. 1995, pp. 98-104. [12] G.J. Tortora, B.R. Funke, and C.L. Case, Microbiology: An Introduction, Eighth Edition, Benjamin Cummings, 2003. [13] “” [14] B. Aguilar-Uscanga and J. François, “A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation,” Letters in Applied Microbiology, vol. 37, 2003, pp. 268-274. [15] “” [16] J.K. Fredrickson, J.M. Zachara, D.L. Balkwill, D. Kennedy, S.W. Li, H.M. Kostandarithes, M.J. Daly, M.F. Romine, and F.J. Brockman, “Geomicrobiology of High-Level Nuclear Waste-Contaminated Vadose Sediments at the Hanford Site, Washington State,” Appl. Environ. Microbiol., vol. 70, Jul. 2004, pp. 4230-4241. [17] M.E. Johnson and J.A. Lucey, “Major Technological Advances and Trends in Cheese,” J. Dairy Sci., vol. 89, Apr. 2006, pp. 1174-1178. [18] E.M. Wise and J.T. Park, “Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 54, Jul. 1965, pp. 75-81. [19] P.S. Raina, F.L. Pollari, G.F. Teare, M.J. Goss, D.A. Barry, and J.B. Wilson, “The relationship between E. coli indicator bacteria in well-water and gastrointestinal illnesses in rural families,” Canadian Journal of Public Health. Revue Canadienne De Santé Publique, vol. 90, Jun. 1999, pp. 172-175. [20] W.J. Dower, J.F. Miller, and C.W. Ragsdale, “High efficiency transformation of E.coli by high voltage electroporation,” Nucl. Acids Res., vol. 16, Jul. 1988, pp. 6127-6145. [21] C.T. Chung, S.L. Niemela, and R.H. Miller, “One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution,” Proceedings of the National Academy of Sciences of the United States of America, vol. 86, Apr. 1989, pp. 2172 -2175. [22] “” [23] “” [24] R.A. Batchelor, P. Alifano, E. Biffali, S.I. Hull, and R.A. Hull, “Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation.,” J. Bacteriol., vol. 174, Aug. 1992, pp. 5228-5236. [25] M.T. Armstrong, S.M. Theg, N. Braun, N. Wainwright, R.L. Pardy, and P.B. Armstrong, “Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts,” FASEB J., vol. 20, Oct. 2006, pp. 2145-2146. [26] R.T. Ellison and T.J. Giehl, “Killing of gram-negative bacteria by lactoferrin and lysozyme.,” Journal of Clinical Investigation, vol. 88, Oct. 1991, pp. 1080-1091. [27] R. Mancini and M. Hunt, “Current research in meat color,” Meat Science, vol. 71, Sep. 2005, pp. 100-121. [28] D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, W. H. Freeman, 2008. [29] H. Holo and I.F. Nes, “High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media,” Appl. Environ. Microbiol., vol. 55, Dec. 1989, pp. 3119-3123. [30] E. Delorme, “Transformation of Saccharomyces cerevisiae by electroporation.,” Appl. Environ. Microbiol., vol. 55, Sep. 1989, pp. 2242-2246. [31] U. Zimmermann, “Electrical breakdown, electropermeabilization and electrofusion,” Reviews of Physiology, Biochemistry and Pharmacology, vol. 105, 1986, pp. 176-256. [32] T. Tsong, “Electroporation of cell membranes,” Biophysical Journal, vol. 60, Aug. 1991, pp. 297-306. [33] S. Lee and Y. Tai, “A micro cell lysis device,” Sensors and Actuators A: Physical, vol. 73, Mar. 1999, pp. 74-79. [34] Q. Ramadan, V. Samper, D. Poenar, Z. Liang, C. Yu, and T. Lim, “Simultaneous cell lysis and bead trapping in a continuous flow microfluidic device,” Sensors and Actuators B: Chemical, vol. 113, Feb. 2006, pp. 944-955. [35] Y. Lin, M. Li, C. Fan, and L. Wu, “A microchip for electroporation of primary endothelial cells,” Sensors and Actuators A: Physical, vol. 108, Nov. 2003, pp. 12-19. [36] U. Zimmermann, P. Scheurich, G. Pilwat, and R. Benz, “Cells with Manipulated Functions: New Perspectives for Cell Biology, Medicine, and Technology,” Angewandte Chemie International Edition in English, vol. 20, 1981, pp. 325-344. [37] T. Kotnik, G. Pucihar, M. Rebersek, D. Miklavcic, and L.M. Mir, “Role of pulse shape in cell membrane electropermeabilization,” Biochimica et Biophysica Acta (BBA) - Biomembranes, vol. 1614, Aug. 2003, pp. 193-200. [38] 吳柏彥, “研究枯草桿菌xylose誘導型系統與生產融合廣效性流感病毒HA抗原片段及重組靈芝免疫調節蛋白及評估對BALB/c小鼠之疫苗效果,” 國立中興大學碩士論文, 2010. [39] 楊景翔, “整合網版印刷與氧化銥電極陣列於微流體晶片以檢測等電點電泳,” 國立中興大學碩士論文, 2007. [40] 洪宗聖, “整合血球破碎及血紅蛋白純化操作於微流體晶片,” 國立中興大學碩士論文, 2010. [41] 蔡佳芸, “利用光聚合凝膠作用在晶片的毛細管中構築梯度之研究,” 國立中興大學碩士論文, 2006. [42] H. Schägger and G. von Jagow, “Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa,” Analytical Biochemistry, vol. 166, Nov. 1987, pp. 368-379. [43] H. Morgan and N.G. Green, AC Electrokinetic: Colloids and Nanoparticles, Research Studies Press Ltd, 2002. [44] Naidu, High voltage engineering, Tata McGraw-Hill, 2009. [45] P. van Solingen and J.B. van der Plaat, “Fusion of yeast spheroplasts.,” J. Bacteriol., vol. 130, May. 1977, pp. 946-947. [46] 彭冠智, “利用地衣芽孢桿菌及枯草桿菌分泌及表層展示源自於枯草桿菌之幾丁聚醣酶,” 國立中興大學碩士論文, 2006. [47] C. de la Rosa and K.V.I.S. Kaler, “Electro-disruption of Escherichia coli bacterial cells on a microfabricated chip,” Conference Proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference, vol. 1, 2006, pp. 4096-4099. [48] J. Suehiro, T. Hatano, M. Shutou, and M. Hara, “Improvement of electric pulse shape for electropermeabilization-assisted dielectrophoretic impedance measurement for high sensitive bacteria detection,” Sensors and Actuators B: Chemical, vol. 109, Sep. 2005, pp. 209-215.
The cellular components such as proteins, DNA, lipids are valuable in modern biotechnology. However, the cell lysis process is necessary for acquiring those materials. In this study, we were inspired form the electroporation technology to develop a microfluidic module that use the pulse waveform for inducing the polarisable particles on the cell membrane to lysis. The microchip with a comb electrodes on glass substrate and PDMS microstructure are fabricated by photolithography. Erythrocyte, yeast and Bacillus were carefully pretreated respectively accroding to their instric characteristics then shocked with designed AC pulse waveform between comb electrodes for cell lysis. The cell wall free erythrocyte can be directly lysed by electrical shock by our desiged AC pulse waveform with the optimal condition at 40 Vpp, 100 k~900 kHz in 5 sec. However, yeast can not be lysed by electrical shock directly because of its cell wall. Therefore, we adopted enzymes prior to the electrical lysis procedure for weakening the cell wall structure. The optimal condition that achieves 91% yeast lysis is electrical shock with designed pulse waveform at 75 Vpp, 50 Hz in 30 sec after incubated with enzymes for 10 min. Electrophoresis results confirm that Bacillus with peptidoglycan cell wall can be weakend by lysozyme and subsequently electrical shock by our module to accelerate the lysis process. The microfluidic module we demonstrated in this study shows the potential that may be integrated with other seperation and analysis modules in the furture to achieve the objective of lab-on-a-chip.

細胞內的各種物質諸如蛋白質、DNA、脂質等,在生物技術上都有非常廣泛的用途,但要取得胞內物質常需先將細胞體之細胞膜及細胞壁破壞掉使內容物流出。本研究啟蒙於基因工程常用的電穿孔轉形技術,利用脈衝波誘導細胞膜上極性分子流動變化導致破裂,發展出一套用於裂解細胞的微流體晶片模組。本研究利用微影製程方式,構築出一組齒梳狀電極和搭配的微通道結構形成晶片主體。所使用的細胞樣品有紅血球、酵母菌、枯草桿菌等等,依照不同標的經過適當的前處理後,利用齒梳狀的電極以設計的特殊交流電波形施以高電位差脈衝電溶細胞。缺乏細胞壁的動物細胞紅血球可利用電擊直接將細胞體裂解,最適電溶條件為:使用本研究設計的特殊交流脈衝波形以電位差40 Vpp,頻率100 k~900 kHz之間電擊5秒可達接近100%的破裂效果。酵母菌因為有堅硬的細胞壁保護,需要先用酵素弱化細胞壁的構造再進行電擊,最適電溶條件為:先使用酵素zymolyase處理10分鐘,再用本研究設計的特殊交流脈衝波形以75 Vpp、50 Hz電擊30秒,可達91%的裂解率。而枯草桿菌同樣有細胞壁保護,先用溶菌酶弱化細胞壁後再用本研究模組進行電擊也能加速菌體的裂解。本研究所開發的微流體模組,可望在未來與分離分析的生物晶片模組相結合,達成實驗室平台晶片的最終目標。
Appears in Collections:食品暨應用生物科技學系

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.