Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35604
標題: 結合圖樣化細胞培養之溶氧電極陣列晶片於細胞呼吸活性的評估
A Dissolved Oxygen Array Chip Integrated with Patternized Cells for Evaluation of Cellular Respiration Activity
作者: 袁佳吟
Yuan, Chia-Yin
關鍵字: Oxygen consumption rate;氧氣消耗率;cellular respiration activity;electrode array;cell pattern;microfluidic system;細胞呼吸活性;電極陣列;細胞圖樣;微流體系統
出版社: 生物產業機電工程學系所
引用: 1.A. J. Cunningham, “Introduction to Bioanalytical Sensors”, New York: John Wiley & SONS, INC, 1998. 2.N. J. Richardson, S. Gardner, D. M. Rawson, “A chemically mediated amperometric biosensor for monitoring eubacterial respiration”, Journal of Applied Bacteriology, 1991, 70, 422-426. 3.Y. Xia, G. M. Whitesides, “Softlithography”, Annual Review Material Science, 1998, 28, 153-84. 4.C. Starr, “BIOLOGY”, Concepts and Applications: 2nd Edition, California: Wadsworth, 1994. 5.L. Bousse, “Whole cell biosensors”, Sensors and Actuators B, 1996, 34, 270-275. 6.B. Alberts, D. Bray, J. Lewis, “Molecular Biology of the Cell”, Garland Publing, Inc., 1994. 7.J. Alderman, J. Hynes, S. M. Floyd, J. Kruger, R. O’Connor, D. B. Papkovsky, “A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing”, Biosensors and Bioelectronics, 2004, 19, 1529-1535. 8.F. G. Gao, A. S. Jeevarajan, M. M. Anderson, “.Long-Term Continuous Monitoring of Dissolved Oxygen in Cell Culture Medium for Perfused Bioreactors Using Optical Oxygen Sensors”, Biotechnology and Bioengineering, 2004, 86, 425-433. 9.Y. Kuang, D. R. Walt, “Detecting Oxygen Consumption in the Proximity of Saccharomyces cerevisiae Cells Using Self-Assembled Fluorescent Nanosensors”, Biotechnology and Bioengineering, 2007, 96, 318-325. 10.T. C. O’Riordan, D. Buckley, V. Ogurtsov, R. O’Connor, D. B. Papkovsky, “A Cell Viability Assay Based on Monitoring Respiration by Optical Oxygen Sensing”, Analytical Biochemistry, 2000, 278, 221–227. 11.T. J. Strovas, J. M. Dragavon, T. J. Hankins, J. B. Callis, L. W. Burgess, M. E. Lidstrom, “Measurement of Respiration Rates of Methylobacterium extorquens AM1 Cultures by Use of a Phosphorescence-Based Sensor”, Applied and Environmental Microbiology, 2006, 72, 1692–1695. 12.T. C. O’Riordan, K. Fitzgerald, G.V. Ponomarev, J. Mackrill, J. Hynes, C. Taylor, D. B. Papkovsky, “Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging”, American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2007, 292, 1613–1620. 13.Y. Torisawa, A. Takagi, Y. Nashimoto, T. Yasukawa, H. Shiku, T. Matsue, “A multicellular spheroid array to realize spheroid formantion, culture, and viability assay on a chip”, Biomaterials, 2007, 28, 559–566. 14.Y. Torisawa, N. Ohara, K. Nagamine, S. Kasai, T. Yasukawa, H. Shiku, T. Matsue, “Electrochemical Monitoring of Cellular Signal Transduction with a Secreted Alkaline Phosphatase Reporter System”, Analytical Chemistry, 2006, 78, 7625-7631. 15.M. Nishizawa, K. Takoh, T. Matsue, “Micropatterning of HeLa Cells on Glass Substrates and Evaluation of Respiratory Activity Using Microelectrodes”, Langmuir, 2002, 18, 3645-3649. 16.T. Kaya, Y. Torisawa, D. Oyamatsu, M. Nishizawa, T. Matsue, “Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring”, Biosensors and Bioelectronics, 2003, 18, 1379-1383. 17.H. Shiku, T. Shiraishi, H. Ohya, T. Matsue, H. Abe, H. Hoshi, M. Kobayashi, “Oxygen Counsumption of Single Bovine Embryos Probed by Scanning Electrochemical Microscopy”, Analytical Chemistry, 2001, 73, 3751-3758. 18.H. Shiku, T. Shiraishi, S. Aoyagi, Y. Utsumi, M. Matsudaira, H. Abe, H. Hoshi, S. Kasai, H. Ohya, T. Matsue, “Respiration activity of single bovine embryos entrapped in a cone-shaped microwell monitored by scanning electrochemical microscopy”, Analytica Chimica Acta, 2004, 522, 51-58. 19.C. C. Wu, Y. Torisawa, H. Shiku, T. Matsue, “Fabrication of miniature Clark oxygen sensor integrated with microstructure”, Sensors and Actuators B, 2005, 110, 342-349. 20.蔡林彥廷,吳靖宙*, “可培養與評估動物細胞活性之微小化Clark式氧氣感測晶片的研發”, 國立中興大學碩士論文, 2007. 21.T. Kitahara, N. Koyama, J. Matsuda, Y. Hirakata, S. Kamihira, S.Kohno, M. Nakashima, H. Sasaki, “Evaluation of Newly Developed Oxygen Meters with Multi-Channels and Disposable Oxygen Electrode Sensors for Antimicrobial Susceptibility Testing”, Biological and Rmaceutical Bulletin , 2003, 26, 1229-1234. 22.J. Karasinski, S. Andreescu, O. A. Sadik, “Multiarray Sensors with Pattern Recognition for the Detection, Classification, and Differentiation of Bacteria at Subspecies and Strain Levels”, Analytical Chemistry, 2005, 77, 7941-7949. 23.S. Andreescu, O. A. Sadik, D. W. McGee, “Autonomous Multielectrode System for Monitoring the Interactions of Isoflavonoids with Lung Cancer Cells”, Analytical Chemistry, 2004, 76, 2321-2330. 24.A. J. Bard, L. R. Faulkner, “Electrochemical methods”, Fundamentals and Applications: 2nd Edition, New York: John Wiley & Sons, Inc, 2001. 25.M. Mrksich, L. E. Dike, J. Tien, D. E. Ingber, G. M. Whitesides, “Using Microcontact Printing to Pattern the Attachment of Mammalian Cells to Self-Assembled Monolayers of Alkanethiolates on Transparent Films of Gold and Silver”, Experimental Cell Research, 1997, 235, 305-313. 26.H. Q. Luo, H. Shiku, A. Kumagai, Y. Takahashi, T. Yasukawa, T. Matsue, “Microcontact printed diaphorase monolayer on glass characterized by atomic force microscopy and scanning electrochemical microscopy”, Electrochemical Communication, 2007, 9, 2703-2708. 27.X. Y. Jiang, R. Ferrigno, M. Mrksich, G. M. Whitesides, “Electrochemical Desorption of Self-Assembled Monolayers Noninvasively Releases Patterned Cells from Geometrical Confinements”, Journal American Chemistry Society, 2003, 125(9),2366-2367. 28.E. Ostuni, R. Kane, C. S. Chen, D. E. Ingber, G.M. Whitesides, “Patterning Mammalian Cells Using Elastomeric Membranes”, Langmuir, 2000, 16(20),7811-7819. 29.A. Revzin, R. G. Tompkins, M. Toner, “Surface Engineering with Poly(ethylene glycol) Photolithography to Create High-Density Cell Arrays on Glass”, Langmuir, 2003, 19(23), 9855-9862. 30.H. Kaji, T. Kawashima, M. Nishizawa, “Patterning Cellular Motility Using an Electrochemical Technique and a Geometrically Confined Environment”, Langmuir, 2006, 22, 10784-10787. 31.A. L. Birkbeck, R. A. Flynn, M. Ozkan, D. Q. Song, M. Gross, S. C. Esener, “VCSEL arrays as micromanipulators in chip-based biosystems”, Biomedicine Microdevice, 2003, 5(1), 47-54. 32.M. Ozkan, T. Pisanic, J. Scheel, C. Barlow, S. Esener, S. N. Bhatia, “Electro-Optical Platform for the Manipulation of Live Cells”, Langmuir, 2003, 19(5), 1532-1538. 33.D. R. Albrecht, G. H. Underhill, T. B. Wassermann, R. L. Sah, S. N. Bhatia, “Probing the role of muticellular organization in three-dimensional microenvironments”, Nature Methods, 2006, 3(5), 369-375. 34.曾煥昌, 張兗君*, “利用微接觸壓印方式分離皮質神經生長錐結構,並探討微圖案培養時細胞聚集現象之成因”,國立清華大學碩士論文, 2006. 35.L. Lauer, C. Klein, A. Offenhausser, “Spot compliant neuronal networks by structure optimized micro-contact printing”, Biomaterials, 2001, 22, 1925-1932. 36.張俊宇, 徐善慧*, “微溝槽表面結構結合ECM改質於許旺細胞排序生長之影響”國立中興大學碩士論文, 2003. 37.H. Kaji, S. Sekine, M. Hashimoto, T. Kawashima, M. Nishizawa, “Stepwise formation of patterned cell co-cultures in silicone tubing”, Biotechnology and Bioengineering, 2007, 98(4), 919-925. 38.P. Sun, F. O. Laforge, M. V. Mirkin, “Scanning electrochemical microscopy in the 21st century”, Physical Chemistry Chemical Physics, 2007, 9, 802–823. 39.C. C. Wu, T. Saito, T. Yasukawa, H. Shiku, H. Abe, H. Hoshi, Y. Matsue, “Microfluidic chip integrated with amperometric detector array for in situ estimating oxygen consumption characteristics of single bovine embryos”, Sensors and Actuator B, 2007, 125, 680-687. 40.M. Koudelka, “Performance characteristics of a planar ‘Clark-type'' oxygen sensor”, Sensors and Actuators, 1986, 9, 249-258 41.H. Suzuki, E. Tamiya, I. Karube, “Fabrication of an oxygen electrode using semiconductor technology”, Analytical Chemistry, 1988, 60, 1078-1080. 42.H. Suzuki, E. Tamiya, I. Karube, “Development of micro-oxygen electrode and its application to micro-glucose sensor”, Proc. MRS Int. Meeting on Advanced Materials, 1988, 14, 115-120. 43.H. Suzuki, N. Kojima, A. Sugama, F. Takei, K. Ikegami, “Disposable oxygen electrodes fabricated by semiconductor techniques and their application to biosensors”, Sensors and Actuators B, 1990, 1, 528–532. 44.H. Suzuki, A. Sugama, N. Kojima, “Miniature Clark-type Oxygen Electrode with a Three-electrode Configuration”, Sensors and Actuators B, 1990, 2, 297-303. 45.H. Suzuki, N. Kojima, A. Sugama, F. Takei, “Development of a miniature Clark-type oxygen electrode using semiconductor technology and its improvement for practical applications”, Sensors and Actuators B, 1990, 2, 185-191. 46.H. Suzuki, A. Sugama, N. Kojima, “Micromachined Clark oxygen electrode”, Sensors and Actuators B, 1993, 10, 91-98. 47.Z. Yang, S. Sasaki, I. Karube, H. Suzuki, “Fabrication of oxygen electrode arrays and their incorporation into sensors for measuring biochemical oxygen demand”, Analytica Chimica Acta, 1997, 357, 41-49 48.H. Suzuki, H. Shiroishi, S. Sasaki, I. Karube, “Microfabricated liquid junction Ag/AgCl reference electrode and its application to a one-chip potentiometric sensor”, Analytical Chemistry, 1999, 71, 5069-5075 49.H. Suzuki, T. Hirakawa, S. Sasaki, I. Karube, “An integrated three-electrode system with a micromachined liquid-junction Ag/AgCl reference electrode”, Analytica Chimica Acta , 1999, 387, 103-112 50.H. Suzuki, T. Hirakawa, , S. Sasaki, I. Karube, “An integrated module for sensing pO(2), pCO(2), and pH”, Analytica Chimica Acta , 2000, 405, 57-65 51.M. Brischwein, E. R. Motrescu, E. Cabala, A. M. Otto, H. Grothe, B. Wolf, “Functional cellular assays with multiparametric silicon sensor chips”, Lab on a Chip, 2003, 3, 234. 52.A. M. Otto, M. Brischwein, A. Niendorf, T. Henning, E. Motrescu, B. Wolf, “Microphysiological testing for chemosensitivity of living tumor cells with multiparametric silicon sensor chips”, Cancer Detection and Prevention, 2003, 27, 291-296. 53.R. Ehret, W. Baumann, M. Brischwein, M. Lehmann, T. Henning, I. Freund, S. Drechsler, U. Friedrich, M. L. Hubert, E. Motrescu, A. Kob, H. Palzer, H. Grothe, B. Wolf, “Multiparametric microsensor chips for screening applications”, Fresenius’ Journal of Analytical Chemistry, 2001, 369, 30-35. 54.T. Henning, M. Brischwein, W. Baumann, R. Ehret, I. Freund, R. Kammerer, M. Lehmann, A. Schwinde, B. Wolf, “Approach to a multiparametric sensor-chip-based tumor chemosensitivity assay”, Anti-Cancer Drugs, 2001, 12, 21-32. 55.Y. Amano, C. Okumura, M. Yoshida, H. Katayama, S. Unten, J. Arai, T. Tagawa, S. Hoshina, H. Hashimoto, H. Ishikawa, “Measuring respiration of cultured cell with oxygen electrode as a metabolic indicator for drug screening”, Human Cell, 1999, 12, 3-10. 56.Y. Torisawa, Y. Nashimoto, T. Yasukawa, H. Shiku, T. Matsue, “Regulation and Characterization of the Polarity of Cells Embedded in a Reconstructed Basement Matrix Using a Three-dimensional Micro-culture System”, Biotechnology and Bioengineering, 2007, 97(3), 615-621.
摘要: 
細胞活性評估晶片是在環境毒性快速檢測與藥物篩選領域中,引人注意的重要工具之一。本研究透過微模造技術圖案化培養細胞與晶片式溶氧電極陣列整合,並結合微流體系統控制細胞外微環境,以發展細胞呼吸活性的量化分析平台。在HeLa cells的呼吸活性量測上,因HeLa cells易攀爬且不易互相聚集的特性,致使HeLa cell不易製作出圖樣化培養,分析其隨機性培養結果,發現10 mM Hepes buffered saline (HBS)+25 mM glucose與HBS+25 mM glucose+1% insulin之測試液,可使HeLa cells呼吸活性較在HBS+25 mM mannitol中分別增加1.13倍及1.29倍。此外,HepG2 cells因易互相聚集重疊生長,可成功製得細胞圖樣化,HepG2 cell在HBS+50 mM glucose與HBS+50 mM glucose+1% insulin的呼吸活性,相較於細胞在無營養源的HBS環境下,分別平均上升1.11倍及1.20倍;若以半球型擴散模型計算,可得氧氣消耗率分別為13.99 ± 4.14×10-14 mol/s,與15.93 ± 5.40×10-14 mol/s,相較於細胞在無營養源的環境下(11.74 ± 3.00×10-14 mol/s),分別上升1.19倍與1.36倍,且其相關係數(R2)皆大於0.83,顯示此晶片可符合半球型擴散模型。未來更期望此系統能再結合pH微感測器與可溫控式培養晶片,構成一可即時(real time)量測細胞活性之多功能檢測平台,以利於環境毒物、臨床分析與藥物篩檢的應用。

The cell activity-estimated chip is one of the most important tools for fast detecting the environmental toxicant and the drug screening. In the study, a cell respiration activity-estimated platform comprising a chip-type dissolved-oxygen-electrode array, a cell pattern formed by micromoloding technology and a microfluidic system capable of controlling the external environment of cells was developed. When measuring the respiration activity of HeLa cells, the cells were randomly cultured to measure the respiration activity due to the properties of fast movement and difficult gathering each other resulting in hardly forming a cell pattern. The cellular respiration activity obtained in 10 mM Hepes buffered saline (HBS) containing 25 mM glucose and 10 mM HBS containing 25 mM glucose+1% insulin was 1.13 and 1.29 times larger than that obtained in 10 mM HBS containing 25 mM mannitol, respectively. In addition, owing to the properties of easy overlap growing of HepG2 cells, the cells can form a cell pattern successfully. The respiration activity of patternized HepG2 cells obtained in HBS containing 50 mM glucose (HBS-glucose) and HBS containing 50 mM glucose+1% insulin (HBS-glucose-insulin) was 1.11 and 1.20 times larger than that obtained in HBS, respectively. The oxygen consumption rate obtained by using the calculation of hemispherical diffusion theory in HBS, HBS-glucose and HBS-glucose-insulin was 11.74×10-14 mol/s, 13.99×10-14 mol/s and 15.93×10-14 mol/s, respectively. The oxygen consumption rate obtained in HBS-glucose and HBS-glucose -insulin was 1.19 and 1.36 times larger than that in HBS, respectively. The squared correlation coefficient (R2) of fitting results was at least larger than 0.83, implying that the measurement of oxygen-electrode array has a good fitting with the hemispherical diffusion model. In the future, we expect that the chip can combine with pH-microsensor and temperature controller to construct a multifunction detection platform for real-time measuring the cellular activity so as to apply to the environment toxicant monitoring, clinical diagnosis and drug screening.
URI: http://hdl.handle.net/11455/35604
其他識別: U0005-2807200915574000
Appears in Collections:生物產業機電工程學系

Show full item record
 

Google ScholarTM

Check


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