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A Dissolved Oxygen Array Chip Integrated with Patternized Cells for Evaluation of Cellular Respiration Activity
|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. 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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.
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