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研究生： 徐溢祥
Hsu, Yi-Hsiang
論文名稱： 電場應用於微藻電穿孔及培養之研究
Applications of electric field on the electroporation and culture of microalgae
指導教授： 王翔郁
Wang, Hsiang-Yu
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2013
畢業學年度： 101
語文別： 中文
論文頁數： 92
中文關鍵詞： 電穿孔 、小球藻 、脂質 、尼羅紅
外文關鍵詞： electroporation, Chlorella vulgaris, lipid, Nile red.
相關次數： 點閱：114  下載：1 
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    • 本研究探討交流電穿孔的頻率及時間對於小球藻細胞染色的影響，並且利用電穿孔將小球藻細胞中油脂及其他分子釋放至細胞外，探討其應用在微藻細胞油脂定量的可能性。
    傳統微藻細胞內油脂含量的測定方法耗時且步驟繁雜，雖然可利用螢光染劑進行油脂含量測量，但某些微藻的細胞膜(壁)較厚，影響螢光染劑進入細胞內的效率，因而影響油脂定量的正確性，因此我們利用電穿孔技術增加細胞膜通透度使染劑可大量進入細胞。本研究藉由施加不同頻率的電場以及不同的電穿孔時間來觀察螢光染色的情形，結果發現10KHz, 2000V/cm的電場可使得固定化的小球藻細胞螢光亮度增加，而利用尼羅紅染色最佳的電穿孔時間為90秒、lipid TOX® Green為20秒。但考慮光褪色會造成觀察上的誤差，吾人利用流動式電穿孔裝置來減少小球藻曝露於激發光下的時間，在實驗的過程中發現流動式電穿孔裝置可釋放細胞內容物的現象，並且發現在頻率為10KHz 時，500V/cm以上的電場可以釋放細胞內容物，而且高細胞濃度的釋放效率較穩定。另外，在進行非流動式電穿孔實驗時，因為電場會導致溶液流動使細胞無法觀察，因此在螢光染色實驗中也探討細胞固定化的方法，利用不同的分散劑將纖維素塗佈在基質表面以固定小球藻細胞，最後發現利用酒精作分散劑有較好的結果。
    除了高電場造成的電穿孔現象以及其應用之外，本研究亦探討弱直流電場對小球藻培養的影響，發現電場可增加小球藻中的葉綠素及其他色素的含量，而油脂含量則與培養起始時的細胞數目有關，起始O.D.值為0.1的小球藻，在電場中脂質累積較多，而起始O.D.值為0.02的小球藻，其脂質反而較未通電的細胞少。

    This study presents the effects of alternating frequency and duration of electric field on the fluorescence labeling of Chlorella vulgaris by two lipid probes: Nile red and Lipid TOX® Green. The application of electroporation on releasing cellular contents is also investigated as well as the feasibility of this technique in quantifying cell compositions.
    Conventional gravimetric methods for quantifying lipid amounts inside microalgae are complicated and time consuming. Fluorescence detection of cellular lipids has been established decades ago; however, it is limited by the inconsistences of fluorescence intensity due to variations in cell membrane compositions. Therefore, this study aims to use electroporation to facilitate the transport of fluorogenic dyes into cells for consistent labeling outcomes. It is found that 10KHz, 2000V/cm was optimal for enhancing the fluorescence labeling of Chlorella vulgaris and optimal durations in electric field were 90 sec for Nile red and 20sec for lipid TOX® Green. To avoid photo-bleaching, we applied flow-through electroporation and discovered that the decay of cell fluorescence was contributed by not only photo-bleaching but also the release of cell contents. When electric field strength was higher than 500V/cm at 10KHz, cell contents can be effectively released and high cell density can provide stable releasing efficiency. Because alternating current electric field can result in dielectrophoretic flow, this study also optimized the cell fixation method to prevent cell movement during observations. Different dispersants were used to disperse cellulose for well surface coating. The results indicate that the mixture of alcohol and cellulose had better abilities for cell fixation.
    In additional to the electroporation of Chlorella vulgaris cells in high electric field strength, this study also investigates the effects of low electric field strength on the cell culture. We found that Chlorella vulgaris have larger amounts of pigment and chlorophyll contents inside electric field. The amount of cellular lipid depended on the starting cell density. When starting O.D.(at 682nm) value was 0.1, Chlorella vulgaris accumulated larger amount of lipid in the electric field; however, when starting OD value was 0.02, they had less cellular lipid amount than those cultured without external electric field.

    摘要 I Abstract II 目錄 IV 圖表目錄 IX 第一章 緒論 1 1-1 前言 1 1-2 研究動機與方法 2 第二章 文獻回顧 4 2-1 藻類含油量偵測分析方法 4 2-2 利用螢光染劑進行細胞油脂定量 5 2-3 電穿孔技術 11 2-4 藻類簡介 16 第三章 實驗方法 20 3-1 玻片清潔 20 3-1-1 實驗藥品 20 3-1-2 藥品配置 20 3-1-3 實驗材料 20 3-1-4 實驗步驟 20 3-2 微電極製備 21 3-2-1 實驗藥品 21 3-2-2 實驗材料 22 3-2-3 實驗儀器 22 3-2-4 實驗步驟 22 3-3 微流道模具製備 26 3-3-1 實驗藥品 26 3-3-2 實驗材料 26 3-3-3 實驗儀器、裝置設置及參數設定 27 3-3-4 實驗步驟 28 3-4 高分子翻模 30 3-4-1 實驗藥品 30 3-4-2 藥品配置 31 3-4-3 實驗儀器 31 3-4-4 實驗步驟 31 3-5 裝置組裝 31 3-5-1 實驗儀器 31 3-5-2 實驗步驟 32 3-6 小球藻實驗固定化及電穿孔實驗 32 3-6-1 實驗藥品 32 3-6-2 藥品配置 33 3-6-3 實驗材料 34 3-6-4 實驗儀器、裝置設置及參數設定 34 3-6-5 實驗步驟 35 3-7 流動式電穿孔裝置及螢光亮度分析實驗 37 3-7-1 實驗藥品 37 3-7-2 藥品配置 38 3-7-3 實驗材料 39 3-7-4 實驗儀器、裝置設置及參數設定 39 3-7-5 實驗步驟 41 3-8 不同生理狀態的細胞螢光訊號分析實驗 43 3-8-1 實驗藥品 43 3-8-2 藥品配置 44 3-8-3 實驗材料 44 3-8-4 實驗儀器、裝置設置及參數設定 44 3-8-5 實驗步驟 44 3-9 以低頻電場釋放小球藻細胞內容物及光譜分析之實驗 45 3-9-1 實驗藥品 45 3-9-2 藥品配置 45 3-9-3 實驗材料 46 3-9-4 實驗儀器、裝置設置及參數設定 46 3-9-5 實驗步驟 47 3-10 通電培養小球藻實驗 48 3-10-1 實驗藥品 48 3-10-2 藥品配置 49 3-10-3 實驗材料 50 3-10-4 實驗儀器、裝置設置及參數設定 50 3-10-5 實驗步驟 51 3-11 分析方法 52 3-11-1 螢光亮度分析方法 52 3-11-2 分光光度計 53 第四章 結果與討論 54 4-1 固定化小球藻實驗 54 4-2 不同頻率的電場下小球藻螢光亮度分析 58 4-3 10KHz 2000V/cm電場下小球藻螢光亮度與時間關係分析 61 4-4 流動式電穿孔裝置及螢光亮度分析 64 4-5 不同生理狀態細胞螢光訊號分析 68 4-6 施加10KHz 2000V/cm電場後小球藻上清液吸收光譜分析 70 4-7 以10KHz 不同強度電場的電穿孔釋放小球藻內容物之研究 73 4-8 通電培養小球藻 76 第五章 結論 80 第六章 未來工作 83 參考文獻 85

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