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  3. 食品暨應用生物科技學系
Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52097
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
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摘要: 
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。
URI: http://hdl.handle.net/11455/52097
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