胡文聰臺灣大學：應用力學研究所呂冠瑩Lu, Kuan-YingKuan-YingLu2007-11-292018-06-292007-11-292018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/62586本論文發表了應用電穿孔技術(electroporation)在具有平面和3D電極的細胞裂解微流體晶片，此研究的目標在對於探討平面及3D電極兩種設計在電穿孔晶片的性能上的差別。 平面電極的形狀為尖頭設計，其中兩電極距離為20 ，寬度為50 ，3D電極刑狀為圓柱形，電極間距同為20 ，直徑及高度都是50 。 實驗所用的電訊號為電脈衝，其中的電壓為10V持續時間為100 ，而這持續時間相較於細胞膜的電容充電時間10-1000ns是相當長的。 同時也應用商用軟體FEMLAB及MABLAB做為電場的模擬計算，而實驗所用的細胞為白血球。 對於電穿孔技術，電極的設計注重的是一個能產生足夠強度且分佈廣電場的電極，而不是一個能產生強度很大但分佈在小區域的電極。 經由FEMLAB的電場模擬以及MATLAB計算有效體積比率，結果顯示3D電極電場不會隨著微流道的高度而衰減且具有較均勻的分佈，另外3D電極的有效體積比率也優於平面電極。 在實驗方面，可發現細胞在3D 電極的形況下，是有多個破孔出現，有別於在平面電極的單個破洞，也發現當細胞出現破孔時，經由Trypan blue的輔助觀察以及在食鹽水稀釋情況下，細胞內液是往外流出的，另外由裂解比率的結果，3D電極也是優於平面電極。This thesis presents microfluidic devices to perform cell lysis using electroporation with planar and 3D electrode designs. The goal of the study is to compare the lysis performance of the two electrode geometries. The planar electrode design is pointed in shape (gap is 20μm, width of base 50μm) and that of 3D electrode are circular cylinders (diameter and height are 50μm, gap between cylinders 20μm), which are fabricated by electroforming. Pulse signal is used for electroporation, with amplitude of 10V and duration of 100μs – long compared to membrane charging time of 10-1000ns. The commercial software FEMLAB and MATLAB are used for simulation of electric field. In the experiment, leukocyte is used as lysis element. Computational results show that the electric field of 3D electrode geometry is, indeed, more uniform than that of planar configuration, resulting in effective volume ratio — volume which electric field is sufficient for cell lysis to net volume — of the 3D geometry is substantially larger than that of the planar design. Also, the efficacy of lysing depends not only on the electric field but, more importantly, on the temporal extend which the cell is exposed to the field strength required for lysis as the cell proceeds downstream. Experimentally, multi-pores are observed on leukocyte lysed with 3D electrodes and just single pore with planar electrodes. This is believed to be due to more uniform electric field of 3D electrodes than that of planar electrodes. Intracellular elements are extracted through the membrane as observed by increased intensity of Trypan blue when diluted by saline solution. We also demonstrate the lysing percentages at same flow velocity to explain the performance of 3D electrode is superior to that of planar electrode.Abstract………………………………………………………………………………..3 中文摘要…………………………………………………………………………….4 Chapter1. Introduction and Literature Review………………………………...5 1.1 Microfluidic system and MEMS technology………………………………….5 1.2 Mechanisms of cell lysis………………………………………………………5 1.3 Literature review………………………………………………………………6 1.4 Motivation and Goal…………………………………………………………..8 Chapter 2. Theory…………………………………………………………………10 2.1 Properties of cell membrane………………………………………………….10 2.2 Electroporation……………………………………………………………….10 2.3 Device Design and Computation of Electric Field...…………………………14 Chapter 3. Chip Fabrication……………………………………………………..19 3.1 Fabrication of planar electrodes and 3D electrodes………………………….19 3.1.1 Surface preparation…………………………………………………...20 3.1.2 Metal deposition……………………………………………………...20 3.1.3 Electrode patterning and protection coating………………………….20 3.1.4 Fabrication of 3D electrodes…………………………………………23 3.2 Microchannel fabrication…………………………………………………….26 Chapter 4. Experimental Setup…………………………………………………29 4.1 Cells preparations…………………………………………………………….29 4.2 Apparatus, procedure and experimental setup………………………………..30 4.2.1 Apparatus……………………………………………………………..30 4.2.2 Procedure and experimental setup……………………………………32 Chapter 5. Experimental Results……………………………………………….34 5.1 Cell lysis by planar electrode at no flow velocity…………………………….34 5.2 Cell lysis by planar and 3D electrodes at low flow velocity………………….38 Chapter 6. Conclusions and Future work……………………………………..40 6.1 Conclusions…………………………………………………………………...40 6.1 Future work…………………………………………………………………...40 Reference…………………………………………………………………………...41964854 bytesapplication/pdfen-US電穿孔細胞裂解微流electroporationcell lysismicrofluidicPDMSthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/62586/1/ntu-94-R92543021-1.pdf