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Title: | 用於細胞抓取並以摩擦奈米發電機遞送shRNA之微孔陣列開發 Development of a Microwell Array for Cell Trapping and shRNA Delivery using Triboelectric Nanogenerator |
Authors: | Che-Yi Li 李哲儀 |
Advisor: | 沈弘俊(Horn-Jiunn Sheen) |
Keyword: | 微孔陣列,細胞抓取,電穿刺,細胞轉染,摩擦奈米發電機, Microwell array,Cell trapping,Electroporation,Transfection,TENG, |
Publication Year : | 2022 |
Degree: | 碩士 |
Abstract: | 本研究利用微機電技術製作出具有不同幾何結構之電穿刺裝置,以結構之電場放大效應提高細胞電穿刺效率,最終達到同時操縱與電穿刺細胞之目的。此裝置亦能夠以摩擦奈米發電機 (triboelectric nanogenerator, TENG) 作為電訊號來源,以達成電穿刺系統之自供電,並用於shRNA 轉染。 為了能有效達成細胞操縱與電穿刺,本研究以高透光性的氧化銦錫玻璃 (Indium tin oxide glass, ITO glass) 作為裝置之正負電極,方便置於螢光顯微鏡上觀察,另於ITO 層設計數種幾何結構,並以微機電製程製造出一體積小、易操作,且方便觀測之電穿刺晶片。透過表面蝕刻,可在用於抓取細胞之孔洞邊緣產生尖端狀之幾何結構,並產生電場放大效應,降低細胞電穿刺之電訊號源所需電壓值,使細胞不易因承受過大電壓而死亡,電穿刺後之細胞存活率可達近80 %。 本研究主要可分為兩部分,分別為細胞操縱與細胞電穿刺。首先,本研究開發之裝置將外接一電訊號源,以負介電泳力 (negative dielectrophoresis, nDEP) 使細胞進入蝕刻出的孔洞內,達成細胞操縱,再藉由調變電訊號強度達成細胞電穿刺之目的。在成功操縱並固定80 % 之細胞後,細胞將染以細胞增殖示蹤螢光試劑 (carboxyfluorescein diacetate succinimidyl ester, CFDA SE) 與鈣黃綠素 (calcein),驗證於電穿刺後,物質可由細胞內而外或由細胞外而內運輸,並找出電穿刺之最適結構設計。接著,將最適裝置接以摩擦奈米發電機,以達成自供電之細胞電穿刺,並以不同電源供應器進行shRNA 轉染,再將轉染後之樣本進行即時聚合酶鏈式反應 (real-time polymerase chain reaction, real-time PCR),以驗證裝置用於細胞轉染之效率。以本研究之電穿刺裝置進行之shRNA 轉染,能夠成功抑制60 % 之細胞基因表達。 In this research, we use microelectromechanical system (MEMS) technology to fabricate devices with different geometric structures. The electric field amplification effect of the device structures is able to improve the efficiency of cell electroporation, and achieve simultaneous cell manipulation and electroporation. Triboelectric nanogenerator (TENG) can be used as an electrical signal source of the devices to achieve self-powered electroporation systems, and be used for shRNA transfection. To effectively achieve cell manipulation and electroporation, we use indium tin oxide glass (ITO glass) with highly transmittance as the positive and negative electrodes of the devices, which is convenient for observation with fluorescence microscope. In addition, several geometric structures are designed on the ITO layer, and electroporation chips which are small in volume, easy to operate and convenient to observe are manufactured by MEMS process. After surface etching, tip-shaped geometric structures can be generated at the edge of the holes used for capturing cells. The structures lead to an electric field amplification effect, which reduces the voltage value required for cell electropuncture, so that the cell won’t be injured due to excessive voltage, to improve cell viability. The cell survival rate after electroporation can reach nearly 80%. This research is divided into two parts, cell manipulation and cell electroporation. In the first part, the devices are connected to an external electrical signal source to make the cells captured into the etched holes with negative dielectrophoresis (nDEP) to achieve cell manipulation, and then achieve cell electroporation by modulating the strength of the electrical signal. After successfully manipulating and fixing 80% of the cells, we stain the cells with carboxyfluorescein diacetate succinimidyl ester (CFDA SE) and calcein to verify that after electroporation, substances can be delivered from inside the cell or from outside the cell, and to find the optimal structural design for electroporation. In the second part, we connect the optimal device to TENG to achieve self-powered cell electroporation, and perform shRNA transfection with different power supplies. After shRNA transfection, we subject transfected samples to real-time polymerase chain reaction (real-time PCR), to verify the efficiency of the device for cell transfection. 60% of the cells' gene expression is successfully inhibited after shRNA transfection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86243 |
DOI: | 10.6342/NTU202202862 |
Fulltext Rights: | 同意授權(全球公開) |
metadata.dc.date.embargo-lift: | 2027-08-29 |
Appears in Collections: | 應用力學研究所 |
Files in This Item:
File | Size | Format | |
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U0001-2608202216094200.pdf Until 2027-08-29 | 5.8 MB | Adobe PDF |
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