- 學位論文
雷射加工孔洞陣列用於 ITO 玻璃之即時細胞電操縱與物質遞送裝置開發
The development of laser engraved wells array on ITO glass for cell electrical manipulation and real-time material delivery
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摘要
本研究成功開發出可以對細胞進行電操縱 (Cell manipulation) 以及電穿刺 (Cell electroporation) 之裝置並可以即時觀察，使用雷射雕刻技術雕刻微孔陣列，微孔陣列在結構上產生不均勻電場，最終透過此裝置可以成功達成細胞電穿刺且能在此裝置進行細胞轉染，且透過流式細胞儀量測細胞在進行轉染後的螢光訊號以及細胞存活率。 本實驗裝置使用高透光性的氧化銦錫玻璃(Indium tin oxide glass, ITO glass) 作為裝置之電極，由於玻璃的高透光性，進行實驗可以即時透過實驗上的倒立式螢光顯微鏡觀察細胞操縱以及穿刺的效果，又由於此裝置僅需使用雷射雕刻即可完成，並且裝置在完成穿刺後將腔體內的樣本收集再充分清洗腔體，因此此裝置具有製備容易、體積小以及實驗重複性高的優勢。電訊號使用方波的交流訊號，過調整輸入電壓、頻率以及輸入時間來改變操縱及穿刺效率。由於雷射雕刻所產生的熱衝擊效應，可以在微孔的邊緣處產生凸起的幾何結構，該區域具有較強的電場，因此可以降低細胞電穿刺所需的電壓，使細胞不易受到過大的電訊號而死亡，最終細胞在完成實驗後保有7成的轉染效率並保有6成的細胞存活率。 本研究分為兩個部分，第一部分為電操縱，實驗會將細胞推移進入細胞執行電穿刺的目標區域，透過施加電操縱訊號，細胞會透過負介電泳力 (Negative dielectrophoresis, nDEP) 的技術將細胞推移至電場較弱的區域。第二部分為細胞電穿刺，透過施加電穿刺訊號，細胞表面會產生親水性的孔洞並使物質遞送至細胞中。實驗首先使用Yo-Pro細胞質染劑作為細胞運輸效率指標，找到最佳的穿刺參數後再使用GFP進行細胞轉染實驗，搭配PI染劑以及流式細胞儀觀察細胞存活率以及轉染效率。
並列摘要
This study successfully developed a device capable of cell manipulation and cell electroporation with real-time observation. The device employs laser engraving technology to create micro-pore arrays, which generate non-uniform electric fields. This device can successfully perform cell electroporation and transfection. Fluorescence signals and cell viability after transfection are measured using flow cytometry. The experimental device uses highly transparent indium tin oxide (ITO) glass as electrodes. Due to the high transparency of the glass, real-time observation of cell manipulation and electroporation can be conducted using an inverted fluorescence microscope. The device is advantageous due to its ease of fabrication, small size, and high repeatability, as it only requires laser engraving. After electroporation, Sample collection and thorough cleaning of the chamber is easy to this device. The electrical signals used are AC square waves, with manipulation and electroporation efficiencies adjusted by varying input voltage, frequency, and duration. The thermal impact from laser engraving creates raised geometric structures at the edges of the micro-pores, which strengthen the electric field in those areas, thereby reducing the voltage needed for cell electroporation. This minimizes cell death caused by excessive electrical signals, resulting in a transfection efficiency of 70% and a cell survival rate of 60%. This study is divided into two parts. The first part involves cell manipulation, where cells are moved into the target area for electroporation. By applying manipulation signals, cells are moved to regions with weaker electric fields through negative dielectrophoresis (nDEP). The second part involves cell electroporation, where applying electroporation signals creates hydrophilic pores on the cell surface, allowing material delivery into the cells. Initially, the experiment uses Yo-Pro dye as an indicator of cell transport efficiency. After determining the optimal electroporation parameters, GFP is used for cell transfection experiments, and PI dye along with flow cytometry is used to observe cell viability and transfection efficiency.
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