- 學位論文
雕刻技術於微流體晶片之開發與驗證 -以磁標記細胞分選應用為例
Development and verification of microfluidic chip using engraving techniques: A case study of magnetic-labeled cell sorting
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摘要
微流體技術廣泛應用於化學分析、生物醫學、環境監測和材料科學等領域，現有的微流體製造技術多樣且複雜，例如雕刻技術、雷射燒蝕、微影製程等技術，不同製造技術對微流體性能會產生不同的影響，其中，雕刻技術在微流體裝置製作中能精確控制微結構的尺寸和形狀，適用於多種材料，且能自動化生產，以有效降低成本和減少材料浪費。本研究運用雕刻技術進行微流體晶片的開發與驗證，並以磁標記細胞分選應用進行性能探討，開發三種不同功能之微流體晶片進行驗證，分別為混合功能、磁分選功能與磁標記細胞分選功能。微流體晶片的製作透過雕刻機對壓克力進行模具的雕刻，使用聚二甲基矽氧烷進行翻模，以製備毫米等級流道大小的微流體晶片。混合功能晶片使用Ansys工程模擬軟體模擬微流體混合效益，並使用染劑進行流體混合參數驗證；磁分選功能晶片透過使用調控輸液流速進行微流體層流參數測試，根據流道內不同顏色液體的層流比例，以找尋最佳輸液參數，而後輸入磁流體進行磁分選測試，以施加不同磁場大小並量測輸出口的磁含量大小，以探討施加磁場控制磁性奈米粒子的最佳參數；磁標記細胞分選晶片使用RT4膀胱癌細胞與B16F10黑色素瘤細胞進行細胞分選驗證，藉由細胞與磁性奈米粒子共培養，使細胞帶有磁性標記，並藉由在流道外施加磁場以進行磁標記細胞分選，並透過細胞計數，以探討與驗證流道之性能。研究結果顯示，在混合晶片測試中輸液流速為400 μL/min時可以達到最佳的混和效率；在磁分選晶片的層流參數測試中，在輸入流道1為70 μl/min時，透過調控輸入流道2流速有最佳的層流比例，磁分選測試中輸入流道1流速為70 μl/min，輸入流道2流速為160 μl/min有最佳的磁分選趨勢，可以分選出90.9%的磁性奈米粒子；在磁標記細胞分選晶片RT4膀胱癌細胞的最佳細胞分選參數為輸液流速25 μl/min，在施加磁場後捕獲率提升了63.86%，B16F10黑色素瘤細胞的最佳細胞分選參數為輸液流速100 μl/min，在施加磁場後捕獲率提升了70.93%。本研究使用雕刻技術成功製作混合功能、磁分選功能與磁標記細胞分選功能的微流體晶片，並應用於磁標記細胞分選，未來將進行雕刻微流體晶片的其他應用驗證，整合多功能之微流體晶片，達到不同領域的技術利用。
並列摘要
Microfluidic technology is widely applied in fields such as chemical analysis, biomedicine, environmental monitoring, and materials science. The existing microfluidic manufacturing techniques are diverse and complex, including engraving technology, laser ablation, and photolithography. Different manufacturing techniques can have various impacts on microfluidic performance. Among them, engraving technology in microfluidic device fabrication can precisely control the size and shape of microstructures, is suitable for various materials, and can automate production to effectively reduce costs and minimize material waste. This study uses engraving technology for the development and validation of microfluidic chips and explores their performance in magnetic labeled cell sorting applications. Three types of microfluidic chips with different functions were developed and validated: mixing function, magnetic sorting function, and magnetic labeled cell sorting function. The microfluidic chip fabrication involved engraving molds on acrylic using an engraving machine and casting with polydimethylsiloxane (PDMS) to prepare microfluidic chips with millimeter-scale channels. The mixing function chip was simulated using Ansys engineering simulation software to model the efficiency of microfluidic mixing, and dye was used to verify fluid mixing parameters. The magnetic sorting function chip tested microfluidic laminar flow parameters by adjusting the infusion flow rates. The optimal infusion parameters were identified based on the laminar flow ratios of different colored fluids within the channels. Magnetic fluid was then introduced for magnetic sorting tests, where different magnetic field strengths were applied, and the magnetic content at the outlet was measured to determine the optimal parameters for controlling magnetic nanoparticles. The magnetic labeled cell sorting chip used RT4 bladder cancer cells and B16F10 melanoma cells for validation. Cells were co-cultured with magnetic nanoparticles to achieve magnetic labeling, and magnetic fields were applied externally to the channels for magnetic labeled cell sorting. Cell counting was used to evaluate and verify the channel performance. The results showed that in the mixing chip test, the optimal mixing efficiency was achieved at an infusion flow rate of 400 μL/min. In the laminar flow parameter test of the magnetic sorting chip, the optimal laminar flow ratio was found when Inlet 1 had a flow rate of 70 μL/min, with optimal magnetic sorting achieved at Inlet 1 flow rate of 70 μL/min and Inlet 2 flow rate of 160 μL/min, resulting in 90.9% sorting of magnetic nanoparticles. The optimal cell sorting parameters for bladder cancer cells on the magnetic labeled cell sorting chip RT4 were an infusion flow rate of 25 μl/min, and the capture rate was increased by 63.86% after applying the magnetic field, and the optimal cell sorting parameters for B16F10 melanoma cells were an infusion flow rate of 100 μl/min, and the capture rate was increased by 70.93% after applying the magnetic field.This study successfully fabricated microfluidic chips with mixing, magnetic sorting, and magnetic labeled cell sorting functions using engraving technology and applied them to magnetic labeled cell sorting. Future work will involve validating other applications of engraved microfluidic chips and integrating multifunctional microfluidic chips to achieve technological applications in various fields.
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