發展透明生物微流體晶片以研究微控制環境下之細胞行為

Abstract

透明生物微流體晶片可利用即時的細胞生長觀察提供必要的資訊以助於細胞研究；本研究發展透明生物微流體晶片來研究細胞在微控制環境下的行為，本研究利用二氧化碳雷射機器加工及LIBWE等，直寫式雷射方法來發展透明微流體晶片；接著本論文成功利用這些方法發展一個具有驅電性或驅化性的透明生物微流體晶來研究細胞行為；此外，本研究亦發展透明導電玻璃圖樣形成技術及細胞圖樣形成技術，此為可應用於細胞行為研究的兩種圖樣形成技術。 透明微流體晶片包含玻璃及壓克力微流體晶片；製作玻璃微流體晶片的四種加工方法，包含二氧化碳雷射機器、使用有機吸收劑的紫外光LIBWE、使用有機吸收劑的可見光LIBWE、使用金屬吸收劑的可見光LIBWE等，皆可以產生無碎裂且高品質的微流道；關於製作壓克力微流體晶片，使用金屬吸收劑的紫外光LIBWE亦可被利用來蝕刻壓克力並且產生高品質的微流道；上述之方法都是直寫式雷射加工，因此整個微流體晶片的發展時間可少於二十四小時；此外，因LIBWE為本研究重要的加工方法，其加工原理也將被深入討論。 本研究提出一個在顯微鏡上可長時間且即時觀察細胞遷移的自主微流體晶片；結合微流道及透明導電玻璃電極，形成一個具有化學濃度梯度且可培養及觀察細胞的相連微腔室。關於和許小姐合作的細胞趨化性研究，我們以超解析率明視野光學顯微術觀測癌細胞絲狀偽足在表皮生長因子濃度梯度下的動態變化，發現在濃度高的表皮生長因子周圍，癌細胞CL1-0的絲狀偽足活動較高。關於和黃慶文小姐合作的細胞趨電性研究，我們利用架在顯微鏡上可長時間做趨電性研究的晶片來觀察肺癌細胞的趨電反應，並以肺癌細胞的高轉移能力（CL1-5）及低轉移能力（CL1-0）來驗證此趨電性晶片的功能。 最後，本論文發展了經由可見光LIBWE的透明導電玻璃圖樣形成技術及經由二氧化碳或紫外光雷射機器加工的細胞圖樣形成技術；經由可見光雷射引發背面蝕刻方法的透明導電玻璃圖樣形成技術所得的結果超越其他正面雷射蝕刻的方法；另外透明導電玻璃圖樣形成技術被應用在製作透明氣體流量計。細胞圖樣形成技術是一種簡單且有效率的技術；此技術是利用在玻璃基材上一層阻隔層來使細胞不貼附，再利用雷射加工移除設定圖案區域，細胞便可貼附及生長在此區域。

Experiments with transparent bio-microfluidic chip can provide essential real-time observation for cell growth and cell studies. Therefore, this study developed a transparent bio-microfluidic chip for study of cell behavior under microcontrolled environment. This study developed transparent microfluidic chips by laser direct-write methods (LDW), which are the laser-induced backside wet etching (LIBWE) systems and CO2 laser machining. Then, this study successfully used these methods to develop a transparent bio-microfluidic chip with chemotaxis or electrotaxis for study of cell behavior. In addition, this study develops two patterning techniques, ITO patterning and cell patterning, to apply in study of cell behaviors. Transparent microfluidic chips include glass and PMMA microfluidic chip. For glass microfluidic chip, four fabrication methods are used for fabricating. These methods, which include modified CO2 laser machining, UV LIBWE using organic absorber, visible LIBWE using organic absorbers and visible LIBWE using metallic absorbers, can produce crack-free and high-quality microchannels. For PMMA microfluidic chip, visible LIBWE using the metallic absorber is used to fabricate and also produces high-quality microchannels. These four fabrication methods are LDW methods, so the overall development time of glass or PMMA microfluidic chip is shortened to less than 24 h. In addition, the mechanism of LIBWE is discussed in depth because the LIBWE in this dissertation is an important fabrication method. This study presents an autonomous microfluidic chip for long-term and real-time observation of cell migration by a microscope. It was combined the microchannels and ITO electrodes to form a closed microchamber with chemical gradient for cell culturing and observation. For chemotaxis collaborated with the Miss Hsu, we used a super-resolution microscopy technique, non-interferometric wide-field optical profilometry (NIWOP), to observe filopodium activity of cells under the EGF gradient. Higher filopodium activity is observed at the side facing higher EGF concentration for single CL1-0 cell subjecting to the EGF gradient. For electrotaxis collaborated with the Miss Huang, we enabled observing the electrotactic response of lung cancer cells by a sealed culture chamber that is suitable for long-term electrotaxis study with a microscope. We used lung cancer cell lines with high and low metastasis potential, CL1–5 and CL1–0, respectively, to demonstrate the function of the electrotactic chip. Finally, this study developed the ITO patterning by visible LIBWE and the cell patterning by CO2 or UV laser machining. The obtained ablation result from ITO patterning by visible LIBWE excels that from front-side laser ablation. The ITO patterning was then utilized in fabricating a transparent gas flow meter. The cell patterning was a simple and effective method for patterning cells on a glass substrate. A passivation layer that is capable of preventing cell adhesion was first coated onto glass surface and then cells adhere and grow cleanly in the laser defined pattern.