本研究旨在結合光阻和氧化銦錫導電玻璃基板，試圖製作出可以同時觀察細胞生長形態、紀錄電生理訊號和給予細胞電流或電壓刺激的多層微流道結構。為達成此一目的，本研究 (1)運用透明之｢氧化銦錫導電玻璃」藉以量測生物電生理訊號，(2)利用犧牲層預先定義細胞柵欄層之基礎結構，並於其上覆蓋負光阻於犧牲層之上形成柵欄層結構，(3)並將具有特殊角度和流道身寬比的細胞引流層堆疊在上，形成用來運送神經元細胞到氧化銦錫電極上。
研究成果如下：(1)本研究成功地在固定面積的基板上製作出｢多層微流道結構」；(2)此一｢多層微流道結構」設計確實能夠量測到斑馬魚心臟電生理訊號，唯未能量測到神經細胞之電生理訊號，原因可能是量測電路設計未臻完善；(3)實驗過程發現老鼠之腦神經細胞能夠在光阻上存活至少14天，成效符合預期目標；(4)微流道引流層之設計確實能夠將固定直徑之微粒導引至預期之細胞柵欄層，唯實驗結果未能如電腦模擬般之效果，可能與製程之參數浮動與實際流體之非理想效應有關；(5)實際以神經細胞進行實測時發現，柵欄層無法成功捕捉到各分支流之細胞，此一結果應是流體流速過快、細胞在通過柵欄層受到擠壓變形，反而得以全數穿過柵欄縫隙。
建議後續之研究可以設法減緩流體流速，具體方法包含減少結構之高度(位能)差異、延長引流道、或是增加流阻等措施，應該可以有效減緩流速。

This study aims to construct a multilayer microfluidic device, with which morphology, and electrophysiological signal could be monitored and recorded simultaneously. The device integrate both photoresist and ITO glass substrate, such that electrical stimulation can also be applied to cells above ITO electrodes.
In order to achieve the above purpose, several processes have been completed. 1. Transparent ITO electrodes were used in order to measure electrophysiological signal. 2. The pillars and cell-trapping gap structure were defined through a positive photoresist, which served as a sacrificial layer. 3. A SU-8 (negative photoresist) was furthermore stacked to form the cell-guiding channels, which have a specific aspect ratio and a branch-channel angle. The multilayer microfluidic device was then fabricated for guiding neurons to ITO electrodes.
Research results are as follows: 1. This study has successfully fabricated the microfluidic device, which should be able to guide and trap living cells on top of the ITO electrodes. 2. The electrophysiological signal of the zebrafish heart has been successfully measured by the device in the study. However, the spike activity of neurons was not able to be detected, probably because the noise of measurement instrument was not well controlled. 3. In the cells cytotoxicity test, the neurons could be cultured on the photoresist for at least 14 days. This proves the biocompatibility of the proposed device. 4. Although the cell-guiding channel could successfully deliver the microbeads to the cell trapping holes, the result was not the same as the computer simulation. This is possibly due to the uncontrollable fabrication variation. 5. When testing the device with the neurons, it was found that the cell-trapping gap failed to trap the neurons. It was found that the neurons was squeezed to pass the cell gap due to the high fluid speed.
The future researcher may pay more attention to slow down the fluid speed, specific methods include reducing the difference of the potential, prolonging the channel length, or increasing the resistance of the fluid in order to effectively slow down the fluid speed.

一、 緒論 1
二、文獻回顧 3
三、多層微流道系統的設計 18
3.1初版微流道結構介紹 18
3.2製作多層微流道裝置的材料 23
1.氧化銦錫玻璃: 23
2.光阻: 24
3.3初版多層微流道測試 26
1.細胞分布不均: 26
3.光罩修改方向: 29
3.4光罩設計修改 29
1.整體光罩布局調整: 30
2. 流體入口與流體出口位置及大小的調整: 32
3. 氧化銦錫電極層: 33
4.犧牲層光罩修改: 34
6.PDMS模具設計: 41
四、多層微流道製作與系統特性量測 46
4.1 多層微流道的製作: 47
1. 第一道光罩，對齊標記(Alignment mark): 47
2. 第二道光罩，氧化铟锡電極和導線製作: 48
3. 第三道光罩，犧牲層: 49
4.第四道光罩，細胞柵欄層: 50
5.第五道光罩，細胞分流層: 51
4.2製程參數問題與調整: 53
1.犧牲層結構變質: 53
4.3 SU-8結構量測: 60
1. 微流道結構全貌: 60
2. 表面輪廓儀量測 (Alpha-Step): 61
3. 掃描電子顯微鏡(Scanning Electron Microscope，SEM): 63
4.4流體測試: 68
1.紅墨水測試: 68
2.細胞分流道測試: 69
五、微流道系統的生物特性量測 75
5.1實驗樣品準備 75
1.樣品清洗: 75
2.初代海馬迴細胞的培養: 76
3.免疫螢光染色: 77
5.2生物相容性 78
5.3多層微流道細胞捕捉測試 80
5.4氧化銦錫電極電性量測 82
1. 氧化銦錫電極阻抗量測: 82
2. 氧化銦錫電極斑馬魚心電圖量測: 82
3. 氧化銦錫電極神經細胞訊號量測: 84
六、結論與未來工作 88
七、文獻回顧 90

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