通常顯微鏡下的細胞培養環境是由玻片或是洋菜盤提供，適合短時間或特定實驗的細胞觀察，但無法提供穩定的環境進行長時間培養。本論文使用可以捕獲單細胞的微流體皮升生物反應器。可以提供營養液連續流動的環境，同時允許營養液進行快速的交換。我們利用su-8黃光微影製程，製作出通道中心高度為1um的模仁。再透過PDMS的軟性微影製程，利用模仁製作出一次性使用的微流體生物反應器。經過切割、打洞、與玻璃接合，將完成的微流體晶片與光學顯微鏡及溫度控制器整合在同一個平台，是一個適合進行連續培養的生物反應器。此外模仁可以重複使用，透過PDMS的軟性微影製程只需要兩天，即可製作出相同的微流體生物反應器。在我們的生物反應器中能成功捕獲單細胞的大腸桿菌，並量測細胞的成長曲線及螢光強度。

Typically slides, agar-pads are applied to provide cell culture environment under microscope. Even though suitable for short time observation, these systems are limited to more complex experiment. In this work, we provide a microfluidic picoliter bioreactor(PLBR). The device allow continuous medium flow enabling constant environment, but also fast medium changes. We use lithography process to make a mold, the center height of channel is 1um. Make a disposable PLBR by the PDMS soft lithography process. Microfluidic chip were cut, punched, assembled, and integrated with microscope and temperature controller. This is a suitable platform for continuous cultivation of microbial organisms. In addition, our su-8 mold can be reused and only need two days to make the same PLBR. In our bioreactor, we can trap E. coli on single cell level, measure cell growth and fluorescence intensity.

目錄
誌謝 i
中文摘要 ii
Abstract iii
目錄 iv
圖目錄 vi
表目錄 vii
一、緒論 1
1-1研究動機 1
1-2文獻回顧 3
1-2-1 恆化器 3
1-2-2 微型恆化器 4
1-2-3 單細胞捕獲 5
1-3微流體晶片介紹 7
1-3-1 光罩 7
1-3-2 模仁 8
1-3-3 軟性微影 9
二、微流體晶片與系統架構 10
2-1 微流體晶片設計與製程 10
2-1-1 光罩設計與製作 10
2-1-2 微流道製程 12
2-1-3 PDMS軟性微影製程 15
2-1-4 微流體晶片製作 15
2-2 實驗系統架構 17
2-2-1 液晶快門與光源控制 17
2-2-2 光學顯微鏡細胞培養箱 18
2-2-3 螢光光源與影像擷取 19
2-2-4 系統整合 20
三、實驗結果 21
3-1 大腸桿菌樣品準備 21
3-2 晶片表面處理 21
3-3 培養條件 21
3-4 實驗結果 22
四、結論 25
附錄 26
參考文獻 29
圖目錄
圖1-1 恆化器組成的示意圖 3
圖1-2 單細胞尺度的微流體恆化器示意圖 4
圖1-3 利用流體力學的方式捕捉單一細胞 5
圖1-4 利用微流控的液滴混合細胞與染劑 5
圖1-5 單細胞捕獲的微流體生物反應器 6
圖1-6 光罩 7
圖1-7 模仁 8
圖1-8 軟性微影示意圖 9
圖2-1 光罩示意圖 11
圖2-2 微流道製程的示意圖 14
圖2-3 軟性微影製程的示意圖 16
圖2-4 微控制器與綠光LED及液晶快門的示意圖 17
圖2-5 光學顯微鏡細胞培養箱 18
圖2-6 螢光光源與影像擷取 19
圖2-7 系統整合架構圖 20
圖3-1 藍綠藻的實驗結果 22
圖3-2 大腸桿菌影像 23
圖3-3 細胞長度對時間的成長曲線 23
圖3-4 大腸桿菌螢光強度對時間的圖 24
圖3-5 綠光條件下的細胞成長曲線 24
圖3-6 暗室條件下的細胞成長曲線 25
圖3-7 不同營養源及抗生素的成長曲線 25
表目錄
表2-1 微流道的製程參數表格 12
表A-2 營養液配置表 29

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