臺灣博碩士論文加值系統

本研究主要嘗試改善微接觸印刷技術在LAPS晶片上創建二維圖案的蛋白質塗層，並進行LAPS電性量測測量。起初的微接觸印刷技術結果顯示出實驗製程良率的問題，並對其製程進行優化。通過新方法旋轉剝離和單軸機械式壓印，可以獲得總面積為7x7mm2的區域和邊長為250 μm的方形膠原蛋白圖案，和設計了帶有“cgu”字母的字體圖案，實現CD為6.25μm的最小圖案。確定了新方法不僅解決了良率問，也為後續實驗提供了穩定的蛋白質塗層。
另外在LAPS上的細胞貼附研究中，細胞在蛋白質塗層上的貼附狀態，確認了蛋白質塗層對細胞有定位的作用。然而，目前LAPS測量還無法區分出晶片表面有蛋白質塗層和無蛋白質塗層的區域，這問題學生目前嘗試用微溝槽結構為替代方案，藉由目前LAPS測量能區分出微溝槽結構，再結合蛋白質塗層對細胞有定位的作用。未來的研究將探索在微溝槽結構上加蛋白質塗層以進一步分析細胞電生理信號。

This research primarily explores using micro-contact printing technology to create two-dimensional protein coating patterns on LAPS chips and to conduct LAPS measurements. The initial micro-contact printing results indicated issues with the quality of the experimental process, which were then optimized. By adopting a new method of rotation peeling and uniaxial mechanical imprinting, we could easily create a total area of 7x7mm2 and a square collagen protein pattern with a width of 250μm. We also designed a font pattern with the letters "cgu" and achieved the smallest pattern with a CD of 6.25μm. The new method resolved the quality issues and provided a stable protein coating for subsequent experiments.
Additionally, in the cell adhesion studies on LAPS, the adhesion status of cells on the protein coating confirmed that the protein coating has a positioning effect on cells. However, current LAPS measurements cannot distinguish between areas on the chip surface with and without protein coating. As a solution to this problem, students are currently trying to use micro-groove structures, which can be identified by current LAPS measurements, combined with the positioning effect of protein coating on cells. Future research will explore adding protein coating to the micro-groove structure to further analyze cell electrophysiological signals.

中文摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 xi
第一章 簡介 1
1.1 背景介紹-生物分子圖案化 1
1.2 現有生物圖案化工藝 3
1.3 微溝槽結構 7
1.4 LAPS之二維化學影像技術回顧 8
1.5 研究動機與方法 9
第二章 微接觸印刷技術之製程優化 16
2.1 簡介 16
2.2 微接觸印刷實驗步驟 16
2.2.1 PDMS 印章製程模板製備 17
2.2.2 PDMS印章製程 17
2.2.3 蛋白質墨水製備 17
2.2.4 LAPS晶片製備 18
2.2.5 微接觸印刷製程 18
2.2.6 微接觸印刷結果 19
2.3 傳統方法結果初步探討與實驗改良設計 19
2.3.1 改變靜置時間 21
2.3.2 磁力式壓印 23
2.3.3 旋轉剝離 24
2.3.4 單軸機械式壓印 25
2.4 新型方法之實驗結果與討論 26
2.5 結論 29
第三章 定向細胞培養和 LAPS 測量之應用 46
3.1 簡介 46
3.2 用LAPS測量LAPS晶片平面上的蛋白質塗層表徵 46
3.2.1 製備蛋白質塗層LAPS晶片 46
3.2.2 LAPS 表徵分析的實驗流程 47
3.2.3 LAPS 表徵分析之實驗結果與討論 47
3.3 細胞在LAPS上的貼附 49
3.3.1 製備蛋白質塗層LAPS晶片 49
3.3.2 SH-SY5Y細胞培養 50
3.3.3 細胞貼附狀態 50
3.3.4 細胞在LAPS上貼附之實驗結果與討論 52
3.4 結果與討論 52
第四章 總結與未來展望 57
4.1 總結 57
4.2 未來展望 57
參考文獻 60

圖1-1 健康受試者和帕金森病患者的Color-coded diffusion-weighted MR images。[12] 10
圖1-2 PDMS印章從黃光微加工，再經過轉印的示意圖[44]。 11
圖1-3 微接觸印刷(Microcontact Printing)高分子薄膜的示意圖[44]。 11
圖1-4電子束光刻(Electron-Beam Lithography)技術的工作原理及其所使用的電子束光阻劑(Trehalose glycopolymer)示意圖[29]。 12
圖1-5 噴墨印刷(Inkjet Printing)技術的工作原理示意圖[45]。 12
圖1-6 微流體(Microfluidics)技術在晶片表面上創建生物分子圖案的示意圖[20] 13
圖1-7 通道結構上染色的MCGC的SEM圖像和CLSM圖像。(a)兩個相連的神經元。(1)沿著通道底部的引導，(2)通道側壁上的引導，以及(3)離開通道的神經突通路。(b)紅色箭頭表示神經突起離開通道的點。黃色箭頭表示未引導的神經突起。藍色箭頭表示神經突重新進入通道。神經突起源於位於通道頂部的細胞簇和體細胞。(c-h)神經突退出、進入和穿過通道的例子。[34] 14
圖1-8 EIS和LAPS感測器的工作原理和典型訊號響應的示意圖[46] 15
圖1-9 研究內容架構圖。 15
圖2-1 (a)通過標準黃光製程所獲得為5 μm SU8的Si晶圓模具，(b)具所需圖案的PDMS 印章。 30
圖2-2 微接觸印刷製程流程與玩成品的螢光示意圖。 30
圖2-3 collagen轉移到基板表面形成圖案。(a)有一層相同圖案的殘影，(b)部分圖案上出現缺口，(c)圖案出現破壞性結構。 31
圖2-4 初步結果探討後應改良實驗的步驟。 31
圖2-5 微接觸印刷流程A、B和C參數說明。 32
圖2-6 微接觸印刷流程A的顯微鏡圖片。(a) collagen在印章上1小時30分鐘後的顯微鏡圖片，（b）collagen轉印後的顯微鏡圖。 33
圖2-7 微接觸印刷流程B的顯微鏡圖片。(a) collagen在印章上1小時後的顯微鏡圖片，（b）collagen轉印後的顯微鏡圖。 33
圖2-8 (a)微接觸印刷流程C轉印前的顯微鏡圖，（b）微接觸印刷流程C轉印後的顯微鏡圖。 34
圖2-9 在微接觸印刷中，將PDMS印章接觸到基板上後，並沒有輕壓過印章，所得到的結果。 34
圖2-10 磁力式壓印方式的示意圖。 35
圖2-11 以流程A作參照，把壓印方式改成磁力式壓印的A’流程。 35
圖2-12 (a)微接觸印刷流程A’轉印前的顯微鏡圖，（b）微接觸印刷流程A’轉印後的顯微鏡圖。 36
圖2-13 失敗的(a)微接觸印刷流程A’轉印前的顯微鏡圖，（b）微接觸印刷流程A’轉印後的顯微鏡圖。 36
圖2-14 流程D用旋轉剝離將多餘的collagen從印章上去掉替代了流程D用氮氣吹掉多餘的collagen。 37
圖2-15 (a) 微接觸印刷流程D轉印前的顯微鏡圖，(b) 微接觸印刷流程D轉印後的顯微鏡圖。 37
圖2-16 (a)為單軸機械式壓印所使用的3D列印機Z軸輔助，(b)為輔助支架概念圖，(c)、(d) 組裝後的示意圖和實體圖。 38
圖2-17 傳統手動微接觸印刷(A組)和新型 3D 列印機控制微接觸印刷(B組)的製程。 39
圖2-18 傳統手動微接觸印刷(A組)和新型 3D 列印機控制微接觸印刷(B組)的示意圖。 40
圖2-19 (a) SU8 模具、(b) PDMS 模具的示意圖和圖片。 41
圖2-20 面積為7 x 7 mm2的列印膠原圖案的圖片。 42
圖2-21 (a)光學顯微鏡下的PDMS模具圖片，(b)傳統模式和(c)螢光顯微鏡下的 3D 列印機控制的膠原反轉圖案。 43
圖2-22 設計了帶有“cgu”字母的字體圖案，線寬的CD從50到6.25μm，研究彎曲圖案的可能性。 43
圖2-23 微接觸印刷所用印章設計圖的(a) layout圖、(b)灰階化後分布圖、(c)不同Threshold值範圍的占比率分布圖。(d)完成微接觸印刷後的螢光照片、(e)灰階化後分布圖、(f)不同Threshold值範圍的占比率分布圖。 44
圖2-24 不同壓印方式結果的失效率比對圖。 45
圖3-1 用單軸機械式壓印Si3N4表面上後和用顯微鏡確認蛋白質塗層的圖片。 53
圖3-2 LAPS表徵分析實驗流程。 53
圖3-3 (a) LAPS測量的示意圖，(b)為壓印在經O2 plasma處理的表面上的圖片，以及雷射對其表面分析路徑，(c) (d)為不壓印與壓印跟經過O2 plasma處理前後對LAPS測量的結果。 54
圖3-4 將細胞加入已含有蛋白質塗層的LAPS晶片，(a)12小時後，(b)17小時後，(c)24小時後，(d)30小時後，(e)36小時後，(f)59小時後固定範圍內的細胞貼附狀況。 55
圖3-5 用ImageJ (a)統計特定區域所貼附的細胞，(b)區域選取示意圖。 56
圖4-1 通過黃光製程所製備的(a-c)各種圖案，和(d)晶片。 59
圖4-2 (a)LAPS對晶片的局部二維影像掃描，以及(b)細胞在晶片表面的生長狀況。 59

表1-1 各項已經開發出的技術在生物圖案化方面優缺點分析[25, 32, 40-43] 10

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