聚二甲基矽氧烷光柵基材表面改質聚電解質多層膜在全血中雷射繞射檢測循環腫瘤細胞與細胞培養光學定量研究

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研究生：	蕭若霜 JO-SHUANG-HSIAO
論文名稱：	聚二甲基矽氧烷光柵基材表面改質聚電解質多層膜在全血中雷射繞射檢測循環腫瘤細胞與細胞培養光學定量研究 Polyelectrolyte Multilayer Grafting to Poly(dimethylsiloxane) Grating Substrate for Detection of Circulating Tumor Cells by Laser Diffraction in Whole Blood and Cell Culture Optical Quantitative Analysis
指導教授：	蘇舜恭 Shuenn-kung Su
口試委員:	陳建光黃昭蓮呂建興
學位類別：	碩士 Master
系所名稱：	工程學院 - 材料科學與工程系 Department of Materials Science and Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	208
中文關鍵詞：	聚二甲基矽氧烷、聚電解質多層膜、循環腫瘤細胞、抗沾黏、細胞培養、雷射
外文關鍵詞：	polydimethysiloxane, layer by layer, circulating tumor cells, antifouling, cell culture, laser, monitor
相關次數：	點閱：315 下載：0
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上一筆

本研究以VLSI技術在矽晶片上製備出洞型有陣序列，再利用聚二甲基矽氧烷(Poly(dimethylsiloxane), PDMS)將矽晶片上的圖案化轉印成為PDMS柱型結構為500nm與1µm的一維以及二維光柵。基材表面進行親水處理、矽烷類自組裝改質、以靜電吸附方式將聚丙烯酸poly(acrylic acid)與支鏈聚乙烯亞胺Branched Polyethylenimine組成聚電解質多層膜塗佈基材上，最後以海藻酸作為聚電解質多層膜之最外層，透過EDC/NHS的反應接上Protein G與上皮細胞黏附分子抗體(Anti-Epithelial Cellular Adhecion Molecule，Anti-EpCAM)用以捕捉血液中具有EpCAM表現之循環腫瘤細胞(Circulating tumor cells，CTCs)，由於血液分離術無法分離CTC與白血球，因此搭配牛血清蛋白(Bovine Serum Albumin，BSA)處理降低白血球沾黏。
依實驗結果得知500 nmPDMS、1µmPDMS分別在聚電解質多層膜第三層(500 nmPDMS-LbL3)、第五層(1µmPDMS-LbL5)時有最佳表面形貌，同時對具有EpCAM表現之循環腫瘤癌細胞捕獲有最大的雷射能量變化，分別為59.95%、49.78%，選其作為後續實驗之基材。基材對具有EpCAM表現之循環腫瘤癌細胞之靈敏度測試，結果顯示其最低偵測極限為2顆CTCs，其雷射能量損失分別為15.42%、11.95%，
以螢光顯微鏡觀察佐證，結果顯示當基材上捕獲2-40顆具有EpCAM表現且大小為15-20µm之循環腫瘤癌細胞時，雷射能量損失隨著細胞顆數不同呈現高度相關性，R2為0.9935、0.9836。臨床試驗中檢測24位患有子宮內膜癌之病人，以雷射能量損失對上癌症指數CA125，其中有10位病人癌症指數CA125呈現正常值以下，仍有捕獲2-12顆CTCs，結果顯示此方法檢測子宮內膜癌靈敏度高於CA125四倍。
細胞培養實驗其結果顯示此基材確實提升細胞培養效果，細胞生長性分別從100%提升至253.33%(500nm柱型)、210.38%(1µm柱型)，最後以海藻酸裂解酶對培養後之基材做釋放，其釋放率達到92.30%(500nm柱型)、94.20%(1µm柱型)。證明此方法可望用於癌症前期診斷及臨床檢體病人手術預後追蹤，且基材捕獲檢體細胞後可直接培養釋放。

In this study, we patterned ordered hole arrays with photoresist on the silicon surface by VLST process. The regular patterns of photoresist were transferred with polydimethylsiloxane (PDMS) to generate ordered 500nm and 1µm pillar arrays of 1D and 2D grating of PDMS.
The grating surfaces were sequentially modified with hydrophilic treatment, silane self-assembly, and electrostatic adsorption is adopted to coat the substrate with two types of polymer, poly (acrylic acid) and branched polyethylenimine. We use alginic acid as the outermost layer of the polyelectrolyte multilayer film. This polymer has a -COOH functional group, which can be use to connect to Protein G and Anti-Epithelial Cellular Adhecion Molecule (Anti-EpCAM) by EDC/NHS reaction. Anti-EpCAM a kind of antibody for immunosorbent of CTC. Finally, bovine serum albumin (BSA) was exploited to educe the fouling of white blood cell (WBCs).
According to the experimental results, we know that the best surface morphology of 500nmPDMS, 1µmPDMS is the third layer (500 nmPDMS-LbL3) and the fifth layer (1µmPDMS-LbL5), and the largest laser energy change captured for CTCs are 59.95% and 49.78%, and they were selected as the substrates for subsequent experiments. The sensitivity test of the substrate to CTCs showed that the detection limit was two CTCs. The laser energy loss were 15.42% and 11.95%, using the fluorescent microscope to observe, and the results showed that when 2-40 CTCs and size of 15-20µm were captured on the substrate, the laser energy loss showed a high correlation with the number of cells, showed R2 0.9935, 0.9836.
In clinical trials, 24 patients have endometrial cancer, and the laser energy loss compared with the cancer index CA125. Among them, 10 patients had a cancer index CA125 below normal, and 2-12 CTCs were still captured. The sensitivity of this method is four times greater than CA125.
The results of the cell culture show that the substrate can enhance the cell culture effect, and the cell growth rate can increase from 100% to 253.33% (500nm PDMS), 210.38% (1µm PDMS). Finally, we used the alginate lyase to release the CTCs. The release rate is 92.30% (500nm PDMS) and 94.20% (1µm PDMS). It proves that this method can be used in pre-cancer diagnosis and surgical prognosis tracking of clinical specimen patients, and the substrate cells can be directly cultured and released after capturing the specimen cells.

目錄
摘要 ..................................................................... I
Abstract ................................................................III
致謝 ....................................................................... V
目錄 ....................................................................... IX
圖目錄 ................................................................... XVII
表目錄 ................................................................... XXVII
緒論 ................................................................... 1
1. 研究背景 ............................................................. 1
2. 研究動機與目的 .................................................. 6
理論與文獻回顧 ........................................................ 8
1. 循環腫瘤細胞 ...................................................... 8
1.1. 循環腫瘤細胞介紹 ....................................................... 8
1.2. 循環腫瘤細胞生物特性標記 ..................................... 10
1.3. 循環腫瘤細胞捕獲 ..................................................... 11
2. 循環腫瘤細胞培養 ............................................. 14
2.1. 細胞培養 .............................................................14
2.2. 三微結構培養...........................................................15
2.3. 細胞貼附行為........................................................... 17
3. 聚二甲基矽氧烷 ................................................. 18
4. 微影製程及翻模 ................................................. 19
5. 自組裝單分子層 ................................................. 22
6. 共價鍵固定法(EDC/NHS reaction) ....................... 24
7. 聚電解質多層法 ................................................. 26
8. 海藻酸(ALG) ....................................................... 29
9. 聚丙烯酸(PAA) ................................................... 31
10. 支鏈聚乙烯亞胺(bPEI) ........................................ 32
11. 重組蛋白與抗體 ................................................. 33
12. 光柵效應 ............................................................ 34
13. 細胞釋放 ............................................................ 36
儀器原理 .................................................................. 39
1. X射線光電子能譜儀(X-ray photoelectron spectroscopy，XPS) ................. 39
2. 原子力顯微鏡(Atomic Force Microscope, AFM)... 42
3. 高解析度場發射掃描式電子顯微鏡(Field-emission scanning electron microscope，FE-SEM) ......................... 47
4. 陣列光束雷射分析儀(BeamMic) ......................... 49
5. 傅立葉轉換紅外線光譜儀 (Fourier-Transform Infrared Spectroscopy, FT-IR) .......................................... 53
6. 表面電位分析儀(Zeta-potential) ......................... 58
7. 電漿蝕刻機(Plasma Machine) ............................. 59
8. 接觸角 (Contact Angle, CA) ................................ 61
9. 雷射掃描式共軛焦顯微鏡 (Laser scanning confocal microscope，LSCM) .......................................... 63
10. 螢光顯微鏡 ........................................................ 65
實驗流程與方法 ....................................................... 68
1. 實驗流程圖 ........................................................ 68
........................................................................ 68
2. 實驗藥品 ............................................................ 69
3. 實驗儀器 ............................................................ 74
4. 實驗步驟 ............................................................ 76
4.1. 微影製程製備具有圖案化光阻層 ............................. 76
4.4.1 表面預處理 .......................................................... 76
4.4.2 光阻塗佈 ............................................................. 77
4.4.3 軟烤 ............................................................... 77
4.4.4 曝光 ............................................................... 77
4.4.5 顯影 .............................................................. 78
4.2. 圖案化轉印於PDMS薄膜 ......................................... 78
4.3. PDMS表面改質 .......................................................... 79
4.3.1 PDMS表面鍍金及親水處理 ............................................ 79
3.3.2 製備自組裝層 ...................................................... 80
3.3.3 聚電解質多層膜 ..................................................... 81
4.4. PDMS表面改質ProtrinG與Anti-EpCAM .................. 82
XIII
4.5. PDMS-anti-EpCAM表面blocking(BSA) ....................... 83
4.6. 白血球抗沾黏實驗 ..................................................... 84
4.6.1 血液樣品處理 ........................................................ 84
4.6.2 白血球接種 .......................................................... 84
4.6.3 白血球固定與染色 ................................................... 84
4.7. 雷射分析儀細胞定量檢測 ......................................... 85
4.7.1 細胞株HCT-116接種基材處理 ...................................... 85
4.7.2 細胞試片螢光染色處理 .................................................. 85
4.8. 病人檢體之循環腫瘤細胞偵測 ................................. 86
4.9. 細胞培養 .......................................................... 86
4.9.1 大腸癌細胞株HCT-116 ................................................... 86
4.9.2 大腸癌細胞株DLD-1 ..................................................... 86
4.9.3 子宮頸癌細胞株HeLa ................................................... 87
4.9.4 細胞接踵至培養基材 .................................................... 87
4.9.5 CCK-8 Assays (Cell Counting kit-8) .................................. 87
4.10. 細胞釋放 .............................................................. 88
4.11. 細胞試片處理 ...................................................... 88
4.11.1 掃描式電子顯微鏡生物試片製作 ................................ 88
結果與討論 .............................................................. 89
1. 圖案化表面分析 ................................................. 89
1.1. 微影製程光阻圖案 ..................................................... 89
1.2. PDMS轉印圖案 .......................................................... 90
2. PDMS圖案化光學性質 ....................................... 92
2.1. 可見光繞射 ........................................................... 92
2.2. 雷射繞射 ............................................................. 93
3. PDMS表面接枝聚電解多層膜分析 .................... 95
3.1. Zeta potential ....................................................... 95
3.2. XPS光譜圖分析 .......................................................... 97
3.2.1 PDMS改質後之Wide Scan分析 .................................. 97
3.2.2 Au能譜分析 .......................................................... 99
3.2.3 Si2p能譜分析 ....................................................... 100
2.3.4 C1s能譜分析 ........................................................ 101
3.2.5 O1s能譜分析 ......................................................... 102
3.3. IR光譜............................................................... 104
3.4. 接觸角親疏水測試 ................................................... 105
3.5. 聚電解質多層膜表面分析 ....................................... 107
3.5.1 500nm柱狀PDMS-LbL ................................................... 108
3.5.2 1μm柱狀PDMS-LbL .................................................... 112
3.6. 聚電解質多層膜之雷射能量分析 ........................... 116
4. 聚電解質多層膜之層數選擇 ............................. 121
4.1. 不同層數造成雷射對細胞之能量損失分析 ............ 121
4.2. 不同層數對細胞貼附及釋放之影響........................ 126
5. 基材抓取細胞之測試 ........................................ 130
5.1. 白血球抗沾黏測試 ................................................... 130
5.1.1 白血球雷射分析結果 .................................................... 130
5.1.2 白血球螢光顯微鏡分析 ................................................ 132
5.2. 基材抓取不同細胞對雷射值之影響........................ 133
5.3. 基材對不同種類細胞之選擇性 ............................... 134
6. 臨床檢體之測試 ............................................... 136
7. 基材培養之細胞生長性測試 ............................. 141
8. 細胞培養 .......................................................... 145
8.1. 螢光成像 ............................................................ 145
8.2. 細胞生長曲線........................................................... 147
8.3. 光柵效應 ............................................................. 148
8.4. 雷射分析結果........................................................... 150
9.1. 螢光成像 ............................................................. 156
9.2. 光柵效應 ............................................................ 157
9.3. 雷射分析結果........................................................... 159
結論 .................................................................... 162
參考文獻 ................................................................... 164


                                

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