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系統識別號 U0026-0102201021083400
論文名稱(中文) 以金奈米粒子修飾之聚雙甲基矽氧烷為基材之反相蛋白質陣列晶片之研發
論文名稱(英文) AuNP-Anchored PDMS Substrate for the Development of Reverse-Phase Protein Array
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 98
學期 1
出版年 99
研究生(中文) 柯秀蓉
研究生(英文) Hsiu-Jung Ko
學號 l3696412
學位類別 碩士
語文別 中文
論文頁數 86頁
口試委員 指導教授-陳淑慧
口試委員-桂椿雄
口試委員-孫亦文
中文關鍵字 聚雙甲基矽氧烷  金奈米粒子  反相蛋白質陣列 
英文關鍵字 PDMS  gold nanoparticles  reverse-phase protein array 
學科別分類
中文摘要 質譜技術與蛋白質晶片技術已成為研究蛋白質體學的重要方法。蛋白質晶片技術可以高效地獲取生物體中的蛋白質訊息,且一次大量的樣品檢驗可減少所需成本。奈米粒子本身具有高的表面積及可與生物分子相容的優點,因此,在本研究中,以金奈米粒子修飾於聚雙甲基矽氧烷基材上,發展以此為基材之反相蛋白質陣列晶片。
我們成功地以血紅素蛋白質做為連結試劑,將金奈米粒子穩定地修飾於晶片表面上,首先針對雌激素受體ERα分析,可得到檢量線 ,線性範圍為44.6至1.67 ng/mL,求得此系統之偵測極限值為0.5 ng/mL。
針對所使用的抗體,也可以藉著反相蛋白質陣列晶片進行快速地篩選,並評估其專一性。更進一步,利用此晶片可以研究MCF-7細胞經過雌激素E2作用後,在不同時間點的蛋白質表現。使用修飾之PDMS基材製作組織晶片,可使組織平整貼附,且具有耐熱、耐有機溶劑等特性。
綜合以上實驗結果,顯示出以金奈米粒子修飾之聚雙甲基矽氧烷為基材之反相蛋白質陣列晶片,具有良好的再現性與穩定性,使用修飾後的PDMS基材製作組織晶片,也有極大的潛力。
英文摘要 Mass spectrometry and protein chip technology have already become important methods for proteomic research. Protein information from organisms can be gained high-efficiently by using protein chip technology. A large number of samples could be screened using protein chips at low cost. On the other hand, nanoparticles have many advantages such as large surface area and high biocompatibility. In this study, we would like to take these advantages associated with gold nanoparticles (AuNPs) in fabricating a reverse-phase protein array on AuNPs-anchored poly (dimethylsiloxane) (PDMS) substrate.
We successfully stabilized and immobilized Au nanoparticles on PDMS surface by using myoglobin as a linking agent. Recombinant estrogen receptor alpha (ERα) was first used to test our system. The calibration curve was estimated to be y=1.1451x + 2.4936 (R2=0.9954) in a linear range from 44.6 to 1.67 ng/mL and the detection limit was determined to be around 0.5 ng/mL.
We demonstrated that such modified PDMS chip could differentiate the specificity of antibodies from different sources at a high screening speed. Furthermore, we chip could be used for protein expression profiling of MCF-7 cells under 17β-estradiol (E2) treatment of different time courses. We further demonstrated that such modified PDMS chip could be used to immobilize tissue pieces smoothly and with good tolerance for heat and organic solvents.
Thus, the results confirmed that our reverse-phase protein array fabricated on AuNP-anchored PDMS substrate exhibited good reproducibility and stability. Furthermore, such modified PDMS chip holds a great potential to fabricate tissue arrays.
論文目次 摘要 I
Abstract II
誌謝 IV
目錄 VI
表目錄 IX
圖目錄 X

第一章 序論
1-1 生物晶片簡介 1
1-2 蛋白質晶片的介紹 5
1-3 金奈米粒子與生物技術的結合 7
第二章 利用金奈米粒子於反相蛋白質陣列中對於雌激素受體之分析
2-1 聚雙甲基矽氧烷(Poly(dimethylsiloxane),PDMS) 9
2-2 雌激素與雌激素受體 10
2-3 PDMS基材之蛋白質晶片及其表面化學 13
2-3.1 實驗藥品與儀器 17
2-3.2 實驗方法 21
2-3.2.1 PDMS蛋白質晶片之製作流程 21
2-3.2.2 PDMS蛋白質晶片之表面修飾 23
2-3.2.3 金奈米粒子溶液之製備 24
2-4 反相蛋白質陣列(Reverse-phase protein array) 26
2-4.1 實驗藥品與溶液配製 29
2-4.2 反相蛋白質陣列之實驗流程 31
2-5 結果與討論 33
2-5.1 修飾金奈米粒子對結果之影響 33
2-5.2 修飾不同層數之金奈米粒子 35
2-5.3 對於雌激素受體(ERα)之分析 37
2-5.4 雌激素受體抗體之專一性測試 39
2-6 結論 44
第三章 利用金奈米粒子於反相蛋白質陣列中對於人類乳腺癌細胞(MCF-7)中雌激素受體之分析
3-1 人類乳腺癌細胞(MCF-7)介紹 45
3-2 材料與實驗方法 45
3-2.1 真實樣品MCF-7與A549細胞之培養 47
3-2.2 MCF-7與A549之全細胞萃取 48
3-2.3 反相蛋白質陣列之實驗流程 49
3-3 結果與討論 51
3-3.1 修飾不同層數之金奈米粒子對於MCF-7強度之比較 51
3-3.2 MCF-7經系列稀釋後之分析 53
3-3.3 MCF-7細胞中其他蛋白質之表現 55
3-3.4 在MCF-7細胞中與雌激素E2作用後之蛋白質表現變化 59
3-3.5 晶片之間的差異性與再現性分析 62
3-4 結論 63
第四章 利用PDMS材質製作組織晶片(Tissue array)
4-1 研究動機 65
4-2 材料與實驗方法 66
4-3 結果與討論 71
4-3.1 不同的PDMS晶片表面修飾對於組織的貼附情形探討 71
4-3.2 不同的表面修飾對於脫臘後組織的貼附情形探討 73
4-3.3 不同厚度之組織陣列切片(Tissue array)在PDMS晶片上之貼附情形探討 76
4-3.4 不同厚度之組織切片在PDMS晶片上經過脫蠟及免疫組織化學染色法後的比較 77
4-4 結論 80
第五章 總結與未來展望 81
參考文獻 83
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