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研究生： 程珮儀
Cheng, Pei-Yi
論文名稱： 藉由空間侷限奈米流體引導蛋白質形成階層式結構
Hierarchical Protein Assembly Driven by Evaporative Spatially-Confined Nanofluids
指導教授： 李介仁
Li, Jie-Ren
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 102
中文關鍵詞： 第一型膠原蛋白 、自組裝 、空間侷限流體 、原子力顯微鏡
外文關鍵詞： Protein, Self-assembly, Spatial confinement, Atomic force microscopy
相關次數： 點閱：79  下載：0 
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    • 在大自然中，生物分子可由多個單體以自組裝的方式組成一個複雜的階層式結構。第一型膠原蛋白是眾多細胞外間質中含量最豐富的蛋白質，可在體內會呈現有方向性的排列並自組裝成階層式結構來做為細胞成長的支架。若能控制第一型膠原蛋白的自組裝並將其應用於仿生材料，這對於生醫工程的研究將會是一大突破。然而蛋白質自組裝是一個極為複雜的行為，許多因素都會影響到自組裝的過程，例如基材的表面特性、 pH值、離子的種類與離子強度等，因此在體外無法像體內一樣能夠精確的控制第
    一型膠原蛋白的自組裝。
    本實驗中我們利用空間侷限的方法使第一型膠原蛋白的自組裝發生在特定的空間中以形成奈米結構，再藉由原子力顯微鏡分析其自組裝後的細部結構。當第一型膠原蛋白溶液被侷限在特定空間中時，於蒸發過程中所產生的毛細作用力會引導第一型膠原蛋白排列成階層式結構，因此可藉由不同結構及形貌的模板來製作出具有複雜結構的第一型膠原蛋白奈米結構。除此之外，當侷限的空間越小時，第一型膠原蛋白的橫向交聯受到壓迫而無法自組裝成有方向性的纖維，透過調整空間侷限的大小能有效控制第一型膠原蛋白纖維的長度。本研究證實了利用空間侷限奈米流體的方法可以控制第一型膠原蛋白的自組裝，並製作出大面積且結構比原本結構還微小的奈米結構。

    Biomolecules are capable of assembling into various hierarchical structures in nature. In particular, type I collagen is the most abundant extracellular matrix (ECM) protein, which can self-assemble into hierarchical scaffold with ordered fibrils for cell growth. Therefore, if the assembly process of type I collagen can be controllable, it is the key breakthrough to design new biomimetic materials for the biomedical applications. However, protein assembly is extremely complicated. Many factors, such as, surface properties, pH value, ion strength and ion species, affect the protein assembly process. As a result, controlling collagen assembly in vitro is not as precise as in vivo. In this research, we used the spatially-confined environment between designed templates and flat substrate to control the collagen assembly process and used AFM to analyze detailed structure. As the collagen solution is spatially-confined in the designed environment, the evaporation process further induces capillary flow to enhance collagen self-assembly, forming hierarchical structures. Different morphology of collagen micro- and nanostructures can be produced by using different templates. However, as the confined space becomes smaller, the lateral cross-linking of collagen molecules is limited, resulting in failure to assemble into fibril. The length of collagen fibril can be regulated by the dimension of spatially-confined environment. Our experiments demonstrate that protein self-assembly can be controlled by using spatially-confined nanofluids, and hierarchical protein structures which are smaller than templates can be produced on large-area substrates.

    中文摘要 I 英文摘要 II 英文延伸摘要 III 致謝 VII 目錄 1 圖目錄 4 第一章、緒論 10 1.1 前言 10 1.2 細胞外間質(Extracellular Matrix, ECM) 10 1.3 膠原蛋白(Collagen) 12 1.3.1 第一型膠原蛋白(Collagen Type I, Col I) 13 1.3.2 第四型膠原蛋白(Collagen Type IV, Col IV) 15 1.4 蛋白質結構奈米製程 16 1.5 原子力顯微鏡(Atomic Force Microscopy, AFM) 20 第二章、利用二氧化矽奈米球模板控制第一型膠原蛋白自組裝 22 2.1 前言 22 2.2 實驗部分 23 2.2.1 實驗材料 23 2.2.2 儀器 23 2.2.3 實驗步驟 24 2.3 結果與討論 26 2.3.1 二氧化矽奈米球模板之結構 26 2.3.2 第一型膠原蛋白在雲母片自組裝之型態 27 2.3.3 第一型膠原蛋白溶液儲存時間對其在二氧化矽奈米球模板上自組裝的影響 28 2.3.4 氯化鉀離子濃度對第一型膠原蛋白在二氧化矽奈米球模板上自組裝的影響 31 2.3.5 二氧化矽奈米球模板尺寸對第一型膠原蛋白自組裝的影響 32 2.3.6 局部濃度差異對第一型膠原蛋白奈米結構的影響 35 2.4 結論 40 第三章、利用 PDMS 模板控制第一型膠原蛋白自組裝 41 3.1 前言 41 3.2 實驗部分 42 3.2.1 實驗材料 42 3.2.2 儀器 43 3.2.3 實驗步驟 44 3.3 結果與討論 48 3.3.1 PDMS 模板之結構 48 3.3.2 第一型膠原蛋白溶液儲存時間對 PDMS 模板控制其在雲母片上自組裝的影響 51 3.3.3 氯化鉀離子濃度對 PDMS 模板控制第一型膠原蛋白在雲母片上自組裝的影響 53 3.3.4 不同空間侷限結構對第一型膠原蛋白自組裝的影響 55 3.3.5 不同寬度之線狀奈米流體對第一型膠原蛋白自組裝的影響 59 3.3.6 空間侷限大小對第一型膠原蛋白自組裝的影響 61 3.3.7 第一型膠原蛋白奈米結構在 PBS 緩衝溶液中之穩定性 67 3.4 結論 75 第四章、利用官能基化表面調節第四型膠原蛋白自組裝 76 4.1 前言 76 4.2 實驗部分 77 4.2.1 實驗材料 77 4.2.2 儀器 78 4.2.3 實驗步驟 79 4.3 結果與討論 83 4.3.1 第四型膠原蛋白溶液之 pH 值與吸附時間對其在 OTS 薄膜上自組裝的影響 83 4.3.2 第四型膠原蛋白濃度對其在 OTS 薄膜上自組裝的影響 87 4.3.3 第四型膠原蛋白在不同基材自組裝之表現 89 4.3.4 表面官能基對第四型膠原蛋白的組裝的影響 91 4.3.5 第四型膠原蛋白在奈米結構上自組裝的型態 95 4.4 結論 98 參考文獻 99

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