臺灣博碩士論文加值系統

本研究主要利用微乳液法（microemulsion method）合成SiO2奈米粒子粒徑平均約為21nm～49nm。經掃瞄式電子顯微鏡（SEM）、穿透式電子顯微鏡（TEM）及原子力顯微鏡（AFM）觀察粒子表面，結果顯示SiO2奈米粒子之粒子型態均勻、表面光滑。以光譜儀掃描確認SiO2奈米粒子能將Tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate（Rubpy）水溶性螢光染料順利包覆於奈米粒子中。進一步比較3-Aminopropyltriethoxysilane（APTES）及Trimethoxysilylpro-
pyldiethylenetriamine （DETA）法修飾奈米粒子表面官能基(NH2)之效能，結果顯示以APTES法之修飾效能較佳，且高DETA法約為3.3倍。將不同粒徑之SiO2-NH2-COOH與山羊抗兔子免疫球蛋白(IgG)抗體聯結製成螢光二氧化矽奈米探針(fluorescent silica nanoprobes, FSP)，並以檢測茄科植物細菌性斑點病原菌（Xanthomonas axonopodis pv.vesicatoria XVT40）為例，利用螢光酵素連結抗體免疫分析儀（FLISA）法進行植物病原菌之偵測，結果顯示當病原菌濃度為103cfu/ml時，FSP亦可檢測出XVT40病原菌抗原之存在；且結果顯示粒徑大小為影響探針之靈敏度之因子之一，粒徑越小其靈敏度亦越高。本研究結果顯示螢光二氧化矽奈米探針具有做為植物病原檢疫診
斷及偵測的潛力。

The study utilized technique of microemulsion to synthesize Silica nanoparticles. The sizes of silica nanoparticles were measured by using Scanning electron microscopy (SEM). Data showed that diameters of nanoparticles were approximate 21～49 nm. We also observed the surface and shape of nanoparticles were relatively smooth and circular via Transmission electron microscope (TEM) and Atomic force microscope (AFM). The silica nanoparticles could be efficiently covered with the fluorescent dye, Tris(2,2’- bipyridyl) dichlororuthenium (II) hexahydrate (Rubpy). Furthermore, compared the methods of APTES with DETA of surface modified, the results showed that APTES were better. It increased more than threefold than which in DETA. The different sizes of fluorescent SiO2-NH2-COOH nanoparticles were further conjugated with Goat anti-rabbit IgG antibodies termed Fluorescent silica nanoprobe (FSP). FSP was tested for the detection of antigens of phytopathogenic bacterium, Xanthomonas axonopodis pv. vesicatoria （XVT40） using fluorescence-linked immunosorbent assay (FLISA). The results showed that varying size nanoparticles of FSP could efficiently detect XVT40 antigens in a concentration 103 cfu/ml. These results demonstrated that the fluorescent silica nanoprobes can be effectively used to detect the
quarantine pathogens on plant diseases.

摘要 I
Abstract III
目錄 V
圖目錄 VIII
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
1.3 研究目的 3
第二章 文獻回顧 4
2.1 奈米材料 4
2.2 奈米材料的製備 5
2.2.1物理方法 5
2.2.2化學方法 5
2.3 微乳液法簡介 6
2.4 奈米材料的應用 8
2.5 二氧化矽簡介 9
2.6 奈米粒子連結生物分子的方式 11
2.6.1 雙功能鍵結 11
2.6.2 靜電吸引力 11
2.6.3 矽烷偶聯劑 12
2.7 茄科細菌性斑點病原菌 12
2.8 ELISA簡介及應用 15
第三章 材料與方法 17
3.1 實驗材料 17
3.2實驗設備 19
3.3 實驗流程規劃 20
3.4. 螢光二氧化矽奈米粒子合成製備 21
3.4.1 不同粒徑螢光二氧化矽奈米粒子 21
3.4.2 含胺基螢光二氧化矽奈米粒子 23
3.5 光譜測定 25
3.6 SEM表面觀察、EDS成分分析及TEM粒徑量測 25
3.7 AFM表面粗糙度量測 25
3.8 螢光猝滅檢測 26
3.9 植物病原細菌全細胞多源抗體製備 26
3.10 化學法表面修飾 28
3.10.1 胺基（SiO2-DETA） 28
3.10.2 醛基（SiO2-DETA-CHO） 29
3.10.3 醛基（SiO2-NH2-CHO） 30
3.10.4 胺基檢測 31
3.10.5 醛基檢測 32
3.10.6 羧基（SiO2-NH2-COOH） 33
3.10.7 羧基檢測 34
3.11 奈米粒子與抗體連結 34
3.11.1 表面修飾醛基之奈米粒子 34
3.11.2 表面修飾羧基之奈米粒子 36
3.12 奈米粒子連結抗體效果檢測 37
3.13 XVT40病原菌檢測 39
第四章 結果與討論 41
4.1 螢光二氧化矽奈米粒子合成製備 41
4.2 光譜測定 43
4.3 SEM表面觀察、EDS成分分析及TEM粒徑量測 44
4.4 原子力顯微鏡（AFM）表面粗糙度量測 53
4.5 螢光猝滅檢測 54
4.6 植物病原細菌全細胞多源抗體 56
4.7 官能基檢測 57
4.7.1 胺基檢測 57
4.7.2 醛基檢測 60
4.7.3 羧基檢測 62
4.8 化學法表面修飾 63
4.8.1 胺基 63
4.8.2 醛基 64
4.8.3 羧基 64
4.9奈米粒子連結免疫球蛋白IgG效果檢測 65
4.10 螢光免疫分析檢測XVT40病原菌 67
第五章 結論 70
參考文獻 73

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