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研究生:沈志豪
研究生(外文):Shen, Chih-Hao
論文名稱:以二氧化矽包覆金奈米棒做為表面增強拉曼標籤偵測沙門氏菌DNA
論文名稱(外文):Silica-Coated Gold Nanorods as Surface-Enhanced Raman Tags for the Detection of a DNA Strand of Salmonella
指導教授:楊子萱楊子萱引用關係
指導教授(外文):Yang, Tzyy-Schiuan
口試委員:陳永恩周禮君
口試委員(外文):Chen, Yung-EnChau, Lai-Kwan
口試日期:2015-07-31
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學暨生物化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:93
中文關鍵詞:表面增強拉曼標籤沙門氏菌金奈米棒
外文關鍵詞:SERS tagsSalmonellaGold nanorod
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表面增強拉曼核殼標籤(Surface-enhanced Raman core-shell tag)具有化學穩定性、表面容易修飾及多重性檢測等優點。根據先前的研究顯示,使用二氧化矽包覆金奈米球與標記分子聚合而成的奈米聚集團粒子(nanoaggregate-embedded bead, NAEB)具有化學穩定性及強烈表面增強拉曼訊號。但金奈米球的聚集程度不易控制,因此訊號強度沒有再現性。單顆金奈米棒所產生的表面增強拉曼散射(SERS)比單顆金奈米球強。本研究使用金奈米棒做為SERS核殼標籤的基材,標記分子的SERS訊號強度約為NAEBs的1/3倍,但可改善先前強度的再現性。本實驗室先前所合成二氧化矽包覆金奈米棒的拉曼標籤使用陽離子界面活性劑cetyltrimethylammonium bromide (CTAB)與矽源前趨物 tetraethyl orthosilicate (TEOS)形成中孔洞的二氧化矽殼層。此殼層再進一步表面修飾DNA時產生破裂。因此本研究進行了殼層穩定性的改善。利用中孔洞的矽殼層當作支架,殼層表面再包覆一層緊密的二氧化矽讓結構穩定,且產生完整平滑表面。藉由調控反應液pH值,中孔洞的二氧化矽殼層外圍成功的包覆一層結構緊密的二氧化矽。經由浸泡於水溶液12小時後TEM影像的對照,外殼層結構無明顯變化,可證實其穩定性。
另一部分研究是以改良後的SERS核殼標籤進行沙門氏菌DNA檢測。核殼標籤使用4-nitrothiophenol(4-NTP)做為標記分子。在SERS核殼標籤表面修飾上探針DNA,金基板上修飾捕捉DNA。利用三明治結構之DNA雜交分析法,加入一段具辨識性沙門氏菌的DNA之後,經由DNA雜交將SERS核殼標籤固定在金基板上。最後檢測金基板上的SERS核殼標籤的SERS訊號做致病菌的檢出。偵測最低DNA濃度可達10-8 M (33-mer 單股DNA)。

Surface-enhanced Raman scattering (SERS) core-shell tags have advantages over fluorescence tags in terms of chemical stability and multiplex detection. They are also easily applied for surface modification. In the previous study, we developed a silica-coated marker-embedded gold nanosphere aggregate, called a nanoaggregate-embedded bead (NAEB), which exhibits measurable single-particle SERS signals. However, the size of aggregation is difficult to control, resulting non-reproducible SERS signals. Since the SERS enhancement of a single gold nanorod (AuNR) is stronger than that of a gold nanosphere, we utilized AuNRs as the substrate for SERS core-shell tags and used them for the analysis of DNA strands.
We had synthesized mesoporous silica-coated single gold nanorods before, but the mesoporous silica shell was destroyed when processing the surface modification by DNA. Therefore, we over-coated a thin layer of dense silica to stabilize the structure and to smooth the surface. The thickness of the dense silica layer was controlled by adjusting pH value of the solution to above 11. From the comparison of TEM images of dense silica shell dispersed in water before and after 12 hrs, the dense silica shell still remained. This demonstrated the stability of the new SERS core-shell tags.
We used improved SERS core-shell tags to detect a special DNA strand that can be used to identify 18 Salmonella pathogenic bacteria. 4-nitrothiophenol (4-NTP) was used as the SERS maker molecule. The surface of SERS core-shell tags were modified by a DNA strand, called the probe DNA, which is complementary to the half segment of the target Salmonella DNA. A gold substrate was planted with another half segment of the complementary DNA, called the capture DNA. The target Salmonella DNA strands were captured by the capture DNA on the Au substrate and then hybridized with the probe DNA on the SERS tags. The SERS signals of immobilized SERS tags were read out for pathogenic assays. The lowest concentration of the target DNA to be detected by this method is 10-8 M (33 mer, strand concentration).

中文摘要 I
Abstract III
目錄 V
圖目錄 IX
表目錄 XII
縮寫表 XIII
第一章 緒論 1
1.1前言 1
1.2沙門氏菌簡介 3
1.3 DNA簡介 4
1.4目前檢測沙門氏菌方法 5
1.4.1 抗原-抗體特異性分析法 5
1.4.2 DNA雜交分析法 8
1.5表面增強拉曼標籤介紹 11
1.5.1 表面增強拉曼標籤應用在生物分析檢測上 11
1.5.2 表面增強拉曼散射標籤 12
1.6 研究動機與目的 14
第二章 實驗原理 16
2.1 拉曼散射簡介 16
2.2 拉曼散射原理 18
2.3 表面增強拉曼散射原理 23
2.3.1 電磁場增強效應(electromagnetic enhancement effect) 24
2.3.2 化學增強效應(chemical enhancement effect) 27
第三章 實驗部分 29
3.1 實驗儀器與藥品 29
3.1.1實驗儀器 29
3.2表面增強拉曼標籤之合成 32
3.2.1 實驗藥品 32
3.2.2 金奈米棒之合成 33
3.2.3 製備中孔洞二氧化矽包覆之金奈米棒 33
3.2.4 製備緻密二氧化矽包覆之金奈米棒 34
3.3表面增強拉曼標籤之表面功能化 35
3.3.1 實驗藥品 35
3.3.2 緩衝溶液配置 36
3.3.3 表面增強拉曼標籤修飾probe DNA 37
3.4 於金基板上製備capture DNA 矩陣 40
3.4.1 實驗藥品 40
3.4.2 製作capture DNA於金基板上 40
3.5 利用三明治結構做DNA雜交分析法 43
第四章 結果與討論 44
4.1表面增強拉曼散射核殼標籤合成 44
4.1.1 金奈米棒性質 44
4.1.2 合成SERS核殼標籤 48
4.1.3 以緻密矽殼層穩定SERS核殼標籤 49
4.1.4 改良後SERS核殼標籤之穩定性測試 53
4.2 表面增強拉曼散射核殼標籤之性質 58
4.2.1不同的表面增強拉曼散射核殼標籤之吸收光譜 58
4.2.2不同的表面增強拉曼核殼標籤之SERS光譜 61
4.3 SERS核殼標籤之表面官能化 63
4.3.1 表面官能化之界達電位 64
4.3.2 修飾DNA在表面增強拉曼標籤之螢光影像 65
4.3.3 不同還原劑去除雙硫鍵之比較 67
4.3.4 SERS核殼標籤在不同鹽類濃度的緩衝溶液的穩定性 69
4.4 利用三明治結構之DNA雜交分析法檢測沙門氏菌DNA 71
第五章 結論 75
參考文獻 76

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