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研究生:黃亦鈺
研究生(外文):Yi-yu Huang
論文名稱:表面增強拉曼光譜於細菌快速檢測應用之技術開發
論文名稱(外文):Rapid Identification of Bacteria Using Surface-Enhanced Raman Spectroscopy
指導教授:江惠華江惠華引用關係
指導教授(外文):Huihua Kenny Chiang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:61
中文關鍵詞:細菌表面增強拉曼光譜學變異種
外文關鍵詞:bacteriaSurface enhancement Raman Scattering (SERS)mutant
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細菌感染現於臨床診斷上多以生化或是分子生物技術辨識。另因細菌感染多以抗生素治療,並以傳統抗生素藥劑感受性試驗決定抗生素的使用,受限於現今技術,各階段步驟多需一至二個工作天,或是更多的時間才能得到檢驗結果。本研究希望能以表面增強拉曼光譜學之技術,配合拉曼光譜學之樣本前處理便利性、量測快速以及表面增強拉曼光譜增強訊號並降低螢光背景之優點,開發高重現性及高敏感度之技術,應用至細菌檢測上。
本實驗利用大腸桿菌及分枝桿菌為樣本,並利用銀奈米粒子包附樣本後量測其表面增強拉曼光譜。本研究結果顯示,此方法已可輕易透過光譜差異分辨兩種細菌,在變異種的光譜量測上,僅得細微差異,但仍可做為辨識之用。現今臨床感染菌株會有不同抗藥性表現,多為基因變化所致,能利用表面增強拉曼光譜辨識變異種,即反應此技術在臨床菌株抗藥性測試之可能性。本實驗在重現性表現上,得一定再現性,表示此法可實際應用。但細菌的表面增強拉曼光譜表現會隨著濃度而變化,高濃度樣本的重現性較高與特徵光譜表現較為豐富,亦即在應用上需固定樣本濃度及樣本數,以得最佳表現。

The detection of bacteria or other microorganism is very important in clinical diagnosis. Conventional protocols for diagnosis are performed by a comparison of morphological characteristics or based on biochemical tests which take few days. Surface-enhanced Raman scattering (SERS) spectroscopy is a very useful method for identifying different microorganisms by their specific fingerprint. This study is using surface enhancement Raman scattering spectroscopy to develop a method for bacteria of different species even mutants identification with the advantage of simple sample preparation, rapid measurement, high reproducibility, lower fluorescence background and high sensitivity.
We used Escherichia coli and mycobacterium in this study, and we can easily indentify Escherichia coli from mycobacterium by their SERS spectrum. The differences between mutants were small but we can still distinguish them. The reproducibility of this method is high and we can measure the valid spectrum for concentration lower to 103 cfu/ml. The spectrum would be different in different sample concentration, it’s important to measure the spectra with fixed concentration to get the best results.

Abstract i
中文摘要 ii
目錄 iii
圖表目錄 v
1. 序論 1
1.1 研究背景 1
1.1.1 細菌構造 1
1.1.2 分枝桿菌(Mycobacterium) 3
1.1.3 大腸桿菌(Escherichia coli) 5
1.1.4 現行微生物檢驗法 7
1.2 文獻回顧 9
1.2.1 拉曼光譜學在細菌檢測上的應用 9
1.2.2 表面增強拉曼光譜學在細菌檢測上的應用 9
1.3 研究目的 11
2. 理論基礎 12
2.1 拉曼散射 12
2.1.1 拉曼散射簡史 12
2.1.2 拉曼散射原理 13
2.2 表面增強拉曼散射(Surface Enhanced Raman Scattering) 18
2.2.1 表面增強拉曼簡史 18
2.2.2 電磁場增益效應 18
2.2.3 化學增益效應 20
3. 材料與方法 22
3.1 儀器簡介 22
3.1.1 拉曼散射光譜儀 22
3.1.2 紫外光/可見光吸收光譜儀 25
3.1.3 穿隧式電子顯微鏡 26
3.2 膠體奈米銀粒子配置 27
3.3 量測前處理 28
3.4 資料處理 30
3.4.1 宇宙射線的移除 30
3.4.2 背景的減贅 30
3.4.3 光譜均一化 30
3.4.4 平均光譜 30
3.4.5 層次數據聚類分析法(Hierarchical clustering analysis, HCA) 31
4. 結果與討論 32
4.1 細菌的一般拉曼光譜與表面增強拉曼光譜差異 32
4.2 奈米銀粒子的重現性測驗 33
4.3 表面增強拉曼光譜對細菌辨識的再現性 36
4.4 不同濃度樣本的表面增強拉曼光譜變化 38
4.4.1 方法一(原始方法) 38
4.4.2 方法二(增加一次清洗步驟) 40
4.4.3 方法三(降低奈米銀濃度至原先的十分之一) 41
4.5 大腸桿菌與分枝桿菌的表面增強拉曼光譜差異 42
4.6 變異種的表面增強拉曼光譜差異 45
5. 結論與未來展望 49
5.1 結論 49
5.2 未來展望 50
參考文獻 51


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