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系統識別號 U0026-2807202022425500
論文名稱(中文) 以表面增強拉曼光譜監測淡水中微囊藻毒之技術發展
論文名稱(英文) Development of Surface-Enhanced Raman Spectroscopy on Monitoring of Microcystins in the Fresh Water
校院名稱 成功大學
系所名稱(中) 環境工程學系
系所名稱(英) Department of Environmental Engineering
學年度 108
學期 2
出版年 109
研究生(中文) 蘇冠臨
研究生(英文) Guan-Lin Su
學號 P56074181
學位類別 碩士
語文別 中文
論文頁數 92頁
口試委員 指導教授-林財富
共同指導教授-陳宣燁
口試委員-黃良銘
口試委員-王根樹
中文關鍵字 藍綠菌  微囊藻毒LR型  表面增強拉曼散射光譜  滴塗沉積拉曼光譜  酵素連結免疫吸附法 
英文關鍵字 Cyanobacteria  Microcystin-LR  Surface-enhanced Raman scattering  Drop coating deposition Raman  ELISA 
學科別分類
中文摘要 全球暖化的加劇以及農、工業快速的發展,使大量氮、磷等營養鹽流入湖泊與水庫等飲用水源進而導致水質優養化。在溫度與營養鹽較高的環境,造成藍綠菌大量生長並引起藻華問題。藍綠菌生長會產生二次代謝物,部分藻類所產生的二次代謝物具有毒性,其中微囊藻毒LR型(Microcystin-LR, MC-LR)為最主要的危害物質之一,會損害人類肝臟功能,同時也具有致癌性。世界衛生組織依據MC-LR的每日容許攝取量(Tolerable daily intake, TDI),將飲用水建議標準制定為1 μg/L。為了評估飲用水的安全性,目前主要檢測的方式採用高效液相層析串聯質譜儀或酵素連結免疫吸附法 (enzyme-linked immunosorbent assay, ELISA)進行分析。但此兩種方法仍存在缺點,前者初期設備與耗材成本高昂,後者穩定性會受到酵素活性影響。因此本研究將應用拉曼光譜儀(Raman spectroscopy)檢測作為環境水體中MC-LR監測的新興方法,根據化合物的結構所產生的特徵拉曼光譜,建立對於MC-LR定性與定量之檢測方法。
本研究使用加熱蒸鍍製備的奈米多孔金膜之基板,相較於室溫蒸鍍的平坦金膜,其粗糙表面上的孔隙具有表面增強拉曼散射(Surface-enhanced Raman scattering, SERS)之熱點效應,能增強樣品的拉曼訊號。並將溶於甲醇之MC-LR標準品,取1 μL滴塗在基板的表面。在乾燥的過程當中,MC-LR液滴會藉由咖啡環效應將分子濃縮於外圍。依據量測結果咖啡環上擁有最強的MC-LR拉曼訊號,可以推測MC-LR分子會因為甲醇蒸發過程中內部產生的毛細流而被堆積在最外圍的咖啡環,因此可以此作為後續拉曼量測之主要區域。再將拉曼光譜儀的曝光時間、狹縫寬度、接收光譜範圍等操作參數最佳化,作為後續拉曼光譜量測MC-LR實驗的依據。
MC-LR標準品分別以632.8 nm及532 nm雷射量測皆可得到明顯的拉曼光譜,並依據濃度與特徵波長的強度建立檢量線。本研究建立的檢量線其偵測範圍1-500 mg/L,最佳決定係數(Coefficient of determination, R^2)大於0.99,具備良好的相關性。環境樣品經固相萃取法(Solid phase extraction, SPE)純化與吹氮濃縮前處理。然而環境水樣中殘留的有機物所產生的螢光訊號以及拉曼散射可能會掩蓋掉MC-LR之拉曼光譜,造成分析結果異常。本研究建立的拉曼檢測方法需在背景干擾較低的環境下方可有較佳MC-LR的拉曼訊號。環境水樣的應用仍須考量更進一步的純化與前處理流程,因此本研究之結論可提供後續相關實驗參考,以避免環境有機物所產生的背景干擾。
英文摘要 Microcystin-LR (MC-LR), one of the most important cyanotoxins, is carcinogenic and may cause damage of human liver. The World Health Organization (WHO) sets the recommended drinking water standard to 1 μg/L of MC-LR, and the detection methods commonly used include high-performance liquid chromatograph tandem mass spectrometry (HPLC/MS-MS) and enzyme-linked immunosorbent assay (ELISA). However, the instrument itself and the consumables for sample pretreatments are expensive for HPLC/MS-MS, and the activity of enzyme for ELISA is unstable. In addition, the time for detection for both methods are relatively long, in the time scale of hours to days. Therefore, a novel and rapid detection technique, Raman spectroscopy, is a possible alternative method for MC-LR monitoring in fresh water.
In this study, Surface-Enhanced Raman Scattering (SERS) substrates, nano-porous gold film prepared by physical vapor deposition at high temperature, was employed to enlarge the Raman signal. One μL of MC-LR standard dissolved in methanol was dripped on the metal coating substrate. Then during the drying process, MC-LR was concentrated when coffee ring was formed on the periphery of the droplets. Under this condition, the strongest MC-LR Raman signal would be present on the coffee ring. After confirming the characteristic peaks and area of measurement, the parameters of Raman spectroscopy such as exposure time, slit, and spectral range, were optimized in this study.
The MC-LR standards was used to determine that the characteristic Raman peaks measured with 632.8 nm and 532 nm lasers. According to the established calibration curves of concentration of MC-LR and intensity of the characteristic peaks, the detection concentrations were determined to be ranged from 1 to 500 mg/L, with high coefficient of determination, R^2>0.9. To measure environmental samples, water samples were purified with solid phase extraction (SPE) and purged with nitrogen blowing. As the fluorescence, present in natural under Raman scattering, overlapped with the Raman spectrum of MC-LR, to be able to apply the method for environmental analysis, more studies are needed to be conduct for reducing the matrix effect on analysis.
論文目次 第一章 緒論 1
1.1 研究緣起 1
1.2 研究目的 2
第二章 文獻回顧 3
2.1 藍綠菌藻華的危害 3
2.1.1 藍綠菌的特性 3
2.1.2 藍綠菌藻華 4
2.1.3 藍綠菌毒素類別 5
2.1.4 微囊藻毒特性 8
2.1.5 藻華之應變措施 9
2.2 微囊藻毒分析方法 12
2.2.1 酵素連結免疫吸附法 12
2.2.2 結合固相萃取與液相層析質譜儀之分析方法 13
2.2.3 拉曼散射光譜之分析方法 13
2.3 拉曼散射光譜 15
2.4 表面增強拉曼散射(Surface-Enhanced Raman Spectroscopy, SERS) 18
2.4.1 電磁增強效應 18
2.4.2 化學增強效應 20
2.4.3 常見之SERS基板 21
2.5 滴塗沉積拉曼散射分析(Drop coating deposition Raman, DCDR ) 22
2.6 環境基質之螢光干擾 23
第三章 實驗設備與方法 24
3.1 實驗流程 24
3.2 純藻培養 26
3.3 藻類的計數 27
3.3.1 實驗設備 27
3.3.2 實驗藥劑 27
3.3.3 操作流程 27
3.3.4 結果分析 28
3.4 酵素連結免疫吸附法(ELISA) 29
3.4.1 實驗設備與藥劑 29
3.4.2 實驗試劑 29
3.4.3 樣品前處理 30
3.4.4 操作流程 30
3.4.5 濃度換算方式 31
3.5 水樣前處理方法-固相萃取法(Solid Phase Extraction, SPE) 32
3.5.1 實驗設備 32
3.5.2 實驗試劑 32
3.5.3 水樣前處理 33
3.5.4 SPE操作流程 33
3.6 表面增強拉曼散射基板製程 34
3.6.1 實驗設備 34
3.6.2 實驗藥劑 35
3.6.3 石英基板清洗 35
3.6.4 加熱蒸鍍金靶材 35
3.7 滴塗沉積拉曼分析法(DCDR) 37
3.7.1 實驗設備與藥劑 37
3.7.2 滴塗沉積法 38
3.7.3 拉曼光譜儀操作步驟 38
第四章 結果與討論 40
4.1 拉曼光譜儀檢測微囊藻毒方法之建立 40
4.1.1 微囊藻毒LR型之拉曼特徵波長 40
4.1.2 拉曼光譜儀系統之雷射選用 43
4.1.3 基板之選用 45
4.1.4 DCDR之咖啡環效應 47
4.1.5 拉曼光譜儀操作參數之最佳化 49
4.2 微囊藻毒LR型檢量線之建立 59
4.2.1 拉曼光譜儀操作參數 59
4.2.2 MC-LR檢量線選用之特徵波長 60
4.2.3 MC-LR檢量線之結果 64
4.3 應用於環境水樣之分析結果 73
4.3.1 環境水樣之準備 73
4.3.2 環境水樣固相萃取純化與濃縮 74
4.3.3 環境水樣以拉曼光譜量測之結果 76
第五章 結論與建議 84
5.1 結論 84
5.2 建議 85
第六章 參考文獻 86
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