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。

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.

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