水溶性發光量子點之製備與其在生化檢測系統之應用

Abstract

本論文探討水溶性量子點 (QD)之備製與其在生物檢測系統之應用，本研究以利用硫醇乙酸、硫醇琥珀酸、硫醇十一酸與2-胺基乙硫醇等四種不同包覆試劑取代傳統有機包覆劑十六羥基-2-癸烯酸合成水溶性CdSe/ZnS殼核量子點，並應用於生化分析。我們分別探討一鍋合成與兩步驟合成水溶性量子點的製程，並藉由紫外-可見光譜儀、螢光光譜儀對所合成量子點進行分析與鑑定發光特性。本論文亦利用X光繞射及穿透式電子顯微鏡測定量子點之晶相微結構及粒徑大小，實驗結果顯示水溶性量子點為六方硫化鋅結構，其粒徑大小為4 nm；經上述包覆劑表面修飾後，傅氏紅外光譜儀分析顯示，CdSe/ZnS量子點在波數1677 cm-1處具有C=O之吸收，此可證明十六羥基-2-癸烯酸確實已成為親水性末端之表面修試劑。 另一方面，本論文也利用傳統的生物鍵結技術，將鏈抗生物素蛋白(SA)與量子點結合以形成QD-SA錯合物，並固定不同濃度之anti-human-IgE-biotin於硝化纖維玻片，並以QD-SAv之螢光作為生物晶片偵測之訊號，再利用共軛焦螢光掃瞄器進行anti-human-IgE-biotin-SA-QD錯合物螢光訊號分析。研究結果顯示利用QD-SA (0.1 mg/ml)錯合物可偵測到濃度範圍為3.3 μg/ml 至 100 μg/ml間之anti-human-IgE-Biotin。因此本研究更確認Qd-SA與biotin之特異性，此將奠定其於應用於生物為感測器的基礎。

The research is attempted to investigate the preparation of water-soluble CdSe/ZnS core/shell quantum dots (QDs) and their applications in the biochemical assays. Different capping agents have been used to replace conventional capping agent hexadecylamine (HDA) to form surface-functionalized and water-soluble QDs, which were then used in biochemical analysis. Employing one-pot and two-pot reactions has performed the synthesis of QDs. Optical characterizations of water-soluble and luminescent QDs have been carried out by using UV-Vis and fluorescence spectroscopy. On the other hand, X-ray diffraction (XRD) and transmission electron microscopy (TEM) were also used to determine the microstructure and particle size distribution of QDs. Our investigations revealed that the CdSe/ZnS QDs crystallized in wurtzite structure with diameter of 4 nm. The wavenumber of 1677 cm-1 attributed to C=O stretching has been observed on the surface of capped QDs, indicating successful replacement of HDA by carboxylic-functionalized capping agents. For the applications of QDs in bio-assays, our method involved a glass plate coated with nitrocellulose (NC), where α-human-IgE-biotin at various concentration was immobilized on an NC plate. Luminscent core-shell CdSe/ZnS QDs-SA conjugates were then used as fluorescent labeling agent to be captured specifically by biotinatedα-human-IgE immobilized in a micro-array. A confocal laser scanner was used to detect the fluorescence signals from theα-human-IgE-biotin-SA QD complex. Experimental findings reveal that fluorescence intensity of QD-SA saturated at QD concentrations above 0.4 mg/ml. Moreover, a calibration curve between the fluorescence intensity and the concentration of α-human-IgE-biotin is plotted. The range of detectable concentrations of biotinlated α-human-IgE was found to be between 3.3 μg/ml and 100μg/ml in the α-human-IgE-biotin-SA QD complex. Therefore, the results were consistent with the binding specificity in a plate-based biochemical assay, as determined using luminescent QDs as a labeling agent.