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標題: | 發展含螢光探針核心結構的水解酵素基質之快速合成方法 The rapid synthesis of a fluorescent probe core for hydrolytic enzymes |
作者: | Tzu-Chen Liu 劉子晨 |
指導教授: | 吳世雄 |
關鍵字: | 核心分子,螢光探針,螢光共振能量傳播,比例測量, core,fluorescent probes,fluorescence resonance energy transfer,ratiometric measurement, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 比例測量 (ratiometric measurement) 是一種可以同時偵測兩種波長螢光強度以及計算兩者比例的量測方式,它可提供準確的數據、甚至是定量的偵測。為了要達成這樣的概念,探針 (probe) 必須要在與目標分子反應後,顯示出大的吸收或是放出波長的改變。這樣的策略,根據螢光共振能量傳播 (fluorescence resonance energy transfer),藉由兩個螢光團的架構,由香豆素 (coumarin) 提供者、環己烷連接橋、螢光素 (fluorescein) 接受者所組成。這樣的架構已經在許多螢光感應器被應用。然而,所有的先前成果,雖然有著相似的結構,卻都是冗長且浪費的從第一步合成自最後一步。所以為了開發一個更有效的模型,在這個研究分為兩個部份。首先,致力於開發「快速」合成螢光核心分子的步驟。接著,藉由在酚上面的兩個羥基,去接上不同的官能集團。最後,這個想法證明是有實用價值的,因為羥基具有化學反應活性,可以接脂肪酸、硫酸鹽、硫酸鹽保護基。進一步的螢光光譜亦證實螢光核心分子的放出波長在接上官能基前後有著大約 40 nm 的差異。此份工作的價格在於建立了一個可以偵測不同水解酵素的平台,並在研究的過程中,發現了利用一從未被開發的再結晶分離羧基螢光素 (carboxyfluorescien) 位向異構物 (regioisomers) 的方法,可以得到超過兩位數克數等級的產物。 A ratiometric measurement, i.e. simultaneously recording of the fluorescence intensities at two wavelengths and calculation of their ratio, is a technique that can provide precise data and even quantitative detection. To carry out this idea, the probe must exhibit a large shift in its emission or excitation spectrum after it reacts with the target molecule. A strategy that is based on fluorescence resonance energy transfer (FRET) by using a two-fluorophore cassette comprised of a coumarin donor, a cyclohexane linker and a fluorescein acceptor has been utilized in many fluorescent sensors. However, with this structural similarity, all of the previous works were done by synthesizing from the first step to the last step, which is wasteful and tedious. Therefore, in order to establish a more efficient model, in this study, our work is separated into two parts. First, we focus on developing a fast procedure of synthesizing fluorescent probe core. Second, we utilize the two hydroxyl groups on the phenols to link with different functional groups. The conclusion shows the results to be practical as the hydroxyl group is chemically reactive and is able to link with fatty acid, sulfate and the protecting group of sulfate. Further fluorescence spectra also confirm that the emission wavelength of the fluorescent probe core differs by around 40 nm before/after the conjugation with functional groups. The value of this work is the establishment of a platform suitable for detecting various hydrolytic enzymes, and during the process of this study, we discovered a method to separate the regioisomers of carboxyfluorescien via fractional recrystallization in a double-digit gram scale. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26673 |
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顯示於系所單位: | 生化科學研究所 |
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