基于硼酸酯的活性氧响应荧光分子探针的构建及应用

发布时间：2018-08-01 18:40

【摘要】：氧气(O_2)对于所有的需氧生物来说都是必需的,在酶或金属离子等的作用下,O_2可以被转化成一系列活性代谢产物,活性氧(ROS)就是其中最重要的一种产物。ROS包括过氧化氢(H202),超氧阴离子(O_2·-),羟基自由基(·OH),单线态氧(1C2),过氧自由基(ROO·),次氯酸及次氯酸盐(HOC1/-OCl)等。许多研究表明,ROS在生命活动中是重要的信号分子,但是,过量的ROS可以导致氧化损伤的累积,这些损伤可能会导致细胞凋亡、老化、癌变等疾病。H202是非常重要的一种ROS,相对于其它的ROS,H202比较稳定。正常状态下,细胞内的H202处于相对低的水平。但是,如果细胞内产生了过量的h2O_2,它将会与低价态的金属离子(如:Fe2+、Cu2+)反应,产生高反应活性的·OH,造成细胞功能紊乱。O_2·-是另一种十分关键的ROS,是其它ROS的重要来源。因此,探究检测h2O_2/O_2·-的有效方法一直处于不断的研究之中。其中,荧光分析因具有操作简单、灵敏度高、选择性好等优点,成为近年来研究的热点。比率型荧光探针是以两个波长处荧光强度的比值作为响应信号,信号强度与探针浓度、光源强度、仪器的灵敏度等因素无关,所以,能够有效地防止背景干扰,提高检测的灵敏度。基于以上背景,我们合成了基于硼酸酯的荧光分子探针,分别实现了对H202的比率型检测和对O_2·-的双功能灵敏性检测。同时基于酶特异性催化氧化反应,比率型聚合物纳米探针可用于能够产生H202的生物基质(如:葡萄糖、胆碱、胆固醇、乳酸)的检测。进而,利用比率型聚合物纳米探针实现了对血清中葡萄糖的检测。论文的具体内容如下:第一章绪论首先本章介绍了 ROS及其相关研究,然后讨论了 H202/02·-及其研究现状。随后介绍了聚合物胶束的相关知识,最后对于本论文研究工作的目的和意义作了表述。第二章基于ARS-硼酸酯衍生物的荧光探针对H202的检测本工作利用硼酸衍生物与ARS结合后可以产生荧光的特性及H202可以将苯硼酸酯转化成苯酚从而使荧光淬灭的性质,实现了对H202的检测。首先,选用带有不同取代基的苯硼酸衍生物与ARS结合,形成ARS-硼酸酯衍生物荧光分子探针,检测结合后的紫外吸收及荧光发射光谱。通过荧光光谱我们计算出了 ARS与苯硼酸衍生物及硼酸(BA)的结合常数。其次,将探针与H2O_2作用,测定反应后的紫外吸收及荧光发射光谱。结果表明,苯硼酸衍生物取代基不同,与ARS结合后形成的探针对H202响应的灵敏度也不同。我们选用有最大响应的ARS-4-羧基-3-氟-苯硼酸(ARS-FPBA)实现了对H202的灵敏性和选择性的检测。第三章自组装比率型聚合物纳米探针对H2O_2的高选择性检测本工作我们构建了一种基于胶束的比率型荧光纳米探针。首先,将FPBA及7-羟基香豆素(HC)通过缩合反应修饰在聚合物两亲性分子二硬脂酰磷脂酰乙醇胺-氨基聚乙二醇(DSPE-PEG-NH2)的亲水端,形成DSPE-PEG-FPBA和DSPE-PEG-HC。PEG-FPBA及PEG-HC是亲水端,DSPE是疏水端。当聚合物加入到水相中时,可形成球状水溶性胶束。然后,将胶束与ARS混合后,ARS与FPBA形成ARS-FPBA而结合在胶束的表面。在405 nm波长光的激发下,HC和ARS-FPBA的荧光发射波长分别在450 nm和600 nm。ARS-FPBA的吸收峰在455 nm,与HC的发射峰(450 nm)有较大重叠,所以,HC与ARS-FPBA之间可以发生荧光共振能量转移(FRET)。将胶束与H202共同孵育后,ARS从胶束上脱离,FRET效率降低,在450 nm处HC的荧光增强,在600 nm处ARS-FPBA的荧光减弱,实现了对H202的比率型荧光检测。此外,我们还实现了对血清中葡萄糖的检测。第四章荧光/比色法双功能探针的合成及其对于O_2·-的灵敏性检测本工作中,通过在二氰亚甲基-4H-苯并吡喃(DCM)分子中引入苯硼酸基团,合成了一种既可以用比色法又可以通过荧光法检测O_2·-的双功能探针。我们通过甲基与醛基之间的缩合反应合成了探针DCM-PBA,并检测了其对O_2·-的响应。与O_2·-作用后,随着O_2·-浓度的增大,探针位于560 nm处的吸光度增加,溶液颜色由黄色变为蓝色。同时,在波长为560 nm光的激发下,探针位于700 nm处的荧光逐渐增强。因此,可以通过比色和荧光的方法来同时实现对O_2·-的检测。
[Abstract]:Oxygen (O_2) is necessary for all aerobic organisms. Under the action of enzymes or metal ions, O_2 can be converted into a series of active metabolites. Reactive oxygen species (ROS), the most important product of which,.ROS includes hydrogen peroxide (H202), superoxide anion (O_2), hydroxyl radical (OH), single state oxygen (1C2), and superoxide self. ROO, hypochloric acid and hypochlorite (HOC1/-OCl). Many studies have shown that ROS is an important signal molecule in life activities. However, excessive ROS can lead to the accumulation of oxidative damage. These damage may lead to cell apoptosis, aging, cancer and other diseases,.H202 is a very important ROS, relative to the other ROS, H202 comparison. Stable. Under normal conditions, the H202 in the cell is relatively low. However, if the cell produces an excessive amount of h2O_2, it will react with the low valence metal ions (such as Fe2+, Cu2+) and produce a highly reactive. OH, causing cell dysfunction.O_2. It is another crucial ROS, an important source of other ROS. The effective methods for detecting h2O_2/O_2 - - have been in constant research. Among them, fluorescence analysis has become a hot topic in recent years because of its advantages of simple operation, high sensitivity and good selectivity. The ratio type fluorescence probe is a response signal with the ratio of fluorescence intensity at two wavelengths, signal intensity and probe concentration, and the intensity of light source. The sensitivity of the instrument is independent, so it can effectively prevent background interference and improve the sensitivity of detection. Based on the above background, we synthesized a fluorescence molecular probe based on borate ester, and realized the ratio detection of H202 and the dual functional sensitivity detection of O_2. The rate type polymer nanoprobe can be used for the detection of biological matrix (such as glucose, choline, cholesterol and lactic acid) that can produce H202. Then, the detection of glucose in serum is realized by using the ratio polymer nanoprobe. The contents of the thesis are as follows: first chapter of the chapter introduced the ROS and its related research, and then discussed it. H202/02 - and its current research status. Subsequently, the related knowledge of polymer micelles was introduced. Finally, the purpose and significance of the research work in this paper were expressed. The second chapter based on the detection of H202 based on the fluorescence probe of borate derivative ARS- in this work, the properties of fluorescence can be produced by the combination of boric acid derivatives with ARS and H202 can be used. H202 was detected by the conversion of benzyl borate to phenol and fluorescence quenching. First, the derivatives of benzboric acid with different substituents were combined with ARS to form a ARS- borate derivative fluorescent molecular probe, which was used to detect the UV absorption and fluorescence emission spectra after the combination. The ARS and benzene were calculated by fluorescence spectra. The binding constants of boric acid derivatives and boric acid (BA) are determined. Secondly, the UV absorption and fluorescence emission spectra of the reaction are determined by the action of the probe and H2O_2. The results show that the substituent of the benzyl boric acid derivatives is different, and the sensitivity of the probe formed after the combination with ARS is different. We choose the ARS-4- carboxyl -3- fluoro benzyl boric acid (A) with the maximum response (A). RS-FPBA) the sensitivity and selectivity of H202 were detected. In the third chapter, the high selectivity detection of H2O_2 by self assembled polymer nano probe, we constructed a ratio based fluorescent nano probe based on micelles. First, the FPBA and 7- hydroxyl coumarin (HC) were modified in the polymer two amphiphilic molecules by condensation reaction. Two the hydrophilic end of the two stearyl phosphatidyl ethanolamine aminopolyethylene glycol (DSPE-PEG-NH2), forming DSPE-PEG-FPBA and DSPE-PEG-HC.PEG-FPBA and PEG-HC are hydrophilic ends and DSPE is the hydrophobic end. When the polymer is added to the water phase, the spherical water-soluble micelles can be formed. Then, the micelle is mixed with ARS and ARS and FPBA form ARS-FPBA to combine in micelle. Surface. Under the excitation of 405 nm wave long light, the fluorescence emission wavelengths of HC and ARS-FPBA are at 450 nm and 600 nm.ARS-FPBA respectively, and the absorption peaks are 455 nm, and the emission peaks of HC (450 nm) are overlapped greatly. Therefore, the fluorescence resonance energy transfer (FRET) can occur between HC and ARS-FPBA. The rate was reduced, the fluorescence of HC was enhanced at 450 nm and the fluorescence of ARS-FPBA was weakened at 600 nm. The ratio of fluorescence to H202 was detected. In addition, we also realized the detection of glucose in the serum. The fourth chapter of the fluorescence / colorimetric dual function probe and the detection of the sensitivity of the O_2. A bifunctional group is introduced into the benzo - DCM molecule, and a bifunctional probe, which can be used to detect O_2 - - by the colorimetric method and the fluorescence method, is synthesized. The probe DCM-PBA is synthesized by condensation reaction between methyl and aldehyde groups. The response of the probe to O_2 - - is detected. After the action of O_2. The probe is increased with the concentration of O_2. The absorbance at 560 nm is increased and the color of the solution changes from yellow to blue. At the same time, the fluorescence of the probe at 700 nm is gradually enhanced under the excitation of the wavelength of 560 nm light. Therefore, the detection of O_2 by colorimetric and fluorescence can be achieved simultaneously.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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