次氯酸双光子荧光探针的合成及其在生物成像中的应用

发布时间：2018-03-23 23:39

本文选题：荧光探针　切入点：双光子荧光　出处：《兰州大学》2017年硕士论文

【摘要】：双光子吸收技术自问世以来一直受到了广泛的关注。与单光子吸收材料相比,双光子吸收材料在分辨率、穿透深度具有显著的优势,可以用于显微成像、微纺织技术、三维数据存储、光限幅、上转换发光、光动力学治疗以及药物靶向释放等诸多领域。特别是双光子显微技术,以近红外的激光为光源对生物样品进行成像,具有穿透性强,空间分辨率高,背景荧光干扰小,以及对生物样品的光损伤较小等优点,在生物医学领域具有广阔的应用前景。然而,传统的双光子材料常常具有大共轭结构,水溶性差、细胞穿透能力差、生物毒性也较大,并不适用于生物成像。因此,设计合成具有较高双光子吸收截面的有机小分子用于生物体内细胞、血管、组织成像,具有重要的研究价值。本文设计合成了两种具有双光子吸收特性的荧光小分子,对其发光性能进行了系统的研究,探索它们在生物成像中的应用。具体的研究内容包括:1、设计合成了一类以寡聚苯乙烯为骨架的双光子次氯酸荧光探针OPV-HOCl,并将其应用于活细胞及组织内的双光子成像。在寡聚苯乙烯骨架上引入次氯酸识别基团——氧硫杂环戊烷,通过1H-NMR、13C-NMR、HRMS对其结构进行了表征,并通过紫外光谱、荧光光谱等进一步研究了该探针对次氯酸的响应性能,测定了其双光子吸收截面。加入次氯酸以后,探针分子末端的氧硫杂环戊烷基团被氧化,并生成醛基。由于分子内强烈的电荷转移导致产物的双光子吸收截面提高了近15倍(从78.9 GM提高到1131.5 GM),因此OPV-HOCl可以作为一个双光子“turn-on”型次氯酸荧光探针。此外,该探针还具有反应速度快、选择性好、pH适用范围宽等优点。MTT实验表明该探针具有较小的细胞毒性。由于该探针优异的次氯酸响应性能和较小的生物毒性,我们成功地将其用于小鼠胶质瘤细胞BV-2中次氯酸的检测,研究表明该探针可以透过细胞膜,并对细胞中外源性次氯酸和脂多糖诱导产生的内源性次氯酸具有高选择性的快速响应。2、合成了具有双光子吸收性质的吲哚四方酸染料SQA-Br,并将其负载于微米级的聚苯乙烯微球中,用于脑中风区域的检测研究。研究表明,聚苯乙烯微球可以为染料提供相对惰性的环境,避免了聚集诱导荧光猝灭,使得染料在水溶液中仍具有良好的发光性能,并且染料在微球中的荧光寿命和量子产率与溶液中相比均有一定程度的提高,因此在生物成像中具有潜在的应用价值。我们将负载四方酸的聚苯乙烯微球注射进入中风小鼠体内,发现微球从中风损伤区域扩散出去并在周围区域富集,可以用于中风损伤区域的初步识别。
[Abstract]:Two-photon absorption technology has been paid more and more attention since it came into being. Compared with single-photon absorption material, two-photon absorption material has obvious advantages in resolution and penetration depth, so it can be used in micro-imaging and micro-textile technology. Three dimensional data storage, optical limiting, up-conversion luminescence, photodynamic therapy and drug targeted release, etc., especially two-photon microscopy, imaging biological samples with near-infrared laser as light source, has strong penetrability. Because of its advantages of high spatial resolution, low background fluorescence interference and low light damage to biological samples, it has broad application prospects in biomedical fields. However, traditional two-photon materials often have large conjugate structures and poor water solubility. Therefore, organic small molecules with high two-photon absorption cross sections are designed and synthesized for biological imaging of cells, blood vessels and tissues. In this paper, two kinds of fluorescent small molecules with two-photon absorption characteristics are designed and synthesized, and their luminescence properties are systematically studied. The specific research contents include: 1, a two-photon hypochlorite fluorescence probe OPV-HOCl with oligopolystyrene as skeleton was designed and synthesized, and applied to two-photon formation in living cells and tissues. Like. Introduction of hypochlorite recognition group-oxo-thiocyclopentane on the framework of oligopolystyrene, The structure of the probe was characterized by 1H-NMR-13C-NMR-HRMS. The response of the probe to hypochloric acid was further studied by UV and fluorescence spectra. The two-photon absorption cross section of the probe was determined after the addition of hypochloric acid. The oxygen-thiocyclopentane group at the end of the probe was oxidized. Due to the strong intramolecular charge transfer, the two-photon absorption cross section of the product was increased by nearly 15 times (from 78.9 GM to 1131.5 GMN), so OPV-HOCl can be used as a two-photon "turn-on" type hypochloric acid fluorescence probe. MTT assay showed that the probe had less cytotoxicity because of its excellent hypochloric acid response and low biotoxicity. We have successfully applied it to the detection of hypochloric acid in mouse glioma cell line BV-2. The results show that the probe can penetrate the cell membrane. And the endogenous hypochloric acid induced by exogenous hypochloric acid and lipopolysaccharide was highly selective and rapid response. SQA-Br2, a two-photon absorption dye, was synthesized and loaded into micrometer polystyrene microspheres. The results show that polystyrene microspheres can provide a relatively inert environment for dyes, avoid agglomeration induced fluorescence quenching, and make the dyes still have good luminescence properties in aqueous solution. Moreover, the fluorescence lifetime and quantum yield of dyes in the microspheres were improved to a certain extent compared with those in the solution. We injected polystyrene microspheres loaded with tetragonal acid into apoplectic mice and found that the microspheres spread out from the wind-damaged area and enriched in the surrounding area. It can be used to identify the area of stroke injury.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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