用于检测活性小分子的快速响应型荧光探针的设计、合成及生物应用

【作者】 刘伟；

【导师】 唐波；

【作者基本信息】 山东师范大学 ， 分析化学， 2016， 硕士

【摘要】 活性小分子在细胞功能的调控、信号传导、新陈代谢等过程中发挥重要作用。有研究发现过量产生的活性小分子与一系列疾病的发生发展紧密相关。因此,实时监测活性小分子在体内的变化,更好的了解它们在细胞中的生物学功能,发展理想的检测工具是十分必要的。目前,荧光探针已经广泛应用于细胞内活性小分子的分析检测,但由于活性小分子本身具有稳态浓度低,反应活性高等特性,且生物体内复杂的环境都给活性小分子的检测带来了一定难度。同时现有探针与活性小分子的反应时间长,这也给在细胞水平监测小分子的真实浓度变化带来了很大困难。因此,设计合成选择性好、快速响应、灵敏度高的荧光探针用于检测活细胞内活性小分子的真实浓度是生命化学学科发展中具有挑战性的前沿课题之一,尤其是对高选择性、快速响应的荧光探针的设计、合成还是非常具有挑战性的。基于此,我们分别发展了三种新型荧光探针,实现了对细胞及活体内HOCl、O2?-及H2O2的快速荧光成像检测。本论文主要包括以下内容:1、基于硒易被氧化还原的特性,利用2,7二溴芴酮作为骨架,设计合成了一种兼具单、双光子荧光性质的荧光增强型探针FO-PSe。该探针FO-PSe具有高选择性、快速响应、可逆等优点。高灵敏的单光子成像和高分辨、深组织穿透深度的双光子成像均表明:探针FO-PSe能够成功地可视化PMA、Zymosan刺激后的小鼠巨噬细胞、斑马鱼和小鼠体内HOCl。因此,探针FO-PSe是一种能够动态可逆地成像观察活细胞及活体内不同深度HOCl水平的理想工具。2、我们通过水热法一步合成了一种以碳点为基体的,兼具单、双光子荧光特性的荧光纳米探针,合成方法简单、所需原料廉价。并且用此方法合成的碳点具有较好的荧光性能、水溶性、光稳定性以及低的细胞毒性。与其它合成碳点的方法相比,此实验过程简便且荧光性能也更好。我们合成的这种基于碳点的纳米探针能够高选择性地、瞬时、可逆地检测O2?-水平的动态变化。单、双光子成像均表明:该碳点能够成功地可视化肝细胞内和小鼠体内O2?-变化,还能够动态可逆地检测凋亡刺激下的细胞内O2?-浓度波动。因此,我们合成的这种基于碳点的纳米探针是一种能够动态可逆地成像观察活细胞及活体内不同深度O2?-水平的理想工具。3、我们设计合成了一种对过氧化氢快速响应的荧光探针TPE-BO,TPE-BO含有四苯乙烯和硼酸酯两个单元。以易得的已合成的两种化合物为起始原料,经过一步简易、高效的合成反应制得该探针,且后处理容易、纯化简便。该荧光探针具有聚集诱导发光性质,能有效地降低探针与过氧化氢的反应时间,而且对过氧化氢具有高选择性,能实现细胞内原位检测过氧化氢的浓度变化。该探针为研究活细胞内过氧化氢的功能及生物学意义提供了一种良好的荧光工具。更多还原

【Abstract】 Reactive small molecule is closely associated with organism physiology, pathology and aging. Abundant evidence suggests that excessively produced reactive small molecule have been implicated in a wide variety of diseases. Therefore, reactive small molecule plays an important role in biology, it is pressing to develop powerful tools for real-time monitor in cellular level.At present, various fluorescence probes has been developed and applied for the reactive small molecule analysis. However, when the probe reacted with the reactive molecule, the recognition reaction is completed after a long times, it has become an important yet complex issue for monitoring reactive molecule concentration level in living cells. If you want to study true concentration of reactive molecule in living cells at specific areas, the slow response probe can not meet the demand. Therefore, development of a rapid and highly selective method for reactive small molecule analysis in living cells is of great significances.Therefore we develop three rapid-response fluorescence probes to detect hypochlorous acid(HOCl), Superoxide anion(O2?-) and hydrogen peroxide(H2O2).The main results of the dissertation were shown as follows:1. We design a novel turn-on probe(FO-PSe) for HOCl was designed based on the 9-fluorenone covalently conjugating with two selenium-containing compounds. The reversible recognition ability of the probe can be achieved through the oxidation–reduction cycles of the selenium center. The 9-fluorenone was selected as a two-photon(TP) fluorophore because of its excellent TP fluorescent performance. In the presence of HOCl, the selenium of the probe was oxidized specifically and the π-electron of the conjugated system redistributed accordingly. Results showed that one-photon and two-photon fluorescence of FO-PSe enhanced markedly in the presence of HOCl. Importantly, the probe FO-PSe was successfully applied to cells, zebrafish and mice to dynamically visualize the changes of the HOCl levels under various stimulations. Compare with previous fluorescent probe for HOCl, the probe combined the merits of deep penetration, less photodamage, reversibility and instantaneity.2. We present a simple but effective carbon dots(CDs)-based sensor for reversible fluorescence imaging of O2?- in live cells and in vivo. The CDs-based sensor is fabricated by one-step strategy, using FO-PSe as the sole precursor. The as-prepared CDs-based sensor is directly functionalized by selenium center in the process of the synthesis, which directly introducing active sites and no any additional surface modification was needed. Moreover, the sensor was successfully applied to imaging of the O2?- levels in mice via two-photon imaging according to depths of various vivo. Due to simplicity and effectivity, the sensor was demonstrated as a useful fluorescent tool for tracking O2?- levels in cells and in vivo. To our knowledge, it is the most simplicity and effectivity CDs-based fluorescent sensor for O2?-. Therefore, it can be employed as a useful fluorescent tool for tracking superoxide anion levels in cells and in vivo.3. Aiming at widespread reaction speed problem of hydrogen peroxide probe, we have designed and synthesized a novel aggregation induced emission(AIE) fluorescence probe TPE-BO with activity based on increased polarity of C-B bonds, which can rapid-response and highly selective monitoring of H2O2 in living cells. The present study provides a fluorescent tool for rapid monitoring of hydrogen peroxide in living cells and we believe that the strategy for improve the electrophilic reaction activity to achieve the rapid fluorescence response will be broadly applicable to the quantitative monitoring active small molecules in biological systems.更多还原