纳米荧光传感器用于细胞及活体内过氧化氢的快速、高灵敏检测

发布时间：2018-04-26 17:12

  本文选题：配位竞争 + 无酶信号放大　； 参考：《山东师范大学》2017年硕士论文

【摘要】：活性氧(ROS)是维持组织和器官的结构和功能稳态的一类非常重要的生物活性分子。其中,过氧化氢(H2O2)不仅是细胞信号转导的第二信使,也是氧化应激的重要标记物。过量产生的H2O2会导致氧化损伤,从而引起衰老和心血管疾病、糖尿病、癌症等。因此,监控细胞及活体内H2O2的浓度变化和时空分布变得尤为重要。荧光成像技术灵敏度高、选择性好、对生物体无损坏,已成为H2O2检测的最佳方法。但是由于H2O2具有寿命短、反应活性较大、浓度低以及对环境敏感等特点,使得研究起来存在一定困难。此外,目前用于检测H2O2的小分子荧光探针大多存在选择性差、响应速度慢,荧光量子产率低、易光漂白等不足,难以真实反映细胞及活体内H2O2的水平变化。因此,迫切需要发展高选择、高灵敏、快速响应的H2O2荧光探针或传感器,从而实现细胞及活体内H2O2实时、动态的可视化示踪。纳米技术的发展恰为实现这一目标提供了契机。纳米材料由于其纳米级别的尺寸,使其具有超常的化学反应活性、分散与团聚能力、催化性能以及吸附能力。因此,可与DNA分子通过π-π堆积、静电力及金属配位等多种分子间作用力组装成纳米荧光探针/传感器,为生物分子的检测提供了有力工具。虽然它们在蛋白质、核酸等大分子检测方面已有很多应用,然而用于小分子H2O2检测成像的研究还鲜有报道。鉴于此,本文综述了荧光探针在H2O2检测方面的研究现状及发展趋势,并基于配位竞争和无酶信号放大机理,利用氧化铈纳米线(CeO2 NWs)和氧化石墨烯(GO)两种纳米材料与DNA和H2O2之间的特殊吸附作用,构建了以下两种H2O2纳米荧光传感器实现了细胞及活体内H2O2的快速、高选择性和高灵敏度的检测:1、基于配位竞争,发展了一种由CeO2 NWs与羧基荧光素(FAM)标记的单链(ss)DNA组装而成的H2O2纳米荧光传感器CeO2 NWs-DNA(CNWD)。由于CeO2 NWs的高荧光猝灭效率,通过磷酸根与Ce4+配位而吸附的DNA的荧光被猝灭;当遇到H2O2时,由于H2O2与Ce4+的更高配位能力,吸附的DNA被竞争下来,荧光恢复。而且CeO2 NWs高长宽比的一维纳米结构使得DNA吸附密度提高,随之灵敏度也得到提高。基于这些优点,所设计的CNWD对H2O2具有瞬时响应和高选择性的特点,且能够对活细胞内的H2O2进行示踪并能对斑马鱼氧化损伤产生的H2O2实时成像。这种传感器提供了一种全新的简便的检测H2O2的方法,对于发现和研究H2O2调控的信号通路尤为重要。更重要的是,通过利用合适的纳米材料和功能化的DNA,这种配位竞争的机理能够用于多种活性小分子的检测。2、基于无酶信号放大,发展了一种组合纳米荧光传感器CeO2 NWs/GO(CG)来对细胞内的H2O2进行高灵敏荧光成像。首先设计合成了3条花菁5(Cy5)标记的、可发生杂交链式反应(HCR)的ssDNA,分别为Flare、发卡型H1和H2。由于CeO2 NWs和GO均具有良好的荧光猝灭能力,且都能吸附ssDNA,因此我们用CeO2 NWs和Flare、GO和H1/H2分别合成了CF以及GH,然后将二者2:1组合为CG纳米荧光传感器。当H2O2存在的时候,可以使CeO2 NWs表面吸附的Flare释放下来,发生荧光的恢复;释放下来的Flare会接着与GO上吸附的H1/H2发生HCR形成长的双链(ds)DNA,由于GO对DNA双链的吸附力远远弱于单链,因此双链上的Cy5也随之远离GO,荧光信号进一步增强。与纳米荧光传感器CNWD相比,CG显著提高了H2O2检测的灵敏度,检测限低至100 nM,可以更好的用于细胞内源性H2O2的检测及荧光成像。这种基于无酶信号放大的方法还为实现细胞内活性小分子的高灵敏检测提供了新的思路。
[Abstract]:Active oxygen (ROS) is a very important class of bioactive molecules to maintain the structure and function homeostasis of tissues and organs. Among them, hydrogen peroxide (H2O2) is not only the second messenger of cell signal transduction, but also an important marker for oxidative stress. Excessive production of H2O2 can lead to oxygen damage and cause aging and cardiovascular disease, diabetes, Therefore, it is very important to monitor the concentration changes and space-time distribution of H2O2 in cells and living bodies. Fluorescence imaging technology has high sensitivity, good selectivity and no damage to the organism. It has become the best method for H2O2 detection. But because H2O2 has the characteristics of short life, large reaction activity, low concentration and environmental sensitivity, it has made research. There are some difficulties. In addition, most of the small molecular fluorescent probes used to detect H2O2 are poor in selectivity, slow response, low fluorescence quantum yield, and easy to light bleaching. It is difficult to truly reflect the level changes of H2O2 in cells and living bodies. Therefore, it is urgent to develop a H2O2 fluorescent probe with high selectivity, high sensitivity and rapid response. The development of nanotechnology provides an opportunity to achieve this goal. Nanomaterials, due to their nanoscale size, make them have abnormal chemical reaction activity, dispersing and reunion capacity, catalytic properties and adsorption capacity. Therefore, it can be used with DNA molecules. Over pion pion accumulation, static electricity and metal coordination and other intermolecular forces are assembled into nanofluorescence probes / sensors, which provide a powerful tool for the detection of biomolecules. Although they have been widely used in the detection of large molecules such as proteins and nucleic acids, there are few reports on the detection of small molecule H2O2 imaging. The current research status and development trend of fluorescent probe in H2O2 detection are reviewed. Based on the mechanism of coordination competition and non enzyme signal amplification, the following two kinds of H2O2 nanofluorescence sensors are constructed by using the special adsorption between two nanomaterials, CeO2 NWs and GO, and DNA and H2O2. The rapid, high selective and highly sensitive detection of H2O2 in the living body: 1, based on coordination competition, a H2O2 nanofluorescence sensor CeO2 NWs-DNA (CNWD), composed of CeO2 NWs and carboxyl fluorescein (FAM) labeled single strand (SS) DNA, was developed. The fluorescence is quenched; when the H2O2 is encountered, the adsorbed DNA is competitive and the fluorescence is restored due to the higher coordination ability of H2O2 and Ce4+. And the CeO2 NWs high width ratio one-dimensional nanostructure makes the DNA adsorption density increase and the sensitivity increases. Based on these advantages, the designed CNWD has instantaneous response and high selectivity to H2O2. It is characterized by the ability to trace the H2O2 in living cells and to real-time imaging of H2O2 in zebrafish oxidative damage. This sensor provides a new and simple method for detecting H2O2, which is particularly important for the discovery and study of the signal pathways regulated by H2O2. More importantly, the use of suitable nanomaterials and functional DN is important. A, the mechanism of the coordination competition can be used to detect.2 for a variety of small active molecules. Based on the non enzyme signal amplification, a combined nano fluorescence sensor CeO2 NWs/GO (CG) is developed for high sensitive fluorescence imaging of H2O2 in the cell. First, 3 cyanine 5 (Cy5) markers are designed and synthesized, and the ssDNA of the hybrid chain reaction (HCR) can occur. Flare, H1 and H2. have good fluorescence quenching ability and can adsorb ssDNA because of CeO2 NWs and GO. Therefore, we use CeO2 NWs and Flare, GO and H1/H2, respectively. The release of the fluorescence is restored; the release of Flare will follow the HCR - shaped double chain (DS) DNA with the HCR - shaped growth of the H1/H2 adsorbed on the GO, because the GO absorbability of the double chain is far weaker than the single strand, so the Cy5 on the double chain is far away from GO and the fluorescence signal is further enhanced. Degree, the detection limit is low to 100 nM, can be better used for cell endogenous H2O2 detection and fluorescence imaging. This method based on no enzyme signal amplification also provides a new way of thinking for high sensitivity detection of small cell active molecules.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;TP212
，

本文编号：1806910