过氧亚硝酸盐纳米发光探针的构建

发布时间：2018-08-06 09:46

【摘要】：活性氧在自然界和生物体中普遍存在,是导致生命体产生疾病、衰老的隐形杀手。为了更好地理解活性氧在生理病理过程中的作用,对活性氧的选择性测定尤为重要。其中,过氧亚硝酸盐是较自由基寿命更长的活性氧物质,但是在生理条件下,其寿命也仅为10 ms。此外,活性氧物质之间还会发生级联反应,化学行为复杂。因此,生物体系中活性氧的选择性测定比较困难。目前,发展最为迅速的是荧光分析法,主要是基于荧光团发生氧化反应后荧光信号的变化来进行测定。当活性氧的氧化性差别不大时,很难实现对某一种活性氧的选择性识别和反应。与荧光分析法相比,化学发光分析法最显著的优势是无需激发光源,从而能够有效避免背景干扰。本论文基于过氧亚硝酸盐体系的化学发光行为,开发了新型过氧亚硝酸盐纳米发光探针,包括蒙脱土纳米片、半导体量子点和碳点。本论文使用静态注射化学发光分析方法,实现了活细胞内过氧亚硝酸盐的高选择性、高灵敏测定。此外,设计合成了对活性氧高稳定的荧光金量子点,有望解决纳米发光探针易被活性氧氧化导致发光猝灭、灵敏度降低的问题,对构建专一性活性氧发光探针具有指导意义。论文主要研究内容如下:(1)开发了一种新型的纳米催化剂—蒙脱土纳米片,用于增敏过氧亚硝酸盐的超微弱化学发光。将蒙脱土纳米颗粒分散在水介质中,通过搅拌获得蒙脱土纳米片,考察了其对过氧亚硝酸盐体系化学发光的影响。研究表明,蒙脱土纳米片能够显著增强过氧亚硝酸盐体系的化学发光。采用透射电子显微镜、原子力显微镜等测试方法对蒙脱土纳米片的形貌进行了表征,并通过活性氧捕获剂、电子自旋共振、化学发光光谱对反应中间体和发光体进行确认,推测了蒙脱土纳米片增敏化学发光的机理。在碱性条件下,蒙脱土纳米片结构中的铁物种能够催化过氧化氢分解,产生羟基自由基,进而与过氧亚硝酸盐反应生成单线态氧,当其回到三线态时产生较强的化学发光。(2)基于活性氧诱导半导体量子点的直接化学发光行为,构建了一种选择性测定过氧亚硝酸盐的化学发光探针。由于过氧亚硝酸盐在生理条件下能够分解同时产生氧化性自由基羟基自由基和还原性自由基超氧阴离子自由基,羟基自由基能够具有向量子点注入空穴的能力,超氧阴离子则具有向量子点注入电子的能力,最终通过电子转移湮灭的机制产生化学发光。在一系列活性氧中,该探针对过氧亚硝酸盐显示出很好的选择性。采用该探针测定过氧亚硝酸盐,线性范围为0.46～46μM,检出限为0.11μM (S/N=3)。通过测定活细胞中的外源性过氧亚硝酸盐,对该探针的实用性进行考察,其测定结果与理论值基本一致。(3)基于碳点表面态发光机制,构建了一种高灵敏度的过氧亚硝酸盐化学发光探针。采用微波法,通过改变合成前体柠檬酸和尿素的比例,对碳点的表面态发光进行了调控。通过傅里叶变换红外光谱和X-射线光电子能谱对碳点的表面官能团进行表征。研究表明,碳点的表面态发光主要来源于表面的C-O基团。随着C-O含量增加,碳点对过氧亚硝酸盐的化学发光响应逐渐增强。通过循环伏安测试对碳点的能级进行评估,探讨了碳点对过氧亚硝酸盐的响应机理。利用该探针测定过氧亚硝酸盐的线性范围为0.01～3.0 μM,检出限为5.0 nM (S/N=3)。该探针表现出很好的生物相容性和低毒性,被成功应用于测定活细胞中外源性和内源性过氧亚硝酸盐。使用该化学发光探针测定活细胞内的过氧亚硝酸盐操作简单、选择性好、灵敏度高。(4)通过改变表面配体的结构,设计合成了一种稳定的红色荧光金量子点,并作为荧光标记物用于高活性氧和长时间细胞标记成像。将牛血清蛋白为配体的蓝色荧光金量子点与二氢硫辛酸为配体的红色荧光金量子点通过碳二亚胺偶联法结合在一起,形成新的金量子点。一方面,通过荧光共振能量转移,使得金量子点的红色荧光增强。另一方面,牛血清蛋白与二氢硫辛酸的共价连接有效地减少了牛血清蛋白分子中的螺旋结构,在金核表面形成稳定的保护层,使得金量子点的耐氧化性和耐酶解能力明显提高。该策略可用于调控金量子点的耐氧化性,以构建稳定的专一性活性氧发光探针。
[Abstract]:Active oxygen is common in nature and living organisms. It is a stealth killer that causes diseases and senescence in life bodies. In order to better understand the role of active oxygen in physiological and pathological processes, selective determination of reactive oxygen species is particularly important. In addition, its life is only 10 ms. in addition, and the reactive oxygen species may also be cascaded and the chemical behavior is complex. Therefore, the selective determination of active oxygen in the biological system is difficult. At present, the most rapid development is the fluorescence analysis method, mainly based on the change of fluorescence signal after the fluorophore oxidation reaction. It is difficult to recognize and react to a certain kind of reactive oxygen species when the oxidation of active oxygen is small. Compared with the fluorescence analysis method, the most significant advantage of the chemiluminescence analysis method is that the light source is not needed, thus the background interference can be avoided effectively. Based on the Chemiluminescence Behavior of the peroxisate system, a new type of chemical luminescence is developed. In this paper, the high selectivity and high sensitive determination of peroxy nitrite in living cells is achieved by static injection chemiluminescence analysis. In addition, a high stable fluorescent gold quantum dot for active oxygen is designed and synthesized, which is expected to solve the nano hair. The optical probe is easily destroyed by reactive oxygen oxidation and leads to the quenching of luminescence. The problem of sensitivity reduction is of guiding significance to the construction of a specific reactive oxygen luminescence probe. The main contents of this paper are as follows: (1) a new nano catalyst, montmorillonite, was developed to sensitize the ultra weak chemiluminescence of nitrite. The effect of Montmorillonite Nanoparticles on the chemiluminescence of peroxy nitrite system was investigated by mixing rice particles in water medium. The results showed that the montmorillonite nanoplates could significantly enhance the chemiluminescence of the peroxy nitrite system. The montmorillonite nanostructures were tested by transmission electron microscopy and atomic force microscopy. The morphology of the tablets was characterized, and the reaction intermediates and luminescent bodies were confirmed by the active oxygen capture agent, electron spin resonance and chemiluminescence spectrum. The mechanism of sensitizing chemiluminescence of montmorillonite nanoscale was speculated. Under alkaline conditions, the iron species in the montmorillonite nanostructure can catalyze the decomposition of hydrogen peroxide and produce hydroxyl free radicals. Based on the reaction of the peroxy nitrite to produce a single state oxygen, a strong chemiluminescence is produced when it returns to the three line state. (2) a chemiluminescence probe for selective determination of peroxy nitrite is constructed based on the Direct Chemiluminescence Behavior of the semiconductor quantum dots induced by reactive oxygen species. The solution produces both the oxidation free radical hydroxyl radical and the reductive free radical superoxide anion radical. The hydroxyl radical can have the ability to inject holes into the quantum dots, and the superoxide anion has the ability to inject electrons into the quantum dots. Finally, the chemiluminescence is produced by the electron transfer annihilation mechanism. In a series of reactive oxygen species, the superoxide anion can produce chemiluminescence. The probe is good selectivity for peroxy nitrite. The determination of peroxy nitrite with this probe is 0.46 ~ 46 mu M and the detection limit is 0.11 mu M (S/N=3). The practicability of the probe is examined by measuring the exogenous peroxy nitrite in living cells. The results are in agreement with the theoretical value. (3) based on the carbon point. A highly sensitive peroxy nitrite chemiluminescence probe was constructed by the surface state luminescence mechanism. The surface state luminescence of the carbon point was regulated by changing the proportion of citric acid and urea by microwave method. The surface functionalities of the carbon point were characterized by Fu Liye transform infrared spectroscopy and X- ray photoelectron spectroscopy. The study shows that the surface state luminescence of the carbon point is mainly derived from the C-O group on the surface. With the increase of C-O content, the chemiluminescence response of the peroxisome is gradually enhanced. The response mechanism of the carbon point to the peroxy nitrite is evaluated by cyclic voltammetry, and the peroxy nitrite is determined by the probe. The linear range of salt is 0.01 ~ 3 M and the detection limit is 5 nM (S/N=3). The probe shows good biocompatibility and low toxicity. It has been successfully applied to the determination of exogenous and endogenous peroxises in living cells. The use of the chemiluminescence probe for the determination of peroxy nitrite in living cells is simple, selective and highly sensitive. (4) a stable red fluorescent quantum dot was designed and synthesized by changing the structure of the surface ligand, and used as a fluorescent marker for high active oxygen and long time cell labeling imaging. The blue fluorescent gold quantum dots with the ligand of bovine serum protein were coupled with the red fluorescent quantum dot of the two hydrogen sulfide octanoic acid as the ligand of the two imide coupling. Together, the new gold quantum dots are formed. On the one hand, the red fluorescence of the gold quantum dots is enhanced by the fluorescence resonance energy transfer. On the other hand, the covalent connection between the bovine serum protein and the two hydrogen lipoic acid effectively reduces the spiral structure in the bovine serum protein molecules and forms a stable protective layer on the surface of the gold nucleus, allowing the gold quantum to be made. This strategy can be used to regulate the oxidation resistance of gold quantum dots to construct stable and specific ROS luminescent probes.
【学位授予单位】：北京化工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

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