基于焦磷酸根和甲醛检测的双光子荧光化学探针的研究

发布时间：2018-07-06 20:26

本文选题：双光子荧光探针 + 比率型　；参考：《安徽大学》2017年硕士论文

【摘要】：近年来,荧光化学探针由于其具有简单方便、灵敏度高、选择性好、实时检测以及良好的生物相容性等优点,被认为是痕量检测生物体内物种如阴阳离子、小分子信号分子以及蛋白质等最有前途的方法之一。荧光探针是通过观察和检测目标样品溶液颜色或者荧光信号的变化来实现对特定样品的定性或定量分析,愈来愈多地被应用于环境检测、生命医学以及材料科学等领域。单光子荧光探针,由于激发波长较短,穿透深度较浅,很难被应用于实现在组织或者活体内跟踪检测目标离子或分子。最近,双光子显微成像(TPM)已经成为一种领先而又成熟的科学技术。在生物研究中,它具有单光子显微成像(OPM)所不具备的很多优点,如近红外激发,更优异的空间定位效果,更深的穿透深度以及更低的光漂白和光致毒性等,成为在生物医学研究中举足轻重的成像技术。此外,比率型的荧光探针,通过测量两种不同发射波长的荧光强度的比值来消除环境因素如浓度、光漂白和背景干扰(pH值、粘度、极性、温度等等)的影响以实现定量检测。具有更加理想的光学信号的双光子荧光探针的开发与应用,已经引起了科研工作者的关注,成为重要的研究领域和方向。焦磷酸根(PPi)是生物体中一种很重要的阴离子,它参与了许多细胞内的生化反应过程,如细胞内ATP水解、DNA和RNA的聚合反应以及其它许多生命代谢过程。此外,PPi的浓度水平还与许多疾病有很大的关系。当PPi水平异常会引起血管钙化,会导致严重的健康问题。而且,医学研究表明患有脱水焦磷酸钙沉积症(CPPD)晶化和软骨钙化的患者体内滑液中有较高浓度的PPi。因此,监测生物体内的PPi是具有重要的意义的。甲醛(FA)是一种常见的致癌和致畸物质,已经被世界卫生组织确定为潜在的变态反应源。作为一种化学污染物,甲醛造成的室内空气、大气环境和食品污染会导致甲醛中毒,造成头晕、头痛或恶心、呕吐,严重的会引起记忆力减退,甚至死亡。在大多数生物有机体中,一些氨基酸和外源性物质,在脱甲基化酶或氧化酶的催化作用下,发生新陈代谢,也会产生内源性的甲醛。在大脑组织内,正常浓度的甲醛通过DNA脱甲基过程,在长期记忆的存储、保存和检索上有着至关重要的作用。然而,人体内的甲醛含量超过一定剂量时,会引起许多疾病,包括阿尔茨海默病、神经源性炎症、过敏性肺炎、哮喘症状,心血管疾病和癌症等。因此,开发一种有效的监测生物体系中甲醛的荧光探针也是一个非常有意义的课题。本文在查阅大量参考文献和深入研究本课题组工作的基础上,分别基于香豆素和喹啉荧光团设计并合成了选择性识别与检测PPi和FA的双光子荧光探针PC和MQAP,并通过核磁氢谱(1HNMR)、核磁碳谱(13CNMR)以及质谱等方法对所得目标化合物进行了结构表征。还进一步研究了它们的光学性质,识别机理和在生物体系中活体成像应用等。一、合成并深入探究了一种基于香豆素母体的用于检测焦磷酸根的具有"ON-OFF-ON"荧光信号的双光子荧光探针PC,通过向香豆素母体框架引入对甲氧基苯乙炔,增加了香豆素母体的共轭结构,从而增加了其有效双光子共轭面积和吸收截面。探针PC的香豆素母体的羧基和二胺甲基吡啶结合后的识别基团,能实现对Cu2+的选择性识别,并形成"ON-OFF"型的荧光淬灭的检测信号。在PCCu配合物溶液中,加入焦磷酸根PPi后,体系的荧光随着PPi浓度增大恢复到之前的荧光强度,形成"OFF-ON"型的荧光检测信号。探针在PCR过程中也得到了成功地应用。由于其细胞毒性小而且细胞通透性良好,可以应用于细胞成像,也第一次应用于PPi的组织成像和斑马鱼活体成像实验。二、设计并合成了一个以喹啉为母体的基于2-aza-cope重排的检测甲醛分子的比率型双光子荧光探针MQAP。我们同样在喹啉母体框架六位上接一个对甲氧基苯乙炔,从而极大地拓展了喹啉的共轭结构,增大了荧光基团的双光子吸收截面。在喹啉母体的醛基位置直接引入烯丙氨基(homoallylamino)作为甲醛的选择性反应基团。探针MQAP的烯丙氨基与甲醛反应形成亚胺中间体,进一步发生2-aza-cope重排和水解,最后生成强吸电子能力的醛基。在醛基和甲氧基的推电子的共同作用下,促进了探针分子的分子内电荷转移(ICT)过程,从而探针的发射荧光光谱表现了红移现象(85nm)。紫外吸收,荧光光谱,理论计算以及高效液相色谱和质谱分析等方法验证了这一机理。探针MQAP的低毒性和较好的生物相容性,可成功应用于在活细胞和斑马鱼活体内的双光子成像。
[Abstract]:In recent years, fluorescent chemical probes have been considered as one of the most promising methods for detecting species such as Yin and Yang, small molecule signal and protein, due to their advantages of simple convenience, high sensitivity, good selectivity, real-time detection and good biocompatibility. The change of the color of the sample solution or the change of the fluorescence signal to realize the qualitative or quantitative analysis of a specific sample, and more and more used in the fields of environmental detection, life medicine and material science. Single photon fluorescence probe is difficult to be applied to the tracking of tissue or in vivo due to the short excitation wavelength and the shallow penetration depth. Detection of target ions or molecules. Recently, two-photon microscopy (TPM) has become a leading and mature science. In biological research, it has many advantages that single photon microscopy (OPM) does not possess, such as near infrared excitation, better spatial positioning effect, deeper penetration depth, and lower photobleaching and photoluminescence. Toxicity, etc., has become an important imaging technique in biomedical research. In addition, the ratio type of fluorescence probe can eliminate the influence of environmental factors such as concentration, photobleaching and background interference (pH value, viscosity, polarity, temperature, etc.) by measuring the ratio of fluorescence intensity of two different emission wavelengths to achieve quantitative detection. The development and application of two photon fluorescence probes for optical signals have attracted the attention of researchers and become an important research field and direction. PPi is a very important anion in the organism. It participates in the biochemical reactions in many cells, such as intracellular ATP hydrolysis, the polymerization of DNA and RNA and its reaction. It has many life metabolic processes. In addition, the concentration level of PPi is also related to many diseases. When abnormal PPi levels cause vascular calcification, it can cause serious health problems. Moreover, medical studies have shown that there is a high concentration of PPi. in synovial fluid in patients with calcium dehydrated calcium phosphate deposition (CPPD) crystallization and cartilage calcification. Monitoring PPi in organisms is of great significance. Formaldehyde (FA) is a common carcinogenic and teratogenic substance that has been identified by the WHO as a potential source of allergy. As a chemical contaminant, formaldehyde caused by indoor air, atmospheric environment and food contamination can lead to formaldehyde poisoning, causing dizziness, headache, or nausea. Vomiting, seriously causing memory loss, and even death. In most biological organisms, some amino acids and exogenous substances metabolize and produce endogenous formaldehyde under the catalysis of demethylation or oxidase. In the brain tissue, the normal concentration of formaldehyde is demethmethylation through DNA, in long-term memory. It has a vital role in storage, storage and retrieval. However, when the content of formaldehyde in the human body exceeds a certain dose, it causes many diseases, including Alzheimer's disease, neurogenic inflammation, allergic pneumonia, asthma symptoms, cardiovascular diseases and cancer. On the basis of consulting a large number of references and studying the work of this group, this paper designs and synthesizes two photon fluorescence probes, PC and MQAP, which are selective identification and detection of PPi and FA based on coumarin and quinoline fluorophores respectively, and through nuclear magnetic hydrogen spectrum (1HNMR), nuclear magnetic carbon spectroscopy (13CNMR) and The structure of the target compounds was characterized by mass spectrometry. The optical properties, recognition mechanism and the application of living body imaging in the biological system were further studied. First, a two photon fluorescent probe PC based on the coumarin matrix for the detection of pyrophosphoric acid with "ON-OFF-ON" fluorescence signal was explored. By introducing Methoxybenzene into the coumarin matrix, the conjugated structure of the coumarin matrix is increased, and the effective two photon conjugation area and the absorption cross section are increased. The recognition group of the carboxyl group of the coumarin matrix of the probe PC and the two amine methyl pyridine can be identified, and the "ON-OFF" type can be formed. In the PCCu complex solution, after the addition of pyrophosphate PPi, the fluorescence of the system is restored to the previous fluorescence intensity as the concentration of PPi is increased to form a "OFF-ON" type of fluorescence detection signal. The probe has also been successfully applied in the PCR process. Cell imaging is also used for the first time in tissue imaging of PPi and in zebrafish living imaging experiments. Two, a ratio type two-photon fluorescence probe, MQAP., based on 2-aza-cope rearrangement based on quinoline, is designed and synthesized. We also connect a pair of methoxybenzenes on the six bit of the quinoline matrix. The earth expands the conjugated structure of quinoline and increases the two-photon absorption cross section of the fluorescent group. The alkenamine (homoallylamino) is introduced directly as the selective reaction group of formaldehyde in the aldehyde group of the quinoline matrix. The allyl amino group of the probe MQAP forms the intermediate body of the imide, and the 2-aza-cope rearrangement and hydrolysis will occur further. Finally The aldehyde group, which produces the strong electron absorption capacity, promotes the intramolecular charge transfer (ICT) process of the probe molecules under the interaction of aldehyde and methoxy electrons. The fluorescence spectra of the probe show the red shift (85nm), UV absorption, fluorescence spectra, theoretical calculation, and high performance liquid chromatography and mass spectrometry analysis. This mechanism, the low toxicity and good biocompatibility of the probe MQAP, can be successfully applied to two-photon imaging in living cells and zebrafish.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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