酞菁红区荧光探针在生物大分子检测中的应用及其用于生物成像的可行性初探

发布时间：2018-11-13 15:39

【摘要】：由于在长波区域具有吸收或发射特性的天然和人工合成的物质极少,因而在进行生物、生化和临床等复杂试样的测定时,使用长波发射荧光试剂可有效避开背景荧光和散射光的干扰,且光漂白作用小,与传统的荧光试剂相比具有很大优越性。能发荧光的酞菁化合物即是一类红区发射的荧光试剂。本论文的工作围绕酞菁红区荧光探针在生物大分子分析及成像检测中的应用而展开,共分五章。第一章：首先对红区及近红外荧光探针的应用进展进行了简要介绍。由于酞菁化合物作为红区荧光试剂的研究构成了本文的主要部分,本章还对酞菁化合物的分子结构和光谱特性以及它在生物模拟酶、光动力治疗、生化分析领域的应用做了介绍。第二章：本章建立了简便、快速测定硫酸软骨素的荧光增强分析法。通过荧光光谱筛选实验发现,具有共轭结构的阳离子头部和长碳链尾部的阳离子表面活性剂可几乎完全猝灭四磺基铝酞菁(Tetrasulfonated Aluminum Phthalocyanine, AlS4Pc)的荧光。而在带有磺基阴离子的硫酸软骨素(Chondroitin Sulfate, CS)存在下,体系荧光显著恢复。以AIS4Pc-阳离子表面活性剂离子缔合物为红区荧光探针,考察了其对CS的荧光恢复响应行为,发现离子缔合物的荧光恢复程度与CS浓度存在良好的相关性,据此实现了复杂样品中CS简便、准确的测定。本章工作分为两部分：由于前期工作筛选出的猝灭效果较好的阳离子表面活性剂有两种,由这两种猝灭剂参与的体系的灵敏度和线性范围不同,各具特色,因此对两个体系分别进行了考察,并建立了两种CS的荧光增强测定法。第三章：本章建立了简便、快速测定溶菌酶的荧光增强分析法。带有强阴离子的黏多糖肝素(Heparin, HP)可诱导阳离子铝酞菁[Tetra(trimethyammionio) Aluminum Phthalocynine, TTMAAlPc]聚集而导致荧光显著猝灭。由于溶菌酶对黏多糖具有催化降解作用,因而可水解HP为小分子片段,从而破坏TTMAAlPc-HP的聚集缔合平衡,使TTMAAlPc被释放,体系荧光将因之恢复。结合荧光光谱与荧光各向异性技术对反应机理进行了探讨。据此建立了溶菌酶测定新方法,并实现了复杂样品中的溶菌酶含量的准确测定。第四章：探讨了红区荧光染料酞菁在指纹成像观测中的应用。考察了几种酞菁染料对潜在指印的成像效果,其中A1(SO2C1)4TSP对于玻片上油指印的染色效果最为理想,进一步的对染料溶剂,浓度,染色方法和时间进行了考察和优化,成像结果令人满意。第五章：为研究红区荧光染料酞菁在细胞中是否有特异性定位,为此首先通过摸索建立了显微注射技术,直接将荧光酞菁染料注射进细胞中,体外培养后观察染料与细胞是否有结合。初步研究的结果表明荧光酞菁化合物具有成为活细胞成像新型荧光探针的潜力。
[Abstract]:Since there are very few natural and synthetic substances with absorption or emission characteristics in the long-wave region, when determining complex samples, such as biological, biochemical and clinical, The use of long wave emission fluorescence reagent can effectively avoid the interference of background fluorescence and scattered light, and the photobleaching effect is small, which is superior to the traditional fluorescent reagent. The fluorescent phthalocyanine compounds are a kind of fluorescent reagents in the red region. This paper focuses on the application of phthalocyanine red region fluorescence probe in biomolecules analysis and imaging detection, which is divided into five chapters. Chapter 1: firstly, the application progress of red region and near infrared fluorescence probe is briefly introduced. Since the study of phthalocyanine compounds as a red region fluorescent reagent constitutes the main part of this paper, this chapter also deals with the molecular structure and spectral characteristics of phthalocyanine compounds, as well as its biological mimic enzyme, photodynamic therapy, The application of biochemical analysis is introduced. Chapter 2: in this chapter, a simple and rapid fluorescence enhanced method for the determination of chondroitin sulfate was established. The fluorescence spectra showed that the cationic surfactants with conjugated structure and long carbon chain tail could almost completely quench the fluorescence of the tetrasulfonyl aluminum phthalocyanine (Tetrasulfonated Aluminum Phthalocyanine, AlS4Pc). In the presence of chondroitin sulfate (Chondroitin Sulfate, CS) with sulfonyl anion, the fluorescence of the system recovered significantly. Using AIS4Pc- cationic surfactant ion-association complex as a red region fluorescence probe, the fluorescence recovery response to CS was investigated. It was found that there was a good correlation between the fluorescence recovery degree of the ion-association complex and the concentration of CS. Based on this, the determination of CS in complex samples was simple and accurate. The work of this chapter is divided into two parts: because there are two kinds of cationic surfactants with better quenching effect, the sensitivity and linear range of the two quenching agents are different and have their own characteristics. Therefore, the two systems were investigated, and two methods of fluorescence enhanced determination of CS were established. Chapter 3: in this chapter, a simple and rapid fluorescence enhanced assay for the determination of lysozyme was established. The strong anionic mucopolysaccharide heparin (Heparin, HP) can induce cationic aluminum phthalocyanine [Tetra (trimethyammionio) Aluminum Phthalocynine, TTMAAlPc] aggregation and lead to fluorescence quenching. Because lysozyme can catalyze the degradation of mucopolysaccharide, HP can be hydrolyzed as a small molecular fragment, thus destroying the aggregation and association equilibrium of TTMAAlPc-HP, releasing TTMAAlPc and restoring the fluorescence of the system. The mechanism of the reaction was discussed by means of fluorescence spectroscopy and fluorescence anisotropy. A new method for the determination of lysozyme was established, and the accurate determination of lysozyme in complex samples was achieved. Chapter 4: the application of fluorescent dye phthalocyanine in fingerprint imaging is discussed. The imaging effects of several phthalocyanine dyes on potential fingerprinting were investigated. The dyeing effect of A1 (SO2C1) 4TSP on oil fingerprinting on glass was the most ideal. The solvent, concentration, dyeing method and time of dye were further investigated and optimized. The imaging results are satisfactory. Chapter 5: in order to study whether the red zone fluorescent dye phthalocyanine has a specific localization in the cell, a microinjection technique was established to directly inject the fluorescent phthalocyanine dye into the cell. After in vitro culture, the binding of dye to cells was observed. The preliminary results show that fluorescent phthalocyanines have the potential to be novel fluorescent probes for living cell imaging.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R927;O657.3

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