【作者】 于法标；

【导师】 唐波；

【作者基本信息】 山东师范大学 ， 分析化学， 2009， 硕士

【摘要】 生物体内存在着各种各样的活性物种(如阳离子、阴离子等),它们有着特殊的生理功能,并对生命过程起着至关重要的影响。如果要详细阐述这些活性小分子物种在生命过程中所发挥的作用、所引起的各种生物学效应和生物学功能,那么就必须对它们进行准确实时地检测。但是由于生物本身环境的多样性和复杂性,对这些活性物种的研究就必须采用选择性好、灵敏度高的分析方法。随着荧光生物分析技术的发展,荧光显微成像的方法比其他侵入性方法提供了更好的灵敏性和操作的简便性,因此,检测生物体内活性物种的可视荧光探针技术也迅速发展起来。细胞内H+在细胞的许多生理过程中起着关键性的作用,例如受体介导的信号转导、酶催化活动、细胞生长和凋亡、离子运输和动态平衡、钙调节、细胞内吞作用、趋化和细胞粘附等。在正常生理状态下,细胞外氢离子浓度维持在一个非常狭窄的范围内,正常值约为40 nmol?L-1(pH值7.40),大约有5 nmol?L-1的浮动范围(pH值7.35-7.45)。pH升高或降低达到0.10-0.20个pH单位即可引起肺心病变和神经系统的问题(如阿尔茨海默氏病),体内较大的pH波动则是致命性的。因此,H+是一个衡量生物体生理变化的最重要的指标之一。氯是人体必需的一种元素,氯在体内主要以离子的形式存在。氯离子在维持生物体内pH平衡、维持细胞外液渗透压和酶催化等生命过程中起着举足轻重的作用。因此对阴离子识别和检测的研究就显得尤为重要。就目前阴离子识别发展状况看来,阴离子与受体相互作用过程中会受到很多因素的干扰,因而设计合成具有特异专一性识别阴离子受体就显得比较困难。阴离子与受体分子之间的相互络合识别的非共价键作用类型主要有:静电引力作用、氢键作用、静电引力与氢键共同作用、金属或路易斯酸配位作用。铁是生物体所必需的过渡金属元素,但细胞内过多的螯合铁却被认为是对细胞致病损伤的一个决定性因子,尤其是细胞胞浆内过负载的亚铁离子能够催化Fenton反应,从而产生氧化性极强的羟基自由基。如果想要对Fe2+的生物学功能作深入性的了解,就必需发展出高选择性、高灵敏度的新型检测方法。对阳离子探针设计合成的机理常见的有:光诱导电子转移、电荷转移激发态、单体-激基缔合、荧光共振能量转移、电子能量转移。为了实现高选择性、高灵敏度快速识别和检测生物体内的H+、Cl-和Fe2+,并使之达到可视化的目的,本论文开展了以下两方面的工作:(一)设计合成了一种近红外中性pH值荧光探针。探针结构采用“荧光团-桥体-受体”设计模式,通过光诱导电子转移机理来操控荧光强度的增减。在结构设计策略基础上,我们选择了具备有高吸光系数的七甲川花菁(Cy)染料作为荧光团,4′-(苄胺基)-2,2′:6′,2′′-三联吡啶(Tpy)为质子受体。通过pH滴定实验,我们发现此探针可用于监测生理pH值微小的波动。经测定,探针的pKa约为7.10。在pH值6.70-7.90范围内,荧光强度对H+浓度变化有强的依赖性和迅速响应灵敏性,并且在此范围内荧光强度和pH值之间有着良好的线性关系。此探针可以在生化体系中有效地避开生物自发荧光和生物内源性物种的干扰。同时探针也展现出高灵敏性、良好的光稳定性和优异的胞膜穿透性等优点。本文成功地实现了对HepG2细胞和HL-7702细胞内H+原位监测成像。(二)设计合成了一种连有三联吡啶基团的磺酸花菁结构的荧光分子探针用于选择性检测生物体内的Fe2+和Cl-。探针的工作原理是基于探针-金属离子-阴离子络合配位和重原子的三重态荧光淬灭效应。探针本身有强的荧光,在氯离子存在下与Fe2+络合后,导致磺酸花菁的荧光猝灭,而且荧光强度的猝灭程度分别与Fe2+的浓度和Cl-的浓度成一定的比例关系。探针在与Fe2+和Cl-反应前后的颜色由蓝色变为紫色,实验结果显示探针对Fe2+和Cl-有高度的选择性。据此,我们分别固定了Fe2+和Cl-浓度,成功地实现了对Fe2+和Cl-的分别检测,探针的荧光强度变化分别与亚铁离子和氯离子的浓度呈现较好的线性关系。探针对介质的酸碱度不敏感,荧光性质稳定。该探针成功用于血浆和血清中Fe2+和Cl-的选择性检测。选择性实验表明该探针是检测生物体内Fe2+和Cl-的理想近红外荧光探针。更多还原

【Abstract】 Intracellular pH plays a pivotal role in many cellular events, including receptor-mediated signal transduction, enzymatic activity, cell growth and apoptosis, ion transport and homeostasis, calcium regulation, endocytosis, chemotaxis, and cell adhesion. Under normal physiological conditions extracellular hydrogen ion concentration is maintained within very narrow limits. The normal value is about 40 nmol?L-1 (pH 7.40) and varies by about 5 nmol?L-1 (pH 7.35-7.45). Deviation by 0.10-0.20 pH units in either direction can cause cardiopulmonary and neurologic problems (e.g., Alzheimer’s disease), and more extreme variations can be fatal. Hence, H+ is one of the most important targets among the species of interests in vivo. Moreover, the fluorescence microscopy provides greater sensitivity and convenience than other invasive methods. These advantages have made fluorescent intracellular pH probes imaging technology developing rapidly.Chlorine is an essential element for creatures, it exists mainly in the form of anion in vivo. Chloride plays a pivotal role in many life processes, such as equilibrium pH values, maintenance extracellular fluid osmotic pressure and enzyme catalysis. So it is important for anion recognition and detection. As far as the development status of anion recognition is concerned, many external influence factors will disturb the recognition process between anion and receptor. Therefore, there are some difficulties for design and synthesis a receptor that is exclusive to an anion. In general, the non-covalent bond association can be divided into four types: electrostatic force, hydrogen bonding, electrostatic force and hydrogen bonding interaction, and metal ions or lewis acids coordination.Iron, a transition metal element, is also essential for lives. However, many studies have shown that intracellular chelating iron is a crucial pathogenic factor for cell damages. The overload iron can catalyze Fenton reaction, and then hydroxyl radical is produced, which is one of the strongest oxidants known. It can lead to irreparable cellular damage. In order to further investigate the biological function of ferrous ion (Fe2+), it is urgent for developing new method with high selectivity and sensitivity to detect Fe2+. There are some familiar mechanisms for design and synthesis ion-probe, such as photoinduced electron transfer, charge-transfer excited states, monomer-excimer, fluorescence resonance energy transfer, and electronic energy transfer.Based on the overall strategy that is fleetly detecting and imaging H+, Cl-, and Fe2+ in vivo with high selectivity and high sensitivity. We carried out two aspects of investigation: First, A near-neutral pH near-infrared (NIR) fluorescent probe utilizing a fluorophore-spacer- receptor molecular framework that can modulate the fluorescence emission intensity through a fast photo induced electron-transfer process was developed. Our strategy was to choose tricarbocyanine (Cy), a NIR fluorescent dye with high extinction coefficients, as a fluorophore, and 4′-(aminomethylphenyl)-2,2′:6′,2′′-terpyridi- ne (Tpy) as a receptor. The pH titration indicated that Tpy-Cy can monitor the minor physiological pH fluctuations with a pKa of～7.10 near physiological pH. The probe responds linearly and rapidly to minor pH fluctuations within the range of 6.70-7.90 and exhibits strong dependence on pH changes. It is shown that the probe effectively avoids the influence of autofluorescence and native cellular species in biological systems and meanwhile exhibits high sensitivity, good photostability, and excellent cell membrane permeability. The real-time imaging of cellular pH and the detection of pH in situ was achieved successfully in living HepG2 and HL-7702 cells by this probe.Second, a probe composed of cyanine (Cy-SO3-) and terpyridine (Tpy) is designed and synthesized to detect Fe2+ and Cl- in vivo. Its working principle is based on probe-metal-anion coordination and heavy atom effect. The strongly fluorescent probe could chelae with Fe2+. When Cl- appeared, the fluorescence quenching and the colour change from blue to purple. The results demonstrated that the probe has high selectivity to Fe2+ and Cl-. The probe responds linearly and rapidly to minor [Fe2+] and [Cl-] variation respectively. The results demonstrate that the probe can detect Fe2+ and Cl- selectively.更多还原