基于荧光传感的分子探针的设计、合成及性能研究
发布时间：2019-06-14 05:29
【摘要】：当具有荧光发射特性的物质受到周围环境(如外来化学、生物物种、温度或酸度等)的影响,发生其荧光发射性能(如光谱和强度等)改变,从而实现对周围环境特性或某种特定物质的识别、响应和检测,常被称为荧光传感。罗丹明类分子探针是基于荧光团结构的变化产生荧光发射,在螺环状态下表现为无色无荧光,在与客体分子作用形成开环结构后,探针发生明显的颜色变化和荧光释放。将罗丹明生色团(氧杂蒽三环分子共轭体系)与识别基团(N、O、S原子)相结合设计出的罗丹明衍生物,是一种能够特异性识别汞离子(Hg2+)的分子探针。本论文在文献调研的基础上,设计并合成了 2种荧光增强型罗丹明B类分子探针N'-3',6'-双(二乙氨基)-3-螺[异吲哚-1,9'-占吨]-2-甲酰胺(RA1)和3',6'-双(二乙氨基)-2-((4-羟基苯亚甲基)氨基)螺[异吲哚-1,9'-占吨]-3-酮(RA2),以期对Hg2+实现可视化检测。借助核磁共振氢谱(1H-NMR)、核磁共振碳谱(13C-NMR)、傅立叶红外光谱(FT-IR)、电子轰击离子源-质谱联用(EI-MS)、紫外-可见吸收光谱(UV-Vis)表征探针的结构。通过紫外-可见吸收光谱法和荧光发射法对分子探针RA1和RA2的光物理性能进行了探索研究。结果表明,2种探针RA1和RA2都对Hg2+有特异性响应,向探针溶液添加Hg2+后,探针在556 nm处出现最大的紫外吸收峰,最大荧光发射波长位于580 nm,发生裸眼可见的颜色变化(由无色到粉红色),在紫外灯照射下可发出明亮的橙黄光。在与其他金属离子共存的CH3CN/H20(1:1,v/v)体系中,接有富电子羟基苯的分子探针RA2更有利于与Hg2+配位并形成1:1的配合物,50 min后荧光强度达到最大值,结合常数为1.74×104(mol/L)-1(R2=0.9904),检测范围 0.14-140 μmol/L,检测限达到0.14 μmol/L,荧光量子产率由0.003升至0.1458,能实现高选择和高灵敏地Hg2+检测。此外,用浸渍法将2种探针制成便携式试纸条(2 cm × 5 cm)和水凝胶块(半径2cm),均可对环境水溶液中的Hg2+产生明显的颜色变化,实现快速检测Hg2+。1,8-萘酰亚胺类化合物是重要的荧光染料之一,该染料量子产率高,可用作与DNA相关的生物标记。1,8-萘酐本身没有荧光,形成1,8-萘酰亚胺结构后可以在短波段范围内发出荧光,但摩尔吸光系数较低,染料颜色较浅。在1,8-萘酰亚胺的苯环上引入给电子基团,形成强烈的分子内推拉电子体系,大大增加了染料的荧光量子产率,同时荧光发射也发生较大的红移。该特性使其成为荧光分子探针的另一种候选者。细胞色素P450(CYP 450)是参与人体生命活动最重要的药物代谢酶之一,其中一类亚型酶CYP1A参与许多内源和外源性物质的生物转化,尤其在致癌物的代谢活化过程中起关键作用。目前,尽管具有高序列同源性的亚型酶之间存在交叉反应,基于分子探针的测定法仍是最广泛应用于评估CYP活性的方法,分子探针的设计大多是基于底物对CYP1A的偏好及其脱烷氧基性能。为了获得对CYP1A具有高选择性的分子探针,本文设计合成了 4种1,8-萘二甲酰亚胺衍生物,并选用6种CYP亚型酶对设计制备的分子探针进行选择筛选,探索制备的探针与CYP1A之间的作用机制:4-甲氧基-1,8-萘二甲酰亚胺(MN)可以轻松进入CYP1A2和CYP1A1的活性腔中,MN末端的羧基可以与CYP1A链上的部分氨基酸形成氢键。结果表明,N-(3-羧基丙基)-4-甲氧基-1,8-萘二甲酰亚胺(NCMN)和N-((2-羧基乙氧基)乙基)-4-甲氧基-1,8-萘二甲酰亚胺(NEMN)2种探针与CYP1A作用后,均发生裸眼可见的颜色变化,测试体系在紫外光的照射下由蓝色变为绿色,这是由于CYP1A催化探针脱甲氧基后,最大荧光发射峰由458 nm红移至552nm。利用比率荧光法(I552nm/I458nm),其中探针NCMN对CYP1A催化脱甲氧基有较好的选择性,探针NEMN对CYP1A有更好的灵敏度(检测范围1.632-150 nM,检测限1.632 nM),响应稳定时间只需10 min。探针NCMN和NEMN对CYP1A检测分别具有良好的选择性和灵敏度。
[Abstract]:when a substance having a fluorescence emission characteristic is affected by the surrounding environment (such as foreign chemical, biological species, temperature or acidity, etc.), its fluorescence emission properties (such as the spectrum and intensity, etc.) change, thereby realizing the identification of the surrounding environment characteristic or a specific substance, The response and detection are often referred to as fluorescence sensing. The rhodamine-based molecular probe is based on the change of the structure of the fluorophore to generate the fluorescence emission, and the rhodamine-based molecular probe is colorless and non-fluorescent in the state of the spiral ring, and after the ring-opening structure is formed with the action of the guest molecule, the probe has obvious color change and fluorescence release. The rhodamine derivative, which is designed by combining the rhodamine chromophoric group (the oxygen hetero-tricyclic molecular eutectic system) with the recognition group (N, O, S atom), is a molecular probe capable of specifically identifying the mercury ions (Hg2 +). On the basis of literature investigation, Two types of fluorescent-enhanced rhodamine B-like molecular probes N '-3',6 '-bis (diethylamino) -3-spiro[isopropylidene-1,9'-(diethylamino) -2-(4-hydroxybenzylidene) amino) spiro[isopropylidene-1,9 '-- T] -3-one (RA2), with a view to visual detection of Hg2 +. The structure of the probe was characterized by nuclear magnetic resonance (1H-NMR), nuclear magnetic resonance (13C-NMR), Fourier infrared (FT-IR), electron bombardment ion source-mass spectrometry (EI-MS), UV-Vis absorption spectrum (UV-Vis). The physical properties of the molecular probes RA1 and RA2 were studied by the UV-Vis absorption spectrum method and the fluorescence emission method. The results showed that both RA1 and RA2 had specific response to Hg2 +. After adding Hg2 + to the probe solution, the maximum UV absorption peak appeared at 556 nm, and the maximum fluorescence emission wavelength was 580 nm, and the visible color change of the naked eye (from colorless to pink) occurred. A bright orange-yellow light can be emitted under the irradiation of an ultraviolet lamp. In the CH3CN/ H20 (1:1, v/ v) system co-existing with other metal ions, the molecular probe RA2, which is connected with the electron-rich hydroxybenzene, is more favorable for coordination with Hg2 + and forms a 1:1 complex, the fluorescence intensity reaches the maximum after 50 min, the binding constant is 1.74-104 (mol/ L) -1 (R2 = 0.9904), the detection range is 0.14-140 & mu; mol/ L, The detection limit reached 0.14. m u.mol/ L, and the fluorescence quantum yield increased from 0.003 to 0.1458, and high selectivity and high sensitivity Hg2 + detection can be achieved. in addition, two probe are made into a portable test strip (2 cm to 5 cm) and a hydrogel block (radius of 2 cm) by an immersion method, The dye quantum yield is high and can be used as a biological marker related to DNA. And the fluorescent quantum yield of the dye is greatly increased, and meanwhile, the fluorescence emission also has a large red shift. This property makes it another candidate for a fluorescent molecular probe. Cytochrome P450 (CYP 450) is one of the most important drug metabolizing enzymes involved in human life activities, one of which is involved in the biotransformation of many endogenous and exogenous substances, especially in the metabolic activation of carcinogens. At present, although there is a cross-reaction between the subtype enzymes with high sequence homology, the molecular probe-based assay is still the most widely used method to assess CYP activity, and the design of the molecular probe is mostly based on the substrate's preference for CYP1A and its dealkylation performance. In order to obtain a molecular probe with high selectivity to CYP1A, four kinds of 1,8-disimethyleneimine derivatives were synthesized, and six kinds of CYP isoforms were used to select the molecular probe prepared by the design, and the mechanism of action between the prepared probe and CYP1A was explored. 4-Methoxy-1,8-Dimethyleneimine (MN) can easily enter the active cavity of CYP1A2 and CYP1A1, and the amino acid groups at the end of the MN can form hydrogen bonds with some of the amino acids on the CYP1A chain. The results showed that after the action of the two probes of N-(3-n-propyl) -4-methoxy-1,8-dimethylamino-imine (NCMN) and N-(( 2-tetrafluoroethoxy) ethyl) -4-methoxy-1,8-dimethylamino-imine (NEMN), the color of the naked eye can be changed. The test system was changed from blue to green under the irradiation of ultraviolet light, due to the fact that the maximum fluorescence emission peak was moved from 458 nm to 552 nm after demethoxy of the CYP1A catalytic probe. Using the ratio fluorescence method (I552nm/ I458nm), the probe NCMN has better selectivity to the catalytic demethoxy of CYP1A, and the probe NEMN has better sensitivity to CYP1A (detection range of 1.632-150 nM, detection limit of 1.632 nM), and the response time is only 10 min. The probe NCMN and NEMN have good selectivity and sensitivity for CYP1A detection, respectively.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;O626
本文编号：2499142
[Abstract]:when a substance having a fluorescence emission characteristic is affected by the surrounding environment (such as foreign chemical, biological species, temperature or acidity, etc.), its fluorescence emission properties (such as the spectrum and intensity, etc.) change, thereby realizing the identification of the surrounding environment characteristic or a specific substance, The response and detection are often referred to as fluorescence sensing. The rhodamine-based molecular probe is based on the change of the structure of the fluorophore to generate the fluorescence emission, and the rhodamine-based molecular probe is colorless and non-fluorescent in the state of the spiral ring, and after the ring-opening structure is formed with the action of the guest molecule, the probe has obvious color change and fluorescence release. The rhodamine derivative, which is designed by combining the rhodamine chromophoric group (the oxygen hetero-tricyclic molecular eutectic system) with the recognition group (N, O, S atom), is a molecular probe capable of specifically identifying the mercury ions (Hg2 +). On the basis of literature investigation, Two types of fluorescent-enhanced rhodamine B-like molecular probes N '-3',6 '-bis (diethylamino) -3-spiro[isopropylidene-1,9'-(diethylamino) -2-(4-hydroxybenzylidene) amino) spiro[isopropylidene-1,9 '-- T] -3-one (RA2), with a view to visual detection of Hg2 +. The structure of the probe was characterized by nuclear magnetic resonance (1H-NMR), nuclear magnetic resonance (13C-NMR), Fourier infrared (FT-IR), electron bombardment ion source-mass spectrometry (EI-MS), UV-Vis absorption spectrum (UV-Vis). The physical properties of the molecular probes RA1 and RA2 were studied by the UV-Vis absorption spectrum method and the fluorescence emission method. The results showed that both RA1 and RA2 had specific response to Hg2 +. After adding Hg2 + to the probe solution, the maximum UV absorption peak appeared at 556 nm, and the maximum fluorescence emission wavelength was 580 nm, and the visible color change of the naked eye (from colorless to pink) occurred. A bright orange-yellow light can be emitted under the irradiation of an ultraviolet lamp. In the CH3CN/ H20 (1:1, v/ v) system co-existing with other metal ions, the molecular probe RA2, which is connected with the electron-rich hydroxybenzene, is more favorable for coordination with Hg2 + and forms a 1:1 complex, the fluorescence intensity reaches the maximum after 50 min, the binding constant is 1.74-104 (mol/ L) -1 (R2 = 0.9904), the detection range is 0.14-140 & mu; mol/ L, The detection limit reached 0.14. m u.mol/ L, and the fluorescence quantum yield increased from 0.003 to 0.1458, and high selectivity and high sensitivity Hg2 + detection can be achieved. in addition, two probe are made into a portable test strip (2 cm to 5 cm) and a hydrogel block (radius of 2 cm) by an immersion method, The dye quantum yield is high and can be used as a biological marker related to DNA. And the fluorescent quantum yield of the dye is greatly increased, and meanwhile, the fluorescence emission also has a large red shift. This property makes it another candidate for a fluorescent molecular probe. Cytochrome P450 (CYP 450) is one of the most important drug metabolizing enzymes involved in human life activities, one of which is involved in the biotransformation of many endogenous and exogenous substances, especially in the metabolic activation of carcinogens. At present, although there is a cross-reaction between the subtype enzymes with high sequence homology, the molecular probe-based assay is still the most widely used method to assess CYP activity, and the design of the molecular probe is mostly based on the substrate's preference for CYP1A and its dealkylation performance. In order to obtain a molecular probe with high selectivity to CYP1A, four kinds of 1,8-disimethyleneimine derivatives were synthesized, and six kinds of CYP isoforms were used to select the molecular probe prepared by the design, and the mechanism of action between the prepared probe and CYP1A was explored. 4-Methoxy-1,8-Dimethyleneimine (MN) can easily enter the active cavity of CYP1A2 and CYP1A1, and the amino acid groups at the end of the MN can form hydrogen bonds with some of the amino acids on the CYP1A chain. The results showed that after the action of the two probes of N-(3-n-propyl) -4-methoxy-1,8-dimethylamino-imine (NCMN) and N-(( 2-tetrafluoroethoxy) ethyl) -4-methoxy-1,8-dimethylamino-imine (NEMN), the color of the naked eye can be changed. The test system was changed from blue to green under the irradiation of ultraviolet light, due to the fact that the maximum fluorescence emission peak was moved from 458 nm to 552 nm after demethoxy of the CYP1A catalytic probe. Using the ratio fluorescence method (I552nm/ I458nm), the probe NCMN has better selectivity to the catalytic demethoxy of CYP1A, and the probe NEMN has better sensitivity to CYP1A (detection range of 1.632-150 nM, detection limit of 1.632 nM), and the response time is only 10 min. The probe NCMN and NEMN have good selectivity and sensitivity for CYP1A detection, respectively.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;O626
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