基于熒光傳感的分子探針的設計、合成及性能研究
發(fā)布時間：2019-06-14 05:29
【摘要】：當具有熒光發(fā)射特性的物質(zhì)受到周圍環(huán)境(如外來化學、生物物種、溫度或酸度等)的影響,發(fā)生其熒光發(fā)射性能(如光譜和強度等)改變,從而實現(xiàn)對周圍環(huán)境特性或某種特定物質(zhì)的識別、響應和檢測,常被稱為熒光傳感。羅丹明類分子探針是基于熒光團結(jié)構(gòu)的變化產(chǎn)生熒光發(fā)射,在螺環(huán)狀態(tài)下表現(xiàn)為無色無熒光,在與客體分子作用形成開環(huán)結(jié)構(gòu)后,探針發(fā)生明顯的顏色變化和熒光釋放。將羅丹明生色團(氧雜蒽三環(huán)分子共軛體系)與識別基團(N、O、S原子)相結(jié)合設計出的羅丹明衍生物,是一種能夠特異性識別汞離子(Hg2+)的分子探針。本論文在文獻調(diào)研的基礎上,設計并合成了 2種熒光增強型羅丹明B類分子探針N'-3',6'-雙(二乙氨基)-3-螺[異吲哚-1,9'-占噸]-2-甲酰胺(RA1)和3',6'-雙(二乙氨基)-2-((4-羥基苯亞甲基)氨基)螺[異吲哚-1,9'-占噸]-3-酮(RA2),以期對Hg2+實現(xiàn)可視化檢測。借助核磁共振氫譜(1H-NMR)、核磁共振碳譜(13C-NMR)、傅立葉紅外光譜(FT-IR)、電子轟擊離子源-質(zhì)譜聯(lián)用(EI-MS)、紫外-可見吸收光譜(UV-Vis)表征探針的結(jié)構(gòu)。通過紫外-可見吸收光譜法和熒光發(fā)射法對分子探針RA1和RA2的光物理性能進行了探索研究。結(jié)果表明,2種探針RA1和RA2都對Hg2+有特異性響應,向探針溶液添加Hg2+后,探針在556 nm處出現(xiàn)最大的紫外吸收峰,最大熒光發(fā)射波長位于580 nm,發(fā)生裸眼可見的顏色變化(由無色到粉紅色),在紫外燈照射下可發(fā)出明亮的橙黃光。在與其他金屬離子共存的CH3CN/H20(1:1,v/v)體系中,接有富電子羥基苯的分子探針RA2更有利于與Hg2+配位并形成1:1的配合物,50 min后熒光強度達到最大值,結(jié)合常數(shù)為1.74×104(mol/L)-1(R2=0.9904),檢測范圍 0.14-140 μmol/L,檢測限達到0.14 μmol/L,熒光量子產(chǎn)率由0.003升至0.1458,能實現(xiàn)高選擇和高靈敏地Hg2+檢測。此外,用浸漬法將2種探針制成便攜式試紙條(2 cm × 5 cm)和水凝膠塊(半徑2cm),均可對環(huán)境水溶液中的Hg2+產(chǎn)生明顯的顏色變化,實現(xiàn)快速檢測Hg2+。1,8-萘酰亞胺類化合物是重要的熒光染料之一,該染料量子產(chǎn)率高,可用作與DNA相關(guān)的生物標記。1,8-萘酐本身沒有熒光,形成1,8-萘酰亞胺結(jié)構(gòu)后可以在短波段范圍內(nèi)發(fā)出熒光,但摩爾吸光系數(shù)較低,染料顏色較淺。在1,8-萘酰亞胺的苯環(huán)上引入給電子基團,形成強烈的分子內(nèi)推拉電子體系,大大增加了染料的熒光量子產(chǎn)率,同時熒光發(fā)射也發(fā)生較大的紅移。該特性使其成為熒光分子探針的另一種候選者。細胞色素P450(CYP 450)是參與人體生命活動最重要的藥物代謝酶之一,其中一類亞型酶CYP1A參與許多內(nèi)源和外源性物質(zhì)的生物轉(zhuǎn)化,尤其在致癌物的代謝活化過程中起關(guān)鍵作用。目前,盡管具有高序列同源性的亞型酶之間存在交叉反應,基于分子探針的測定法仍是最廣泛應用于評估CYP活性的方法,分子探針的設計大多是基于底物對CYP1A的偏好及其脫烷氧基性能。為了獲得對CYP1A具有高選擇性的分子探針,本文設計合成了 4種1,8-萘二甲酰亞胺衍生物,并選用6種CYP亞型酶對設計制備的分子探針進行選擇篩選,探索制備的探針與CYP1A之間的作用機制:4-甲氧基-1,8-萘二甲酰亞胺(MN)可以輕松進入CYP1A2和CYP1A1的活性腔中,MN末端的羧基可以與CYP1A鏈上的部分氨基酸形成氫鍵。結(jié)果表明,N-(3-羧基丙基)-4-甲氧基-1,8-萘二甲酰亞胺(NCMN)和N-((2-羧基乙氧基)乙基)-4-甲氧基-1,8-萘二甲酰亞胺(NEMN)2種探針與CYP1A作用后,均發(fā)生裸眼可見的顏色變化,測試體系在紫外光的照射下由藍色變?yōu)榫G色,這是由于CYP1A催化探針脫甲氧基后,最大熒光發(fā)射峰由458 nm紅移至552nm。利用比率熒光法(I552nm/I458nm),其中探針NCMN對CYP1A催化脫甲氧基有較好的選擇性,探針NEMN對CYP1A有更好的靈敏度(檢測范圍1.632-150 nM,檢測限1.632 nM),響應穩(wěn)定時間只需10 min。探針NCMN和NEMN對CYP1A檢測分別具有良好的選擇性和靈敏度。
[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.
【學位授予單位】：東南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3;O626
本文編號：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.
【學位授予單位】：東南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3;O626
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