CN115261015B - 一种基于ICT原理检测N2H4和Cu2+的双通道荧光探针及其制备方法和应用 - Google Patents
一种基于ICT原理检测N2H4和Cu2+的双通道荧光探针及其制备方法和应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
本发明公开一种新型有效的用于双通道特异性识别内N2H4和Cu2+的荧光探针及其制备方法和应用,荧光探针为(E)‑2‑(3‑(4‑(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯;双识别位点的修饰显着提高了荧光探针应用潜力;可在各种干扰物存在下对N2H4和Cu2+的识别具有优越的选择性,对常见生物分子有着很强的抗干扰能力;可进入HeLa细胞和斑马鱼体内并与体内N2H4和Cu2+迅速发生反应,产生可肉眼分辨的荧光淬灭现象,能够清晰地观察N2H4和Cu2+在活性细胞中的分布情况;不仅可对胞内N2H4和Cu2+选择性识别,且能在各种活细胞的生长环境下高灵敏度的定量检测N2H4和Cu2+。
Description
技术领域
本发明属于有机小分子荧光探针领域,具体的涉及以(E)-3- (4-(二乙氨基)苯基)-1-(2-羟基苯基)丙-2-烯-1-酮为荧光母体用于N2H4和Cu2+检测的双通道荧光探针及其制备方法和应用。
背景技术
肼(N2H4),也称为联氨,是一种无色油状液体,在环境条件下具有类似氨的刺激性气味。其结构式包含两个具有孤对电子的氮原子和四个可取代的氢原子。因此,由于这种所谓的α效应,它具有很强的还原性、高反应性碱度和良好的水溶性。肼(N2H4) 也是一种非常重要的化学品,广泛用作喷气发动机和火箭燃料,抗氧化剂,还原剂、催化剂、燃料电池反应物、高分子交联剂和扩链剂、CO2清除剂、以及农药和医药的原料。然而,N2H4的大量使用也因其高毒性给人类健康和环境安全带来了重大风险。暴露于N2H4皮肤接触或吸入会导致暂时失明、头晕、恶心、过敏和灼伤。此外,不可忽视的是,N2H4可以与水以任意体积比轻松混溶,形成水合肼。这很容易导致人类和其他生物通过饮用水缓慢摄入 N2H4,造成长期损害。并且已经证实,长期接触N2H4会导致肝损伤、代谢异常、高脂血症、DNA损伤、肝毒性、甚至癌症。US-EPA和 IARC建议N2H4的低阈值(TLV)为10ppb(0.31μM)。因此,N2H4的选择性和灵敏监测以简单方便的方法引起了广泛关注。包括流动注射在内的各种分析方法已应用于N2H4的测定,电化学传感,电催化剂,色谱,化学发光。然而,这些方法往往操作繁琐,检测时间长,仪器昂贵,无法实现实时分析和生物成像。相反,荧光探针技术以其灵敏度高、特异性好、操作方便、检测过程短、成本低、可在体内可视化和成像等独特优势而受到广泛关注。
Cu2+是一种人体必需的微量元素,在细胞呼吸、骨形成和神经功能调节等多种基本生理过程中发挥着至关重要的作用。人体内缺乏Cu2+会增加患冠心病的风险。另一方面,高浓度的Cu2+对环境有毒有害。Cu2+的超载可引起一些疾病,因为它具有毒性、氧化作用并取代其他金属离子作为各种辅助因子。它还与阿尔茨海默病、帕金森病、酶催化反应、朊病毒病、威尔逊病和家族性肌萎缩侧索硬化症。因此,开发高选择性、灵敏、快速的Cu2+化学传感器非常重要。迄今为止,已提出多种信号机制并将其用于Cu2+的光学检测。由于Cu2+的顺磁性具有内在的荧光猝灭特性,因此存在少量的Cu2+荧光“开启”探针,例如具有席夫碱结构的有机荧光分子,基于罗丹明和其他荧光探针的闭饱和螺环具有保护官能团。相比之下,Cu2+的比色或“关闭”荧光探针相对容易实现且更复杂。特别是,已经报道了许多用于的简单“肉眼”或比色荧光探针。褪色和荧光猝灭或从紫外到可见光区域的颜色变化都不够醒目。因此,明显的吸收光谱偏移(>100nm)和覆盖可见区域的颜色变化更为理想。
发明内容
1.要解决的技术问题
本发明要解决的第一个技术问题是开发出一种抗干扰能力强且可特异性识别N2H4和Cu2+的双通道荧光探针,该荧光探针可区分 N2H4和Cu2+与其他分析物。
本发明要解决的第二个技术问题是提供一种可选择性N2H4和 Cu2+的双通道荧光探针的制备方法。
本发明要解决的第三个技术问题是提供上述可选择性N2H4和 Cu2+的双通道荧光探针的应用方法。
2.技术方案
为解决上述问题,本发明采取如下技术方案:
一种基于分子内电荷转移原理用于双通道特异性检测N2H4和 Cu2+的荧光探针,所述荧光探针为(E)-2-(3-(4-(二乙氨基) 苯基)丙烯酰)苯基吡啶甲酸酯,其化学结构式如式(I)所示:
本发明还提供了上述荧光探针的制备方法,包括以下步骤:
(1)(1)荧光母体(E)-3-(4-(二乙氨基)苯基)-1-(2- 羟基苯基)丙-2-烯-1-酮合成具体操作步骤如下:
①将2,4-二乙氨基苯甲醛和2-羟基苯乙酮使用EtOH作溶剂混合均匀;
②向步骤①所得的混合溶液中添加KOH水溶液,并在室温下搅拌24小时;
③待步骤②中反应完成后用稀盐酸中和反应混合物,直到将 pH值调整到7.0;
④将步骤③所得的混合物通过抽吸过滤收集沉淀,并用乙醇洗涤三次,所得到的残留物利用洗脱机通过硅胶层析进一步纯化,得到荧光基团(E)-3-(4-(二乙氨基)苯基)-1-(2-羟基苯基) 丙-2-烯-1-酮;
(2)荧光探针(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰) 苯基吡啶甲酸酯合成具体操作步骤如下:
①使用二氯甲烷为溶剂将荧光基团(E)-3-(4-(二乙氨基) 苯基)-1-(2-羟基苯基)丙-2-烯-1-酮和2-吡啶甲酸溶解并混合均匀;
②向步骤①所得的混合溶液中加入4-二甲氨基吡啶和1-(3二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;
③将步骤②中所得的混合溶液在室温下搅拌过夜,用旋转蒸发器除去溶剂,用硅胶柱纯化得到荧光探针(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯。
本发明提供了上述荧光探针对Cu2+和N2H4的识别影响因素筛选方法,包括以下步骤:
(1)将所述的荧光探针用不同溶剂配制成浓度为10μM的工作溶液,所述溶剂分别为二甲基亚砜、N,N-二甲基甲酰胺、乙腈、甲醇、乙醇、丙酮、乙酸乙酯和水;向不同溶剂配制好的10μM的荧光探针溶液中分别对应加入200μM Cu2+和N2H4,每种溶液设置三个平行;反应完全,得到48份反应物,分别对48份反应物进行荧光强度测定;荧光探针检测Cu2+体系中使用乙腈效果最好;荧光探针检测N2H4体系中使用二甲基亚砜效果最好,最后选择乙腈为溶剂检测Cu2+,二甲基亚砜为溶剂检测N2H4;
(2)将所述的荧光探针用不同比例的二甲基亚砜与4-羟乙基哌嗪乙磺酸配制成浓度为10μM的荧光探针溶液,所述乙腈和二甲基亚砜比例分别为10%,20%,30%,40%,50%,60%,70%,80%,90%, 100%;向使用不同比例乙腈和二甲基亚砜配制好的10μM的荧光探针溶液中分别对应加入200μM Cu2+和N2H4,每种溶液设置三个平行;反应完全,得到60份反应物,分别对60份反应物进行荧光强度测定;结果表明荧光探针检测Cu2+体系中4-羟乙基哌嗪乙磺酸与乙腈比例为3:7最佳,荧光探针检测N2H4体系中4-羟乙基哌嗪乙磺酸与二甲基亚砜比例为2:8最佳;
(3)将所述的荧光探针用缓冲液配制成浓度为10μM的荧光探针溶液,所述Cu2+检测体系工作液由体积比为3:7的4-羟乙基哌嗪乙磺酸和乙腈配制成,N2H4检测体系工作液由体积比为2:8的4- 羟乙基哌嗪乙磺酸和二甲基亚砜配制成;设置缓冲溶液pH为2,3, 4,5,6,6.5,7,7.4,8,9,10,11,12;向不同pH浓度为 10μM的近红外荧光探针溶液中分别对应加入100μM Hg2+,每种溶液设置三个平行;反应完全,得到78份反应物,分别对78份反应物进行荧光强度测定;结果表明N2H4和Cu2+检测体系的最适pH均在7.4附近。
本发明还提供了上述荧光探针的应用,上述荧光探针用于双通道特异性检测N2H4和Cu2+。
进一步地,所述的荧光探针用于环境溶液介质中N2H4和Cu2+特异性的检测方法,操作步骤如下:
(1)将所述的荧光探针配制成浓度为10μM的4-羟乙基哌嗪乙磺酸和二甲基亚砜缓冲液;所述缓冲液中4-羟乙基哌嗪乙磺酸和二甲基亚砜的体积比为3:7,缓冲液的pH值为7.4;
(2)向配制好的10μM的荧光探针溶液中分别加入200μM 的分析物溶液,包括生物体内常见氨基酸:半胱氨酸,天冬氨酸,色氨酸,酪氨酸,组氨酸,谷氨酸,苏氨酸;各种金属离子:碘化钾,氯化镍,硝酸钙,氟化钠,氯化镁,氯化钴,氯化锰,氯化钡,氯化铵和氯化银,反应完全后进行荧光强度测定;
(3)通过对荧光强度变化的研究,表明该荧光探针可以与N2H4和Cu2+进行特异性反应产生显著的荧光变化,即所述荧光探针可以特异性双通道识别N2H4和Cu2+。
进一步地,所述的荧光探针用于检测人体宫颈癌组织HeLa细胞中N2H4和Cu2+分布的检测方法,操作步骤如下:
(1)将所述荧光探针配制成浓度为20μM的缓冲液;分为a,b, c,d四组,a组为空白对照组:未被任何处理的HeLa细胞,作为a组检测物;b组为N2H4和Cu2+对照组:用50μM的N2H4和Cu2+分别孵育HeLa细胞30分钟,作为b组检测物;c组为荧光探针对照组:用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯孵育HeLa细胞30分钟,作为c组检测物;d组为实验组:将HeLa细胞用N2H4和Cu2+分别处理30min,然后用HEPES缓冲溶液洗涤多余的N2H4和Cu2+3次,再用探针溶液处理30min;
(2)通过a,b,c,d组荧光成像结果显示(E)-2-(3-(4- (二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯能够进入细胞中,并与细胞中的N2H4和Cu2+进行反应,细胞成像能够很明显的看出 N2H4和Cu2+在细胞中的分布情况。
进一步地,所述的荧光探针用于斑马鱼中N2H4和Cu2+的检测方法,操作步骤如下:
(1)将所述的荧光探针配制成浓度为20μM的工作液;分为a, b,c,d四组,a组为空白对照组:未被任何处理的三日龄斑马鱼,作为a组检测物;b组为N2H4和Cu2+对照组:用50μM的N2H4和Cu2+分别孵育三日龄斑马鱼30分钟,作为b组检测物;c组为荧光探针对照组:用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯孵育三日龄斑马鱼30分钟,作为c组检测物;d组为实验组:将三日龄斑马鱼用N2H4和Cu2+分别处理30min,然后用HEPES 缓冲溶液洗涤多余的N2H4和Cu2+3次,再用探针溶液处理30min;
(2)结果显示在28℃下用(E)-2-(3-(4-(二乙氨基) 苯基)丙烯酰)苯基吡啶甲酸酯处理过的斑马鱼显示出明显的荧光,而同时使用荧光探针与N2H4和Cu2+处理的斑马鱼产生一定的荧光淬灭现象;荧光成像结果表明(E)-2-(3-(4-(二乙氨基) 苯基)丙烯酰)苯基吡啶甲酸酯能够进入斑马鱼体内并与N2H4和 Cu2+反应,产生荧光淬灭,从而达到检测的效果。
3.有益效果
1、本发明中的荧光探针具有合成路线短、制作原料易得,成本低廉,产率理想,反应条件温和以及稳定性强等优点。
2、本发明中的荧光探针中双通道识别的设计显着提高了荧光探针对N2H4和Cu2+的选择性和灵敏度。
3、本发明中的荧光探针不与生物体内常见其他分析物反应,且在常见分析物的干扰下可有效的检测N2H4和Cu2+,即该荧光探针抗干扰能力强;不仅如此,该荧光探针能在各种活细胞的生长环境下有效检测外源性N2H4和Cu2+。
4、本发明中的荧光探针具有在不同环境水样中定量检测N2H4和Cu2+的潜力。
5、本发明中的荧光探针的logP=5.12,表明该荧光探针是亲脂性化合物,具有较好的细胞膜通透性。不同浓度的细胞毒性试验表明该荧光探针具有较低的细胞毒性。
6、本发明中的荧光探针可有效的特异性识别生物体内的N2H4和Cu2+,并成功应用于HeLa细胞及斑马鱼成像中。
附图说明
图1为在(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯的工作液中加入N2H4和Cu2+反应的紫外光谱(a)及荧光发射光谱(b);
图2为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯与N2H4和Cu2+反应产物的高分辨质谱图;
图3为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯与N2H4和Cu2+反应溶剂和溶剂比例的筛选;
图4为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯与N2H4和Cu2+反应溶液的pH影响;
图5为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯识别N2H4和Cu2+的特异性和抗干扰性研究;
图6为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯识别N2H4和Cu2+的灵敏度研究;
图7为不同浓度(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰) 苯基吡啶甲酸酯对细胞生存率影响的柱状图;
图8为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯在HeLa细胞中的荧光显微成像图;
图9为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯在3日龄斑马鱼中孵育的荧光显微成像图。
具体实施方式
本发明公开了一种用于双通道识别N2H4和Cu2+的荧光化学传感器及其制备方法与应用。该荧光化学传感器的特征在于它有两个识别位点,其中C=C和羰基作为N2H4识别位点,荧光化学传感器上三个氧原子为Cu2+识别位点。所报告荧光化学传感器上双识别位点能与体系中 N2H4和Cu2+发生特异性反应,使荧光化学传感器的荧光发生变化,从而实现对N2H4和Cu2+的特异性检测。
下面结合附图和实施例对本发明作进一步详细的说明。
实施例1
制备用于双通道特异性检测N2H4和Cu2+的荧光探针
所述荧光探针为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯,且制备过程如下:
具体地,所述荧光探针的制备方法,包括以下步骤:
(1)将2,4-二乙氨基苯甲醛(12mmol,1.79g)和2-羟基苯乙酮(13.2mmol,1.80g)使用EtOH(30mL)作溶剂混合均匀;向上述混合溶液中添加KOH(50mmol,2.8g)水溶液,并在室温下搅拌24小时;反应完成后用稀盐酸中和反应混合物,直到将pH值调整到7.0;通过抽吸过滤收集沉淀,并用乙醇洗涤三次,所得到的残留物利用洗脱机通过硅胶层析进一步纯化,得到(E) -3-(4-(二乙氨基)苯基)-1-(2-羟基苯基)丙-2-烯-1-酮(3.1 g,87%)。
上述洗脱机应用过程中所需的洗脱液为正己烷和乙酸乙酯,且溶剂比例为5:1。
核磁共振氢谱:1H NMR(600MHz,DMSO-d6)δ13.16(s,1H), 8.22(dd,J=8.0,1.6Hz,1H),7.79(d,J=15.1Hz,1H), 7.70(d,J=9.0Hz,3H),7.53–7.47(m,1H),7.00–6.89(m,2H),6.69(d,J=8.6Hz,2H),1.10(t,J=7.0Hz,6H)。
核磁共振碳谱:13C NMR(151MHz,DMSO-d6)δ193.51,162.71, 150.45,147.02,136.09,132.20,130.72,121.51,121.00, 119.28,118.13,114.58,111.69,111.05,44.40,44.31,40.17, 39.74,12.90。
高分辨质谱:HRMS(ESI,m/z)计算值[C19H21NO2+H]+:296.1651, 发现值:296.1648。
(2)使用二氯甲烷为溶剂将荧光基团(E)-3-(4-(二乙氨基)苯基)-1-(2-羟基苯基)丙-2-烯-1-酮(2mmol)和2-吡啶甲酸(3mmol)溶解并混合均匀;向上述混合溶液中加入4-二甲氨基吡啶和1-(3二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(2mmol);溶液在室温下搅拌过夜。用旋转蒸发器除去溶剂,用硅胶柱纯化得到荧光探针(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯(0.63g,79%)。
上述荧光探针在纯化过程中所需的洗脱液为二氯甲烷和甲醇,溶剂比例为30:1。
核磁共振碳谱:13C NMR(151MHz,DMSO-d6)δ189.65, 163.68,150.47,150.08,148.93,147.02,146.39,138.04, 133.12,132.82,131.42,130.30,128.27,126.87,126.13, 123.99,121.10,119.10,111.57,44.25,40.59,40.45,12.87。
核磁共振氢谱:1H NMR(600MHz,DMSO-d6)δ8.76(dt,J= 4.6,1.5Hz,1H),8.15(dt,J=7.8,1.1Hz,1H),7.98(td,J =7.7,1.7Hz,1H),7.82(dd,J=7.7,1.7Hz,1H),7.68–7.62 (m,2H),7.47–7.37(m,5H),7.12(d,J=15.5Hz,1H),6.62 –6.58(m,2H),3.36(q,J=7.1Hz,4H),1.07(t,J=7.0Hz, 6H)。
高分辨质谱:HRMS(ESI,m/z)计算值[C25H24N2O3+H]+:401.1865,发现值:401.1848。
上述制得的荧光探针为(E)-2-(3-(4-(二乙氨基)苯基) 丙烯酰)苯基吡啶甲酸酯,且对N2H4、Cu2+检测原理如下:
具体地,本发明所述的荧光探针双通道识别N2H4和Cu2+机理如下:
(1)(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯特异性识别N2H4机理:首先N2H4上其中一个N原子攻击荧光探针上的羰基氧原子发生加成反应形成C=N键,然后另一个N原子进攻(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯上的C=C键,发生加成环化反应生成五元环,该过程通过异构化消耗大部分激发能量,导致荧光量子产率显着降低,从而产生荧光淬灭现象,该机制也通过高分辨质谱被证实(ESI,m/z: 437.1924);
(2)(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯特异性识别Cu2+机理:探针和Cu2+之间的相互作用模式通过1HNMR和HNMR进行了检测。探针溶液中加入Cu2+前后的核磁共振氢谱并未发生大的改变,这意味着探针没有和Cu2+进行反应或者和Cu2+产生物理络合作用。而添加Cu2+后,探针Cu2+体系溶液中产生荧光淬灭,并且荧光探针的结构存在一些能够与Cu2+配位的杂原子(O等),暗示探针和Cu2+之间的相互作用模式为络合作用。为了验证探针以什么形式与Cu2+络合,通过高分辨质谱对体系中分子量进行了探究(图1)。结果显示Cu2+能够同时与荧光探针上O原子相互作用络合产生复合物(ESI MS[M+H]+,m/z:486.5925),荧光探针分子内电荷转移过程被阻断产生荧光猝灭现象,从而实现对Cu2+的特异性检测。
实施例2
用于溶液体系中荧光探针对N2H4和Cu2+双通道识别影响因素筛选
(1)将实施例1中所制备的荧光探针用不同溶剂配制成浓度为10μM的工作溶液,所述溶剂分别为二甲基亚砜(DMSO)、N,N- 二甲基甲酰胺(DMF)、乙腈(Acetonitrile)、甲醇(Methanol)、乙醇(EtOH)、丙酮(Acetone)、乙酸乙酯(EA)和水(H2O);向不同溶剂配制好的10μM的荧光探针溶液中分别对应加入200μM Cu2+和N2H4,每种溶液设置三个平行;反应完全,得到48份反应物,分别对48份反应物进行荧光强度测定。荧光探针检测Cu2+体系中乙腈效果;荧光探针检测N2H4体系中使用DMSO效果最好,最后选择乙腈为溶剂检测Cu2+,DMSO为溶剂检测N2H4。
(2)将实施例1中所制备的荧光探针用不同比例的二甲基亚砜与4-羟乙基哌嗪乙磺酸(HEPES)配制成浓度为10μM的荧光探针溶液,所述乙腈和DMSO比例分别为10%,20%,30%,40%,50%, 60%,70%,80%,90%,100%;向使用不同比例乙腈和DMSO配制好的10μM的荧光探针溶液中分别对应加入200μM Cu2+和N2H4,每种溶液设置三个平行;反应完全,得到60份反应物,分别对60份反应物进行荧光强度测定(图2)。结果表明荧光探针检测Cu2+体系中4-羟乙基哌嗪乙磺酸(HEPES)与乙腈比例为3:7最佳,荧光探针检测N2H4体系中4-羟乙基哌嗪乙磺酸(HEPES)与二甲基亚砜比例为2:8最佳。
(3)将实施例1中所制备的荧光探针用缓冲液配制成浓度为 10μM的荧光探针溶液,所述Cu2+检测体系工作液由体积比为3:7 的4-羟乙基哌嗪乙磺酸(HEPES)和乙腈配制成,N2H4检测体系工作液由体积比为2:8的4-羟乙基哌嗪乙磺酸(HEPES)和二甲基亚砜配制成;设置缓冲溶液pH为2,3,4,5,6,6.5,7,7.4,8,9, 10,11,12;向不同pH浓度为10μM的近红外荧光探针溶液中分别对应加入100μM N2H4和Cu2+,每种溶液设置三个平行;反应完全,得到78份反应物,分别对78份反应物进行荧光强度测定(图3)。结果表明N2H4和Cu2+检测体系的最适pH均在7.4附近,考虑到后续在生物体内进行成像实验,因此选择生理pH为7.4作为后续实验溶液的pH。
实施例3
用于溶液体系中N2H4和Cu2+双通道识别的有效性探究
用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液与实施例1制备的荧光探针配制成浓度为10μM的荧光探针检测液,选择性的对溶液中的N2H4和Cu2+进行检测;具体操作过程如下:
向配制好的10μM的荧光探针溶液分别加入100μM的N2H4和Cu2+,图4的结果显示荧光探针本身荧光很强,而在加入N2H4和Cu2+后产生不同程度的荧光淬灭现象,结果表明该荧光探针可有效识别溶液体系中N2H4和Cu2+。
实施例4
用于环境溶液介质中N2H4和Cu2+的特异性检测
将实施例1中制备的荧光探针配制成浓度为10μM的4-羟乙基哌嗪乙磺酸(HEPES)和二甲基亚砜(DMSO)缓冲液;所述Cu2+检测体系工作液由体积比为3:7的4-羟乙基哌嗪乙磺酸(HEPES)和乙腈配制成,N2H4检测体系工作液由体积比为2:8的4-羟乙基哌嗪乙磺酸(HEPES)和二甲基亚砜配制成;向配制好的10μM的荧光探针溶液中分别加入100μM的分析物溶液,包括生物体内常见氨基酸:半胱氨酸(Cys),天冬氨酸(Asparticacid),色氨酸(Tryptophan),酪氨酸(Tyrosine),组氨酸(Histidine),谷氨酸(Glutamicacid),苏氨酸(Threonine);各种金属离子:碘化钾(KI),氯化镍(NiCl2),硝酸钙(Ca(NO3)2),氟化钠(NaF),氯化镁(MgCl2),氯化钴(CoCl2),氯化锰(MnCl2),氯化钡(BaCl2),氯化铵(NH4Cl)和氯化银(AgCl),每种溶液设置三个平行;反应完全,得到114份反应物,分别对114 份反应物进行荧光强度测定(图5);通过对荧光强度变化的研究,表明该荧光探针可以与N2H4和Cu2+进行特异性反应产生显著的荧光变化,即所述荧光探针可以特异性双通道识别N2H4和Cu2+。为了模拟细胞中的复杂环境,我们还进行了竞争实验,通过添加潜在的干扰物来确定荧光探针的抗干扰能力。结果表明荧光探针具有很强的抗干扰能力。
实施例5
荧光探针对N2H4和Cu2+识别灵敏度探究
将实施例1中制备的荧光探针配制成浓度为10μM的4-羟乙基哌嗪乙磺酸(HEPES)和二甲基亚砜(DMSO)缓冲液;所述Cu2+检测体系工作液由体积比为3:7的4-羟乙基哌嗪乙磺酸(HEPES)和乙腈配制成,N2H4检测体系工作液由体积比为2:8的4-羟乙基哌嗪乙磺酸(HEPES)和二甲基亚砜配制成;向配制好的10μM的荧光探针溶液中分别逐渐添加浓度为0-500μM的N2H4和Cu2+,每组三个平行,共得到90分反应物,分别对90份反应物进行荧光强度测定(图6)。结果表明荧光强度与N2H4和Cu2+浓度成反比,具有良好的线性关系,也就是说所述荧光探针能够在液体介质中定量检测N2H4和Cu2+。
实施例6
荧光探针对HeLa细胞的细胞毒性探究
将一系列探针溶液的浓度梯度(10,20,30和40μM)加入到含有HeLa细胞(每孔5×104个细胞)的96孔培养板中,并将未经处理的HeLa细胞作为对照。然后,孵育24小时后,通过CCK-8 检测验证HeLa细胞的细胞活力(图7)。结果表明荧光探针浓度在40μM时,HeLa存活率也在80%以上,表明该荧光探针具有低细胞毒性,可用于下一步活体成像应用。
实施例7
HeLa细胞内N2H4和Cu2+荧光成像
用于人体宫颈癌HeLa细胞中N2H4和Cu2+检测时,用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液与本发明所述荧光探针配制成浓度为20μM的工作溶液;
具体操作过程如下:分为a,b,c,d四组,a组(空白对照):未被任何处理的HeLa细胞,作为a组检测物;b组(N2H4和Cu2+对照):用50μM的N2H4和Cu2+分别孵育HeLa细胞30分钟,作为b 组检测物;c组(荧光探针对照):用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯(20μM)孵育HeLa细胞 30分钟,作为c组检测物;d组(实验组):将HeLa细胞用N2H4和Cu2+(50μM)分别处理30min,然后用HEPES缓冲溶液洗涤多余的N2H4和Cu2+3次,再用探针溶液(20μM)处理30min;通过a,b, c,d组荧光成像结果显示(E)-2-(3-(4-(二乙氨基)苯基) 丙烯酰)苯基吡啶甲酸酯(20μM)能够进入细胞中,并与细胞中的N2H4和Cu2+进行反应,细胞成像能够很明显的看出N2H4和Cu2+在细胞中的分布情况(图8)。
实施例8
斑马鱼体内内N2H4和Cu2+荧光成像
用于斑马鱼体内N2H4和Cu2+成像时,用4-羟乙基哌嗪乙磺酸 (HEPES)/二甲基亚砜(DMSO)缓冲液与本发明所述的荧光探针配制成浓度为20μM的工作溶液;
具体操作过程如下:分为a,b,c,d四组,a组(空白对照):未被任何处理的三日龄斑马鱼,作为a组检测物;b组(N2H4和Cu2+对照):用50μM的N2H4和Cu2+分别孵育三日龄斑马鱼30分钟,作为b组检测物;c组(荧光探针对照):用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯(20μM)孵育三日龄斑马鱼30分钟,作为c组检测物;d组(实验组):将三日龄斑马鱼用N2H4和Cu2+(50μM)分别处理30min,然后用HEPES缓冲溶液洗涤多余的N2H4和Cu2+3次,再用探针溶液(20μM)处理30min;结果显示在28℃下用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰) 苯基吡啶甲酸酯处理过的斑马鱼显示出明显的荧光,而同时使用荧光探针与N2H4和Cu2 +处理的斑马鱼产生一定的荧光淬灭现象(图 9);荧光成像结果表明(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯能够进入斑马鱼体内并与N2H4和Cu2+反应,产生荧光淬灭,从而达到检测的效果。
本技术领域中的普通技术人员应当认识到,以上的实施例仅是用来说明本发明,而并非用作为对本发明的限定,只要在本发明的实质精神范围内,对以上所述实施例的变化、变型都将落在本发明的权利要求范围内。
Claims (7)
1.一种基于分子内电荷转移原理用于双通道特异性检测N2H4和Cu2+的荧光探针,其特征在于:所述荧光探针为(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯,其化学结构式如式(І)所示:
式(І)。
2.权利要求1所述的荧光探针的制备方法,其特征在于,包括以下步骤:
(1)荧光母体(E)-3-(4-(二乙氨基)苯基)-1-(2-羟基苯基)丙-2-烯-1-酮合成具体操作步骤如下:
①将2,4-二乙氨基苯甲醛和2-羟基苯乙酮使用EtOH作溶剂混合均匀;
②向步骤①所得的混合溶液中添加KOH水溶液,并在室温下搅拌24小时;
③待步骤②中反应完成后用稀盐酸中和反应混合物,直到将pH值调整到7.0;
④将步骤③所得的混合物通过抽吸过滤收集沉淀,并用乙醇洗涤三次,所得到的残留物利用洗脱机通过硅胶层析进一步纯化,得到荧光基团(E)-3-(4-(二乙氨基)苯基)-1-(2-羟基苯基)丙-2-烯-1-酮;
(2)荧光探针(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯合成具体操作步骤如下:
①使用二氯甲烷为溶剂将荧光基团(E)-3-(4-(二乙氨基)苯基)-1-(2-羟基苯基)丙-2-烯-1-酮和2-吡啶甲酸溶解并混合均匀;
②向步骤①所得的混合溶液中加入4-二甲氨基吡啶和1-(3 二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;
③将步骤②中所得的混合溶液在室温下搅拌过夜,用旋转蒸发器除去溶剂,用硅胶柱纯化得到荧光探针(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯。
3.权利要求1所述的荧光探针对Cu2+和N2H4识别影响因素的筛选方法,其特征在于,包括以下步骤:
(1)将权利要求1所述的荧光探针用不同溶剂配制成浓度为10μM的工作溶液,所述溶剂分别为二甲基亚砜、N,N-二甲基甲酰胺、乙腈、甲醇、乙醇、丙酮、乙酸乙酯和水;向不同溶剂配制好的10μM的荧光探针溶液中分别对应加入200μM Cu2+和N2H4,每种溶液设置三个平行;反应完全,得到48份反应物,分别对48份反应物进行荧光强度测定;荧光探针检测Cu2+体系中使用乙腈效果最好;荧光探针检测N2H4体系中使用二甲基亚砜效果最好,最后选择乙腈为溶剂检测Cu2+,二甲基亚砜为溶剂检测N2H4;
(2)将权利要求1所述的荧光探针用不同比例的二甲基亚砜与4-羟乙基哌嗪乙磺酸配制成浓度为10μM的荧光探针溶液,所述乙腈和二甲基亚砜比例分别为10%,20%,30%,40%,50%,60%,70%,80%,90%,100%;向使用不同比例乙腈和二甲基亚砜配制好的10μM的荧光探针溶液中分别对应加入200μM Cu2+和N2H4,每种溶液设置三个平行;反应完全,得到60份反应物,分别对60份反应物进行荧光强度测定;结果表明荧光探针检测Cu2+体系中4-羟乙基哌嗪乙磺酸与乙腈比例为3:7最佳,荧光探针检测N2H4体系中4-羟乙基哌嗪乙磺酸与二甲基亚砜比例为2:8最佳;
(3)将权利要求1所述的荧光探针用缓冲液配制成浓度为10 μM的荧光探针溶液,所述Cu2+检测体系工作液由体积比为3:7的4-羟乙基哌嗪乙磺酸和乙腈配制成,N2H4检测体系工作液由体积比为2:8的4-羟乙基哌嗪乙磺酸和二甲基亚砜配制成;设置缓冲溶液pH为2,3,4,5,6,6.5,7,7.4,8,9,10,11,12;向不同pH浓度为10μM的近红外荧光探针溶液中分别对应加入100μM Hg2+,每种溶液设置三个平行;反应完全,得到78份反应物,分别对78份反应物进行荧光强度测定;结果表明N2H4和Cu2+检测体系的最适pH均在7.4附近。
4.权利要求1所述的荧光探针的应用,其特征在于,所述荧光探针用于N2H4和Cu2+双通道的特异性检测。
5.根据权利要求4所述的荧光探针用于环境溶液介质中N2H4和Cu2+特异性的检测方法,其特征在于,操作步骤如下:
(1)将权利要求1所述的荧光探针配制成浓度为10μM的4-羟乙基哌嗪乙磺酸和二甲基亚砜缓冲液;所述缓冲液中4-羟乙基哌嗪乙磺酸和二甲基亚砜的体积比为3:7,缓冲液的pH值为7.4;
(2)向配制好的10μM的荧光探针溶液中分别加入200μM的分析物溶液,包括生物体内常见氨基酸:半胱氨酸,天冬氨酸,色氨酸,酪氨酸,组氨酸,谷氨酸,苏氨酸;各种金属离子:碘化钾,氯化镍,硝酸钙,氟化钠,氯化镁,氯化钴,氯化锰,氯化钡,氯化铵和氯化银,反应完全后进行荧光强度测定;
(3)通过对荧光强度变化的研究,表明该荧光探针可以与N2H4和Cu2+进行特异性反应产生显著的荧光变化,即所述荧光探针可以特异性双通道识别N2H4和Cu2+。
6.根据权利要求4所述的荧光探针用于检测人体宫颈癌组织HeLa细胞中N2H4和Cu2+分布的检测方法,其特征在于,操作步骤如下:
(1)将权利要求1所述荧光探针配制成浓度为20μM的缓冲液;分为a,b,c,d四组,a组为空白对照组:未被任何处理的HeLa细胞,作为a组检测物; b组为N2H4和Cu2+对照组:用50μM的N2H4和Cu2+分别孵育HeLa细胞30分钟,作为b组检测物;c组为荧光探针对照组:用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯孵育HeLa细胞30分钟,作为c组检测物;d组为实验组:将HeLa细胞用N2H4和Cu2+分别处理30 min,然后用HEPES缓冲溶液洗涤多余的N2H4和Cu2+3次,再用探针溶液处理30 min;
(2)通过a,b,c,d组荧光成像结果显示(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯能够进入细胞中,并与细胞中的N2H4和Cu2+进行反应,细胞成像能够很明显的看出N2H4和Cu2+在细胞中的分布情况。
7.根据权利要求4所述的荧光探针用于斑马鱼中N2H4和Cu2+的检测方法,其特征在于,操作步骤如下:
(1)将权利要求1所述的荧光探针配制成浓度为20μM的工作液;分为a,b,c,d四组,a组为空白对照组:未被任何处理的三日龄斑马鱼,作为a组检测物; b组为N2H4和Cu2+对照组:用50μM的N2H4和Cu2+分别孵育三日龄斑马鱼30分钟,作为b组检测物;c组为荧光探针对照组:用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯孵育三日龄斑马鱼30分钟,作为c组检测物;d组为实验组:将三日龄斑马鱼用N2H4和Cu2+分别处理30 min,然后用HEPES缓冲溶液洗涤多余的N2H4和Cu2+3次,再用探针溶液处理30 min;
(2)结果显示在28℃下用(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯处理过的斑马鱼显示出明显的荧光,而同时使用荧光探针与N2H4和Cu2+处理的斑马鱼产生一定的荧光淬灭现象;荧光成像结果表明(E)-2-(3-(4-(二乙氨基)苯基)丙烯酰)苯基吡啶甲酸酯能够进入斑马鱼体内并与N2H4和Cu2+反应,产生荧光淬灭,从而达到检测的效果。
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