CN105859733B - 一种双光子荧光探针及其制备方法和用途 - Google Patents

一种双光子荧光探针及其制备方法和用途 Download PDF

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CN105859733B
CN105859733B CN201610272352.1A CN201610272352A CN105859733B CN 105859733 B CN105859733 B CN 105859733B CN 201610272352 A CN201610272352 A CN 201610272352A CN 105859733 B CN105859733 B CN 105859733B
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孟祥明
韦新洁
朱满洲
邵宗龙
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Abstract

本发明公开了一种双光子荧光探针及其制备方法和用途，其中双光子荧光探针是以香豆素与罗丹明为母体，其结构由以下表示：

Description

一种双光子荧光探针及其制备方法和用途
一、技术领域
本发明涉及一种双光子荧光探针及其制备方法和用途。
二、背景技术
钯及其化合物是合成药物分子有效的催化剂，钯优秀的催化能力使其被广泛用作有机合成中的催化剂。钯催化反应是合成药物分子有效的催化剂而被大量应用，但是在最终产品中会残留大量的钯，随着药物的服用而进入人体。在汽车催化转化器中，钯被用来催化转化有害气体，减少废物排放。不幸的是，钯也会随着尾气排放到环境中，增加环境中钯的含量，进而增加进入人体的风险。同时，钯离子可以与含硫氨基酸、蛋白质、DNA和维生素B6等生物大分子和生化试剂作用，扰乱多种细胞过程，钯还可以诱发一系列的毒性效应进而导致严重的皮肤和眼睛刺激。
荧光化学传感器区别于传统检测生物体内生物小分子含量的检测工具，其高灵敏度、高选择性、易于合成、廉价以及良好的生物应用特点，使其已经逐渐成为了生命科学和环境科学中主要的检测工具。通过探针与生物体内的活性物质的相互作用，显示出对应的荧光信号变化，能够更有效地帮助人们从微观角度出发来观察和了解生命活动。在生命科学以及环境科学等研究领域，设计且合成高灵敏度、高选择性的有机小分子荧光化学传感器已经成为人们的热衷研究的课题之一。然而，在过去的研究中单光子荧光化学传感器得到了快速的发展，但其在激发波长短，激发能量大，生物毒性强上的劣势会导致一系类不足，阻碍了荧光传感器的前进和发展。如：荧光淬灭，在生物细胞或组织中有自荧光的干扰，组织穿透深度小(<100μm)等。相比之下，双光子荧光探针在激发波长长，激发能量低，组织穿透能力深，空间分辨率高，较小的荧光干扰背景和光散射，以及双光子延长细胞、组织显微成像时间上的特殊优势，使得双光子荧光化学传感器更利于人们观察研究生命过程。
三、发明内容
本发明旨在提供一种双光子荧光探针及其制备方法和用途，所要解决的技术问题是通过分子设计遴选合适的荧光探针结构，以实现双光子成像定性检测细胞中的钯离子(Ⅱ)，具有选择性专一、灵敏度高、检测浓度低的优点，细胞毒性测试表明本发明荧光探针对细胞几乎没有毒性作用。
本发明双光子荧光探针，是以香豆素为母体，简称荧光探针或荧光探针分子(RN3)，其结构由下式表示：
本发明双光子荧光探针的制备方法，包括如下步骤：
(1)将7-((4-甲氧基苯基)乙炔)-3-羧酸-香豆素(1g，3mmol)、1-羟基苯并三唑(0.65g，4.5mmol)和EDC·HCl(0.65g，3.3mmol)加入史莱克瓶中，在无水无氧条件下加入30mL N,N-二甲基甲酰胺，室温搅拌反应24h；反应结束后先加50mL二氯甲烷将反应物溶解，再用30mL水萃取，得到有机相，减压蒸馏旋干得中间体1；
(2)将罗丹明B(2g，4.16mmol)置于三颈烧瓶中，加入100ml乙醇并加热至回流，待罗丹明完全溶解后，缓慢滴加二乙烯三胺(0.646g，6.26mmol)，加热至回流反应24h；待反应完降至室温，向反应液中加入步骤(1)制备得到的中间体1，室温搅拌反应15h；反应完成后向反应液中加入50mL二氯甲烷将反应物溶解，再用30mL水萃取，得到有机相，通过柱层析200-300目硅胶(洗脱液乙酸乙酯：甲醇＝10:1，v/v)得到目标产物RN3 0.6g，产率25％。
本发明荧光探针分子的合成过程如下：
本发明双光子荧光探针在定性检测细胞中钯离子(Ⅱ)时作为检测试剂使用，检测方法如下：
将本发明荧光探针分子溶于DMSO中制得1mM的母液，取250μL的该母液于10mL容量瓶中，再用DMSO定容，配制成25μM的检测试剂。向所述检测试剂中加入1倍当量的钯离子(Ⅱ)，可见紫外在550nm左右出现一个明显的吸收(图2)，检测415-700nm范围内的荧光光谱变化，随着时间的变化，可以观察到，515nm处的发射峰逐渐减弱，575nm发射峰逐渐增强(图3)。
本发明荧光探针检测钯离子(Ⅱ)的机理是荧光探针分子本身具有两个荧光基团，分别是香豆素和罗丹明，是两种常见的荧光基团，这两种基团的激发波长分别在380nm和510nm左右，发射波长分别位于510nm和550nm处。当柔性链区域未与钯离子(Ⅱ)螯合时，395nm激发波长能够为香豆素基团提供能量，使其在515nm发出荧光。与钯离子(Ⅱ)螯合后，由于螯合作用钯离子(Ⅱ)使罗丹明基团发生开环反应，罗丹明能够吸收到来自香豆素的发射峰所传递的能量，从而发出罗丹明的荧光(575nm)，荧光的最大发射峰从515nm红移至575nm(斯托克斯位移约为55nm)。
本发明荧光探针分子结构简单，作用位点和荧光基团为一整体。本发明荧光探针分子与钯离子(Ⅱ)有明确的作用位点，本发明通过胺基与钯离子(Ⅱ)反应后形成稳定螯合物。本发明荧光探针分子以荧光的变化来检测钯离子(Ⅱ)，并且能在可见光下看出前后溶液颜色的变化，溶液颜色从黄色变成红色。本发明荧光探针分子与钯离子(Ⅱ)作用后，在紫外灯下，肉眼就可以看出其荧光变化，荧光颜色从黄绿色光变成橘黄色光，操作简单，快速灵敏。本发明荧光探针分子选择性专一，灵敏度高，检测浓度低。
四、附图说明
图1是本发明荧光探针分子与钯离子(Ⅱ)的反应过程图。
图2是将本发明荧光探针分子(25μM)加入钯离子(Ⅱ)的紫外随着时间变化光谱图。
图3是将本发明荧光探针分子(25μM)加入钯离子(Ⅱ)的荧光浓度光谱图。
图4本发明荧光探针分子的双光子荧光共聚焦成像照片，荧光探针分子(10μM)在细胞培养30分钟后，用PBS缓冲液(pH7.4)冲洗，其中(a)、(e)分别为反应前、反应后CHO细胞在515nm处荧光共聚焦成像。(b)、(f)分别为反应前、反应后HeLa细胞在575nm处的荧光共聚焦成像。(c)、(g)是HeLa细胞的明场，(d)是(a)、(b)、(c)的叠加，(h)是(e)、(f)、(g)的叠加。从细胞成像可以看出，反应前，荧光探针分子RN3在515nm处有荧光，575nm处无荧光。用钯离子(Ⅱ)处理后，荧光探针分子RN3在515nm处荧光减弱，575nm处出现一个明显的荧光。
五、具体实施方式
下面通过实施例对本发明做进一步说明。
实施例1：荧光探针分子RN3的合成
1、将7-((4-甲氧基苯基)乙炔)-3-羧酸-香豆素(1g，3mmol)、1-羟基苯并三唑(0.65g，4.5mmol)和EDC·HCl(0.65g，3.3mmol)加入史莱克瓶中，在无水无氧条件下加入30mL N,N-二甲基甲酰胺，室温搅拌反应24h；反应结束后先加50mL二氯甲烷将反应物溶解，再用30mL水萃取3次，得到有机相，减压蒸馏旋干得中间体1；
2、将罗丹明B(2g，4.16mmol)置于三颈烧瓶中，加入100ml乙醇并加热至回流，待罗丹明完全溶解后，缓慢滴加二乙烯三胺(0.646g，6.26mmol)，加热至回流反应24h；待反应完降至室温，向反应液中加入步骤1制备得到的中间体1，室温搅拌反应15h；反应完成后向反应液中加入50mL二氯甲烷将反应物溶解，再用30mL水萃取3次，得到有机相，通过柱层析200-300目硅胶(洗脱液乙酸乙酯：甲醇＝10:1，v/v)得到目标产物RN3 0.6g，产率25％。
1H NMR(400MHz,)δ8.86,8.80,7.84,7.83,7.61,7.59,7.53,7.50,7.48,7.46,7.44,7.43,7.41,7.40,7.38,7.09,7.07,6.93,6.90,6.46,6.44,6.38,6.38,6.30,6.29,6.28,6.27,3.85,3.41,3.40,3.36,3.34,3.32,3.31,3.29,3.27,2.67,2.49,2.48,2.46,1.18,1.16,1.14,-0.00.
13C NMR(400MHz,)δ169.86,160.99,160.43,154.27,153.61,153.32,148.82,147.28,140.45,133.52,132.39,129.64,129.49,128.77,128.16,127.97,126.05,123.81,122.77,118.81,118.34,118.07,114.22,108.14,105.79,97.76,94.96,87.08,77.34,77.23,77.02,76.70,55.39,53.44,47.88,47.68,44.37,39.95,12.61,0.00.
实施例2：荧光探针分子的荧光测试
将本发明荧光探针分子溶于DMSO中制得1mM的母液，取250μL的该母液于10mL容量瓶中，再用DMSO定容，配制成25μM的检测试剂。向所述检测试剂中加入1倍当量的钯离子(Ⅱ)，可见紫外在550nm左右出现一个明显的吸收(图2)，检测415-700nm范围内的荧光光谱变化，随着时间的变化，可以观察到，515nm处的发射峰逐渐减弱，575nm发射峰逐渐增强(图3)。
当柔性链区域未与钯离子(Ⅱ)螯合时，395nm激发波长能够为香豆素基团提供能量，使其在515nm发出荧光。与钯离子(Ⅱ)螯合后，由于螯合作用钯离子(Ⅱ)使罗丹明基团发生开环反应，罗丹明能够吸收到来自香豆素的发射峰所传递的能量，从而发出罗丹明的荧光(575nm)，荧光的最大发射峰从515nm红移至575nm(斯托克斯位移约为55nm)。
实施例3：细胞成像测试
293FT细胞由DEME(invitrogen)培养液培养，成像前一天，HeLa细胞放于平底表面皿中，成像时HeLa细胞和10μM的荧光探针RN3的DMSO溶液于37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液或培养液充分洗涤后，用荧光共聚焦成像，得图4。向上述含荧光探针的细胞培养液中加入(10μM)钯离子(Ⅱ)溶液，在37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液或培养液充分洗涤后，再进行双光子荧光共聚焦成像。从图中可以看出，加入钯离子(Ⅱ)前，515nm有荧光；加入钯离子(Ⅱ)后，575nm荧光明显增强，荧光的最大发射峰从515nm红移至575nm(斯托克斯位移约为55nm)。

Claims (4)

1.一种双光子荧光探针，其特征在于其结构式如下：
2.一种权利要求1所述的双光子荧光探针的制备方法，其特征在于包括如下步骤：
(1)将7-((4-甲氧基苯基)乙炔)-3-羧酸-香豆素1g、1-羟基苯并三唑0.65g和EDC·HCl0.65g加入史莱克瓶中，在无水无氧条件下加入N,N-二甲基甲酰胺，室温搅拌反应24h；反应结束后先加二氯甲烷将反应物溶解，再用水萃取，得到有机相，减压蒸馏旋干得中间体1；
(2)将罗丹明B 2g置于三颈烧瓶中，加入乙醇并加热至回流，待罗丹明完全溶解后，缓慢滴加二乙烯三胺0.646g，加热至回流反应24h；待反应完降至室温，向反应液中加入步骤1制备得到的中间体1，室温搅拌反应15h；反应完成后向反应液中加入二氯甲烷将反应物溶解，再用水萃取，得到有机相，通过柱层析200-300目硅胶得到目标产物。
3.根据权利要求2所述的制备方法，其特征在于：
柱层析时的洗脱液为乙酸乙酯和甲醇按体积比10:1混合得到。
4.一种权利要求1所述的双光子荧光探针的用途，其特征在于：权利要求1所述的双光子荧光探针在制备用于定性检测细胞中钯离子(Ⅱ)的检测试剂中的用途。
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