CN108299450A - 一种检测生物硫醇的荧光探针 - Google Patents

一种检测生物硫醇的荧光探针 Download PDF

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CN108299450A
CN108299450A CN201810070839.0A CN201810070839A CN108299450A CN 108299450 A CN108299450 A CN 108299450A CN 201810070839 A CN201810070839 A CN 201810070839A CN 108299450 A CN108299450 A CN 108299450A
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fluorescence
probe
biological thiol
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宋相志
苏远安
杨雷
熊海青
张帆
韩金量
张赟
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Abstract

本发明涉及一种检测生物硫醇的荧光探针，属于荧光探针领域。其分子结构如下：该探针分子没有荧光，与生物硫醇作用后发出蓝色荧光。本发明所述的探针分子不仅可以定性分析生物硫醇，而且可以实现快速、定量检测生物硫醇，在生物化学等领域具有重要的应用价值。

Description

一种检测生物硫醇的荧光探针
本发明涉及一种基于香豆素型生物硫醇荧光探针的制备及其在检测生物硫醇方面的研究，属于荧光探针领域。
背景技术
生物硫醇是生物体内含巯基化合物的总称，广泛地存在于生物体内，最具代表性的生物硫醇为三种巯基化合物：半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)。巯基化合物是生物体酶发挥作用后和生理活动中的重要信号分子，同时也可以调节细胞的正常氧化还原状态，在生物体的生理活动中具有重要的功能。生物体内巯基化合物的浓度异常与许多种疾病有关，Cys的缺乏与生长缓慢、肝损伤、毛发褪色、皮肤病、昏睡以及水肿等疾病有关；血浆中Hcy浓度过高与心血管疾病、阿兹海默症和骨质疏松症有关；GSH能够保护机体免受氧化及自由基介导的氧应激损伤，其浓度与白血病、癌症及艾滋病等有关。因此，检测生物体内巯基化合物浓度对研究细胞功能和进行疾病早期诊断具有重要的意义。相对于其它传统的检测技术，荧光分析法具有操作简单、选择性好、灵敏度高等优点，尤其可以用于细胞及生物活体成像，因此近年来利用荧光探针来检测离子及生物分子的研究受到了广泛的关注。
发明内容
本发明目的在于提供一种检测生物硫醇的荧光探针，其结构式如下：合成路线如下：
具体合成方法如下：(a)在25mL的圆底烧瓶中加入400mg化合物1、295mg化合物2，再加入10mL甲苯，氩气保护回流反应24小时，抽滤，用10mL石油醚洗涤滤饼，烘干得到210mg灰白色粉末为化合物3，产率：54.5％。(b)在25mL圆底烧瓶中加入50mg化合物3、三乙胺35μL冰水浴30分钟，再将63.8mg 2,4-二硝基苯磺酰氯加入到烧瓶中，继续保持冰水浴，2小时后旋干溶剂进行柱层析，得到34.2mg黑色粉末为探针，产率35.1％。
本发明的荧光探针测试方法如下，将探针分子溶解在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，室温下进行测试。可以对生物硫醇进行定性、定量检测，具体实施方法在实施实例中详细介绍。
本发明的荧光探针的响应机理如下：生物硫醇与探针分子作用后，生物硫醇的巯基进攻磺酰酯基，探针分子脱去一个二氧化硫分子、2,4-二硝基苯硫醇分子，释放出染料分子。溶液从黄色变成无色，从没有荧光变成有荧光，实现检测生物硫醇。探针分子的响应过程如下：
本发明的荧光探针没有荧光，与生物硫醇响应后的荧光发射峰在409nm处。探针与生物硫醇完全响应后409nm处荧光强度显著增强。
本发明所述的探针分子合成路线简单，操作简单，成本较低，可以实现快速检测生物硫醇。
附图说明
图1为本发明的荧光探针在氘代DMSO中核磁共振氢谱，横坐标化学位移，纵坐标为强度。
图2为本发明的荧光探针在氘代DMSO中核磁共振碳谱，横坐标化学位移，纵坐标为强度。
图3为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与浓度均为50μM三种不同生物硫醇作用后荧光强度与时间关系，横坐标为时间，纵坐标为荧光强度。
图4为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度半胱氨酸作用后荧光光谱图，横坐标为波长，纵坐标为荧光强度。
图5为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度半胱氨酸作用后在409纳米处荧光强度与半胱氨酸浓度关系，横坐标为半胱氨酸浓度，纵坐标为荧光强度。
图6为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度半胱氨酸作用后在409纳米处荧光强度与半胱氨酸浓度线性关系，横坐标为半胱氨酸浓度，纵坐标为荧光强度。
图7为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度同型半胱氨酸作用后荧光光谱图，横坐标为波长，纵坐标为荧光强度。
图8为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度同型半胱氨酸作用后在409纳米处荧光强度与同型半胱氨酸浓度关系，横坐标为同型半胱氨酸浓度，纵坐标为荧光强度。
图9为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度同型半胱氨酸作用后在409纳米处荧光强度与同型半胱氨酸浓度线性关系，横坐标为同型半胱氨酸浓度，纵坐标为荧光强度。
图10为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度谷胱甘肽作用后荧光光谱图，横坐标为波长，纵坐标为荧光强度。
图11为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度谷胱甘肽作用后在409纳米处荧光强度与谷胱甘肽浓度关系，横坐标为谷胱甘肽浓度，纵坐标为荧光强度。
图12为本发明的荧光探针(1.0×10-5mol/L)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中，与不同浓度谷胱甘肽作用后在409纳米处荧光强度与谷胱甘肽浓度线性，横坐标为谷胱甘肽浓度，纵坐标为荧光强度。
具体实施实例
实施例1：化合物3的合成
在25mL的圆底烧瓶中加入400mg化合物1、295mg化合物2，再加入10mL甲苯，氩气保护回流反应24小时，抽滤，用10mL石油醚洗涤滤饼，烘干得到210mg灰白色粉末为化合物3，产率，54.5％。
实施例2：探针的合成
在25mL圆底烧瓶中加入50mg化合物3、三乙胺35μL冰水浴30分钟，再将63.8mg 2,4-二硝基苯磺酰氯加入到烧瓶中，继续保持冰水浴，2小时后旋干溶剂进行柱层析，得到34.2mg黑色粉末为探针，产率35.1％。1H NMR(400MHz,DMSO)δ9.13(d,J＝1.8Hz,1H),8.66(dd,J＝8.7,1.9Hz,1H),8.55(d,J＝8.7Hz,1H),6.98(s,1H),5.85(s,1H),3.29(s,4H),2.71(t,J＝6.2Hz,2H),2.65(t,J＝5.9Hz,2H),1.92–1.79(m,4H).13C NMR(101MHz,DMSO)δ161.21(s),158.19(s),152.24(s),151.18(s),148.46(s),147.55(s),134.31(s),130.99(s),128.30(s),121.76(s),119.85(s),119.22(s),106.06(s),101.65(s),97.28(s),49.75(s),49.20(s),27.35(s),20.97(s),20.31(s),20.04(s).
实施案例3：本发明荧光探针检测生物硫醇研究
将探针溶pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中,配制成1.0×10-5mol/L的溶液，加入三种生物硫醇可以迅速观测到409纳米处的荧光变化，检测时间短，可以实现定性、定量检测生物硫醇。

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1.一种检测生物硫醇的荧光探针，其结构为：
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108912134A (zh) * 2018-07-17 2018-11-30 商丘师范学院 一种高灵敏检测半胱氨酸的荧光探针及制备方法、应用
CN109912612A (zh) * 2019-04-04 2019-06-21 郑州大学 一种基于分子逻辑门识别Cys、GSH和HOCl荧光探针的合成、制备及其应用
CN110615786A (zh) * 2019-07-17 2019-12-27 浙江工业大学 用于检测粘度的近红外荧光化合物及其制备与应用
CN112209923A (zh) * 2020-10-14 2021-01-12 中南大学 一种高选择性检测Cys/Hcy和GSH的近红外荧光探针的合成和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIAO-BO WANG，等: "A novel reaction-based fluorescent turn-on probe for biothiols and itsapplication in cell imaging", 《SENSORS AND ACTUATORS B》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108912134A (zh) * 2018-07-17 2018-11-30 商丘师范学院 一种高灵敏检测半胱氨酸的荧光探针及制备方法、应用
CN109912612A (zh) * 2019-04-04 2019-06-21 郑州大学 一种基于分子逻辑门识别Cys、GSH和HOCl荧光探针的合成、制备及其应用
CN109912612B (zh) * 2019-04-04 2021-05-11 郑州大学 一种基于分子逻辑门识别Cys、GSH和HOCl荧光探针的合成、制备及其应用
CN110615786A (zh) * 2019-07-17 2019-12-27 浙江工业大学 用于检测粘度的近红外荧光化合物及其制备与应用
CN112209923A (zh) * 2020-10-14 2021-01-12 中南大学 一种高选择性检测Cys/Hcy和GSH的近红外荧光探针的合成和应用

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Application publication date: 20180720