CN114634473B - 一种能够快速高效检测生物硫醇的荧光探针及其制备方法 - Google Patents
一种能够快速高效检测生物硫醇的荧光探针及其制备方法 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种荧光探针及其制备方法,具体涉及一种能够快速高效检测生物硫醇的荧光探针及其制备方法,属于荧光探针技术领域。
背景技术
生物硫醇主要包括谷胱甘肽(GSH)、半胱氨酸(Cys)和高半胱氨酸(Hcy),是一类含巯基的氨基酸,参与了人体细胞中多种氧化还原反应及信号传导过程,在生理和病理过程中具有重要的作用。当体内生物硫醇含量过高时,会导致心血管疾病、中风、骨软弱症,甚至形成阿尔茨海默症及精神分裂症等多种不可逆转的疾病。了解生物硫醇的生物学功能以及在体内的分布规律,将有助于有效控制体系中生物硫醇紊乱造成的生物损伤,从而及时诊断并治疗相关疾病。因此,开发高效、快速的检测食品和生物样品中生物硫醇的含量及动态变化的探针,有助于疾病的诊断与治疗,具有重要应用价值。
常见的生物硫醇检测方法有同位素法、色谱法、免疫学法等。采用这些方法检测生物硫醇时,不仅所用的仪器设备昂贵,而且检测耗时长、样品预处理复杂,并且在生物体内具有多种不确定干扰因素,对检测过程及结果造成巨大干扰。最关键的是,这些方法不能够实现生物体系原位实时监测,而且微环境的变化极易影响硫醇及蛋白巯基的氧化还原态。相比较而言,荧光分析检测技术因具有简便性、可视性、快速高效性以及高灵敏度和高选择性等优点,在样品的原位可视化检测方面已趋于成熟,尤其是在无损、实时监测这一方面具有突出优势。
目前,关于检测生物硫醇的荧光探针已经有了很多报道,但大都存在响应时间较长、灵敏度相对较低的问题,在生物样品中的局限性较大。因此,开发一种合成方法简单,具有高选择性和高灵敏度,尤其能够快速检测生物硫醇的荧光探针,将其应用于食品以及细胞和活体中生物硫醇的检测,具有一定的挑战性和研究意义。
发明内容
为解决现有技术的不足,本发明的第一个目的在于:提供一种结构稳定、能够快速高效的检测生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的荧光探针;本发明的第二个目的在于:提供一种合成路线简单、产率高的制备上述荧光探针的方法。
为了实现上述第一个目标,本发明采用如下的技术方案:
一种能够快速检测生物硫醇的荧光探针,所述生物硫醇为谷胱甘肽、半胱氨酸和高半胱氨酸,其特征在于,所述荧光探针为3-甲磺酰基-7-二乙氨基香豆素,结构如式I所示:
式I。
为了实现上述第二个目标,本发明采用如下的技术方案:
一种制备式1所述的能够快速检测生物硫醇的荧光探针的方法,其特征在于,包括以下步骤:
Step1:将3-溴-7-二乙氨基香豆素溶于N,N-二甲基甲酰胺中,不断搅拌并滴加甲硫醇钠的水溶液,于室温下充分反应,待反应完全后,将反应液逐滴滴入去离子水中,静置过夜,析出固体初产物;
Step2:将所得固体初产物过滤、去离子水反复洗涤,然后干燥,得到中间产物3-甲硫基-7-二乙氨基香豆素;
Step3:将中间产物3-甲硫基-7-二乙氨基香豆素溶于二氯甲烷中,不断搅拌并分批加入间氯过氧苯甲酸的二氯甲烷溶液,于室温下充分反应,待反应完全后,柱层析分离,得到式I所示荧光探针。
优选的,在Step1中,所述3-溴-7-二乙氨基香豆素与甲硫醇钠的摩尔比为1:1~5。
优选的,在Step3中,所述3-甲硫基-7-二乙氨基香豆素与间氯过氧苯甲酸的摩尔比为1:2~5。
优选的,在Step1和Step3中,用薄层色谱监测反应进程。
本发明的有益之处在于:
(1)本发明提供的荧光探针,结构稳定,有利于室温下长期保存;
(2)本发明提供的荧光探针,在510nm处有较弱的荧光发射峰,加入生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)后,510nm处的荧光强度明显增强,而加入其他常见氨基酸后,510nm处的荧光发射强度几乎没有变化,因此在检测生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的过程中不会受到其他氨基酸的干扰,具有高选择性;
(3)本发明提供的荧光探针,在生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)存在下,2min即可表现出显著的荧光增强,并且能够达到最大值,响应速度显著提升,灵敏度较高,具有快速高效的优势,可用于食品、细胞和活体内生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的快速高效检测;
(4)本发明提供的制备荧光探针的方法,合成路线简单,产率高,易于大量获取荧光探针。
附图说明
图1是式I所示荧光探针的合成路线图;
图2是式I所示荧光探针(10μM)检测谷胱甘肽、半胱氨酸和高半胱氨酸(浓度均为100μM)的荧光发射光谱图,其中,激发波长为385nm,最大发射波长为510nm,溶剂为PBS缓冲溶液;
图3是式I所示荧光探针(10μM)在不同时间检测谷胱甘肽、半胱氨酸和高半胱氨酸的荧光发射强度图,其中,激发波长为385nm,最大发射波长为510nm,溶剂为PBS缓冲溶液,温度为室温;
图4是式I所示荧光探针(10μM)选择性检测生物硫醇的荧光强度变化图,其中,0为式I所示荧光探针,1~15分别为胱氨酸、苏氨酸、丙氨酸、天冬氨酸、组氨酸、甘氨酸、酪氨酸、缬氨酸、丝氨酸、苯丙氨酸、抗坏血酸、脯氨酸、葡萄糖、硫化钠和硫氢化钠(浓度均为100μM),16~18分别为谷胱甘肽、高半胱氨酸和半胱氨酸(浓度均为100μM),激发波长为385nm,最大发射波长为510nm,溶剂为PBS缓冲溶液;
图5是式I所示荧光探针检测HepG2细胞内生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的共聚焦显微镜照片,其中a为HepG2细胞在37℃孵育5min的明场;b为HepG2细胞与式I所示荧光探针(浓度为10μM,溶剂为PBS缓冲溶液)在37℃孵育5min的明场;c为HepG2细胞在37℃孵育5min的荧光场;d为HepG2细胞与式I所示荧光探针(浓度为10μM,溶剂为PBS缓冲溶液)在37℃孵育5min的荧光场。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
一、检测生物硫醇的荧光探针的结构
本发明提供的检测生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的荧光探针,以7-二乙氨基香豆素为荧光团,将甲磺酰基接枝于该荧光团的3位,得到3-甲磺酰基-7-二乙氨基香豆素,结构式具体如下所示:
式I。
二、检测生物硫醇的荧光探针的制备方法
本发明提供的制备式I所示检测生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的荧光探针的方法,合成路线如图1所示。
在本具体实施例中,制备式I所示检测生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的荧光探针的方法,具体包括以下步骤:
Step1:将3-溴-7-二乙氨基香豆素(1mmol,297mg)完全溶于N,N-二甲基甲酰胺(10mL)中,不断搅拌并滴加甲硫醇钠(3mmol,210mg)的水溶液,于室温下充分反应,用薄层色谱监测反应进程,待反应完全后,将反应液逐滴滴入去离子水(20mL)中,静置过夜,析出固体初产物;
Step2:将所得固体初产物过滤、去离子水反复洗涤,然后干燥,得到中间产物3-甲硫基-7-二乙氨基香豆素;
Step3:将中间产物3-甲硫基-7-二乙氨基香豆素(1mmol,263mg)溶于二氯甲烷(20mL)中,不断搅拌并分批加入间氯过氧苯甲酸(m-CPA,3mmol,520mg)的二氯甲烷溶液,于室温下充分反应,用薄层色谱监测反应进程,待反应完全后,柱层析分离,得到最终产物。
经检测,Step2得到的中间产物的核磁共振氢谱和碳谱分别如下:
1H NMR(300MHz,CDCl3)δ7.48(dd,J=9.0,2.3Hz,1H),6.56(dd,J=9.0,2.2Hz,1H),6.46(d,J=2.2Hz,1H),5.80(d,J=2.1Hz,1H),3.51-3.27(m,4H),2.49(d,J=2.1Hz,3H),1.45-1.07(m,6H);
13C NMR(75MHz,CDCl3)δ160.64,157.40,154.44,150.71,124.60,108.31,107.07,100.37,97.25,44.69,13.60,12.39。
由上述核磁共振氢谱和碳谱可知,中加产物为3-甲硫基-7-二乙氨基香豆素,结构如式III所示。
经检测,Step3得到的最终产物的核磁共振氢谱和碳谱分别如下:
1H NMR(500MHz,CDCl3)δ7.99(d,J=9.2Hz,1H),6.72(s,H),6.66(dd,J=9.3,2.6Hz,1H),6.57(d,J=2.6Hz,1H),3.45(q,J=7.1Hz,4H),3.19(s,3H),1.24(t,J=7.1Hz,6H);
13C NMR(126MHz,CDCl3)δ160.13,157.23,151.38,151.11,126.23,110.83,109.61,101.90,98.08,44.92,43.64,12.39。
由上述核磁共振氢谱和碳谱可知,最终产物为式I所示荧光探针。
另外,经计算,Step2得到的中间产物的产率为62%,Step3得到的最终产物的产率为49%。
可见,本发明提供的制备荧光探针的方法,合成路线简单,产率高,易于大量获取荧光探针。
三、检测生物硫醇的荧光探针的特点及性能
1、在水溶液中的荧光发射情况
用具体实施例制备得到的式I所示荧光探针(浓度为10μM,溶剂为PBS缓冲溶液)检测生物硫醇谷胱甘肽、半胱氨酸和高半胱氨酸(浓度均为10μM,溶剂均为PBS缓冲溶液),激发波长为385nm,最大发射波长为510nm,得到的荧光发射光谱图如图2所示。
由图2可知:式I所示荧光探针在510nm处有较弱的荧光发射峰,加入生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)后,510nm处的荧光强度明显增强,强度达到40倍以上。
这说明:式I所示荧光探针能够实现水溶液中生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的检测。
2、检测速度
用具体实施例制备得到的式I所示荧光探针(浓度为10μM,溶剂为PBS缓冲溶液)在室温条件下、于不同时间分别检测谷胱甘肽、半胱氨酸和高半胱氨酸(浓度均为100μM,溶剂均为PBS缓冲溶液),激发波长为385nm,最大发射波长为510nm,得到的荧光发射光谱图如图3所示。
由图3可知:在室温条件下,当加入谷胱甘肽(GSH)、半胱氨酸(Cys)和高半胱氨酸(Hcy)时,随着时间的推移,式I所示荧光探针在2min内均有明显的荧光强度增强,并且稳定性很好,可达到最大值。
这说明:式I所示荧光探针能够实现生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)的高效快速检测,能够快速检测样品中的生物硫醇,具有明显的优势。
3、选择性
用具体实施例制备得到的式I所示荧光探针(浓度为10μM,溶剂为PBS缓冲溶液)分别检测胱氨酸、苏氨酸、丙氨酸、天冬氨酸、组氨酸、甘氨酸、酪氨酸、缬氨酸、丝氨酸、苯丙氨酸、抗坏血酸、脯氨酸、葡萄糖、硫化钠、硫氢化钠、谷胱甘肽、高半胱氨酸和半胱氨酸(浓度均为10μM,溶剂均为PBS缓冲溶液),激发波长为385nm,最大发射波长为510nm,得到的荧光发射光谱图如图4所示。
由图4可知:在385nm激发波长的激发下,式I所示荧光探针(图中0)在510nm处有较弱的荧光发射峰;加入生物硫醇谷胱甘肽、半胱氨酸和高半胱氨酸(图中16~18)后,510nm处的荧光强度显著增强,强度达到40倍以上;加入其他常见氨基酸(胱氨酸、苏氨酸、丙氨酸、天冬氨酸、组氨酸、甘氨酸、酪氨酸、缬氨酸、丝氨酸、苯丙氨酸、抗坏血酸、脯氨酸)以及葡萄糖、硫化钠和硫氢化钠(分别对应图中1~15)后,510nm处的荧光强度不发生变化。
这说明:式I所示荧光探针能够排除外界环境的干扰,选择性检测生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸),对生物硫醇具有良好的选择性和灵敏度,更利于食品和生物样品的检测分析。
四、检测生物硫醇的荧光探针的应用场景
用具体实施例制备得到的式I所示荧光探针检测HepG2细胞内生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸),激发波长405nm,收集波长480~580nm,得到的共聚焦显微镜照片如图5所示。其中:
第一列为对照组,分别为HepG2细胞在37℃孵育5min的明场(a)和荧光场(c);
第二列为实验组,分别为HepG2细胞与式I所示荧光探针(浓度为10μM,溶剂为PBS缓冲溶液)在37℃孵育5min的明场(b)和荧光场(d)。
由图5可以看出:加入式I所示荧光探针后,荧光成像照片中的荧光强度显著增强。
这说明:式I所示荧光探针可以检测细胞中的生物硫醇(谷胱甘肽、半胱氨酸和高半胱氨酸)。
综上,本发明提供的式I所示荧光探针适用于实际样品中生物硫醇的快速高效检测,不受待测环境及样品的干扰,生物兼容性强,可用于食品、细胞以及活体中生物硫醇的实时原位成像,有利于深入探究生物硫醇在生物体内的氧化还原态以及动力学和生物学效应。
需要说明的是,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无法对所有的实施方式予以穷举。凡是属于本发明技术方案所引申出的显而易见变化或变动仍处于本发明的保护范围之列。
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