CN105038762B - 一种检测过氧化氢的比率型荧光探针及其应用 - Google Patents
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Abstract
本发明公开了一种检测过氧化氢的比率型荧光探针,为类花青素衍生物,其化学结构式如式(1)所示,具体为2‑(3‑(7‑二乙胺基)香豆素)‑4‑(2‑苯甲基)‑7‑二乙胺基‑苯并吡喃高氯酸盐。本发明的探针廉价易得,使用方便,可以特异性与过氧化氢发生反应,生成荧光发射能力更强的香豆素衍生物,在检测过氧化氢过程中不会受到其他相关氧化物的干扰,对过氧化氢具有良好的选择性,能对活细胞中的过氧化氢进行准确检测。
Description
技术领域
本发明涉及一种检测过氧化氢的比率型荧光探针及其应用,属于分析化学技术领域。
背景技术
活性氧(ROS)是生物体内含氧自由基和含氧非自由基的总称,在很多生理和病理过程中起着非常重要的作用。生物体内的氧化应激、炎症等生理和病理过程,通常会经过酶促和非酶促反应产生各种活性氧。近代生物医学研究表明,体内产生的活性氧和许多疾病的发生、发展和机体的老化过程有着密切的关系。作为一种重要的活性氧,过氧化氢(H2O2)作为一种重要的信号分子,参与各种各样的信号传导过程,也是氧化应激等相关疾病的标记物。细胞内,细胞因子、生长因子和神经递质等激活白细胞氧化酶,催化细胞周围环境中的氧生成过氧化氢。生物体内适量水平的过氧化氢对生物正常的生理过程有益,参与细胞内蛋白质的可逆氧化,以及调剂蛋白质的磷酸化到基因表达等细胞过程。但是,体内过氧化氢浓度的异常与人类的许多疾病密切相关,包括癌症、心血管疾病和神经系统性疾病等。因此,检测生物体内的过氧化氢的产生与动态变化对于调查研究相关疾病的发生、发展具有重要的作用。
传统的过氧化氢检测方法包括滴定法、紫外可见分光光度法、化学发光法、电化学法(极谱法、电流滴定法、恒电位法)、色谱法等,这些方法通常会引起待测样品的损失,或者是不适用于生物体内过氧化氢的检测。荧光成像由于具有高灵敏度、高选择性和实时监测,为检测生物体内的过氧化氢提供了一种可行的方法。目前已开发的过氧化氢荧光探针大都侧重于荧光增敏机制,但是这种方法易受激发光强度、细胞内溶液pH、探针浓度等因素的影响,因此具有一定的局限性。相比之下,比率型荧光探针依靠双发射波长的比值提供信号,可有效的解决这些问题的干扰。但检索发现,有关检测过氧化氢的比率型荧光探针及其应用还鲜见报道。
发明内容
针对现有技术的不足,本发明要解决的问题是提供一种检测过氧化氢的比率型荧光探针及其应用。
本发明所述的检测过氧化氢的比率型荧光探针,为类花青素衍生物,其特征在于:所述荧光探针的化学结构式如式(1)所示:
其中:R1=OH或N(Et)2;R2=H,OH或N(Et)2。
上述的检测过氧化氢的比率型荧光探针,优选是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐。
上述检测过氧化氢的比率型荧光探针以如下方法制备:
(1)中间体1制备:2-(4-二乙胺基-邻羟基苯基)苯甲酸的制备:将间二乙胺基苯酚和邻苯二甲酸酐按照1:1的摩尔比溶于20ml甲苯中,氮气保护下回流6小时,冷制60℃之后加入30ml 36%NaOH水溶液,继续反应2小时,然后冷制室温,1N的HCl调节pH为5,过滤,烘干得到浅灰色产品。
(2)中间体2制备:2-(4-羟基-邻羟基苯基)苯甲酸的制备:将荧光素溶于30ml36%NaOH水溶液中,100℃下加热6小时,1N的HCl调节pH为5,过滤,烘干得到浅灰色产品。
(3)荧光探针的制备:将中间体1或2与3-乙酰基香豆素衍生物按照1:1的摩尔比例加入到6ml浓硫酸中,90℃下反应12小时,冷至室温后,倒入到冰水之中,加入70%的高氯酸,过滤,所的固体使用柱层析(洗脱剂:甲醇与二氯甲烷体积比为1:15)提纯即得所述荧光探针。
本发明所述的检测过氧化氢的比率型荧光探针在检测活细胞中过氧化氢中的应用。
本发明所述荧光探针可以应用于活细胞内的过氧化氢含量的定量分析。该探针作为过氧化氢的特异性探针,能和探针发生氧化插氧反应,进而水解成荧光更强、且波长变短的香豆素衍生物。通过定量检测溶液所发射的双荧光的比值,可以确定细胞中的过氧化氢的含量。具体测定方法为:25℃条件下,在含有5%甲醇的PBS(pH=7.4,20mM)中,以5μM类花青素衍生物为特异性探针,将过氧化氢的PBS溶液的PBS溶液加入到探针体系中,测定溶液的荧光强度,根据两个荧光强度比值作为过氧化氢含量的评价指标。
具体的,本发明所述荧光探针在检测活细胞中过氧化氢的应用实例:
将人肺癌A549细胞加入铺有盖玻片的培养皿中,并置于二氧化碳培养箱中培养,36小时后对铺有并长满细胞的盖玻片的培养皿冲洗,加入5μM本发明所述的比率型过氧化氢荧光探针后37℃下培养40分钟。之后,使用PBS冲洗,制片,在激光共聚焦显微镜下观察细胞,进行荧光成像。并列的,向铺有并长满细胞的盖玻片的培养皿中加入50μM浓度的过氧化氢,37℃下培养1小时,制片,在共聚焦显微镜下观察细胞,进行荧光成像。
本发明提供了一类用于特异性识别过氧化氢的探针,其与过氧化氢反应之后可生成具有荧光发射能力更强的香豆素衍生物,并依靠两个荧光发射峰的比值为信号,可以有效解决周围环境对探针信号的干扰。该反应具有高选择性,能够广泛用于检测活细胞中的过氧化氢。
本发明的有益效果是:所制备的类花青素衍生物的比率型荧光探针能够与过氧化氢进行特异性反应,特别是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐,通过荧光信号的产生对过氧化氢进行定性检测,能够对活细胞中的过氧化氢进行定量检测。此外该荧光探针廉价易得,可经化学合成获得,且合成工艺简单易行。进一步的,本发明提供的用于检测过氧化氢的荧光探针易于制备、使用方便,能够基于双荧光强度的比值快速检测活细胞中的过氧化氢。
附图说明
图1是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐的1H NMR图谱。
图2是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐的13C NMR图谱。
图3是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐对不同物质的选择性图谱。
其中:1至11分别为:探针、过氧化氢、次氯酸钠、一氧化氮、半胱氨酸、羟基自由基、过氧化二叔丁基、过氧化二叔丁醇、谷胱甘肽、维生素C和硝酸钠。
图4是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐与过氧化氢的线性关系数据。
图5是本发明所述的荧光探针对细胞内过氧化氢监测的照片。
其中:A、B和C依次为探针(10μM)在细胞中绿色通道、红色通道和明场下的成像,D、E和F依次为探针(10μM)和过氧化氢(200μM)在细胞中绿色通道、红色通道和明场下的成像。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,其中所述检测过氧化氢的比率型荧光探针以2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐为例,但不仅限于此。
实施例1:2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐的制备:
将间二乙胺基苯酚(1.65g,10mmol)和邻苯二甲酸酐(1.48g,10mmol)溶于20ml甲苯中,氮气保护下回流6小时,冷制60℃之后加入30ml 36%NaOH水溶液,继续反应2小时,然后冷制室温,1N的HCl调节pH为5,过滤,烘干,重结晶得到浅灰色中间体1,即为:2-(4-二乙胺基-邻羟基苯基)苯甲酸。
将中间体1(1.56g,5mmol)和与3-乙酰基-7-乙二胺基-香豆素(1.29g,5mmol)加入到6ml浓硫酸中,90℃下反应12小时,冷至室温后,倒入到冰水之中,加入70%的高氯酸,过滤,所的固体使用柱层析提纯,得到深蓝色产品,即为:2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐。鉴定该探针的1H NMR图谱见图1,13C NMR图谱见图2。
1H NMR(MeOD-CDCl3,v:v=1:1,400MHz):δ8.90(s,1H),8.20(s,1H),8.10(d,1H),7.67(m,3H),7.31(m,2H),7.17(s,1H),6.88(m,2H),6.50(s,1H),3.56(m,8H),1.28(t,12H).
13C NMR(MeOD-CDCl3,v:v=1:1,400MHz):δ158.90,158.21,158.09,154.80,154.41,145.66,132.71,130.27,129.92,129.46,128.16,115.58,111.76,111.26,109.64,106.56,96.38,96.17,45.37,12.10.HRMS(ESI):[M+H]+m/z 537.2384,calcd forC33H33N2O5 +537.2651。
实施例2:2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐对不同物质的选择性鉴定
预先准备10份5mL的5mM 2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐PBS溶液(含5%甲醇),然后分别向所述10份体系中依次加入50μL浓度为10mM的过氧化氢、次氯酸钠、一氧化氮、半胱氨酸、羟基自由基、过氧化二叔丁基、过氧化二叔丁醇、谷胱甘肽、维生素C和硝酸钠的PBS溶液。进行荧光检测(λEx=380nm);计算各体系中471nm和693nm处的荧光强度比值I471/I693,结果见图3。其中显示,所述2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐对过氧化氢选择性明显增强。
实施例3:2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐与过氧化氢的线性关系
配制1mL浓度为0~5mM梯度的过氧化氢的PBS溶液体系,然后均分别加入9mL浓度为50μM的探针PBS溶液(含5%甲醇),进行荧光检测(λEx=380nm,λEm=471nm和693nm),计算各体系中荧光强度,以及471nm和693nm处的荧光强度比值I471/I693,建立荧光强度与过氧化氢浓度标准曲线,标准曲线见图4。
本发明所述荧光探针2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐的检测下限为0.079μM(S/N=3)。
实施例4:本发明所述检测过氧化氢的比率型荧光探针的应用
荧光探针2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐本身在471nm处有微弱的荧光,但其与过氧化氢反应的产物在471nm处具有很强的荧光。
将人肺癌A549细胞加入铺有盖玻片的培养皿中,并置于温度为37℃的5%二氧化碳培养箱中,在采用DMEM培养基(含10%小牛血清)以及100μg/ml的双抗进行培养,36小时后对铺有并长满细胞的盖玻片的培养皿采用不含血清的DMEM培养基冲洗3次,加入10μM本发明所述的检测过氧化氢的比率型荧光探针后37℃下培养40分钟。
使用PBS冲洗铺有并长满细胞的盖玻片的培养皿3次,制片,在激光共聚焦显微镜下观察细胞,进行荧光成像。
并列的,向铺有并长满细胞的盖玻片的培养皿中加入200μM浓度的过氧化氢,37℃下培养1小时,制片,在共聚焦显微镜下观察细胞,进行荧光成像。
结果见图5。
Claims (1)
1.一种检测过氧化氢的比率型荧光探针在检测活细胞中过氧化氢中的应用,其中所述比率型荧光探针是2-(3-(7-二乙胺基)香豆素)-4-(2-苯甲酸)-7-二乙胺基-苯并吡喃高氯酸盐。
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