CN107954941A - 一种特异性识别汞离子的荧光探针 - Google Patents

一种特异性识别汞离子的荧光探针 Download PDF

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CN107954941A
CN107954941A CN201711186181.1A CN201711186181A CN107954941A CN 107954941 A CN107954941 A CN 107954941A CN 201711186181 A CN201711186181 A CN 201711186181A CN 107954941 A CN107954941 A CN 107954941A
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宋相志
谭倩
廖立德
苏远安
任晓杰
杨雷
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Abstract

本发明公开了一种可以特异性识别汞离子的荧光探针。其分子结构式如下：该探针分子是能够选择性、灵敏性检测Hg2+的红色荧光探针。这个探针检测限为14.4nM，斯托克斯位移为130nm。而且这个探针还可以检测细胞内的Hg2+。

Description

一种特异性识别汞离子的荧光探针
技术领域
本发明属于化学分析检测技术领域，具体涉及一种可以特异性识别Hg2+的荧光探针，具体研究了该探针的制备方法以及在体外和活细胞内检测Hg2+的应用。
背景技术
汞是一种运用在电子、化工、制药、生物等方面必不可少的重金属元素，是所有的重金属中对人类和环境毒害最大的。汞广泛分布于土壤、水、空气中。并可以通过冶金、使用化石燃料、焚烧固体垃圾等各种人类活动和火山爆发等自然活动的方式将汞排放到环境中。汞通常以汞单质、离子、络合物和有机汞的形式存在。水溶性的汞离子可以通过细菌转化成甲基汞(CH3HgX,X＝Cl-,AcO-等)，甲基汞随后可以被生物体吸收，通过食物链方式在人体内富集。由于生物放大作用，汞的危害呈现生物富集和生物放大作用。在伊拉克、日本就有甲基汞中毒导致流行病的事件。无机汞及其衍生物可以无障碍地通过皮肤、粘膜。由于汞离子具有很强的结合性，容易与酶和蛋白质中的巯基发生反应。汞的积累会使细胞功能紊乱，从而导致一系列前脑、神经系统、肾脏、认知、有丝分裂、内分泌、运动各个方面疾病。即使只摄取少量的汞，也会对人体产生严重或者慢性伤害，环保组织规定了饮用水、食物中最大汞含量为2ppb(0.01μM)。因此研发快速、高效的方法检测环境和生物体内Hg2+含量很有必要。迄今为止，各种传统应用于检测Hg2+的分析技术包括：高效液相(HPLC)、毛细管电泳(CE)、原子吸收光谱(AAS)、诱导耦合质谱(ICP-Mass)。然而这些传统的分析方法有着一些不可忽略的弊端，例如：耗时长、样品前处理复杂等。最近几十年，大量基于蛋白质、核酸、DNA、纳米粒子、有机荧光团、比色法、聚合物的特异性检测汞离子的方法相继被研发出来。荧光探针在检测痕量汞离子方面相比其他方法具有独特的优势，因其具有良好选择性、灵敏性、操作简单、可以用于生物成像。发射波长在红外或者近红外区域的荧光染料因为可以减少背景干扰、减少对生物样品的光损伤以及具有深的组织穿透能力等优势，在生物成像方面更受欢迎。
发明内容
本发明目的之一是提供一种合成简单、反应条件温和、成本较低的荧光探针合成方法；目的之二是提供一种灵敏度高、选择性好，抗干扰能力强，斯托克斯位移130nm，发射波长在625nm，能够对体外或者活细胞内的Hg2+进行快速监测或者细胞成像的荧光探针。
本发明解决问题采取的技术方案为，其分子结构式如下：合成路线如下：
具体合成方法如下：
(a)468mg化合物1、1504mg 1,2-二溴乙烷溶于30mL乙腈中，边搅拌边加入1112mg碳酸钾，氩气保护回流反应20h。抽滤，滤饼用30mL二氯甲烷洗涤，旋干溶剂，粗产品柱层析体提纯，得到化合物2，暗黄色固体120mg，产率17.6％。
(b)120mg化合物2溶于5mL DMSO，80mg叔丁醇钾加入到反应液中。氩气保护，室温搅拌6h。反应结束后，将反应液倒入到150ml去离子水中，用50mL乙酸乙酯萃取，重复3次，合并有机相并用无水硫酸钠干燥，旋干，柱层析体提纯，得到化合物3，油状物60mg，产率60.7％。
(c)60mg化合物3、30mg丙二腈溶于10mL色谱甲醇中，加入15μL哌啶。氩气保护、室温搅拌8h。停止反应，旋干溶剂，余物进行柱层析分离提，得到探针，红色粉末45mg，产率63.6％。
本发明的荧光探针的作用机理如下：由于烯醚部分的光致转移发光(PET)效应导致探针分子几乎没有荧光发射。汞离子可以特异性与烯醚键进行加成反应，再脱去一个有机汞分子，形成一个过渡态，然后经历一个合环反应产生染料。探针的响应过程如下：
本发明的荧光探针在测试体系为乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)，室温下进行测试。可以对Hg2+进行定量测试，具体实施方法在实施例中介绍。
本发明的荧光探针与Hg2+响应后的荧光发射峰在625nm处，完全响应后荧光强度增强了102倍。
本发明所述的探针分子合成路线简单，操作简单，成本较低，对Hg2+的选择性好、抗干扰能力强，斯托克斯位移大，发射波长在近红外，该荧光探针在生物诊断、分析化学、环境科学等领域具有实际的应用价值。
附图说明
图1为本发明荧光探针的选择性，荧光探针(1.0×10-5mol/L)在乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)中，与不同物质响应后的荧光光谱，横坐标为波长，纵坐标为荧光强度。
图2为本发明荧光探针的抗干扰能力，Hg2+与其它干扰物共存时，与荧光探针(1.0×10-5mol/L)在乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)中，与Hg2+响应后的荧光强度比值(I/I0)柱状图。
图3为本发明的荧光探针(1.0×10-5mol/L)在乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)中，与不同浓度Hg2+响应后的荧光光谱变化，横坐标为波长，纵坐标为荧光强度。
图4为本发明的荧光探针(1.0×10-5mol/L)pH在乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)中，与Hg2+浓度的线性关系，横坐标为浓度，纵坐标为荧光强度。
图5为本发明的荧光探针(1.0×10-5mol/L)在乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)中，与Hg2+响应过程中荧光强度随时间的变化，横坐标为时间，纵坐标为荧光强度。
图6为本发明的荧光探针(1.0×10-5mol/L)在不同pH的乙醇和20mMpH＝7.4的HEPESNa混合溶液(体积比为3比7)中，与Hg2+响应前后的荧光强度，横坐标为pH，纵坐标为荧光强度。
图7为本发明的荧光探针(1.0×10-5mol/L)在不同条件下的细胞(Hela细胞)成像图(a)探针和Hg2+溶液处理的细胞中明场成像(b)探针和Hg2+溶液处理的细胞中荧光成像中荧光成像(c)探针和Hg2+溶液处理的细胞中复合场成像(d)探针在细胞中明场成像(e)探针在细胞中荧光成像(f)探针在细胞中复合场成像。
具体实施实例
实施例1：中间产物化合物2的合成
468mg化合物1、1504mg 1,2-二溴乙烷溶于30mL乙腈中，边搅拌边加入1112mg碳酸钾，氩气保护回流反应20h。抽滤，滤饼用30mL二氯甲烷洗涤，旋干溶剂，粗产品柱层析体提纯，得到化合物2，暗黄色固体120mg，产率17.6％。HRMS(EI)m/z:理论值C15H21BrN2O2[M+Na]+,363.0684；实验值,363.0572.1HNMR(500MHz,CDCl3)δ10.22(s,1H),7.01(s,1H),6.04(s,1H),4.35(t,J＝6.2Hz,2H),3.67(t,J＝6.2Hz,2H),3.55–3.50(m,2H),3.42(q,J＝7.1Hz,2H),3.34(q,J＝7.1Hz,2H),3.22–3.17(m,2H),1.24(t,J＝7.1Hz,3H),1.18(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δc 186.83,157.01,143.22,114.81,108.59,94.72,69.68,47.77,45.74,45.32,44.79,29.21,10.76,9.96。
实施例2：化合物3的合成
120mg化合物2溶于5mL DMSO，将80mg叔丁醇钾加入到反应液中。氩气保护，室温搅拌6h。反应结束后，将反应液倒入到150ml去离子水中，50mL×3乙酸乙酯萃取，合并有机相用无水硫酸钠干燥，旋干，柱层析体提纯，得到化合物3，油状物60mg，产率60.7％。HRMS(EI)m/z:理论值C15H20N2O2[M+Na]+,283.1422；实验值,283.1281.1H NMR(400MHz,CDCl3)δ10.08(s,1H),7.01(s,1H),6.66(dd,J＝13.8,6.1Hz,1H),6.11(s,1H),4.68(dd,J＝13.8,1.9Hz,1H),4.41(dd,J＝6.1,1.9Hz,1H),3.53(dd,J＝5.7,4.3Hz,2H),3.44–3.38(m,2H),3.38–3.33(m,2H),3.23(dd,J＝5.7,4.3Hz,2H),1.22(t,J＝5.8Hz,3H),1.19(t,J＝5.7Hz,3H).13C NMR(101MHz,CDCl3)δc 186.69,154.75,150.03,142.95,130.97,115.43,107.58,98.53,94.11,47.56,45.74,45.27,44.68,10.66,9.93。
实施例3：探针的合成
60mg化合物3、30mg丙二腈溶于10mL色谱甲醇中，加入15μL哌啶。氩气保护、室温搅拌8h。停止反应，旋干溶剂，余物进行柱层析分离提，得到探针，红色粉末45mg，产率63.6％。HRMS(ESI)m/z:理论值C18H20N4O[M+1]+,308.1631；实验值,308.1310.1H NMR(500MHz,CDCl3)δ7.91(s,1H),7.54(s,1H),6.55(dd,J＝13.7,6.0Hz,1H),6.09(s,1H),4.79(dd,J＝13.7,2.0Hz,1H),4.53(dd,J＝6.0,2.0Hz,1H),3.64–3.54(m,2H),3.45(q,J＝7.2Hz,2H),3.37(q,J＝7.1Hz,2H),3.30–3.24(m,2H),1.28–1.21(m,6H).13C NMR(101MHz,CDCl3)δc153.44,150.06,148.35,144.32,130.77,117.15,116.00,110.68,107.12,97.29,96.37,68.80,47.99,46.05,45.57,44.45,10.96,9.76。
实施例4：探针的合成
将探针溶于乙醇和20mM pH＝7.4的HEPESNa混合溶液(体积比为3比7)中，配制成1.0×10-5mol/L的溶液，向溶液中加其它选择项物质(NH4 +、Ba2+、Ca2+、Cd2+、Cr3+、Co2+、Al3+、K+、Mg2 +、Mn2+、Na+、Ni2+、Pb2+、Fe3+、Zn2+、Cu2+、Fe2+、Sn2+)。加人Hg2+之后溶液荧光明显增强，该荧光探针对Hg2+表现出高灵敏性、高选择性。当Hg2+与干扰物质(NH4 +、Ba2+、Ca2+、Cd2+、Cr3+、Co2+、Al3 +、K+、Mg2+、Mn2+、Na+、Ni2+、Pb2+、Fe3+、Zn2+、Cu2+、Fe2+、Sn2+)共存时，探针对Hg2+响应表现出强抗干扰能力。该探针分子与Hg2+响应速度快，半个小时内可以观察到荧光的变化。探针分子在pH为7至11的范围内都可以对Hg2+选择性识别，可以在细胞内检测Hg2+。

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1.一种特异性识别汞离子的荧光探针，其结构为：
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108997255A (zh) * 2018-07-30 2018-12-14 河南师范大学 一种乙烯基醚类Hg2+荧光探针及其制备方法和应用
CN109485619A (zh) * 2018-11-13 2019-03-19 河南师范大学 一种乙烯基醚类Hg2+荧光探针及其制备方法和应用

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Publication number Priority date Publication date Assignee Title
CN103242195A (zh) * 2013-04-28 2013-08-14 中南大学 一种荧光增强检测汞离子探针的合成及其应用
CN106946801A (zh) * 2016-01-06 2017-07-14 中南大学 一种特异性识别半胱氨酸的新型荧光探针的制备与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103242195A (zh) * 2013-04-28 2013-08-14 中南大学 一种荧光增强检测汞离子探针的合成及其应用
CN106946801A (zh) * 2016-01-06 2017-07-14 中南大学 一种特异性识别半胱氨酸的新型荧光探针的制备与应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108997255A (zh) * 2018-07-30 2018-12-14 河南师范大学 一种乙烯基醚类Hg2+荧光探针及其制备方法和应用
CN109485619A (zh) * 2018-11-13 2019-03-19 河南师范大学 一种乙烯基醚类Hg2+荧光探针及其制备方法和应用

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