CN106588910A - 一种双功能荧光探针、制备方法及应用 - Google Patents
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Abstract
本发明公开了一种双功能荧光探针，同时提供其制备方法及应用。该荧光探针可高选择性和高灵敏度检测HOCl，同时可对细胞内的线粒体进行定位。本发明的双功能荧光探针具有细胞内次氯酸检测和线粒体定位双功能，所述的探针具有如式1所示的结构：其中R为H、CH3或T‑Bu，探针为2‑(5‑烷基‑4‑醛基‑2‑羟基苯基)苯并噻唑的衍生物。
Description
技术领域
本发明涉及一种荧光探针、制备方法及应用，更具体地说涉及一种双功能荧光探针、制备方法及应用。
背景技术
HOCl/OCl-是一种强氧化剂，这样的性质使其在日常生活中常常扮演者漂白剂和消毒剂的重要角色。而在生物体中，HOCl/OCl-又是活性氧物种(ROS)的重要一员，在免疫抵抗微生物感染和炎症过程中发挥着极其重要的作用。在生物体中，H2O2和Cl-在髓过氧化物酶(MPO)的催化作用下产生OCl-，这是内源性OCl-的主要来源。研究结果表明，当细胞内的OCl-浓度水平不能维持在正常的生理水平范围内时，将会导致生物体的多种疾病，比如关节炎、类风湿性关节炎、动脉粥样硬化和肿瘤等。由此可见，维持细胞内正常的OCl-浓度水平对很多细胞功能是至关重要的。而与其他形式的检测方法相比，荧光探针则拥有选择特异性好、灵敏度高、易操作、即时检测和响应迅速等众多优势。这些优点使得其在化学、生物医学等学科领域都有着较为广泛的应用，尤其是在生物医学领域，荧光探针不但可以用于体外分析还可以用于活体的影像学研究。
近年来，能特异性检测HOCl/OCl-的小分子荧光探针被大量报道。然而，能够高准度定位细胞内线粒体的荧光探针却很少，而且其中许多探针水溶性差、灵敏度低、响应时间长、pH的变化对检测的效果影响较大；另有一些探针生物毒性大、细胞膜渗透能力差，这些缺陷很大程度上影响了探针的应用。因此，开发出能克服这些缺陷的新型荧光探针是非常必要的。
发明内容
本发明所要解决的技术问题是：克服现有技术存在的不足，提供一种双功能荧光探针，同时提供其制备方法及应用。该荧光探针可高选择性和高灵敏度检测HOCl，同时可对细胞内的线粒体进行定位。
本发明的技术构思如下：该基于2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑的衍生物探针可选择性与OCl-离子反应并发生蓝移现象，且在0-10μM OCl-离子浓度范围内，460nm处荧光强度和615nm处荧光强度的比值(I460nm/I615nm)与OCl-离子浓度有着较好的线性关系。本发明人首次制备2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑衍生物并首次将其用于OCl-离子的选择性检测，并对细胞内的线粒体进行定位，从而解决上述技术问题。
本发明解决其技术问题的技术方案如下：
本发明的双功能荧光探针具有细胞内次氯酸检测和线粒体定位双功能，所述的探针具有如式1所示的结构：
其中R为H、CH3或T-Bu，探针为2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑的衍生物。
本发明上述的双功能荧光探针的制备方法，其包括以下步骤：
将2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑和1,4-二甲基吡啶碘化物在乙醇溶液中反应，并逐滴加入哌啶，反应生成制得的2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑衍生物即为双功能荧光探针。
本发明上述的制备方法，其进一步的技术方案是所述的反应是在油浴条件下剧烈搅拌，反应温度60-70℃，反应时间为6h或以上。
本发明上述的制备方法，其更进一步的技术方案还可以是所述的反应结束后进行纯化，纯化步骤为：将反应液减压浓缩，粗产物在甲醇和乙醚的混合溶液中重结晶，之后对固体过滤、真空干燥即得双功能荧光探针。
本发明上述的制备方法，其更进一步的技术方案还可以是所述的2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑、1,4-二甲基吡啶碘化物和哌啶的用量质量比为1-4:1。
本发明上述的荧光探针在氯酸根离子含量的检测及细胞中线粒体的定位中有很好的应用。
本发明具有以下有益效果：
1)本发明荧光探针在pH＝7.4的缓冲溶液中呈暗红色且荧光为红色，与ClO-反应后发生蓝移现象，红色荧光逐渐消失，蓝色荧光出现。
2)采用本发明荧光探针后，检测灵敏度高，对次氯酸根的检测限可达到10-7M。
3)本发明荧光探针仅与ClO-发生荧光反应，对其它离子均无反应，具有很好的选择性和特异性。具有适宜的荧光发射波长(615nm)。
4)本发明荧光探针制备工艺简单易行，易于规模化生产。
附图说明
图1为本发明实施例2荧光探针对ClO-的选择特异性。
图2为本发明实施例2荧光探针与各种离子反应的荧光变化图。
图3为本发明实施例3荧光探针与ClO-反应的I460nm/I615nm增量图。
图4为本发明实施例3荧光探针对ClO-浓度的荧光强度(I460nm/I615nm)工作曲线。
图5为本发明实施例3荧光探针对0-10μM ClO-的荧光强度(I460nm/I615nm)线性关系图。
图6为本发明实施例4荧光探针对细胞内线粒体的高准确定位细胞图。
图7为本发明实施例1荧光探针的核磁氢谱表征图。
图8为本发明实施例1荧光探针的质谱表征图。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
实施例1 制备HClO检测和线粒体定位的荧光探针
将269mg 2-(5-甲基-4-醛基-2-羟基苯基)苯并噻唑和235mg 1,4-二甲基吡啶碘化物溶于60ml的乙醇溶液，然后将哌啶1.0mMo l逐滴加入到上述溶液中；在油浴条件下，剧烈搅拌反应过夜；反应结束后，先对反应液进行减压浓缩，然后用甲醇和乙醚的混合溶液对粗产品进行重结晶，最后对固体过滤、真空干燥得到黄褐色固体393mg，即为HClO检测和线粒体定位的荧光探针纯品(1H-NMR图和高分辨质谱图见图7、图8)。所得荧光探针纯品实测分子量为486.37。
本实施例工艺路线：
实施例2 HClO检测和线粒体定位的荧光探针与各种离子反应的光谱性质
称取4.9mg实施例1制得HClO检测和线粒体定位的荧光探针，配成浓度为1mM的10mL CH3CN溶液，作为母液。
荧光光谱测试：将30μL上述母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中，然后分别加入各种离子：H2O2,NO,.O2 -,ONOO-,ROO.,1O2,.OH，ClO-，使离子终浓度为500μM，荧光探针终浓度为10μM。在398nm激发光波长下即时测试其荧光发射光谱。激发与发射的狭缝宽度为5/10nm。所得I460nm/I615nm的柱状图如图1所示。将上述所配置溶液用365nm紫外灯照射，观察其荧光变化，结果见图2。
以上结果表明：
(1)实施例1制得荧光探针本身在溶液呈暗红色切荧光为红色，但随次氯酸根离子的加入，该探针在615nm处红色荧光逐渐消失，在460nm处蓝色荧光逐渐增强。
(2)实施例1制得荧光探针对ClO-具有高度的选择性和特异性，并且在上述条件下，能够从H2O2,NO,.O2 -,ONOO-,ROO.,1O2,.OH等常见重活性氧离子中区分出ClO-。
实施例3 HClO检测和线粒体定位的荧光探针与ClO-反应产物的光谱性质
将30μL实施例2中的母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中，然后加入不同当量的ClO-，使荧光探针的终浓度为10μM，金离子终浓度分别为0μM、0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、0.9mM、1.0mM。离子加入后，即时测量其荧光发射光谱。荧光发射光谱测定时以398nm激发波长；激发与发射的狭缝宽度为5/10nm。所得荧光强度I460nm/I615nm增量图见图3；以I460nm/I615nm的数据制作工作曲线，结果见图4。
该实验结果表明，反应后I460nm/I615nm随次氯酸根离子浓度的增加而增加；反应后I460nm/I615nm与0-10μM范围内的次氯酸根离子浓度呈较好的线性关系，可以用于次氯酸根离子含量的定量分析检测,以I460nm/I615nm与0-10μM范围内的次氯酸根离子浓度线性关系曲线见图5。
实施例4 HClO检测和线粒体定位荧光探针对细胞内线粒体的准确定位。
取探针母液30μL加入到1mL的培养基中，此时探针的浓度为30μM，37℃条件下培养HeLa细胞30min；之后加入相同剂量的商业化线粒体定位染料健那绿B，同样培养30min；最后用共聚焦显微镜对HeLa细胞进行细胞成像实验，结果如图6所示，图(A)表示明场下HeLa细胞的形态；图(B)表示商业化染料健那绿B在HeLa细胞中的荧光成像图；图(C)表示探针在HeLa细胞中的荧光成像图；图(D)表示(B)和(C)的定位复合图；图(E)表示(A)和(D)的复合图。经计算，图(D)的pearson定位系数Rr高达0.97，证明探针对细胞内的线粒体具有非常好的定位效果。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
Claims (6)
1.一种双功能荧光探针，其特征在于所述的探针具有细胞内次氯酸检测和线粒体定位双功能，所述的探针具有如式1所示的结构：
其中R为H、CH3或T-Bu。
2.一种如权利要求1所述的双功能荧光探针的制备方法，其特征在于包括以下步骤：
将2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑和1,4-二甲基吡啶碘化物在乙醇溶液中反应，并逐滴加入哌啶，反应生成制得的2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑衍生物即为双功能荧光探针。
3.根据权利要求2所述的制备方法，其特征在于所述的反应是在油浴条件下剧烈搅拌，反应温度为66-70℃，反应时间为6h或以上。
4.根据权利要求2所述的制备方法，其特征在于所述的反应结束后进行纯化，纯化步骤为：将反应液减压浓缩，粗产物在甲醇和乙醚的混合溶液中重结晶，之后对固体过滤、真空干燥即得双功能荧光探针。
5.根据权利要求2所述的制备方法，其特征在于所述的2-(5-烷基-4-醛基-2-羟基苯基)苯并噻唑、1,4-二甲基吡啶碘化物和哌啶的用量质量比为1-4：1。
6.一种如权利要求1所述的荧光探针在氯酸根离子含量的检测及细胞中线粒体的定位中的应用。
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