CN108409791A - 一种检测h2o2的荧光探针及其制备方法 - Google Patents
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Abstract
本发明属于过氧化氢分析检测技术领域，具体涉及一种检测H2O2的荧光探针及其制备方法，其结构式如图所示，包括罗丹明衍生物和磷脂，一定量的罗丹明内酰胺与磷脂在一定条件下发生缩合反应得到目标化合物，合成过程原料简单易得、反应条件温和、操作简单，所得检测H2O2的荧光探针具有水溶性好、灵敏度高、光稳定性好、荧光量子产率高、波长长等优点，而且反应后的副产物不会对生物体产生危害，可广泛应用于生物细胞及药物合成。
Description
技术领域
本发明属于过氧化氢分析检测技术领域，具体涉及一种检测H2O2的荧光探针及其制备方法。
背景技术
过氧化氢是一种重要的活性氧物质，在生物机体活动和外界环境中有着极其重要的应用。在自然环境中，过氧化氢可以用来净化自来水，在生物进行生命活动的时候，过氧化氢在细胞代谢过程中发挥着免疫标志的功能。生物新陈代谢过程中产生的过氧化氢(H2O2)是生命活动所必需的，过氧化氢在生理过程中有着重要作用。最近几年的研究表明，过氧化氢可以以信号分子的角色对生理和病理过程进行调节，与细胞的增殖分化以及迁移过程息息相关，除此之外，过氧化氢在很多病理过程中例如炎症、机体防御等方面也发挥着重要作用。但是很多疾病也是由于过量的过氧化氢而引起的，例如：癌症、阿尔察默病等疾病。因此实时定量的检测生物机体和外界环境中的过氧化氢具有非常重要的意义。
针对过氧化氢的检测，现已有多种方式，包括氧化还原敏感荧光蛋白、纳米管、超极化、超声、质谱、PET、化学发光，以及过氧化氢反应性小分子荧光探针。值得注意的是，小分子荧光探针由于具有高敏感、对组织无损伤等特性，越来越引起研究人员的关注。然而，现有的市售的荧光探针，如二氯二氟荧光素、二氢罗丹明等，对ROS缺乏特异性。
为解决这个问题，Chang等(JAmChemSoc,2004,126:15392-15393.)报道了以荧光素为发光团，以硼酸酯基团作为荧光反应位点的荧光素类的双硼酸酯荧光探针，该探针对过氧化氢的响应比其他活性氧化合物高500倍以上，可以检测到人体胚肾细胞内微摩尔级的过氧化氢，反应机制如下。然而，芳基硼酸酯类探针对过氧化亚硝基阴离子(ONOO-)的反应活性高于过氧化氢，在生物体内脱靶反应严重，这一缺点限制了其广泛应用。同时，该类探针反应后的副产物硼酸的生物学效应未知。
因此，为了解决荧光探针对ROS缺乏特异性，水溶性差等问题，设计合成对过氧化氢具有高度特异性，即选择性好、灵敏度高、抗干扰能力强，同时，其反应后的产物可以对过氧化氢造成的氧化应激损伤起到一定的保护性效果的新型荧光探针具有重要的意义。
发明内容
为了解决荧光探针对ROS缺乏特异性，水溶性差等问题，本发明一种检测H2O2的荧光探针及其制备方法。
一种检测H2O2的荧光探针，其结构式如图1所示。
进一步的，检测H2O2的荧光探针结构式中基团R1，R2为烷基基团。
进一步的，检测H2O2的荧光探针结构式中n为0，2，3，...n。
进一步的，制备上述的检测H2O2的荧光探针，其合成方法如下：
合成罗丹明B内酰胺中间体：称取2-3g罗丹明染料，将其溶于有机溶剂，接着将胺类化合物溶液逐滴加入到罗丹明溶液中，加热至50-60℃，回流反应2-7h后得到罗丹明B内酰胺中间体；
合成检测H2O2的荧光探针：称取1-2mmol罗丹明B内酰胺，将其溶于有机溶剂，接着将磷脂溶液逐滴加入到罗丹明B内酰胺溶液中，加热至60-85℃，回流，薄层层析跟踪至反应结束，反应结束后通过柱层析分离纯化得到目标化合物。
进一步的，有机溶剂为甲醇、乙醇、乙腈。
进一步的，合成中间体的反应温度为60℃，反应时间为6h。
进一步的，罗丹明B内酰胺与磷脂的摩尔比为1:1.0-2.0。
进一步的，合成目标化合物的反应温度为80℃。
进一步的，柱层析分离纯化溶剂为二氯甲烷与乙醇。
本发明基于罗丹明染料和磷脂具有较好的水溶性，设计出一种特殊结构的检测H2O2的荧光探针。一定量的罗丹明内酰胺与磷脂在一定条件下发生缩合反应得到检测H2O2的荧光探针，合成过程原料简单易得，反应条件温和，操作简单，所得检测H2O2的荧光探针具有灵敏度高，光稳定性好，荧光量子产率高，波长长等优点，而且反应后的副产物不会对生物体产生危害，可广泛应用于生物细胞及药物合成。
附图说明
图1为检测H2O2的荧光探针的结构式；
图2为检测H2O2的荧光探针的选择性测试；
图3为检测H2O2的荧光探针对3eq的H2O2和5eq其它干扰离子的荧光发射光谱图；
图4为检测H2O2的荧光探针对不同H2O2浓度的荧光发射光谱图；
图5为检测H2O2的荧光探针的荧光线性范围。
具体实施方式
如图1所示，一种检测H2O2的荧光探针的反应方程式，具体实施方案如下：
实施例一
一种检测H2O2的荧光探针的设计，基团R1与R2为CH3，n为2的检测H2O2的荧光探针。
一种检测H2O2的荧光探针的合成方法，制备上述的罗丹明B荧光探针，其合成方法如下：
合成罗丹明B内酰胺中间体：称取2g罗丹明B染料，将其溶于乙醇溶剂，接着将乙二胺溶液逐滴加入到罗丹明B溶液中，加热至50℃，回流反应6h，反应过程中溶液由枣红色变成橙黄色，得到罗丹明B内酰胺中间体；
合成检测H2O2的荧光探针：取1mmol罗丹明B内酰胺，将其溶于乙醇溶剂，接着将1.2mmol磷脂溶液逐滴加入到罗丹明B内酰胺溶液中，加热至80℃，回流，薄层层析跟踪至反应结束，反应结束后通过柱层析分离纯化得到目标化合物，柱层析分离纯化溶剂乙醇与二氯甲烷体积比为1:200。
实施例二
一种检测H2O2的荧光探针的设计，基团R1为CH3、R2为C2H5，n为2的检测H2O2的荧光探针。
一种检测H2O2的荧光探针的合成方法，制备上述的罗丹明B荧光探针，其合成方法如下：
合成罗丹明B内酰胺中间体：称取2g罗丹明B染料，将其溶于乙醇溶剂，接着将乙二胺溶液逐滴加入到罗丹明B溶液中，加热至50℃，回流反应6h，反应过程中溶液由枣红色变成橙黄色，得到罗丹明B内酰胺中间体；
合成检测H2O2的荧光探针：取1mmol罗丹明B内酰胺，将其溶于乙醇溶剂，接着将1.2mmol磷脂溶液逐滴加入到罗丹明B内酰胺溶液中，加热至80℃，回流，薄层层析跟踪至反应结束，反应结束后通过柱层析分离纯化得到目标化合物，柱层析分离纯化溶剂乙醇与二氯甲烷体积比为1:200。
实施例三
一种检测H2O2的荧光探针的设计，基团R1与R2为C2H5，n为2的检测H2O2的荧光探针。
一种检测H2O2的荧光探针的合成方法，制备上述的罗丹明B荧光探针，其合成方法如下：
合成罗丹明B内酰胺中间体：称取2g罗丹明B染料，将其溶于乙醇溶剂，接着将乙二胺溶液逐滴加入到罗丹明B溶液中，加热至50℃，回流反应6h，反应过程中溶液由枣红色变成橙黄色，得到罗丹明B内酰胺中间体；
合成检测H2O2的荧光探针：取1mmol罗丹明B内酰胺，将其溶于乙醇溶剂，接着将1.2mmol磷脂溶液逐滴加入到罗丹明B内酰胺溶液中，加热至80℃，回流，薄层层析跟踪至反应结束，反应结束后通过柱层析分离纯化得到目标化合物，柱层析分离纯化溶剂乙醇与二氯甲烷体积比为1:200。
实施例四
如图2所示，往探针溶液中分别加入5倍当量的PO4 3-、Cl-、Br-、NO3 -、NO2 -、SO4 2-、Ac-、ClO3 -、I-、CO3 2-离子时，探针溶液为无色，在580nm处荧光强度也没有发生变化，可见探针保持原来的结构，能够稳定存在于这些溶液体系中。当探针溶液中加入H2O2后，溶液由无色变为红色，在580nm处荧光强度明显增强，出现一个强的发射峰。这是由于H2O2与探针反应使得罗丹明的螺环打开，产生荧光发射。
实施例五
如图3所示，以最大发射峰位置的荧光强度为纵坐标，金属离子为横坐标，首先往探针溶液中分别加入5倍当量的干扰离子(PO4 3-、Cl-、Br-、NO3 -、NO2 -、SO4 2-、Ac-、ClO3 -、I-、CO3 2-)，再加入3倍当量的H2O2，溶液从无色状态马上变为红色，测试其荧光强度，由图可知，在其他干扰离子的存在下，探针对H2O2仍具有很好的识别能力。
实施例六
如图4和图5所示，配制10份5mL的5μM探针溶液，分别加入0-50μM的H2O2，进行荧光检测(λEx＝520nm)，计算各体系中荧光强度，通过分析580nm处的荧光强度与H2O2浓度的关系，评估探针对H2O2的响应性能。图4表明随着H2O2浓度的增大，溶液的荧光强度逐渐增强，直到最大发射强度不在变化。图5表明探针在H2O2浓度2×10-6mol/L～3×10-5mol/L范围内呈线性关系，线性相关系数为R2＝0.9787，而且探针对H2O2的检测限为3.18×10-8mol/L。
上述仅为本发明的优选具体实施方式，但本发明的设计构思并不局限于此，凡利用此构思对本发明进行非实质性的改动，均应属于侵犯本发明保护范围的行为。
Claims (9)
1.一种检测H2O2的荧光探针，其特征在于：检测H2O2的荧光探针结构式如下所示：
2.根据权利要求1所述检测H2O2的荧光探针，其特征在于：所述检测H2O2的荧光探针结构式中基团R1，R2为烷基基团。
3.根据权利要求1所述检测H2O2的荧光探针，其特征在于：所述检测H2O2的荧光探针结构式中n为0，2，3，...n。
4.一种检测H2O2的荧光探针的合成方法，其特征在于：制备权利要求1所述的检测H2O2的荧光探针，其合成方法如下：
步骤一：合成罗丹明B内酰胺中间体，称取2-3g罗丹明染料，将其溶于有机溶剂，接着将胺类化合物溶液逐滴加入到罗丹明溶液中，加热至50-60℃，回流反应2-7h后得到罗丹明B内酰胺中间体；
步骤二：合成检测H2O2的荧光探针，称取1-2g罗丹明B内酰胺，将其溶于有机溶剂，接着将磷脂溶液逐滴加入到罗丹明B内酰胺溶液中，加热至60-85℃，回流，薄层层析跟踪至反应结束，反应结束后通过柱层析分离纯化得到目标化合物。
5.根据权利要求4所述检测H2O2的荧光探针的合成方法，其特征在于：所述有机溶剂为甲醇、乙醇、乙腈。
6.根据权利要求4所述检测H2O2的荧光探针的合成方法，其特征在于：所述合成中间体的反应温度为60℃，反应时间为6h。
7.根据权利要求4所述检测H2O2的荧光探针的合成方法，其特征在于：所述罗丹明B内酰胺与磷脂的摩尔比为1:1.0-2.0。
8.根据权利要求4所述检测H2O2的荧光探针的合成方法，其特征在于：所述合成目标化合物的反应温度为80℃。
9.根据权利要求4所述检测H2O2的荧光探针的合成方法，其特征在于：所述柱层析分离纯化溶剂为二氯甲烷与乙醇。
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| US4988616A (en) * | 1986-06-10 | 1991-01-29 | Bayer Aktiengesellschaft | Method for detecting hydrogen peroxide employing triaryl- and trihetarylmethane derivatives as redox indicators |
| JP2008209361A (ja) * | 2007-02-28 | 2008-09-11 | Nobuaki So | 脂質膜局在型蛍光プローブ |
| CN104017569A (zh) * | 2014-05-23 | 2014-09-03 | 苏州科技学院 | 一种含罗丹明内酰胺基团的小分子pH荧光探针及合成方法 |
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| US4988616A (en) * | 1986-06-10 | 1991-01-29 | Bayer Aktiengesellschaft | Method for detecting hydrogen peroxide employing triaryl- and trihetarylmethane derivatives as redox indicators |
| JP2008209361A (ja) * | 2007-02-28 | 2008-09-11 | Nobuaki So | 脂質膜局在型蛍光プローブ |
| CN104017569A (zh) * | 2014-05-23 | 2014-09-03 | 苏州科技学院 | 一种含罗丹明内酰胺基团的小分子pH荧光探针及合成方法 |
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