CN107286151A - 一种基于咔唑的双光子荧光探针及其制备方法和用途 - Google Patents
一种基于咔唑的双光子荧光探针及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种基于咔唑的双光子荧光探针及其制备方法和用途，其中基于咔唑的双光子荧光探针的结构式如下：本发明双光子荧光探针分子在亚硫酸氢根(HSO3 ‑)与其他干扰试剂共存的体系中，表现出专一性响应和高的灵敏度。细胞毒性测试表明该探针对于细胞几乎没有什么毒副作用，双光子共聚焦荧光显微成像实验表明该探针对MCF‑7细胞渗透性好，适用于细胞线粒体内HSO3 ‑的双光子荧光成像和定性检测。
Description
一、技术领域
本发明涉及一种双光子荧光探针及其制备方法和用途，具体地说是一种基于咔唑的双光子荧光探针及其制备方法和用途，以实现双光子成像定性检测细胞线粒体内的HSO3 -，具有选择性专一、灵敏度高、检测浓度低的优点。
二、背景技术
SO2是重要的大气污染来源之一，还会在生理浓度下产生生物效应。气态SO2能转化成其在中性水溶液中的亚硫酸盐/亚硫酸氢盐(SO3 2-/HSO3 -＝1:3)衍生物。内源性SO2及其衍生物几乎参与各种生理过程，包括血管舒张，降压作用，血管平滑肌的抑制和心脏通道功能的调节。然而亚硫酸盐的生物学作用知之甚少，因为SO2及其衍生物不能在体内直接检测到。用于测定生物样品中亚硫酸盐/亚硫酸氢盐的最常用方法，即高效液相色谱(HPLC)，需要复杂的样品处理，不适合实时和长期检测生物亚硫酸盐/亚硫酸氢盐。因此，需要开发一种快速，便捷且可靠的生物体中亚硫酸盐/亚硫酸氢盐水平的实时检测方法。
线粒体是哺乳动物细胞中至关重要的一个细胞器，它在细胞凋亡的调控中(细胞程序性死亡)和某些疾病如癌症细胞凋亡异常反应特征中起重要作用。线粒体是细胞内RSS(活性硫物质)和ROS(活性氧)的主要来源。此外，SO2作为活性硫物质之一，具有重要的生理作用，因此监测线粒体中的SO2衍生物是特别有意义和有价值的。
荧光化学探针由于具有灵敏度高、选择性好、易合成、廉价以及良好的生物应用等特点，已成为生命科学和环境科学中主要的检测工具。目前，大部分检测细胞中HSO3 -的荧光探针均为单光子荧光探针，单光子荧光探针一般具有自荧光干扰大，激发波长小导致对细胞的光毒性大，容易发生荧光自淬灭等缺点。与单光子荧光探针相比，双光子荧光探针具有很多明显的优点，如：细胞光毒性小，不会引起荧光自淬灭，时间空间分辨率高，组织渗透深度大。因而双光子荧光探针已经作为科学家们研究的一个重要课题。咔唑基团不仅具有大的共轭体系和共平面性能，而且具有良好的光稳定性、低毒性，因此，咔唑可以成为一个优秀的双光子荧光团，通过结构修饰应用于双光子成像定性检测细胞线粒体内的HSO3 -。
三、发明内容
本发明旨在提供一种基于咔唑的双光子荧光探针及其制备方法和用途，所要解决的技术问题是通过分子设计得到一种合适的荧光探针结构，以实现双光子成像定性检测细胞线粒体内的HSO3 -。本发明荧光探针具有选择性专一、灵敏度高、检测浓度低的优点，细胞毒性测试表明本发明荧光探针对细胞几乎没有毒性作用。
本发明基于咔唑的双光子荧光探针，简记为MBCB，是以咔唑为母体，其结构式如下：
本发明基于咔唑的双光子荧光探针的制备方法，包括如下步骤：
将3-苯并噻唑-6-甲酰基-N-乙基咔唑(0.53g，1.5mmol)和15mL甲醇加入100mL圆底烧瓶中，加热搅拌至原料溶解，随后加入2,3-二甲基苯并噻唑-3-鎓碘化物(0.44g，1.5mmol)，并滴加2-3滴哌啶，70℃下回流反应4h；反应结束后静置冷却，分层后过滤得到红色粗产物，用二氯甲烷：甲醇＝4:1(v/v)的混合溶剂重结晶得到目标产物0.63g，产率67％。
本发明双光子荧光探针MBCB的合成过程如下：
本发明双光子荧光探针的用途，是在检测细胞中线粒体内的HSO3 -时作为检测试剂的应用，检测方法如下：
将本发明双光子荧光探针溶于DMSO中制得1mM的母液，取100μL的该母液于10mL容量瓶中，再用甘油:PBS＝2:8的溶剂定容，配制成10μM的检测试剂。检测试剂分别在316nm和480nm有吸收峰；加入20倍当量的HSO3 -后，MBCB位于316nm和480nm的吸收峰逐渐降低，且在306nm和351nm出现两个新的吸收峰(图1)。10μM检测试剂加入20倍当量的各种阴离子、活性氧物种和氨基酸后(图2a)，检测375-675nm范围内的荧光光谱变化，可以看出MBCB仅对HSO3 -和SO3 2-有明显的荧光增强现象，具有专一性响应；当随着HSO3 -(0-500μM)的不断加入，可以观察到600nm处的荧光发射峰强度逐渐减弱，且434nm处的荧光逐渐增强。在加入20倍当量的HSO3 -后，荧光强度趋于饱和(图2b)。10μM检测试剂在加入HSO3 -的浓度范围为0-6μM时，其荧光强度与HSO3 -的浓度之间有很好的线性关系，检测浓度低至2.67×10-10M(图3)。
本发明双光子荧光探针的结构简单，易于合成，利用Schiff base反应将作用位点和荧光基团通过C＝N键连接为一整体。本发明双光子荧光探针以荧光颜色的变化来检测HSO3 -，与HSO3 -作用后，在紫外灯下，肉眼就可以看出其荧光变化，荧光颜色从红色变成蓝光，操作简单，快速灵敏。本发明双光子荧光探针对HSO3 -具有专一性荧光响应，灵敏度高，检测浓度低。
四、附图说明
图1是10μM荧光探针加入20倍当量的HSO3 -前后的紫外吸收光谱图。
图2是10μM荧光探针加入20倍当量的各种金属离子、阴离子、活性氧物种和氨基酸前后的荧光光谱图(2a)。10μM荧光探针加入HSO3 -(0-500μM)的荧光滴定光谱图(2b)，2b插图是荧光最大发射峰强度I434与HSO3 -浓度之间的散点图。
图3是10μM荧光探针在加入HSO3 -(0-6μM)后的荧光强度与浓度的线性关系图。
图4是10μM荧光探针的双光子吸收截面数值。
图5是在不同含量(0μM、5μM、10μM、15μM)的荧光探针分子的作用下的MCF-7细胞存活率图。
图6是10μM荧光探针分子在MCF-7细胞中加入100μM HSO3 -前后的双光子荧光成像照片。在740nm激发下，蓝色通道中荧光发射收集范围420-460nm，红色通道中荧光发射收集范围是570-610nm。图a、b是只加10μM荧光探针MBCB的双通道成像，图e、f是加荧光探针10μMMBCB和100μMHSO3 -的双通道成像，图d、h是MCF-7细胞的明场，图c是a、b的叠加，图g是e、f的叠加。)
图7是10μM荧光探针分子在MCF-7细胞中加入100μM HSO3 -后的线粒体定位成像照片。其中图a，b分别为绿色通道和红色通道下的荧光共聚焦成像。图c是MCF-7细胞的明场，图d是a、b、c的叠加，图e是图d中单个细胞的荧光强度剖面图，图f是MBCB和Mitochondria@Tracker Red FM强度的相关性分布图，重叠系数为0.93。
图8是10μM荧光探针分子在活体斑马鱼中加入100μM HSO3 -前后的双光子荧光成像照片。在740nm激发下，蓝色通道中荧光发射收集范围420-460nm，红色通道中荧光发射收集范围是570-610nm。图a、b是只加10μM探针MBCB的双通道成像，图d、e是加探针10μMMBCB和100μMHSO3 -的双通道成像，图c是a、b与明场的叠加，图f是c、d与明场的叠加。)
五、具体实施方式
下面通过实施例对本发明做进一步说明。
实施例1：荧光探针分子MBCB的合成
将3-苯并噻唑-6-甲酰基-N-甲基咔唑(0.53g，1.5mmol)加入100mL圆底烧瓶中，加入15mL甲醇，加热搅拌至原料溶解后，再加入2,3-二甲基苯并噻唑-3-鎓碘化物(0.44g，1.5mmol)，并滴加2-3滴哌啶，70℃下回流反应4h；反应结束后静置冷却，分层后过滤得到红色粗产物，用二氯甲烷：甲醇＝4:1(v/v)的混合溶剂重结晶两次得到目标产物0.63g，产率67％。
1H NMR(400MHz,DMSO-d6):δ9.22(s,1H),9.04(s,1H),8.42(m,2H),8.30–8.19(m,3H),8.15(t,J＝12.6Hz,2H),8.07(d,J＝8.1Hz,1H),7.89(m,3H),7.79(t,J＝7.6Hz,1H),7.57(t,J＝7.4Hz,1H),7.47(t,J＝7.4Hz,1H),4.60(q,J＝6.6Hz,2H),4.41(s,3H),1.42(t,J＝7.0Hz,3H).δ171.87,167.93,150.04,142.82,142.02,141.94,134.29,129.18,128.06,127.41,126.58,126.21,125.96,125.19,125.09,124.07,123.28,122.96,122.78,122.50,122.36,122.23,119.60,116.49,110.81,110.64,110.45,37.76,36.16,13.87.
实施例2：荧光探针分子的光谱测试
将本发明双光子荧光探针溶于DMSO中制得1mM的母液，取100μL的该母液于10mL容量瓶中，再用甘油:PBS＝2:8的溶剂定容，配制成10μM的检测试剂。检测试剂分别在316nm和480nm有吸收峰；加入20倍当量的HSO3 -后，MBCB位于316nm和480nm的吸收峰逐渐降低，且在306nm和351nm出现两个新的吸收峰(图1)。10μM检测试剂加入20倍当量的各种阴离子、活性氧物种和氨基酸后(图2a)，检测375-675nm范围内的荧光光谱变化，可以看出MBCB仅对HSO3 -和SO3 2-有明显的荧光增强现象，具有专一性响应；当随着HSO3 -(0-500μM)的不断加入，可以观察到600nm处的荧光发射峰强度逐渐减弱，且434nm处的荧光逐渐增强。在加入20倍当量的HSO3 -后，荧光强度趋于饱和(图2b)。10μM检测试剂在加入HSO3 -的浓度范围为0-6μM时，其荧光强度与HSO3 -的浓度之间有很好的线性关系，检测浓度低至2.67×10-10M(图3)。
实施例3：荧光探针分子的双光子性能测试
利用双光子诱导荧光测量技术，测试荧光探针分子(MBCB)的双光子吸收截面，从图4可以看出，双光子激发波长在740nm时，荧光探针分子的最大吸收截面是138GM。说明探针可以应用于双光子生物成像。
实施例4：细胞毒性测试
MTT(3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐)实验是根据已报道的文章操作进行细胞毒性测试。分别在同一批细胞中加入0，5，10，15的荧光探针分子，条件是在37℃、含5％CO2的细胞培养箱中孵育24小时，根据细胞存活度的公式：细胞存活率％＝OD570(样品)/OD570(对照组)×100，可算得细胞存活率(图5)。从图6中我们可以看出，浓度为10μM时，细胞存活率还有93％左右，当探针浓度达到15μM时，细胞存活率仍然有约88％，说明了本发明荧光探针分子对细胞无明显毒性作用，因此可以用来检测细胞中线粒体内的HSO3 -。
实施例5：细胞成像测试
MCF-7细胞由DEME(invitrogen)培养液培养，成像前一天，MCF-7细胞放于玻底培养皿中，成像时MCF-7细胞和10μM的荧光探针MBCB的DMSO溶液于37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液或培养液充分洗涤后，用双光子荧光共聚焦成像，420-460nm通道荧光微弱(图6a)，570-610nm通道有明显荧光(图6b)。向上述含荧光探针的细胞培养液中加入(100μM)HSO3 -，在37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液或培养液充分洗涤后，再进行双光子荧光共聚焦成像，420-460nm通道内荧光明显增强(图6d)，570-610nm通道内荧光减弱(图6e)。图d、h是MCF-7细胞的明场，图c是a、b的叠加，图g是e、f的叠加。从图6中可以看出，探针可用于细胞内HSO3 -的双光子荧光成像。
实施例6：细胞定位测试
MCF-7细胞由DEME(invitrogen)培养液培养，成像前一天，MCF-7细胞放于激光共聚焦皿中，成像时MCF-7细胞和10μM的荧光探针MBCB的DMSO溶液于37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液洗涤3次后，再往培养皿中加入0.5μM商品化线粒体染色剂Mitochondria@Tracker Red FM溶液继续孵育0.5小时，用中性的PBS缓冲溶液洗涤3次后，再往培养皿中加入100μM HSO3 -溶液继续孵育0.5小时，用中性的PBS缓冲溶液洗涤3次后，用双光子荧光共聚焦成像，设置绿色通道tracker1，激发波长为740nm，发射波段为420-460nm，这个通道用来接受探针分子MBCB加入HSO3 -后发射的荧光。设置红色通道tracker2，激发波长为579nm，发射波长为580-620nm，这个通道用来接收商品化线粒体染色剂Mitochondria@Tracker Red FM发射的荧光。其中图a，b分别为绿色通道和红色通道下的荧光共聚焦成像。图c是MCF-7细胞的明场，图d是a、b、c的叠加，图e是图d中单个细胞的荧光强度剖面图，图f是MBCB和Mitochondria@Tracker Red FM强度的相关性分布图，重叠系数为0.93。从图7可以看出，MBCB绝大多数定位在线粒体内，可用于细胞线粒体内HSO3 -的双光子荧光成像和定性检测。
实施例7：斑马鱼成像测试
25℃条件下，将两条5天的斑马鱼分别置于灭菌的pH为7.4的10mM PBS缓冲溶液中培养。向其中一条斑马鱼中加入10μM探针培养0.5小时，用灭菌的pH为7.4的10mM PBS缓冲溶液清洗3遍，最后将斑马鱼置于盖玻片上并用麻醉剂MS-222固定好。向另一条斑马鱼中加入100μMHSO3 -培养0.5小时，用灭菌的pH为7.4的10mM PBS缓冲溶液清洗3遍，再加入10μM探针培养0.5小时，用灭菌的pH为7.4的10mM PBS缓冲溶液清洗3遍，最后将斑马鱼置于盖玻片上并用麻醉剂MS-222固定好。将它们分别进行双光子荧光共聚焦成像实验。在740nm激发下，蓝色通道中荧光发射收集范围420-460nm，红色通道中荧光发射收集范围是570-610nm。图a、b是只加10μM探针MBCB的双通道成像，蓝色通道中荧光微弱，红色通道有明显荧光。图d、e是加探针10μMMBCB和100μMHSO3 -的双通道成像，蓝色通道中荧光明显增强，红色通道中荧光减弱。图c是a、b与明场的叠加，图f是d、e与明场的叠加。)从图8可以看出，MBCB可用于活体斑马鱼中HSO3 -的双光子荧光成像和定性检测。
Claims (4)
1.一种基于咔唑的双光子荧光探针，其特征在于其结构式为：
2.一种权利要求1所述的基于咔唑的双光子荧光探针的制备方法，其特征在于包括如下步骤：
将3-苯并噻唑-6-甲酰基-N-乙基咔唑0.53g和甲醇15mL加入反应器中，加热搅拌至原料溶解，随后加入2,3-二甲基苯并噻唑-3-鎓碘化物0.44g，并滴加2-3滴哌啶，70℃下回流反应4h；反应结束后静置冷却，分层后过滤得到红色粗产物，重结晶得到目标产物。
3.根据权利要求2所述的方法，其特征在于：
重结晶所用溶剂为二氯甲烷和甲醇按体积比4:1的比例混合得到。
4.一种权利要求1所述的基于咔唑的双光子荧光探针的用途，其特征在于：
所述双光子荧光探针在定性检测细胞中线粒体内的HSO3 -时作为检测试剂的应用。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108484590A (zh) * | 2018-05-18 | 2018-09-04 | 安徽大学 | 一种基于咔唑的双光子粘度荧光探针及其制备方法和用途 |
| CN110003173A (zh) * | 2019-04-26 | 2019-07-12 | 安徽大学 | 一种基于咔唑的双光子极性荧光探针及其制备方法和用途 |
| CN110156773A (zh) * | 2019-05-22 | 2019-08-23 | 济南大学 | 一种检测二氧化硫的比率型荧光探针及其应用 |
| CN111253935A (zh) * | 2019-12-24 | 2020-06-09 | 安徽大学 | 一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203512A (zh) * | 2015-09-16 | 2015-12-30 | 山西大学 | 一种咔唑类荧光探针及其制备方法和应用 |
| CN106674195A (zh) * | 2016-12-26 | 2017-05-17 | 安徽大学 | 一种苯并吲哚衍生物双光子荧光探针及其制备方法和用途 |
-
2017
- 2017-06-15 CN CN201710450453.8A patent/CN107286151B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203512A (zh) * | 2015-09-16 | 2015-12-30 | 山西大学 | 一种咔唑类荧光探针及其制备方法和应用 |
| CN106674195A (zh) * | 2016-12-26 | 2017-05-17 | 安徽大学 | 一种苯并吲哚衍生物双光子荧光探针及其制备方法和用途 |
Non-Patent Citations (1)
| Title |
|---|
| 王奇: "基于SO2、ClO-检测的线粒体靶向双光子荧光探针的研究", 《安徽大学硕士学位论文》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108484590A (zh) * | 2018-05-18 | 2018-09-04 | 安徽大学 | 一种基于咔唑的双光子粘度荧光探针及其制备方法和用途 |
| CN108484590B (zh) * | 2018-05-18 | 2021-02-26 | 安徽大学 | 一种基于咔唑的双光子粘度荧光探针及其制备方法和用途 |
| CN110003173A (zh) * | 2019-04-26 | 2019-07-12 | 安徽大学 | 一种基于咔唑的双光子极性荧光探针及其制备方法和用途 |
| CN110156773A (zh) * | 2019-05-22 | 2019-08-23 | 济南大学 | 一种检测二氧化硫的比率型荧光探针及其应用 |
| CN111253935A (zh) * | 2019-12-24 | 2020-06-09 | 安徽大学 | 一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途 |
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