CN114249760A - 三通道同时区分次氯酸和过氧化氢的荧光探针的合成与应用 - Google Patents
三通道同时区分次氯酸和过氧化氢的荧光探针的合成与应用 Download PDFInfo
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Abstract
本发明公开了一种通过绿、红、黄三种不同的荧光发射信号同时区分检测次氯酸(HOCl)和过氧化氢(H2O2)的荧光分子探针，该分子探针的化学结构式如下：
。该荧光探针能在同一检测条件下区分HOCl、H2O2及二者共存。探针与HOCl反应后在350 nm激发波长下发射504 nm的绿光，与H2O2反应后在430 nm激发波长下发射640 nm的红光，与两者共同反应(HOCl和H2O2)后在440 nm激发波长下发射550 nm的黄光。该探针可通过三通道同时顺序性检测HOCl和H2O2及共存条件下的HOCl和H2O2，具有灵敏度高、选择性好、斯托克斯位移大等优势，在分析化学、生命科学、环境科学等技术领域有着巨大的应用前景。
Description
技术领域
本发明属于分析化学技术领域，具体涉及一种检测次氯酸和过氧化氢荧光探针的合成，以及该探针在环境中定量分析次氯酸和过氧化氢，在细胞、组织和活体等生物***中同时区分成像次氯酸和过氧化氢的应用。
背景技术
活性氧(Reactive oxygen species, ROS)是多种生理过程中必不可少的信使分子(J. Amer. Chem. Soc., 2017, 139, 6911−6918),包括衰老、信号转导、细胞氧化还原稳态和防御病原体入侵等(Spectrochim. Acta. A Mol. Biomol. Spectrosc., 2021,246, 118927)。过氧化氢(H2O2)和次氯酸(HClO)是两种重要的活性氧，在许多生理和病理过程中发挥重要作用。有证据表明，过量的HClO与各种人类疾病有关，如心血管疾病、神经退行性变、关节炎和癌症(Anal. Chem., 2017, 89, 10384−10390)；此外，细胞内异常产生的H2O2参与各种病理过程，如糖尿病、癌症、心血管疾病和神经退行性变等(Anal. Chem.,2016, 88, 8019–8025)。由于这两种活性氧其中一种含量的变化可能会引起另一种活性氧的关联变化，而且很多疾病的发生都与它们的含量变化密切相关。因此，开发高效的检测方法/技术用于同时区分检测HOCl和H2O2这两种活性氧物种，了解其生物学功能具有非常重要的意义。基于小分子荧光探针的荧光成像技术由于其实时、灵敏、无创等特点，已成为一种有效的可视化生物***中生物分子时空分布的方法(Anal. Chem., 2016, 88, 8019–8025)。尽管小分子活性氧次氯酸(HOCl)和过氧化氢(H2O2)具有相似的化学性质，但也发展了一些了单/双荧光通道荧光探针用于检测HOCl或者H2O2 (如J. Am. Chem. Soc., 2017,139, 6911–6918、Chem. Sci., 2018, 9, 8207–8212、Anal. Chem., 2020, 92, 3262–3269、Chem. Commun., 2018, 54, 9238–9241等)。只有极少数荧光探针可同时检测HOCl和H2O2这两种活性氧，尚未实现三通道同时区分HOCl、H2O2及其共存条件下的HOCl和H2O2。
发明内容
鉴于上述情况，克服一些现有技术的不足，本发明的目的在于提供一种利用三通道荧光信号同时区分检测次氯酸和过氧化氢及其共存的荧光分子探针，能利用不同的荧光信号区分成像细胞等生物***中的次氯酸和过氧化氢，为分析检测等领域提供一些分析检测方法和思路。本发明的目的还在于提供一种制备方法简单、多检测活性氧荧光分子探针的合成与应用方法。
本发明解决问题采取的具体技术方案为，三通道同时区分次氯酸和过氧化氢的荧光探针的合成与应用，其探针的化学结构式如下：
同时区分次氯酸和过氧化氢的荧光探针，其特征在于，所述荧光分子探针的制备方法包括以下步骤：
步骤1. 合成11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H）-酮
步骤2. 合成荧光探针4-（11-乙基-2-氧代-2,11-二氢吡喃并[2,3-b]吩噻嗪-3-基）-1-（4-（4,4,5,5-四甲基-1,3,2-二氧杂环-2-基）苄基）吡啶-1-溴化铵
a．在氮气保护条件下，将11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H）-酮与4-溴甲基苯硼酸频哪醇酯加入无水乙腈中，在100℃回流搅拌12小时，得红褐色溶液，
b．待反应冷至室温后，过滤，得到红褐色固体4-（11-乙基-2-氧代-2,11-二氢吡喃并[2,3-b]吩噻嗪-3-基）-1-（4-（4,4,5,5-四甲基-1,3,2-二氧杂环-2-基）苄基）吡啶-1-溴化铵。
本发明的荧光分子探针同时区分检测次氯酸和过氧化氢使用方法：无特殊说明，通常在室温下将探针分子溶解在有机相和水相体积比为5:5的环境下用于分析检测，有机相为二甲基亚砜(DMSO)，水相为pH = 7.4的磷酸盐缓冲溶液(PBS)和分析物的水溶液。
本发明的荧光分子探针同时区分次氯酸和过氧化氢具体特征如下：该分子荧光探针用二甲基亚砜(DMSO)溶解，探针分子溶解在有机相和水相(5:5,v/v)溶液中，探针与次氯酸反应后在350 nm激发波长下发射504 nm的绿光，与过氧化氢反应后在430 nm激发波长下发射640 nm的红光，与两者共同反应后在440 nm激发波长下发射550 nm的黄光。因此实现特定激发与荧光发射信号检测特定分析物，还可以利用第三个荧光通道实现二者的顺序性和共存检测。上述荧光分子探针实现了在同一检测条件下同时区分检测次氯酸和过氧化氢，对其他活性氧、活性硫、常见的氨基酸、金属离子和活性氮等均无明显响应。探针检测对HOCl和H2O2的检测限分别低至51.11 nM和39.45 nM。因此，本发明公开的荧光分子探针能够实现三通道同时区分次氯酸和过氧化氢。
附图说明
图1为本发明的荧光探针的核磁共振氢谱图。
图2为本发明所述的荧光探针检测次氯酸和过氧化氢的荧光光谱图。
图3为本发明的荧光探针同时通过绿、红、黄三颜色荧光成像RAW 264.7（巨噬细胞）细胞中细胞内源性的次氯酸(HOCl)和过氧化氢(H2O2)荧光成像图。
具体实施方式
下面结合附图对本发明做进一步的说明。
本发明所述的荧光分子探针的合成路线如下式所示：
实施例1. 合成11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H）-酮
I．将1.0 g (3.69 mmol) 10-乙基-2-羟基-10H-吩噻嗪-3-甲醛与608.81 mg(3.69 mmol) 4-吡啶乙酸乙酯加入10 mL无水乙醇中，在95℃搅拌8-12小时，得红色溶液，
II．待反应冷至室温后，过滤，得到红色固体11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H-酮）1.0 g，产率为73.0%。
实施例2. 合成荧光探针4-（11-乙基-2-氧代-2,11-二氢吡喃并[2,3-b]吩噻嗪-3-基）-1-（4-（4,4,5,5-四甲基-1,3,2-二氧杂环-2-基）苄基）吡啶-1-溴化铵
A．在氮气保护条件下，将1.0 g (2.68 mmol) 11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H）-酮与797.4 mg (2.68 mmol) 4-溴甲基苯硼酸频哪醇酯加入15 mL无水乙腈中，在100℃回流搅拌12小时，得红褐色溶液，
B．待反应冷至室温后，过滤，得到红褐色固体4-（11-乙基-2-氧代-2,11-二氢吡喃并[2,3-b]吩噻嗪-3-基）-1-（4-（4,4,5,5-四甲基-1,3,2-二氧杂环-2-基）苄基）吡啶-1-溴化铵1.5 g，产率为83.3%。
实施例3. 荧光分子探针在体外环境中区分检测次氯酸和过氧化氢的应用
本发明所述的荧光分子探针检测次氯酸和过氧化氢的光谱性质实验：将探针溶解在二甲基亚砜(DMSO)中配置成浓度为1 mM的探针溶液，分别配置浓度为1 mM的次氯酸和过氧化氢水溶液。具体测试方式为：取20 μL的1 mM的探针溶液，980 μL的分析纯DMSO和所需量的PBS缓冲水溶液，所需量次氯酸/过氧化氢水溶液于2 mL的样品管中，所有测试均保持有机相和水相的体积比为5:5(每一个测试样品总体积为2 mL)。例如当要求测试次氯酸浓度为20 μM时探针与次氯酸反应后的荧光强度，配制样品情况为：取20 μL 1 mM的探针溶液，980 μL的分析纯DMSO，960 μL的PBS缓冲溶液，和40 μL的1 mM的次氯酸水溶液于2 mL的样品管中，室温下震荡摇匀15分钟后即可用350 nm的激发波长测量其荧光发射强度，其他测试操作与上述步骤类似。该探针对次氯酸和过氧化氢的检测限分别低至51.11 nM和39.45 nM。因此，本发明公开的荧光分子探针能够实现对两者的高灵敏区分检测。
实施例4. RAW 264.7（巨噬细胞）细胞中内源性次氯酸和过氧化氢三通道荧光成像分析
将RAW 264.7细胞传代至共聚焦皿细胞培养基中，在标准生长条件下培养24小时后，加入适量探针(5 μM)继续在标准生长条件下培养30分钟，然后在共聚焦荧光显微镜下照相，分别用绿色、红色、黄色荧光通道进行荧光成像RAW 264.7细胞内源性的次氯酸和过氧化氢，从图3可以看出，本发明的荧光探针在细胞中能发射绿、红和黄色荧光，说明探针能够检测细胞中的次氯酸和过氧化氢以及二者的共同检测，成功实现了细胞中内源性HOCl和H2O2的三通道荧光成像分析。
本发明提供的同时区分次氯酸和过氧化氢的荧光探针实现了基于同一探针、同一检测条件下利用探针与次氯酸和过氧化氢发生不同的化学反应，生成不同的荧光物质，从而在特定激发波长下发射绿色、红色、黄色三种颜色的荧光，达到同时区分检测次氯酸和过氧化氢的目的，成功实现三通道同时荧光成像细胞中内源性的次氯酸和过氧化氢。希望为今后的活性氧荧光探针的发展提供一些思路。尽管本发明的内容已经通过上述优选实施例作了详细的介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，具有本文所述技术特征的同时区分次氯酸和过氧化氢的荧光探针，均落入本专利的保护范围。
Claims (3)
1.三通道同时区分次氯酸和过氧化氢的荧光探针，其特征在于，所述的荧光分子探针的化学结构式如下所示：
2.如权利要求1所述的荧光探针的合成，其特征在于，所述荧光分子探针的制备方法包括以下步骤：
步骤1. 合成11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H）-酮
步骤2. 合成荧光探针4-（11-乙基-2-氧代-2,11-二氢吡喃并[2,3-b]吩噻嗪-3-基）-1-（4-（4,4,5,5-四甲基-1,3,2-二氧杂环-2-基）苄基）吡啶-1-溴化铵
a．在氮气保护条件下，将11-乙基-3-（吡啶-4-基）吡喃[2,3-b]吩噻嗪-2（11H）-酮与4-溴甲基苯硼酸频哪醇酯加入无水乙腈中，在100℃回流搅拌12小时，得红褐色溶液，
b．待反应冷至室温后，过滤，得到红褐色固体4-（11-乙基-2-氧代-2,11-二氢吡喃并[2,3-b]吩噻嗪-3-基）-1-（4-（4,4,5,5-四甲基-1,3,2-二氧杂环-2-基）苄基）吡啶-1-溴化铵。
3.如权利要求1所述的荧光探针的应用，其特征在于，所述的荧光探针能够制备在环境中定量分析HOCl和H2O2，在细胞、组织和活体中同时区分成像HOCl和H2O2的器件的应用。
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