CN114195811B - 双功能荧光探针的合成及其同时区分检测过氧化氢和HClO的应用 - Google Patents
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Abstract
本发明公开了一种通过红、绿两种不同的荧光发射信号同时区分过氧化氢(H2O2)和次氯酸(HClO)的双功能荧光分子探针，该荧光探针的化学结构式如下：
。该荧光探针能在同一检测条件下利用探针与H2O2和HClO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红、绿两种颜色的荧光，达到同时区分检测H2O2和HClO的目的。探针与H2O2反应后在560 nm激发波长下发射648 nm的红光，与HClO反应后在427 nm激发波长下发射548nm的绿光。该探针检测H2O2和HClO具有灵敏度高、选择性好、区分度大等优势，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，具体涉及一种双通道同时区分过氧化氢(H2O2)和次氯酸(HClO)的荧光探针的合成，以及该探针在环境中同时定量检测H2O2和HClO，双通道荧光成像细胞内源性H2O2和HClO的应用。
背景技术
活性氧(ROS，包括H2O2，O2 -和1O2)在正常生理条件下保持一定浓度范围，在维持细胞形态和基本功能方面起着重要作用(Anal.Chem.,2014,86,9970–9976)。活性氧作为内源性代谢产物被认为是氧化应激的指标。其中过氧化氢(H2O2)是最重要的ROS之一。当H2O2以低浓度(<0.7μM)受控生成时，作为细胞内第二信使可以激活信号通路以刺激细胞增殖，分化和迁移。与其他信使不同的是，过氧化氢(H2O2)作为一种氧化剂，在应激或受到外源性化学物质刺激的条件下，过氧化氢会异常产生，导致氧化应激，产生的ROS可以攻击细胞结构或生物分子，例如蛋白质，脂质体和DNA，这些与衰老，阿尔茨海默氏病和癌症有关(Analyst,2017,142,4522-4528)。一般认为，HClO可通过髓过氧化物酶(MPO)催化过氧化氢和氯离子在生物体中产生(Anal.Chem.,2014,86,671–677)。作为活性氧中的代表性物质之一，HClO在日常生活和体内起着不可替代的作用。一方面，HClO用作漂白剂，消毒剂，除臭剂等。另一方面，过量的HClO会引起心血管疾病，关节炎，动脉硬化和癌症等多种疾病。因此，对体内HClO的准确检测具有重要意义。目前针对检测二者的荧光探针已有报道，但是只能检测其中一种或区分度较小。在本发明中，我们通过对香豆素类荧光团的修饰改造，开发了一种同时区分H2O2和HClO的双功能荧光探针，具有检测灵敏度高、选择性好、区分度大等优势，在环境分析、生物成像中具有较好的应用前景。
发明内容
鉴于上述情况，克服现有技术的不足，本发明的目的在于提供一种同时区分H2O2和HClO的荧光分子探针，为分析检测、环境科学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、双功能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，双功能荧光探针的合成及其同时区分检测H2O2和HClO的应用，其探针的化学结构式如下：
同时区分H2O2和HClO双功能荧光探针，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9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮；
a.在氮气保护条件下，将9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶基[3,2,1-ij]喹啉-11-酮和适量的劳森试剂加入适量干燥重蒸的甲苯中，110℃搅拌12小时，得红色溶液，
b.将步骤a中红色反应液旋干，乙酸乙酯重结晶，得红色固体即9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮；
步骤2.合成(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈
a.在氩气保护且避光条件下，将2-(嘧啶-4-基)乙腈和1.2倍当量的NaH在无水乙腈中常温搅拌30分钟，再加入9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮的乙腈溶液，室温搅拌2小时，最后加入硝酸银反应90分钟终止反应；
b.将反应液柱层析纯化，得紫色固体即(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈；
步骤3.合成所述的荧光探针
将步骤2合成的(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈和4-溴甲基苯硼酸频哪醇酯1:1当量在在乙腈溶剂中80℃回流12小时，析出固体，过滤洗涤干燥，得墨蓝色固体即所述的荧光探针。
本发明的双功能荧光分子探针同时区分H2O2和HClO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，有机相为乙腈(ACN)，水相为pH＝7.4的磷酸盐缓冲溶液(PBS)和分析物的水溶液。
本发明的双功能荧光探针检测H2O2和HClO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，与H2O2在37℃反应30分钟后，在560nm激发波长下发射648nm的强红色荧光；与HClO室温反应15分钟后，在427nm激发波长下发射548nm的强绿色荧光。探针本身并无明显荧光。因此实现特定激发与荧光发射信号检测特定分析物，当两种活性氧物质均存在时，利用不同的激发和荧光发射信号也能很好地将两者区分。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H2O2和HClO，对NAC,Gly,Ala,His,Met,Thr,Lys,Asp,Glu,Pro,Ser,O2 -,1O2,ONOO-,TBHP,·OH,NO,n-Butylamine,aniline等氨基酸、含硫衍生物及其他活性氧物质均无明显响应，对H2O2和HClO两者的检测限分别低至0.136μM和0.28μM。因此，本发明公开的双功能荧光分子探针能够实现对两者的高灵敏区分检测。
附图说明
图1为本发明的双功能荧光探针的核磁共振氢谱图。
图2为本发明的双功能荧光探针同时通过红、绿两种颜色荧光成像RAW 264.7(巨噬细胞)细胞内源性H2O2和HClO的荧光成像图。
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9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮
I.在氮气保护条件下，将2.00g(6.47mmol)9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶基[3,2,1-ij]喹啉-11-酮和劳森试剂3.92g(9.72mmol)1:1.5当量加入20mL干燥重蒸甲苯中，110℃搅拌12小时，得红色溶液；
II.将步骤a中红色反应液旋干，乙酸乙酯重结晶，得红色固体，即9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮1.4g，产率为66.54％。
实施例2.合成(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈
i.在氩气保护且避光条件下，将2-(嘧啶-4-基)乙腈0.47g(4.0mmol)和等当量的NaH(0.16g，4.00mmol)在无水乙腈中常温搅拌30分钟，再加入9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮1.00g(3.07mmol)的乙腈溶液，室温搅拌2小时，最后加入硝酸银0.68g(4.0mmol)反应90分钟终止反应；
ii.将反应液柱层析纯化，得紫色固体即(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈；1.00g，产率为79.28％。
实施例3.合成所述的荧光探针
将1.00g(2.44mmol)(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈和0.72g(2.44mmol)4-溴甲基苯硼酸频哪醇酯1:1当量在在乙腈溶剂中80℃回流12小时，析出固体，过滤洗涤干燥，得墨蓝色固体即所述的荧光探针0.70g，产率为45.9％。
实施例4.荧光分子探针在体外环境中区分检测H2O2和HClO的应用
本发明所述的双功能荧光分子探针光谱性质实验：将探针溶解在二甲基亚砜(DMSO)中配置成浓度为1mM的探针溶液，分别配置浓度为1mM的HClO和H2O2水溶液。具体测试方式为：取20μL 1mM的探针溶液，980μL的分析纯乙腈，所需量的PBS缓冲水溶液和所需的1mM的HClO/H2O2水溶液于2mL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2mL)，例如当要求测试H2O2浓度为10μM时探针与H2O2反应后的荧光强度，配制样品情况为：取20μL 1mM的探针溶液，980μL的分析纯乙腈，20μL1mM的H2O2水溶液和980μL的PBS缓冲溶液于2mL的样品管中，37℃下震荡摇匀30分钟后即可用560nm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。该探针分子实现了用不同的激发波长和荧光发射信号区分检测H2O2和HClO这两种活性氧物质，具有很高的灵敏度，对H2O2和HClO两者的检测限分别低至0.136μM和0.28μM，非常适合活细胞内源性H2O2和HClO的成像和分析。
实施例5.RAW 264.7(巨噬细胞)内源性H2O2和HClO双通道荧光成像分析将RAW264.7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(5μM)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，分别用红色和绿色荧光通道进行荧光成像RAW 264.7细胞内源性H2O2和HClO，从图2可以看出，本发明的双检测荧光探针成功实现细胞中内源性和外源性的H2O2和HClO双通道荧光成像分析，在生物化学、分析检测等领域极具应用价值。
本发明的双功能荧光探针的合成及其同时区分H2O2和HClO的应用，发展了一种高效简单的双功能检测活性氧物质的荧光探针，可用于同时区分检测H2O2和HClO，基于同一探针、同一检测条件下利用探针与H2O2和HClO发生不同的化学反应，生成不同的荧光物质，进而在特定激发波长下发射红、绿两种颜色的荧光，达到同时区分检测的目的，成功实现双通道同时荧光成像细胞中内源性的H2O2和HClO。希望为今后的双通道及多通道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双功能荧光探针的合成及其同时区分检测H2O2和HClO的应用，均落入本专利的保护范围。
Claims (3)
1.同时区分H2O2和HClO的双功能荧光探针，其特征在于，所述的荧光分子探针的化学结构式如(1)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9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮；
a.在氮气保护条件下，将9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶基[3,2,1-ij]喹啉-11-酮和的劳森试剂加入干燥重蒸的甲苯中，110℃搅拌12小时，得红色溶液，
b.将步骤a中红色反应液旋干，乙酸乙酯重结晶，得红色固体即9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮；
步骤2.合成(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈；
a.在氩气保护且避光条件下，将2-(嘧啶-4-基)乙腈和1.2倍当量的NaH在无水乙腈中常温搅拌30分钟，再加入9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶并[3,2,1-ij]喹啉-11-硫酮的乙腈溶液，室温搅拌2小时，最后加入硝酸银反应90分钟终止反应；
b.将反应液柱层析纯化，得紫色固体即(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈；
步骤3.合成权利要求1所述的荧光探针
将步骤2合成的(E)-2-(嘧啶-4-基)-2-(9-(三氟甲基)-2,3,6,7-四氢-1H，5H，11H-吡喃并[2,3-F]吡啶[3,2,1-ij]喹啉-11-亚乙基)乙腈和4-溴甲基苯硼酸频哪醇酯1:1当量在在乙腈溶剂中80℃回流12h，析出固体，过滤洗涤干燥，得墨蓝色固体即权利要求1所述的荧光探针。
3.如权利要求1所述的荧光探针用于制备器件的应用，其特征在于，制备的器件能够在环境中检测H2O2和HClO，以及在细胞内同时区分成像H2O2和HClO。
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