CN106967102B - 一种基于罗丹明衍生物的过氧化氢增强型荧光探针 - Google Patents
一种基于罗丹明衍生物的过氧化氢增强型荧光探针 Download PDFInfo
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Abstract
本发明公开了一种基于罗丹明衍生物的过氧化氢增强型荧光探针，其化学结构式如式（1）所示。本发明的探针廉价易得，使用方便，可以特异性与过氧化氢发生反应，生成荧光发射能力更强的罗丹明衍生物，在检测过氧化氢过程中不会受到其他相关氧化物的干扰，对过氧化氢具有良好的选择性，能对活细胞和活动物中的过氧化氢进行准确检测。。
Description
技术领域
本发明涉及一种基于罗丹明衍生物的过氧化氢荧光探针，属于分析化学技术领域。
背景技术
活性氧（ROS）在生物体内普遍存在，是含氧自由基和含氧非自由基的总称，在许多生理和病理过程中起着重要的作用。活性氧通常由生物体内的氧化应激、炎症等生理和病理过程的酶促和非酶促反应产生。过氧化氢（H2O2），作为一种重要的活性氧，是一种重要的信号分子，广泛参与生物体系内各种各样的信号传导过程，同时也是氧化应激等相关疾病的标记物。生物体内的过氧化氢的产生与细胞因子、生长因子和神经递质等激活白细胞氧化酶相关。适量水平下，生物体内的过氧化氢对生物正常的生理过程有益，可参与细胞内蛋白质的可逆氧化，以及调剂蛋白质的磷酸化到基因表达等众多细胞过程。但是，生物体内过氧化氢浓度的异常与人类的许多疾病密切相关，包括癌症、心血管疾病、阿兹海默症等。因此，检测生物体内的过氧化氢水平对于调查研究相关疾病的发生、发展具有重要的作用。
传统的过氧化氢检测方法包括滴定法、化学发光法、电化学法（极谱法、电流滴定法、恒电位法）、比色法等。但是，这些方法通常会破坏测样品，耗费高，不适用于生物体内过氧化氢的实时检测。相比之下，荧光成像具有不破坏样品、高灵敏度、高选择性和实时监测等特点，可为检测生物体内的过氧化氢提供了一种可行的方法。目前，已开发的过氧化氢荧光探针大都侧重于蓝色或绿色荧光团，但是这种方法使用的激发波长较短，不适合小动物活体成像，因此具有一定的局限性。相比之下，红色荧光探针依靠具有波长较长的红色荧光的提供提供信号，可适用于活体成像。
发明内容
针对现有技术的不足，本发明提供了一种检测过氧化氢的红色荧光探针。
本发明所述的检测过氧化氢的荧光探针，为罗丹明衍生物，其特征在于：所述荧光探针的化学结构式如式（1）所示：
（1）
上述检测过氧化氢的比率型荧光探针以如下方法制备：
。
（1）中间体2制备：将中间1（1 mmol）、N,N-双(三氟甲磺酰基)苯胺（1 mmol）和N,N-二异丙基乙胺（2 mmol）溶于5 ml DMF中，室温下反应6小时，加入5 ml水，使用5ml乙酸乙酯萃取，将油相真空干燥得粗产品。使用柱层析（洗脱剂：乙酸乙酯：石油醚 = 1:5）对粗产品进行纯化，得纯品中间体2（产率73%）。
（2）荧光探针（化合物3）的制备：将中间体2（1 mmol）、双联频哪醇硼酸酯（1mmol）、 1,1-双(二苯基膦)二茂铁二氯化钯二氯甲烷络合物（0.2 mmol）和醋酸钾（1.5mmol）溶于 10 ml甲苯中，氮气保护下回流6 h。反应结束之后，真空除溶剂，得粗产品。使用柱层析（洗脱剂：乙酸乙酯：石油醚 = 1:4）对粗产品进行纯化，得探针纯品（产率69%）。
本发明所述的基于罗丹明衍生物的过氧化氢荧光探针在检测活细胞中过氧化氢中的应用。
本发明所述荧光探针可以应用于活细胞内的过氧化氢含量的定量分析。该探针作为过氧化氢的特异性探针，能和探针发生氧化反应，进而生成荧光更强的罗丹明衍生物。通过定量检测溶液所发射的荧光的强度，可以确定细胞中的过氧化氢的含量。具体测定方法为：25℃条件下，在荧光探针的PBS（含有5%甲醇，pH = 7.4， 20 mM）溶液中，将过氧化氢的PBS溶液加入到探针体系中，测定溶液的荧光强度，根据荧光作为过氧化氢含量的评价指标。
具体的，本发明所述荧光探针在检测活细胞和活动物中过氧化氢的应用实例：
（1）将HeLa细胞加入铺有盖玻片的培养皿中，并置于二氧化碳培养箱中培养，36小时后对铺有并长满细胞的盖玻片的培养皿冲洗，加入5 μM本发明所述的过氧化氢荧光探针后37℃下培养40分钟。之后，使用PBS冲洗，制片，在激光共聚焦显微镜下观察细胞，进行荧光成像。并列的，向铺有并长满细胞的盖玻片的培养皿中加入50 μM浓度的过氧化氢，37℃下培养1小时，制片，在共聚焦显微镜下观察细胞，进行荧光成像。
（2）首先向小鼠腹腔内注射甲苯噻嗪（10 mg. kg-1）和氯胺酮（80 mg. kg-1），然后注射本发明所述的过氧化氢荧光探针（50 nmol），随后进行小鼠成像实验。在此小鼠基础上，向小鼠腹腔注射100 μM过氧化氢，20 min之后进行小鼠成像实验。
本发明提供了一类用于特异性识别过氧化氢的探针，其与过氧化氢反应之后可生成具有荧光发射能力更强的罗丹明衍生物，并依靠长波长的荧光发射峰的强度为信号，可以用于小鼠成像。该探针可发射长波长荧光，能够广泛用于检测活细胞和小鼠中的过氧化氢。
本发明的有益效果是：所制备的罗丹明衍生物的荧光探针能够与过氧化氢进行特异性反应，通过荧光信号的产生对过氧化氢进行定性检测，能够对活细胞中的过氧化氢进行定量检测。此外该荧光探针廉价易得，可经化学合成获得，且合成工艺简单易行。进一步的，本发明提供的用于检测过氧化氢的荧光探针易于制备、使用方便，能够快速检测活细胞和活动物中的过氧化氢。
附图说明
图1是荧光探针的核磁氢谱图谱。
图2是荧光探针的液质数据（LC-MS）图谱。
图3是荧光探针对不同物质的选择性图谱。其中，相关分析物为：次氯酸钠、一氧化氮、半胱氨酸、羟基自由基、过氧化二叔丁基、过氧化二叔丁醇、谷胱甘肽、维生素C和硝酸钠。
图4是荧光探针与过氧化氢的线性关系数据。
图5是本发明所述的荧光探针对细胞内过氧化氢监测的照片。
其中：A、B和C依次为探针（10 μM）在细胞中明场照片、红色通道照片、明场和红色通道叠加照片；D、E和F依次为探针（10 μM）和过氧化氢（200 μM）在细胞中明场照片、红色通道照片、明场和红色通道叠加照片。
图6是本发明所述的荧光探针对小鼠内过氧化氢监测的照片。其中，A为注射探针的小鼠成像照片；B为注射探针和过氧化氢的小鼠成像照片。
具体实施方式
下面结合实施例和附图对本发明做进一步说明。
实施例1
荧光探针的制备：
将中间1（1 mmol）、N,N-双(三氟甲磺酰基)苯胺（1 mmol）和N,N-二异丙基乙胺（2mmol）溶于5 ml DMF中，室温下反应6小时，加入5 ml水，使用5ml乙酸乙酯萃取，将油相真空干燥得粗产品。使用柱层析（洗脱剂：乙酸乙酯：石油醚 = 1:5）对粗产品进行纯化，得纯品中间体2（产率73%）。将中间体2（1 mmol）、双联频哪醇硼酸酯（1 mmol）、 1,1-双(二苯基膦)二茂铁二氯化钯二氯甲烷络合物（0.2 mmol）和醋酸钾（1.5 mmol）溶于 10 ml甲苯中，氮气保护下回流6 h。反应结束之后，真空除溶剂，得粗产品。使用柱层析（洗脱剂：乙酸乙酯：石油醚 = 1:4）对粗产品进行纯化，得探针纯品（产率69%）。荧光探针的核磁氢谱和液质数据见图1和图2。
实施例2
荧光探针对不同物质的选择性鉴定
预先准备10份5 mL的 5 μM荧光探针PBS溶液(含5%甲醇)，然后分别向所述10份体系中依次加入50 μL浓度为100 μM的过氧化氢、次氯酸钠、一氧化氮、半胱氨酸、羟基自由基、过氧化二叔丁基、过氧化二叔丁醇、谷胱甘肽、维生素C和硝酸钠的PBS溶液。进行荧光检测（λEx = 580 nm）；计算各体系中638 nm处的荧光强度I638，结果见图3。其中显示，所述荧光探针对过氧化氢有较好的选择性。
实施例3
荧光探针与过氧化氢的线性关系
配制1 mL浓度为梯度的过氧化氢的PBS溶液体系，然后均分别加入9 mL浓度为50μM的探针PBS溶液(含5%甲醇)，进行荧光检测（λEx = 580 nm, λEm= 638 nm），计算各体系中荧光强度，以及638 nm处的荧光强度I638，建立荧光强度与过氧化氢浓度标准曲线，标准曲线见图4。本发明所述荧光探针的检测下限为0.071 μM (S/N = 3)。
实施例4
本发明所述检测过氧化氢的比率型荧光探针的应用
荧光探针在638 nm处有微弱的荧光，但其与过氧化氢反应的产物在638 nm处具有很强的荧光。将HeLa细胞加入铺有盖玻片的培养皿中，并置于温度为37℃的5%二氧化碳培养箱中，在采用DMEM培养基（含10%小牛血清）以及100 μg/ml的双抗进行培养，36小时后对铺有并长满细胞的盖玻片的培养皿采用不含血清的DMEM培养基冲洗3次，加入10 μM本发明所述的检测过氧化氢的荧光探针后37℃下培养40分钟。使用PBS冲洗铺有并长满细胞的盖玻片的培养皿3次，制片，在激光共聚焦显微镜下观察细胞，进行荧光成像。并列的，向铺有并长满细胞的盖玻片的培养皿中加入200 μM浓度的过氧化氢，37℃下培养1小时，制片，在共聚焦显微镜下观察细胞，进行荧光成像。细胞成像数据见图5。
检测小鼠体内过氧化氢含量的成像实验：鼠腹腔内注射甲苯噻嗪（10 mg. kg-1）和氯胺酮（80 mg. kg-1），然后注射本发明所述的过氧化氢荧光探针（50 nmol），随后进行小鼠成像实验。在此小鼠基础上，向小鼠腹腔注射100 μM过氧化氢，20 min之后进行小鼠成像实验。小鼠成像实验见图6。
Claims (2)
1.一种基于罗丹明衍生物的过氧化氢增强型荧光探针，其特征在于：所述荧光探针的化学结构式如式（1）所示：
。
（1）
2.一种权利要求1所述的基于罗丹明衍生物的过氧化氢增强型荧光探针的制备方法，其特征在于，采用以下步骤：
（1）中间体2制备：将1 mmol中间体1、1 mmol N,N-双(三氟甲磺酰基)苯胺和2 mmol N,N-二异丙基乙胺溶于5 ml DMF中，室温下反应6小时，加入5 ml水，使用5ml乙酸乙酯萃取，将油相真空干燥得粗产品；使用洗脱剂：乙酸乙酯：石油醚 = 1:5柱层析对粗产品进行纯化，得纯品中间体2；
（2）荧光探针化合物3的制备：将1 mmol中间体2、1 mmol双联频哪醇硼酸酯、0.2 mmol1,1-双(二苯基膦)二茂铁二氯化钯二氯甲烷络合物和1.5 mmol醋酸钾溶于 10 ml甲苯中，氮气保护下回流6 h；反应结束之后，真空除溶剂，得粗产品，使用洗脱剂为乙酸乙酯：石油醚 = 1:4柱层析对粗产品进行纯化，即得；
化学反应式如下：。
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