CN108102645A - 一种用于次氯酸根离子检测的荧光探针的制备及应用 - Google Patents
一种用于次氯酸根离子检测的荧光探针的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种用于次氯酸根离子检测的荧光探针的制备及应用。本发明采用氟硼荧染料作为荧光母体，在氟硼荧母体的8号位引入肟基作为次氯酸根响应基团；荧光探针在次氯酸根存在下，光物理性质发生变化。本发明所述荧光探针能够实现对次氯酸根离子的高选择性、高灵敏度检测，为构建一种高选择性、高灵敏度检测次氯酸根离子的化学生物传感器提供了可能。
Description
技术领域
本发明涉及一种可用于次氯酸根离子检测的荧光探针的制备，以及其在次氯酸根离子化 学生物传感领域的用途。
背景技术
反应性活性氧是一类在生物体内广泛存在的重要的生物活性物质。次氯酸根(ClO-)离 子便是一种常见的活性氧物种。在生物学领域，次氯酸根在微生物的免疫防御以及炎症中也 发挥着重要作用。在中性粒细胞中，在髓过氧化物酶的催化作用下，过氧化氢和细胞内的氯 离子发生作用生成次氯酸根离子。但是髓过氧化物酶的紊乱表达等因素会导致生物体内次氯 酸根浓度水平的失常，这会引发严重的组织损伤和相应的疾病，如动脉粥状硬化、风湿性关 节炎甚至肿瘤的生成。因此，通过检测生物体内异常的次氯酸根浓度可以预测特定的疾病。
目前常用于次氯酸根检测的方法有电化学分析法、分光光度法和化学发光法等。与传统 检测方法不同，生物发光成像技术通过采用高灵敏的荧光探针进行发光标记从而对生物体内 的次氯酸根进行检测。由于荧光探针具有高灵敏、高分辨和多维的数据采集和实时成像等优 点，生物发光成像已经成为一种实时、非侵入性的次氯酸根检测方法。次氯酸根在生物细胞 中浓度变化较快，所以检测次氯酸的荧光探针需要一定的响应速度，以达到对生物体系次氯 酸根进行实时检测的目的。生物体内存在大量的不同类型的活性氧物种(如超氧自由基、羟 基自由基、过氧化氢、单线态氧等)，在其他干扰离子或分子存在的条件下，专一性地对体 内次氯酸根进行识别，需要探针分子具有较好的抗干扰性。
发明内容
本发明通过分子结构设计，提供一种具有响应速度快、选择性好、灵敏度高的次氯酸荧 光探针。一种用于次氯酸根离子检测的荧光探针，其特征在于，氟硼荧母体的8号位引入肟 基，具有如下结构通式：
其中，R1和R3分别为具有1至6个碳的烷基链或氢，R2为具有1至6个碳的烷基链、氢或氰基；
其中，R4为含不多于2个碳的烷基链或氢，R5至R8为具有1至6个碳的烷基链或氢。
合成路线具体如下：
具体过程如下：
(1)将化合物1(吡咯或吲哚衍生物，如反应路线中所示)(10mmol)和0.7mL乙 酰氧基乙酰氯(6.5mmol)溶解在10mL干燥无氧的二氯甲烷中，避光反应1小时后，待恢 复到室温加入3.5mL N,N-二异丙基乙基胺(21mmol)；反应30分钟后加入2.49mL三氟 化硼-***络合物(19mmol),反应1小时后停止反应，减压旋干溶剂。用硅胶柱分离，洗脱 剂配比为石油醚/二氯甲烷＝1:1，得到固体产物2；
(2)将产物2(1mmol)和84mg氢氧化锂(2mmol)溶解在50mL四氢呋喃 和水的混合溶剂中(体积比为1:1)。用TCL板检测反应，反应完全后，减压旋干溶剂。用硅胶 柱分离,洗脱剂配比为二氯甲烷/乙酸乙酯＝2:1，得到固体产物3；
(3)将产物3(0.2mmol)溶于20mL干燥无氧的二氯甲烷中，将127.2mg(0.3 mmol)戴斯-马丁氧化剂溶于20mL干燥无氧的二氯甲烷中，用注射器将前者在冰浴条件 下缓慢加到后者，全部加完后在45℃条件下反应5小时。用饱和硫代硫酸钠淬灭反应，用饱 和食盐水萃取三次，加入无水硫酸钠干燥，减压蒸馏除去溶剂。用硅胶柱分离，洗脱剂配比 为石油醚/二氯甲烷＝10:1，得到固体产物4；
(4)称取产物4(0.12mmol)和16.7mg盐酸羟胺(0.24mmol)加入到单口瓶中，加 入10mL无水乙醇，80℃条件下冷凝回流反应2小时。减压旋干溶剂。用硅胶柱分离，洗脱 剂配比为石油醚/二氯甲烷＝1:1，得到固体产物5即荧光探针。
所述荧光探针可应用于生物体系中次氯酸根的检测，检测包含荧光检测、细胞成像和和 目视比色。
所述荧光探针应用于检测次氯酸根时，荧光探针本身无荧光或荧光很弱，在可见光区发 生颜色改变，目视比色，可用于定性检测次氯酸根。
所述次荧光探针应用于检测次氯酸根时，以波长495nm光进行激发，随着次氯酸根含 量的逐渐增加，其在572nm处的荧光峰值逐渐增强；检测细胞中次氯酸时，激发波长为515 nm，发射光检测范围为570-620nm。在一定浓度范围内，荧光强度与次氯酸浓度呈线性正 相关，从而实现定量测定次氯酸的浓度。
有益效果
探针的合成相对简单；实现了对次氯酸根离子的快速检测，并且选择性好，抗其他分子 干扰能力强；用肉眼就可以观察到溶液颜色的变化，紫外灯下同样可以观察到荧光颜色变化， 是一种具有生色传感功能的荧光探针；基于其特异性且显著的颜色变化，该试剂可作为显示 生物细胞内次氯酸根离子存在的专一性指示剂，可进行实时定性的检测；通过时间分辨技术 可对溶液、细胞和活体中次氯酸根离子进行定性检测。故而，本发明是一种简单、快速、灵 敏的次氯酸根离子特异性检测试剂，在生物分子检测领域具有广阔的应用前景。
附图说明
图1是本发明所述荧光探针A的结构式；
图2为次氯酸根离子对本发明所述荧光探针A的吸收滴定图；
图3为次氯酸根离子对本发明所述荧光探针A的荧光滴定图；
图4为次氯酸根离子对本发明所述荧光探针A的荧光滴定数据拟合图；
图5为本发明所述荧光探针A对常见活性氧物种的选择性荧光光谱；
图6为本发明所述荧光探针A对常见活性氧物种的选择性柱状图数据；
图7为本发明所述荧光探针A在细胞培养后加入次氯酸钠前后的共聚焦成像图；
图8为本发明所述荧光探针A加入次氯酸钠前后在可见光和紫外光下的对比图；
图9为本发明所述荧光探针A在细胞培养后加入次氯酸钠前后的荧光寿命成像图。
具体实施方式
下面通过具体实施例对本发明作进一步的说明，但本发明的保护内容不局限于以下。
本发明实施例所用的原料均为已知化合物，可有市场购得，或按照现有技术的方法合成 得到。
实施例1：荧光探针A的制备。
本实施例采用了如下制备路线：
(1)将1ml 2,4-二甲基吡咯(10mmol)和0.7ml乙酰氧基乙酰氯(6.5mmol)溶解 在10ml干燥无氧的二氯甲烷中，避光反应1小时后,待恢复到室温加入3.5ml N,N-二异丙 基乙基胺(21mmol)；反应30分钟后加入2.49ml三氟化硼-***络合物(19mmol)，反应1 小时后停止反应，减压旋干溶剂。用硅胶柱分离，洗脱剂配比为石油醚/二氯甲烷＝1:1，得 到240mg金黄色固体产物A2，产率为15％。
(2)将320mg产物2(1mmol)和84mg氢氧化锂(2mmol)溶解在50mL四氢 呋喃和水的混合溶剂中(体积比为1:1)。用TCL板检测反应，反应完全后，减压旋干溶剂。用 硅胶柱分离，洗脱剂配比为二氯甲烷/乙酸乙酯＝2:1，得到189mg黄色固体产物A3，产率 为68％。
(3)将55.6mg产物3(0.2mmol)溶于20ml干燥无氧的二氯甲烷中，将127.2mg(0.3mmol)戴斯-马丁氧化剂溶于20ml干燥无氧的二氯甲烷中，用注射器将前者在冰浴条件下缓慢加到后者，全部加完后在45℃条件下反应5小时。用饱和硫代硫酸钠淬灭反应,用饱 和食盐水萃取三次，加入无水硫酸钠干燥，减压蒸馏除去溶剂。用硅胶柱分离，洗脱剂配比 为石油醚/二氯甲烷＝10:1，得到39.2mg黄色固体产物A4，产率为71％。
(4)称取33.2mg产物4(0.12mmol)和16.7mg盐酸羟胺(0.24mmol)加入到单口 瓶中，加入10ml无水乙醇，80℃条件下冷凝回流反应2小时。减压旋干溶剂，用硅胶柱分 离，洗脱剂配比为石油醚/二氯甲烷＝1:1，得到30.7mg金色固体产物A5即荧光探针A(结 构式如图1所示)，产率为88％。1H NMR(400MHz,CD3CN):δ9.70(s,1H),8.25(s,1H),6.17 (s,2H),2.47(s,6H),2.17(s,6H).13C NMR(101MHz,CD3CN)δ157.67,145.25,144.01,132.57,132.18,122.29,16.23,14.81.GC-MS测得分子量为273。
实施例2：所述荧光探针A在溶液中对次氯酸根离子的检测。
配置实施例1中制备的荧光探针A的溶液，溶剂为二甲基亚砜，浓度为10-3mol/L，测试时用乙醇和水(体积比为1:1)的混合溶剂稀释到10-5mol/L。
在室温条件下，用紫外—可见吸收光谱仪测试荧光探针溶液滴加次氯酸钠溶液过程中吸 收强度的变化，其吸收滴定光谱图如图2所示。从图2中可以看到，随着体系中次氯酸根离 子浓度的增加(0-50μM)，所述荧光探针在510nm左右处的吸收强度逐渐变弱，而560nm 左右处的吸收强度逐渐加强。
在室温条件下，用荧光光度计测试荧光探针溶液滴加次氯酸钠溶液过程中荧光强度的变 化，其荧光滴定图和滴定数据拟合图分别如图3和图4所示。从图3中可以看到，随着体系 中次氯酸根离子浓度的增加(0-50μM)，所述荧光探针在572nm左右处的吸收强度逐渐增 强。以次氯酸钠浓度为横坐标，以荧光强度为纵坐标作图，得图4，随着NaClO浓度浓度增加，荧光强度显著增加。由图可以看出0-30μM浓度范围内，荧光强度与NaClO浓度呈 线性正相关。
实施例3：所述荧光探针A对其它活性氧物种的响应
分别配置含过氧化氢、一氧化氮、单线态氧(1O2)、超氧阴离子(O2 ·-)、过氧化物自由基(ROO·)和羟自由基(·OH)的溶液，浓度为5ⅹ10-5mol/L，加入10-5mol/L的探针溶 液，反应30分钟后检测溶液的荧光发射光谱变化，结果如图5所示。根据不同活性氧物种 的荧光强度作图，如图6所示。由图5与图6看可以发现，荧光探针对多种竞争分子有较强 的抗干扰性，对荧光探针在572nm处的荧光几乎没有影响，而次氯酸钠溶液的加入使荧光 探针在572nm处的荧光显著增强。
实施例4：所述荧光探针A在细胞成像中的应用
配置实施例1中制备的荧光探针A的溶液，溶剂为二甲基亚砜，浓度为10-3mol/L，在细胞成像实验中取该母液稀释到PBS缓冲溶液中，浓度为10-5mol/L。
将培养好的HeLa细胞用PBS缓冲溶液洗三次，然后用上述配好的所述荧光探针溶液培 养15分钟后，加入次氯酸钠水溶液继续培养15分钟。参照组则只用所述荧光探针溶液培养 15分钟。明场成像和荧光成像用共聚焦显微镜观测。共聚焦显微镜激发波长为515nm，收 集波段通道为530-630nm区域。图7为本发明所述荧光探针在细胞培养后加入次氯酸钠溶 液前后的共聚焦成像图。其中，图7A、图7B、图7C为加入荧光探针培养后的HeLa细胞 成像图，其中图7A为收集波段通道为530-630nm的成像照片，图7B为明场照片，图7C 为叠加场照片。图7D、图7E、图7F为加入次氯酸钠溶液培养后的HeLa细胞成像图，其 中图7D为收集波段通道为530-630nm的成像照片，图7E为明场照片，图7F为叠加场照 片。从图7可以看出，只用所述荧光探针溶液培养的HeLa细胞，在530-630nm通道荧光 信号较弱；而加入次氯酸钠溶液之后，在该通道有很强的荧光信号。明场观察可以看到很好 的细胞形态，证明在整个实验过程中细胞活性良好。
细胞成像实验表明，所述荧光探针A可以很好的穿透细胞膜，用作活细胞中次氯酸根 离子的检测。
实施例5：所述荧光探针A通过目视比色法定性检测次氯酸根离子
配置实施例1中制备的荧光探针的溶液，溶剂为二甲基亚砜，浓度为10-3mol/L，用乙 醇和水(体积比为1：1)的混合溶剂稀释到10-5mol/L，向装有2mL探针溶液的比色皿中 加入5ⅹ10-5mol/L次氯酸钠溶液，含1mL探针溶液的比色皿作为对比，两者之间颜色存 在差异，目视比色法定性检测次氯酸根离子。图8A为365nm紫外灯下发光情况，图8B为 自然光下的颜色情况。
实施例6：所述荧光探针A在细胞荧光寿命成像中的应用
配置实施例1中制备的荧光探针A的溶液，溶剂为二甲基亚砜，浓度为10-3mol/L，在细胞成像实验中取该母液稀释到PBS缓冲溶液中，浓度为10-5mol/L。
在96孔板中培养六组HeLa细胞，用PBS缓冲溶液洗三次，然后用上述配好的所述荧光探针溶液培养15分钟后，加入不同浓度次氯酸钠水溶液继续培养15分钟。参照组则只用所述荧光探针溶液培养15分钟。荧光寿命成像效果如图9所示，A图为参照组，B至F图 中次氯酸钠浓度分别为2ⅹ10-6mol/L、6ⅹ10-6mol/L、10-5mol/L、2ⅹ10-5mol/L和3ⅹ10-5 mol/L。由图可知，在加入不同浓度次氯酸钠溶液后有明显的荧光寿命变化。
Claims (7)
1.一种用于次氯酸根离子检测的荧光探针，其特征在于，氟硼荧母体的8号位引入肟基，具有如下结构通式：
其中，R1和R3分别为具有1至6个碳的烷基链或氢，R2为具有1至6个碳的烷基链、氢或氰基；
其中，R4为含不多于2个碳的烷基链或氢，R5至R8为具有1至6个碳的烷基链或氢。
2.如权利要求1所述的荧光探针的制备方法，其特征在于，合成路线如下：
3.如权利要求2所述的荧光探针的制备方法，其特征在于，具体如下：
(1)将10mmol化合物1吲哚或吡咯衍生物，和0.7mL乙酰氧基乙酰氯6.5mmol溶解在10mL干燥无氧的二氯甲烷中，避光反应1小时后，待恢复到室温加入3.5mL N,N-二异丙基乙基胺21mmol；反应30分钟后加入2.49mL三氟化硼-***络合物19mmol,反应1小时后停止反应，减压旋干溶剂；用硅胶柱分离，洗脱剂配比为石油醚/二氯甲烷＝1:1，得到固体产物2；
(2)将1mmol产物2和84mg氢氧化锂2mmol溶解在50mL四氢呋喃和水的混合溶剂中，体积比为1:1；用TCL板检测反应，反应完全后，减压旋干溶剂；用硅胶柱分离,洗脱剂配比为二氯甲烷/乙酸乙酯＝2:1，得到固体产物3；
(3)0.2mmol将产物3溶于20mL干燥无氧的二氯甲烷中，将127.2mg即0.3mmol戴斯-马丁氧化剂溶于20mL干燥无氧的二氯甲烷中，用注射器将前者在冰浴条件下缓慢加到后者，全部加完后在45℃条件下反应5小时；用饱和硫代硫酸钠淬灭反应，用饱和食盐水萃取三次，加入无水硫酸钠干燥，减压蒸馏除去溶剂；用硅胶柱分离，洗脱剂配比为石油醚/二氯甲烷＝10:1，得到固体产物4；
(4)称取0.12mmol产物4和16.7mg盐酸羟胺0.24mmol加入到单口瓶中，加入10mL无水乙醇，80℃条件下冷凝回流反应2小时；减压旋干溶剂；用硅胶柱分离，洗脱剂配比为石油醚/二氯甲烷＝1:1，得到固体产物5即荧光探针。
4.如权利要求1所述的荧光探针的应用，其特征在于，所述荧光探针应用于生物体系中次氯酸根的检测，检测包含荧光检测、细胞成像和和目视比色；
所述荧光探针应用于检测次氯酸根时，荧光探针本身无荧光或荧光很弱，在可见光区发生颜色改变，目视比色，用于定性检测次氯酸根。
5.如权利要求1所述的荧光探针的应用，其特征在于，当荧光探针应用于检测次氯酸根时，以波长495nm光进行激发，随着次氯酸根含量的逐渐增加，其在572nm处的荧光峰值逐渐增强；检测细胞中次氯酸时，激发波长为515nm，发射光检测范围为570-620nm；在一定浓度范围内，荧光强度与次氯酸浓度呈线性正相关，从而实现定量测定次氯酸的浓度。
6.如权利要求1所述的荧光探针的应用，其特征在于，所述探针基于时间分辨荧光技术检测溶液中的次氯酸根离子。
7.如权利要求1所述的荧光探针的应用，其特征在于，所述探针基于荧光寿命成像技术检测细胞及活体中的次氯酸根离子。
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