CN112142718B - 用于检测次氯酸的近红外荧光分子探针、制备方法及用途 - Google Patents
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Abstract
本发明公开了用于检测次氯酸的近红外荧光分子探针、制备方法及用途。该探针分子式为C34H35N2O2S+，结构式如下。该探针以二甲氨基硫代甲酯基团作为响应基团检测次氯酸具有非常优异的选择性，吸收和发射均在近红外区具有较强的生物体穿透性，检测高效。
Description
技术领域
本发明涉及荧光分子探针、制备方法及用途，特别涉及用于检测次氯酸的近红外荧光分子探针、制备方法及用途。
背景技术
活性氧(ROS)在信号转导、炎症、癌变和神经退行性损伤等生物学过程中起着多种作用。次氯酸(HOCl)是一种重要的ROS，内源性HOCl由过氧化氢(H2O2)和氯离子(Cl-)通过髓过氧化物酶(MPO)的催化生成，虽然浓度较低，但在免疫防御入侵细菌和其他病原体方面起着重要作用。大量研究表明，体内HOCl过量涉及多种疾病，如炎性疾病、心血管疾病、肾病、急性肺损伤、囊性纤维化、神经退行性疾病，甚至癌症。因此实现对细胞内次氯酸浓度变化的实时监测是非常必要的。
许多用于HOCl检测和成像的荧光探针被开发出来。然而，它们中很少能靶向活细胞线粒体，而活细胞线粒体是实时和精确传感的关键。目前荧光探针与次氯酸的响应方式很多，本探针以二甲氨基硫代甲酯基团作为识别基团检测次氯酸，具有良好的选择性和灵敏性，但是如何得到性能更加优异的分子探针，还需要进一步研究。
发明内容
发明目的：本发明目的是提供选择性强、相应快速的用于检测次氯酸的近红外荧光分子探针。
本发明另一目的是提供所述用于检测次氯酸的近红外荧光分子探针的制备方法及用途。
技术方案：本发明提供一种用于检测次氯酸的近红外荧光分子探针，其分子式为C34H35N2O2S+，结构式如下：
所述用于检测次氯酸的近红外荧光分子探针的制备方法，包括如下步骤：
(1)在惰性气体保护条件下，将无水二氯甲烷加入到化合物1中，搅拌；
(2)加入三乙胺，搅拌；
(3)用无水二氯甲烷将二甲氨基硫代甲酰氯溶解，注射到(2)的处理后的反应液中，50-60℃搅拌回流，反应；
(4)反应液进行萃取，取有机溶液层，蒸干溶剂，即得，
进一步地，所述化合物1与二氯甲烷的体积比1mmol∶(10-15mL)。
进一步地，所述化合物1、二甲氨基硫代甲酰氯、三乙胺的摩尔比1∶(2-4)∶(0.5-1)。
进一步地，所述步骤(3)中反应结束的时机为反应溶液变为蓝紫色。
进一步地，反应液中加入碳酸氢钠溶液，静置分液，取下层有机溶液层。
所述的用于检测次氯酸的近红外荧光分子探针在检测HOCl中的用途。
进一步地，所述HOCl为溶液中、细胞中或者活体中的HOCl。
本发明荧光分子其母核为半花菁类近红外荧光团，能吸收近红外的光并发出波长更长的荧光。原理为半花菁母核与识别基团以共价键结合组成大的给电子-受电子共轭体系，分子体系内发生由给电子基团到电子受体的分子内电荷转移过程，探针分子在荧光光谱中表现出特定的发射峰。当探针分子与待测物质相作用时，连接两基团的共轭结构被破坏，荧光光谱中的发射峰将发生红移。从而实现对次氯酸的精准检测。并且，荧光分子探针的近红外特性可以使得检测的深度以及分辨率得到提高。通过细胞以及活体成像实验显著性效果，可以看到该探针应用于临床之中的潜力。
有益效果：本发明具有如下优势：
1、本发明以二甲氨基硫代甲酯基团作为响应基团检测次氯酸具有非常优异的选择性。并且其近红外吸收与荧光的发射的特性使其能很好的运用于活体成像等。
2、本发明的近红外荧光探针吸收和发射均在近红外区具有较强的生物体穿透性，还能减少生物体自发荧光的干扰。
3、本发明的近红外荧光探针具有较小的生物毒性。
4、本发明的近红外荧光探针具有较好的细胞成像效果。
附图说明
图1是本发明近红外荧光探针的质谱、核磁氢谱表征(氘代甲醇)；
图2是本发明近红外荧光探针与次氯酸响应的紫外吸收以及荧光变化图；
图3是本发明近红外荧光探针的MTT实验数据图(图中横坐标单位为μM)；
图4是本发明近红外荧光探针的共聚焦显微镜细胞成像图。
具体实施方式
实施例1
检测次氯酸近红外荧光探针的制备：
将一定量的化合物1加入到圆底烧瓶中，在氮气保护下缓慢加入无水二氯甲烷，搅拌均匀，继续加入一定量的三乙胺，搅拌5分钟，用无水二氯甲烷将二甲氨基硫代甲酰氯溶解，注射到化合物1的二氯甲烷溶液中。油浴锅预热50-60℃，安装回流装置。搅拌回流反应约2h，待溶液变为蓝紫色，冷却体系，将瓶中反应液倒入分液漏斗中加入碳酸氢钠溶液，充分混摇之后，静置分液漏斗，待分液漏斗中溶液分层之后，取下层溶液。将下层溶液倒入干净的分液漏斗，加入蒸馏水充分混摇之后，静置分液漏斗，待分液漏斗中溶液分层之后，取下层溶液。用减压旋转蒸馏的方法将溶剂蒸干得蓝紫色固体。
所述化合物1与二氯甲烷的比例1mmol∶12mL。
所述化合物1与二甲氨基硫代甲酰氯和三乙胺的摩尔比1∶3∶0.5。
所得探针经纯化处理后，经过核磁氢谱及质谱检测得图1，核磁和质谱均无明显杂峰，即所得探针纯度较高。(1H NMR(400MHz，Methanol-d4)δ8.92(d，J＝15.3Hz，1H)，8.41(d，J＝8.5Hz，1H)，8.18(d，J＝8.9Hz，1H)，8.12(d，J＝8.2Hz，1H)，7.86(d，J＝8.9Hz，1H)，7.77(ddd，J＝8.3，6.9，1.3Hz，1H)，7.65(t，J＝7.6Hz，1H)，7.55-7.51(m，1H)，7.34(s，1H)，7.27(d，J＝2.2Hz，1H)，7.06(dd，J＝8.4，2.2Hz，1H)，6.69(d，J＝15.2Hz，1H)，4.62(d，J＝5.6Hz，2H)，3.46(d，J＝22.0Hz，6H)，2.85-2.78(m，5H)，2.12(d，J＝2.3Hz，7H)，2.02-1.97(m，2H)，1.59(t，J＝7.3Hz，4H).；MS(ESI+)：calcd for C34H35N2O2S+[M+H]+535.24，found535.3)
实施例2
与实施例1的不同之处在于：所述化合物1与二氯甲烷的比例1mmol∶10mL。
所述化合物1与二甲氨基硫代甲酰氯和三乙胺的摩尔比1∶2∶0.5。
实施例3
与实施例1的不同之处在于：所述化合物1与二氯甲烷的比例1mmol∶15mL。
所述化合物1与二甲氨基硫代甲酰氯和三乙胺的摩尔比1∶4∶1。
实施例4
近红外荧光探针与次氯酸体外响应紫外吸收以及荧光发射测量实验：
称取5.35mg探针溶于2mL DMSO，配置成5mM探针母液。称取16.53mg NaOCl·5H2O溶于10mL水，配置成10mM NaOCl母液。准备12个5mL的离心管，依次在每个离心管加入3mLPBS缓冲液(pH＝7.4)后，再加入3μL的探针母液，此时每个离心管探针浓度为5μM。待充分混匀后在每个离心管中分别加入0、3、6、9、12、15、18、21、24、27、30、33μL的NaOCl母液，反应10分钟后，依次加入比色皿中进行紫外吸收以及荧光发射的检测(激发光波长为650nm)，所得数据经origin软件处理得图3，708nm处紫外吸收峰随HOCl的加入呈线性增高，732nm处荧光强度随HOCl加入逐渐升高，表明探针在溶液中与次氯酸的响应效果好。
实施例5
近红外荧光探针与RAW264.7细胞进行MTT生物相容性实验：
在96孔板的外围一圈加入PBS溶液，在剩余内部孔中铺满RAW264.7细胞，培养12小时。称取5.35mg探针溶于2mL生物用DMSO溶液，配置成5mM生物用探针母液。配置5mg/mL MTT溶液。准备6个5mL的离心管，选其中一个加入6.4μL母液和4mL培养基，依次梯度稀释使6个离心管中探针的浓度依次为0、5、10、15、20、25μM。将6个离心管中溶液吸取190μL依次横排等浓度加入6排孔中孵育24h后，在每个孔中加入10μL MTT溶液。继续孵育4小时，倒掉液体中培养基，在每个孔中均加入100μL DMSO溶液。待充分溶解后，进行紫外吸收检测(检测波长为490nm)。用origin进行数据处理得图3，浓度高达25μM的HOCl孵育细胞24h，细胞存活率高于80％，表明探针的生物适用性较好，细胞毒性小。
实施例6
近红外荧光探针与次氯酸响应的共聚焦显微镜细胞实验：
在三个共聚焦皿中铺适当浓度的RAW264.7细胞，培养至细胞状态优良，更换新鲜培养基。其中一组共聚焦皿用2μg/mL Lps孵育6小时，另一组用2μg/mL Lps孵育6小时后，再用NAC孵育6小时，最后一组不做处理。12小时后，用10μM探针孵育30分钟。多聚甲醛溶液固定10分钟，倒出后用PBS清洗一遍，加入DAPI染色液染色5分钟，PBS缓冲液清洗3次，加入1mL多聚甲醛溶液固定保存。共聚焦显微镜拍摄图片如图4，对照组荧光强度较弱；Lps刺激细胞产生HOCl后成像，荧光强度较强且成像清晰；NAC消耗掉Lps刺激产生的HOCl后成像，荧光强度变弱。三组细胞图成像清晰，核质分明，表明探针在细胞中能有很好的成像效果。
Claims (8)
1.一种用于检测次氯酸的近红外荧光分子探针，其分子式为C34H35N2O2S+，结构式如下：
2.权利要求1所述的用于检测次氯酸的近红外荧光分子探针的制备方法，其特征在于：包括如下步骤：
(1)在惰性气体保护条件下，将无水二氯甲烷加入到化合物1中，搅拌；
(2)加入三乙胺，搅拌；
(3)用无水二氯甲烷将二甲氨基硫代甲酰氯溶解，注射到(2)的处理后的反应液中，50-60℃搅拌回流，反应；
(4)反应液进行萃取，取有机溶液层，蒸干溶剂，即得，
3.根据权利要求2所述的用于检测次氯酸的近红外荧光分子探针的制备方法，其特征在于：所述化合物1与二氯甲烷的体积比1mmol∶(10-15mL)。
4.根据权利要求2所述的用于检测次氯酸的近红外荧光分子探针的制备方法，其特征在于：所述化合物1、二甲氨基硫代甲酰氯、三乙胺的摩尔比1∶(2-4)∶(0.5-1)。
5.根据权利要求2所述的用于检测次氯酸的近红外荧光分子探针的制备方法，其特征在于：所述步骤(3)中反应结束的时机为反应溶液变为蓝紫色。
6.根据权利要求2所述的用于检测次氯酸的近红外荧光分子探针的制备方法，其特征在于：所述萃取的方法为：反应液中加入碳酸氢钠溶液，静置分液，取下层有机溶液层。
7.权利要求1所述的用于检测次氯酸的近红外荧光分子探针在制备检测HOCl药物中的用途。
8.根据权利要求7所述的用途，其特征在于：所述HOCl为溶液中、细胞中或者活体中的HOCl。
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