CN114478612A - 一种基于硅罗丹明的脑部次氯酸检测荧光探针及其制备方法和应用 - Google Patents
一种基于硅罗丹明的脑部次氯酸检测荧光探针及其制备方法和应用 Download PDFInfo
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- CN114478612A CN114478612A CN202210047525.5A CN202210047525A CN114478612A CN 114478612 A CN114478612 A CN 114478612A CN 202210047525 A CN202210047525 A CN 202210047525A CN 114478612 A CN114478612 A CN 114478612A
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Abstract
本发明涉及生物医药技术领域,具体公开了一种基于硅罗丹明的脑部次氯酸检测荧光探针及其制备方法和应用。本发明公开了基于硅罗丹明的脑部次氯酸检测荧光探针的结构式;本发明还具体公开了基于硅罗丹明的脑部次氯酸检测荧光探针的制备方法及其溶液光谱性质,细胞中的外源性与内源性的次氯酸的检测,通过制造小鼠神经炎症模型特异性检测小鼠脑部次氯酸。本发明的荧光探针利用甲酰肼基团作为反应位点,通过硅罗丹明特有的开关环机制实现对次氯酸的专一性检测,具有近红外发射、细胞毒性低、选择性好、抗光漂白能力强、可穿透血脑屏障等优点,使得利用小分子探针对神经炎症等脑部疾病进行脑部实时成像成为可能。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种基于硅罗丹明的脑部次氯酸检测荧光探针及其制备方法 和应用。
背景技术
活性氧(ROS)在细胞信号传导和维持生物体内平衡的等方面起到不可或缺的作用,参与生物体的各项 生理活动。次氯酸(HOCl)作为一种重要的活性氧,主要来源是在髓过氧化物酶的帮助下通过Cl-和H2O2的 过氧化产生的。次氯酸在脑部的异常积累会导致脑部组织损伤,诱导产生神经炎症,大脑中的持续炎症环 境是阿尔茨海默病、帕金森病、脑损伤、癫痫等神经退行性疾病的重要标志。因此检测脑部的次氯酸含量 对于研究神经退行性疾病的根本原因具有重要意义。
荧光成像技术是通过非侵入性方式研究生物体的有力工具。小分子荧光探针通过与待测物的相互作用 引起其荧光的变化,具有较高的灵敏度与选择性。然而,许多小分子探针由于吸收与发射波长短,无法避 免生物自荧光带来的影响,同时抗光漂白能力弱,限制了其在体内的应用。
血脑屏障是神经胶质细胞与脑部毛细血管壁形成的屏障,其作用隔离有害物质进入大脑,但它的存在 也给小分子探针在脑部的应用带来困难,许多性能优良的探针,由于无法穿透血脑屏障,无法用于脑部炎 症的实时监测。
因此,亟需开发一种新型荧光探针,能够解决发射波长短、抗光漂白能力差、无法穿透血脑屏障等问 题,从而实现脑部次氯酸的实时荧光检测。
发明内容
为了解决次氯酸荧光探针检测所面临的问题,本发明提供了基于硅罗丹明的脑部次氯酸检测荧光探 针,该荧光探针利用甲酰肼作为次氯酸反应位点,通过硅罗丹明的开关环机制实现对次氯酸的荧光成像, 在保证小分子探针对次氯酸的检测能力的同时,此发明探针解决了小分子探针穿过血脑屏障这一关键问 题,从而实现对脑部神经炎症的检测成像,为神经炎症的病理研究和早期检测提供了有效解决方案。
本发明还提供了上述基于硅罗丹明的脑部次氯酸检测荧光探针及其制备方法及其在溶液、细胞及小鼠 活体脑部检测次氯酸中的应用。
本发明为解决上述技术问题采用如下技术方案,一种基于硅罗丹明的脑部次氯酸检测荧光探针及其制 备方法和应用,其特征在于该荧光探针的结构式如下:
本发明所述的基于硅罗丹明的脑部次氯酸检测荧光探针,其特征在于具体步骤为:
步骤S1:在0℃下向氢化钠的四氢呋喃悬浮液中加入50mmol 3-溴苯胺,其中氢化钠含量为125mmol 于60%矿物油分散体中,四氢呋喃含量为100mL;将反应物在0℃下搅拌0.5小时,然后加入125mmol 碘乙烷,将混合物在室温下搅拌24小时;用水淬灭反应,将反应混合物用二氯甲烷萃取三次;取有机相 用无水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比40:1的石油醚/二氯甲烷作为洗脱剂,得到黄色油 状物化合物1,其结构式如下:
步骤S2:将30mmol化合物1与60mL无水乙醚加入至干燥的250mL圆底烧瓶中,磁力搅拌5min 使其溶解,随后将13.1mL摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液缓慢滴加至反应液中,滴加完毕 后于0℃反应2小时,再将18mmol二氯二甲基硅烷溶于10mL无水乙醚中并缓慢滴加至反应液中,滴加 完毕后反应至室温搅拌过夜,加入50mL水猝灭反应,将反应混合物用二氯甲烷萃取三次;取有机相用无 水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比80:1的石油醚/乙酸乙酯作为洗脱剂,得到黄色油状物 化合物2,其结构式如下:
步骤S3:将1.09mmol化合物2、5.46mmol邻醛基苯甲酸、0.17mmol溴化铜加入到100mL玻璃厚 壁耐压管中,于140℃加热搅拌反应5小时后自然冷却至室温,再将反应混合物溶于50mL二氯甲烷中, 用二氯甲烷萃取三次;取有机相用无水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比20:1的石油醚/乙 酸乙酯作为洗脱剂,得到淡黄色固体化合物3,其结构式如下:
步骤S4:将3mmol三氯氧磷缓慢加入到10mL含有1mmol化合物3的1,2-二氯乙烷溶液中;上述 混合溶液在80℃反应3小时,然后减压浓缩;所得产物直接用10mL无水二氯甲烷溶解,然后将3mmol 甲酰肼和200μL二异丙基乙胺溶于5mL无水二氯甲烷并将其缓慢加入到反应体系中。然后将混合溶液在 30℃下反应10小时。减压浓缩所得溶液,随后通过柱色谱法纯化,用体积比50:1的二氯甲烷/甲醇作为洗 脱剂得到目标荧光探针化合物SiR-FH。
本发明的有益效果在于:
本发明合成的荧光探针可实现对次氯酸的快速灵敏响应,且具有高选择性与低细胞毒性及低溶血性, 探针具有近红外发射荧光性质,增加组织穿透深度。同时解决穿过血脑屏障这一关键问题,将探针用于脑 部神经炎症的检测成像,对于研究神经退行性疾病的根本原因具有重要意义。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本发明的实施例,并与说明书一起 用于解释本发明的原理。
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要 使用的附图作简单地介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动性的前提下, 还可以根据这些附图获得其他的附图。
图1是实施例1制得的探针SiR-FH在加入不同浓度次氯酸后的荧光光谱图;
图2是实施例1制得的探针SiR-FH在加入不同浓度次氯酸后的紫外可见吸收光谱图;
图3是实施例1制得的探针SiR-FH在发射波长为670nm处的荧光强度随次氯酸浓度(0-60μM)变化的 关系曲线图;
图4是实施例1制得的探针SiR-FH对不同离子和分子的选择性荧光光谱图;
图5是实施例1制得的探针SiR-FH对次氯酸响应的机理;
图6是实施例1制得的探针SiR-FH对次氯酸响应的质谱图;
图7是实施例1制得的探针SiR-FH对次氯酸的响应时间;
图8是实施例1制得的探针SiR-FH与商用探针Rhodamin123在细胞内的荧光共聚焦成像的光稳定性 变化曲线图;
图9是实施例1制得的探针SiR-FH与商用探针Rhodamin123在0,100,200,300s激光照射后的荧 光共聚焦图像;
图10是实施例1制得的不同浓度的探针SiR-FH在Hela细胞(a)和Raw264.7细胞(b)下的细胞毒性;
图11是实施例1制得的不同浓度的探针SiR-FH的溶血图,图(a)为不同浓度SiR-FH的溶血率,图(b)为不同浓度SiR-FH溶血图;
图12是实施例1制得的探针SiR-FH检测HeLa细胞中不同浓度外源性次氯酸(0μM,50μM)荧光成像 图,图(b)是通过imageJ对图(a)进行的量化表征;
图13是实施例1制得的探针SiR-FH检测Raw264.7细胞中脂多糖诱导的内源性次氯酸荧光成像图;
图14(a)是实施例1制得的探针SiR-FH检测小鼠神经炎症的活体成像图,图(b)是实施例1制得的探针 SiR-FH检测小鼠神经炎症的柱状图表征;
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围 仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
荧光探针化合物SiR-FH的合成
(1)化合物1的合成
在0℃下向氢化钠的四氢呋喃悬浮液中加入50mmol 3-溴苯胺,其中氢化钠含量为125mmol于60% 矿物油分散体中,四氢呋喃含量为100mL;将反应物在0℃下搅拌0.5小时,然后加入125mmol碘乙烷, 将混合物在室温下搅拌24小时;用水淬灭反应,将反应混合物用二氯甲烷萃取三次;取有机相用无水硫 酸钠干燥;将粗产物用硅胶柱纯化,用体积比40:1的石油醚/二氯甲烷作为洗脱剂,得到黄色油状物化合 物1,产率73%。1H NMR(400MHz,Chloroform-d)δ7.06(t,J=7.9Hz,1H),6.80(s,1H),6.76(d,J=7.4Hz, 1H),6.60(d,J=7.6Hz,1H),3.35(q,J=7.0Hz,4H),1.18(t,J=7.1Hz,6H),其合成路线如下:
(2)化合物2的合成
将30mmol化合物1与60mL无水乙醚加入至干燥的250mL圆底烧瓶中,磁力搅拌5min使其溶解, 随后将13.1mL摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液缓慢滴加至反应液中,滴加完毕后于0℃反 应2小时,再将18mmol二氯二甲基硅烷溶于10mL无水乙醚中并缓慢滴加至反应液中,滴加完毕后反应 至室温搅拌过夜,加入50mL水猝灭反应,将反应混合物用二氯甲烷萃取三次;取有机相用无水硫酸钠干 燥;将粗产物用硅胶柱纯化,用体积比80:1的石油醚/乙酸乙酯作为洗脱剂,得到黄色油状物化合物2,产 率37.82%。1H NMR(400MHz,Chloroform-d)δ7.25(t,J=7.7Hz,2H),6.96–6.82(m,4H),6.74(d,J=7.0 Hz,2H),3.36(q,J=7.0Hz,8H),1.16(t,J=7.0Hz,12H),0.56(s,6H)。其合成路线如下:
(3)化合物3的合成
将1.09mmol化合物2、5.46mmol邻醛基苯甲酸、0.17mmol溴化铜加入到100mL玻璃厚壁耐压管 中,于140℃加热搅拌反应5小时后自然冷却至室温,再将反应混合物溶于50mL二氯甲烷中,用二氯甲 烷萃取三次;取有机相用无水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比20:1的石油醚/乙酸乙酯作 为洗脱剂,得到淡黄色固体化合物3,产率47%。1HNMR(400MHz,Chloroform-d)δ7.99(d,J=7.6Hz,1H), 7.70–7.64(m,1H),7.56(d,J=3.3Hz,1H),7.43–7.34(m,1H),6.93(d,J=7.6Hz,2H),6.74(s,2H),6.50(d,J =8.7Hz,2H),3.36(dd,J=13.5,6.8Hz,8H),1.18(t,J=6.8Hz,12H),0.64(d,J=7.4Hz,6H)。其合成路线如 下:
(4)化合物SiR-FH的合成
将3mmol三氯氧磷缓慢加入到10mL含有1mmol化合物3的1,2-二氯乙烷溶液中;上述混合溶液在 80℃反应3小时,然后减压浓缩;所得产物直接用10mL无水二氯甲烷溶解,然后将3mmol甲酰肼和200 μL二异丙基乙胺溶于5mL无水二氯甲烷并将其缓慢加入到反应体系中。然后将混合溶液在30℃下反应 10小时。减压浓缩所得溶液,随后通过柱色谱法纯化,用体积比50:1的二氯甲烷/甲醇作为洗脱剂得到目 标荧光探针化合物SiR-FH,产率27%。1H NMR(400MHz,Chloroform-d)δ8.01(d,J=7.1Hz,1H),7.78– 7.60(m,1H),7.55–7.49(m,2H),7.09(d,J=7.0Hz,1H),6.80(s,2H),6.56(s,4H),3.37(q,J=6.3Hz,8H), 1.19(t,J=6.9Hz,12H),0.53(d,J=18.4Hz,6H);13C NMR(101MHz,DMSO)δ166.57,165.02,153.65,146.19,136.52,135.48,134.14,132.00,129.85,128.55,125.61,124.40,123.54,114.69,114.27,73.27,43.87, 39.98,12.91,0.70,0.13,-0.14;HRMS(ESI):calcd for[M+H]527.283774,found 527.283679,合成路线如下:
实施例2荧光探针SiR-FH与次氯酸的光谱学变化
探针SiR-FH在次氯酸存在下,紫外-可见光或荧光光谱的变化。取实施例1制备的SiR-FH荧光探针 溶于二甲基亚砜(DMSO)中,制成10mM储备液,从储备液中取出20μL用去离子水稀释至2mL。次氯酸 用次氯酸钠溶液进行配制,其浓度用292nm处的吸光度进行标定。以620nm为激发光。在室温下从640 到800nm扫描荧光发射光谱,荧光光谱如图1所示,紫外吸收光谱如图2所示。随着次氯酸加入,在670 nm处的荧光和652nm处的吸收逐渐增强,其荧光强度与次氯酸浓度的关系如图3所示,在60μM以内范 围内呈线性关系,(R2=0.9909)。此外,SiR-FH对次氯酸的检测限为0.226μM。
实施例3荧光探针SiR-FH对不同分子或离子的选择性
将浓度为10μM的SiR-FH加入各种干扰物质测试其荧光强度变化,如图4所示,干扰物分别为 H2O2,·OH,1O2,·O2 -,等活性氧,NO,NO2 -等活性氮,H2S,HSO3 -,GSH,Cys等活性硫,Na+,K+, Ca2+,Mg2+,Fe3+,Co2+,Ni2+,Cu2+,Zn2+等金属离子。测试结果表明,探针SiR-FH对包括活性氧、活 性氮、活性硫、金属离子在内的干扰物均无荧光响应,而次氯酸的加入使探针SiR-FH的荧光显著增强。 表明探针SiR-FH对次氯酸的特异性响应。
实施例4荧光探针SiR-FH的反应机理
荧光探针SiR-FH的反应机理如图5所示,SiR-FH探针分子异构化生成C=N键,并在次氯酸的作用 下发生加成反应,产生一个在同一个碳原子上含有两个羟基的高度不稳定的中间体。进一步分子内脱水, 最终N-氯甲酰肼从分子上发生裂解,最终生成开环的羧酸罗丹明。将荧光探针SiR-FH与次氯酸进行反应 后通过质谱进行机理验证,质谱如图6所示;
实施例5荧光探针SiR-FH对次氯酸的响应时间
在荧光探针SiR-FH溶液中分别加入100μΜ的次氯酸,在分光光度计的时间模式下,测定探针在670 nm处的荧光强度随时间的变化情况,结果如图7。从图中可知,在20s内探针对次氯酸的荧光响应逐渐 稳定,具有较好的响应速度。
实施例6荧光探针SiR-FH的抗光漂白性
通过连续激光照射以表征SiR-FH的光稳定性,同时与商业染料Rhodamin123进行对比。对比结果如 图8所示,激光照射300s后,SiR-FH仍然具有很强的荧光强度,而Rhodamin123荧光强度不足原始强 度的40%。分别拍摄在0,100,200,300s激光照射后SiR-FH和Rhodamin123的共聚焦荧光图像,如图 9,结果表明与商业染料相比,SiR-FH具有出色的光稳定性,可提供长期的细胞成像能力。
实施例7荧光探针SiR-FH的细胞毒性及溶血性
使用MTT分析法测定SiR-FH的细胞毒性。如图10所示,向Hela细胞和Raw264.7细胞中加入不 同浓度的探针后,细胞仍表现出较高的存活率。表明探针SiR-FH具有较低的细胞毒性。
溶血是指红细胞膜破裂或出现小孔使得血红蛋白逸出。红细胞游离液随着溶血的同时透明度增加,呈 深红色,且在540nm处有紫外吸收。故可以通过探针与红细胞孵育后细胞的破裂情况来评估材料的生物 相容性。如图11,在浓度为200μM及以下时,SiR-FH不会使红细胞破裂,上清液澄清,溶血率低至5% 以下。表明探针SiR-FH具有良好的生物相容性。
实施例8荧光探针SiR-FH检测细胞中外源性和内源性的次氯酸
将探针SiR-FH进行细胞中外源性次氯酸的共聚焦成像实验。将四组Hela细胞分别与含有探针 SiR-FH(10μM)的DMEM培养液共同孵育60分钟,然后向第二、三、四组细胞中分别加入含不同浓度次 氯酸的DMEM培养液(10μM,30μM,50μM),再继续孵育30分钟。如图12所示,仅用SiR-FH孵育 的细胞荧光很弱,在加入次氯酸的细胞中,随着次氯酸浓度的增加,细胞中的荧光强度逐渐增强。表明探 针SiR-FH对次氯酸有着良好的响应。
接下来进行探针SiR-FH对细胞中内源性次氯酸的检测。如图13所示,a组将Raw264.7细胞用 SiR-FH(10μM)单独培养作为对照组。b组细胞先用LPS(1μg/mL)预处理,再加入10μM探针进行孵育。c 组作为外源性对照组,首先用10μM探针培养细胞,然后加入次氯酸(50μM)继续培养。脂多糖LPS可刺 激Raw264.7细胞产生氧化应激,从而生成过量的次氯酸。共聚焦显微图像显示,与对照组a组相比,加 入LPS刺激的b组产生了明显的荧光增强。证明了SiR-FH和SiR-NSPh对细胞内源性次氯酸具有良好的 成像能力。以上实验证明了探针SiR-FH是活细胞中外源性和内源性次氯酸成像的良好工具。
实施例9荧光探针SiR-FH监测神经炎症小鼠脑中内源性的次氯酸水平变化
选定2只6周的C57BL/6小鼠,其中1号老鼠为空白对照注射100μL生理盐水;2号小鼠通过腹腔注 射100μL脂多糖(2mg/kg)诱导脑部神经炎症。24小时后,通过尾静脉注射探针100μL SiR-FH(0.2mg/kg)到两只小鼠体内,异氟烷麻醉后使用小动物活体成像仪进行荧光成像,结果如图14所示。从图中可知, 左侧1号空白小鼠脑部发出微弱荧光,而被脂多糖诱导产生神经炎症的右侧2号小鼠脑部出现明显荧光信 号,表明神经炎症诱导产生了脑部次氯酸。次氯酸进一步作用于探针SiR-FH从而产生信号。以上结果表 明荧光探针SiR-FH能够透过血脑屏障并对次氯酸进行识别进一步实现活体中神经炎症的监测。
以上所述仅是本发明的具体实施方式,使本领域技术人员能够理解或实现本发明。对这些实施例的多 种修改对本领域的技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或 范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所述的这些实施例,而是要符合 与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
2.一种基于硅罗丹明的脑部次氯酸检测荧光探针的制备方法,其特征在于具体步骤为:
步骤S1:在0℃下向氢化钠的四氢呋喃悬浮液中加入50mmol 3-溴苯胺,其中氢化钠含量为125mmol于60%矿物油分散体中,四氢呋喃含量为100mL;将反应物在0℃下搅拌0.5小时,然后加入125mmol碘乙烷,将混合物在室温下搅拌24小时;用水淬灭反应,将反应混合物用二氯甲烷萃取三次;取有机相用无水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比40:1的石油醚/二氯甲烷作为洗脱剂,得到黄色油状物化合物1,其结构式如下:
步骤S2:将30mmol化合物1与60mL无水乙醚加入至干燥的250mL圆底烧瓶中,磁力搅拌5min使其溶解,随后将13.1mL摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液缓慢滴加至反应液中,滴加完毕后于0℃反应2小时,再将18mmol二氯二甲基硅烷溶于10mL无水乙醚中并缓慢滴加至反应液中,滴加完毕后反应至室温搅拌过夜,加入50mL水猝灭反应,将反应混合物用二氯甲烷萃取三次;取有机相用无水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比80:1的石油醚/乙酸乙酯作为洗脱剂,得到黄色油状物化合物2,其结构式如下:
步骤S3:将1.09mmol化合物2、5.46mmol邻醛基苯甲酸、0.17mmol溴化铜加入到100mL玻璃厚壁耐压管中,于140℃加热搅拌反应5小时后自然冷却至室温,再将反应混合物溶于50mL二氯甲烷中,用二氯甲烷萃取三次;取有机相用无水硫酸钠干燥;将粗产物用硅胶柱纯化,用体积比20:1的石油醚/乙酸乙酯作为洗脱剂,得到淡黄色固体化合物3,其结构式如下:
步骤S4:将3mmol三氯氧磷缓慢加入到10mL含有1mmol化合物3的1,2-二氯乙烷溶液中;上述混合溶液在80℃反应3小时,然后减压浓缩;所得产物直接用10mL无水二氯甲烷溶解,然后将3mmol甲酰肼和200μL二异丙基乙胺溶于5mL无水二氯甲烷并将其缓慢加入到反应体系中。然后将混合溶液在30℃下反应10小时。减压浓缩所得溶液,随后通过柱色谱法纯化,用体积比50:1的二氯甲烷/甲醇作为洗脱剂得到目标荧光探针化合物SiR-FH。
3.一种权利要求1所述的基于硅罗丹明的脑部次氯酸检测荧光探针的应用,其特征在于,用于溶液中荧光性质检测。
4.一种权利要求1所述的基于硅罗丹明的脑部次氯酸检测荧光探针的应用,其特征在于,在细胞成像检测中的应用,其中包括外源性次氯酸细胞成像、内源性次氯酸细胞成像及抗激光漂白中的应用。
5.一种权利要求1所述的基于硅罗丹明的脑部次氯酸检测荧光探针的应用,其特征在于,所述荧光探针可以穿透血脑屏障。
6.一种权利要求1所述的基于硅罗丹明的脑部次氯酸检测荧光探针的应用,其特征在于,用于检测或监测动物脑内的次氯酸水平或水平的变化。
7.一种权利要求1所述的基于硅罗丹明的脑部次氯酸检测荧光探针的应用,其特征在于,在制备用于脑部次氯酸检测试剂方面的应用。
8.一种权利要求1所述的基于硅罗丹明的脑部次氯酸检测荧光探针的应用,其特征在于,在制备神经炎症诊断试剂方面的应用。
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