CN105482807A - 具有溶酶体定位功能的硅基罗丹明铜离子荧光探针及其制备方法和应用 - Google Patents
具有溶酶体定位功能的硅基罗丹明铜离子荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种具有溶酶体定位功能的硅基罗丹明铜离子荧光探针,其结构如式(I)所示;所述的荧光探针能够选择性识别铜离子,且具有定位于细胞中溶酶体的功能;优点:本发明的硅基罗丹明铜离子探针能够特异性定位于细胞的溶酶体中,并能够实现对溶酶体内铜离子的监测。该探针的激发和发射波长均位于近红外光区,在应用于生物成像时可以降低生物体自身荧光背景的干扰,且穿透能力强,对活体组织或细胞的损伤较小,可以满足近红外荧光检测的要求。
Description
技术领域
本发明涉及化学和生物成像技术领域,具体地说,是具有溶酶体定位功能的硅基罗丹明铜离子荧光探针及其制备方法和应用。
背景技术
铜元素是生物体必须的微量元素,也是人体内含量排在锌铁之后、位居第三的过渡元素,广泛分布于生物组织中,对于中枢神经系统、免疫系统、骨骼及内脏组织的发育和功能有着重要影响。铜元素是许多金属蛋白和酶的重要组成部分、如金属硫蛋白、环氧合酶COX、细胞色素C氧化酶、铜-锌超氧化物歧化酶等,参与生物体内的细胞呼吸、抗氧化、神经递质及结缔组织的生物合成、天然色素的形成等过程。生物体内铜含量的失衡会导致蛋白质、脂类、DNA和其它生物分子的氧化性损伤,导致生长和代谢的紊乱,并可能引发Menkes、Wilson、Parkinson和Alzheimer等疾病。
溶酶体是真核细胞中由单层膜包被的囊状结构,溶酶体内pH(≈4.5)低于胞浆内pH(≈7.0),内含多种水解酶和内分泌蛋白,能对细胞经吞噬作用、胞吞作用等吸收的大分子进行消化分解。溶酶体还在胆固醇的动态平衡、质膜的修复、骨组织的重塑、病原体防御等过程中发挥重要作用。溶酶体能够多方面调控细胞的凋亡过程,与肿瘤的发生发展有着密切的联系。
溶酶体与生物体铜离子的代谢有着密切的关系,例如肝细胞通过溶酶体可以将细胞内多余的铜离子运送到细胞外,最后进入胆汁中排出体外,因此检测溶酶体的铜离子有着重要的生理意义。荧光检测法具有灵敏度高、响应时间短等优点,被广泛应用于细胞的成像研究中,但由于溶酶体内特殊的酸性环境,对探针的稳定性、酸性条件下的识别性能等提出了更高的要求,因此特异性检测溶酶体内铜离子的荧光探针鲜有报道。CongWu等人报道了一个基于罗丹明的含六元螺环的铜离子探针(Org.Lett.,2012,14,4198-4201),但没有用于细胞成像实验中;XiaoboWang等人报道了一个基于ICT机理的铜离子荧光探针,并且具有溶酶体靶向功能(Chem.Commun.,2013,49,11263-11265),但是该探针属于荧光减弱型探针,灵敏度较差,且检测波长不能够满足近红外检测的要求。专利中也有许多关于铜离子荧光探针的报道,例如专利CN102127421A、CN103664971A、CN104087285A、CN101914102A等,这些专利中报道的铜离子探针均无法定位于细胞内溶酶体,且检测波长均无法满足近红外检测的要求。中国专利201510010745.0,公开日2015年1月公开了一种用于细胞内铜离子荧光成像的纳米探针及其制备方法,该探针具有尺寸均一、结果稳定、毒性低、生物相容性好的优点。中国专利201410552491.0,公开日2014年10月公开了一种基于罗丹明B的Cu2+探针及其制备方法,该探针在pH=7.2的HEPES缓冲溶液中为无色,加入铜离子后,迅速变为红色,可通过肉眼直接观测颜色变化从而测定水样中部低于6μM的铜离子。以上所述荧光探针都能较好的特异性识别铜离子,但其以荧光素和罗丹明为母体结构,激发波长和发射波长在可见光区,且不具备溶酶体特异性定位功能。我们知道,细胞溶酶体内为酸性环境,对探针的稳定性、酸性条件下的识别性能等提出了更高的要求,因此设计合成具有溶酶体靶向的铜离子荧光探针具有良好的应用前景。
发明内容
本发明的目的是针对现有技术中的不足,提供一种具有溶酶体定位功能的硅基罗丹明铜离子荧光探针。
本发明的再一的目的是,提供如上所述硅基罗丹明铜离子荧光探针的制备方法。
本发明的另一的目的是,提供如上所述硅基罗丹明铜离子荧光探针的用途。
为实现上述目的,本发明采取的技术方案是:
一种具有溶酶体定位功能的硅基罗丹明铜离子荧光探针,所述的荧光探针为SiR-Cu,其结构如式(I)所示;所述的荧光探针能够选择性识别铜离子(Cu2+),且具有定位于细胞中溶酶体的功能;
所述的荧光探针检测pH值为4.5-9.0;优选为5-7;最优为6。
所述的荧光探针最大激发波长为667nm,最大发射波长为680nm。
为实现上述第二个目的,本发明采取的技术方案是:
一种制备如上所述具有式(I)结构的硅基罗丹明铜离子荧光探针的方法,包括如下步骤:
1)将2-NH2-SiR与CSCl2反应生成2-NCS-SiR;
2)将2-NCS-SiR与肼反应生成探针SiR-Cu;
具体制备方法如下:
1)将4.0当量CSCl2溶于干燥CH2Cl2中,冰浴,将1.0当量2-NH2-SiR溶于CH2Cl2中,缓慢加入CSCl2的CH2Cl2溶液中,再加入4.0当量NaOH,冰浴下反应并慢慢升至室温,反应4小时后,加入0.1M碳酸钠水溶液,继续反应0.5小时,CH2Cl2萃取,CH2Cl2层用无水硫酸钠干燥,蒸干溶剂,得固体2-NCS-SiR;
2)将固体2-NCS-SiR用乙腈溶解,缓慢加入20.0当量85%水合肼,室温下反应4小时,蒸干溶剂,CH2Cl2萃取,无水硫酸钠干燥,硅胶柱层析,得探针SiR-Cu。
为实现上述第三个目的,本发明采取的技术方案是:
如上所述硅基罗丹明铜离子荧光探针在检测细胞溶酶体内铜离子及细胞溶酶体荧光成像中的应用。所述的检测为体外样品检测。
如上所述的硅基罗丹明铜离子荧光探针在制备铜离子检测试剂中的应用。所述的试剂可特异性定位于细胞中溶酶体。所述铜离子为Cu2+。
如上所述的硅基罗丹明铜离子荧光探针在制备细胞染料、生物染色剂、生物分子或生物粒子的铜离子荧光标记物中的应用,所述的铜离子荧光标记物具有溶酶体定位功能。
本发明优点在于:
本发明设计并合成出一种新型硅基罗丹明铜离子探针,该探针能够特异性定位于细胞的溶酶体中,并能够实现对溶酶体内铜离子的检测。该探针的激发和发射波长均位于近红外光区,在应用于生物成像时可以降低生物体自身荧光背景的干扰,且穿透能力强,对活体组织或细胞的损伤较小,可以满足近红外荧光检测的要求。
附图说明
附图1为本发明的探针SiR-Cu的核磁氢谱图。
附图2为本发明的探针SiR-Cu的核磁碳谱图。
附图3为本发明的探针SiR-Cu在不同pH条件下激发光波长为620nm时,680nm处的荧光变化图。
附图4为本发明的探针SiR-Cu在pH为7.4条件下加入不同当量铜离子的荧光光谱图。
附图5为本发明的探针SiR-Cu在pH为5.0条件下加入不同当量铜离子的荧光光谱图。
附图6为本发明的探针SiR-Cu在pH为5.0与7.4条件下选择性实验结果。
附图7为本发明的探针SiR-Cu对人正常肝细胞(LO2)染色激光共聚焦荧光成像效果图。
附图8为本发明的探针SiR-Cu对人肝癌细胞(HepG2)激光共聚焦荧光成像效果图。
附图7、附图8中,(a)均表示激发光为633nm时激光共聚焦荧光成像效果图;(b)均表示激发光为488nm时激光共聚焦荧光成像效果图;(c)均表示细胞明场效果图;(d)均表示(a)、(b)、(c)的叠加效果图。
附图9为探针Rh-Cu在不同pH下激发波长540nm,检测波长588nm荧光变化图。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明公开的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1探针SiR-Cu的合成
在100mL茄形反应瓶中,加入原料2-NH2-SiR400mg,用20mLCH2Cl2溶解,冰浴,磁力搅拌。将CSCl2402mg溶于20mLCH2Cl2中,缓慢滴加到反应瓶中,滴加完毕后,加入NaOH140mg,冰浴下反应并缓慢升至室温,继续反应4小时。加入0.1M碳酸钠水溶液,继续反应0.5小时,CH2Cl2萃取三次,合并CH2Cl2层,用水洗涤两次,饱和NaCl溶液洗涤一次,无水硫酸钠干燥,过滤,蒸干溶剂。固体用乙腈溶解,缓慢加入85%水合肼1.06mL,室温下反应4小时,蒸干溶剂,加入水50mL,CH2Cl2萃取三次,合并CH2Cl2层,用水洗涤两次,饱和NaCl溶液洗涤一次,无水硫酸钠干燥,过滤,蒸干溶剂,硅胶柱层析,洗脱剂石油醚:乙酸乙酯=10:1,得探针SiR-Cu。其核磁1HNMR(300MHz,CDCl3)δ0.50(s,3H),0.53(s,3H),1.13(t,12H,J=7.2Hz),3.32(q,8H,J=7.2Hz),4.58(s,2H),6.47-7.09(m,9H),8.86(s,1H);13CNMR(75MHz,CDCl3)δ-0.37,-0.10,12.66,44.10,72.55,113.30,113.48,114.50,123.34,127.23,129.94,130.20,130.53,131.12,134.00,136.82,145.69,170.86;高分辨质谱HRMS(ESI)calcd.forC30H40N5SSi[M+H]+:530.2768,found:530.2773。
实施例2在不同pH下的荧光变化情况测定
1、探针SiR-Cu高标溶液配制
准确秤取探针SiR-Cu2mg,用乙腈溶剂,配置成浓度为0.5mM的高标溶液。
2、探针SiR-Cu自身在不同pH下荧光变化情况测定
移液器吸取10μL探针母液分别加入2000μL不同pH(2.0,3.0,4.0,4.5,5.0,6.0,7.0,7.4,8.0,9.0,10.0,11.0,12.0)的HEPES缓冲液(20.0mM,含20%乙腈)中,30分钟后测其荧光值,激发波长620nm,检测波长680nm。荧光效果见附图3。
3、探针SiR-Cu在铜离子存在时在不同pH下荧光变化情况测定
移液器吸取10μL探针母液分别加入2000μL不同pH(2.0,3.0,4.0,4.5,5.0,6.0,7.0,7.4,8.0,9.0,10.0,11.0,12.0)的HEPES缓冲液中,再分别加入等摩尔量的铜离子,30分钟后测其荧光值,激发波长620nm,检测波长680nm。荧光效果见附图3。
实施例3探针SiR-Cu在pH为7.4条件下的荧光光谱测定
微量移液器吸取10μL探针高标溶液,加入2000μLHEPES缓冲液中(pH=7.4),再在该缓冲液中加入不同当量的铜离子(0,0.25,0.5,1.0,1.25,1.5,2.0,3.0,5.0,7.5,10.0,15.0,20.0),30分钟后测其荧光值。激发波长620nm,检测波长650-800nm。荧光效果见附图4。
实施例4探针SiR-Cu在pH为5.0条件下的荧光光谱测定
测定方法同实施例3,只是所用的HEPES缓冲液pH为5.0。荧光效果见附图5。
实施例5探针SiR-Cu在pH为7.4条件下选择性实验
微量移液器吸取10μL探针高标溶液,加入2000μLHEPES缓冲液中(pH=7.4),再在该缓冲液中加入不同的干扰离子(100equiv.K+;100equiv.Ca2+;100equiv.Ba2+;100equiv.Mg2+;100equiv.Zn2+;10equiv.Fe3+;1equiv.Co2+;10equiv.Ni2+;10equiv.Pb2+;1equiv.Hg2+;10equiv.Mn2+;10equiv.Cd2+;10equiv.Ag+.),室温下孵育30分钟后测其荧光值。激发波长620nm,检测波长680nm。检测结果见附图6。
实施例6探针SiR-Cu在pH为5.0条件下选择性实验
具体方法同实施例6,只是缓冲液pH为5.0。检测结果见附图6。
实施例7探针SiR-Cu活细胞荧光成像实验
1.细胞培养
试验细胞:选用人正常细胞(LO2)和人肝癌细胞(HepG2);
细胞培养条件:使用含10%FBS、0.1mg/ml链霉素和100U/mL的青霉素的DMEM培养基,在含CO2、95%空气,37℃恒温,饱和湿度的细胞培养箱中培养细胞。细胞聚合度达到90%时,吸出培养基并用PBS溶液清洗细胞2次,使用0.25%胰蛋白酶消化2分钟,吸出胰蛋白酶,加入培养基吹散细胞,将细胞以1:3比例传代到培养皿,每天更换一次培养基。
2.试验用荧光染料的配置
将探针SiR-Cu用DMSO配置成1.0MMmM的溶液备用。
3.细胞染色方法
将LO2细胞以2×105密度接种于激光共聚焦培养皿中,在含5%CO2、95%空气,37℃恒温,饱和湿度的细胞培养箱中培养细胞24h后,弃去培养基并用PBS清洗1遍。在2.0mL新鲜培养基中加入探针SiR-Cu溶液10μL,混合均匀后加入培养皿中,培养箱中孵育40分钟,弃去培养基,用PBS缓冲液洗涤三遍。再在2.0mL新鲜培养基中加入DND-26(1.0mM)2μL,混合均匀后加入培养皿中,培养箱中再孵育30分钟,弃去培养基,用PBS缓冲液洗涤三遍,激光共聚焦显微镜下观察,激发光源488nm和633nm。激光共聚焦荧光成像图见附图7。探针SiR-Cu对HepG2细胞染色方法同对LO2细胞染色方法一致,激光共聚焦荧光成像图见附图8。
4.细胞染色实验结果
细胞染色结果见附图7、附图8(成像效果转化为灰度模式),可见探针SiR-Cu可以透过细胞膜进入细胞并与DND-26定位于相同的细胞器—溶酶体,并且可以对细胞溶酶体的铜离子进行监测与成像。
对比例
发明人在实验过程中还合成了具有以下结构的铜离子探针Rh-Cu,并检测不同pH对其荧光强度的影响。
具体实验方法如下:
1、探针Rh-Cu高标溶液配置
准确称取探针Rh-Cu约2mg,用乙腈溶剂,配置成浓度为0.5mM的高标溶液。
2、Rh-Cu自身在不同pH下荧光变化情况测定
移液器吸取10μL探针母液分别加入2000μL不同pH(2.0,3.0,4.0,4.5,5.0,6.0,7.0,7.4,8.0,9.0,10.0,11.0,12.0)的HEPES缓冲液(20.0mM,含20%乙腈)中,30分钟后测其荧光值,激发波长540nm,检测波长588nm。
3、探针Rh-Cu在铜离子存在时在不同pH下荧光变化情况测定
移液器吸取10μL探针母液分别加入2000μL不同pH(2.0,3.0,4.0,5.0,6.0,7.0,7.4,8.0,9.0,10.0,11.0,12.0)的HEPES缓冲液中,再分别加入等摩尔量的铜离子,30分钟后测其荧光值,激发波长540nm,检测波长588nm。
检测结果如下,结果显示:Rh-Cu激发发射波长不能满足近红外检测的要求,且当缓冲液pH小于7时,随着酸性的增强,其荧光背景逐渐增强(见图9),检测灵敏度大大降低,不适于酸性条件下Cu2+的检测。本发明中硅原子的引入,不但使探针的光谱红移至近红外光区,还增强了螺环的稳定性,在低pH条件下,荧光背景不会增加,因此可用于溶酶体中Cu2+的选择性成像。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.一种具有溶酶体定位功能的硅基罗丹明铜离子荧光探针,其特征在于,所述的荧光探针为SiR-Cu,其结构如式(I)所示;所述的荧光探针能够选择性识别铜离子,且具有定位于细胞中溶酶体的功能;
2.根据权利要求1所述的具有溶酶体定位功能的硅基罗丹明铜离子荧光探针,其特征在于,所述的荧光探针检测pH值为4.5-9.0。
3.根据权利要求1所述的具有溶酶体定位功能的硅基罗丹明铜离子荧光探针,其特征在于,所述的荧光探针最大激发波长为667nm,最大发射波长为680nm。
4.一种制备权利要求1所述的具有式(I)结构的硅基罗丹明铜离子荧光探针的方法,其特征在于,所述方法包括如下步骤:
1)将2-NH2-SiR与CSCl2反应生成2-NCS-SiR;
2)将2-NCS-SiR与肼反应生成探针SiR-Cu;
5.根据权利要求4所述的方法,其特征在于,进一步包括:
1)将4.0当量CSCl2溶于干燥CH2Cl2中,冰浴,将1.0当量2-NH2-SiR溶于CH2Cl2中,缓慢加入CSCl2的CH2Cl2溶液中,再加入4.0当量NaOH,冰浴下反应并慢慢升至室温,反应4小时后,加入0.1M碳酸钠水溶液,继续反应0.5小时,CH2Cl2萃取,CH2Cl2层用无水硫酸钠干燥,蒸干溶剂,得固体2-NCS-SiR;
2)将固体2-NCS-SiR用乙腈溶解,缓慢加入20.0当量85%水合肼,室温下反应4小时,蒸干溶剂,CH2Cl2萃取,无水硫酸钠干燥,硅胶柱层析,得探针SiR-Cu。
6.权利要求1所述的硅基罗丹明铜离子荧光探针在检测细胞溶酶体内铜离子及细胞溶酶体荧光成像中的应用。
7.根据权利要求6所述的应用,其特征在于,所述的检测为体外样品检测。
8.权利要求1所述的硅基罗丹明铜离子荧光探针在制备铜离子检测试剂中的应用。
9.根据权利要求要求8所述的应用,其特征在于,所述的试剂可特异性定位于细胞的溶酶体。
10.权利要求1所述的硅基罗丹明铜离子荧光探针在制备细胞染料、生物染色剂、生物分子或生物粒子的铜离子荧光标记物中的应用,所述的铜离子荧光标记物具有溶酶体定位功能。
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