CN112898963B - 一种检测粘度的荧光探针及其制备方法和应用 - Google Patents
一种检测粘度的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及有机小分子荧光探针技术领域,具体涉及一种检测粘度的荧光探针及其制备方法和应用,尤其涉及一种长波发射的检测粘度的荧光探针及其制备方法和应用。
背景技术
生物体的流动特性使得粘度成为生物系统中十分重要的一个微环境参数,粘度对于生物体中物质运输、信号传递等生理过程有着十分重要的作用。细胞水平粘度异常与许多疾病有关,例如阿尔兹海默症、粥状动脉硬化、肿瘤等。但是传统的测定粘度的方法例如旋转粘度计、毛细管粘度计测定只能对宏观条件下的生物环境进行测试,而对于单个细胞内微环境内的粘度变化的测定却无法实现。而荧光探针分子检测生物体内微环境的方法因其方便快捷、检测灵敏度高、对细胞损伤小以及可以实现生物体内实时监测等优点被广泛关注。利用荧光探针检测生物系统中的粘度的方法可以很好的对细胞内微环境的粘度进行检测,从而达到很好的疾病诊断与治疗的目的。
目前已经有比较多的对荧光探针的研究,但是大部分粘度荧光探针都具有短波长荧光发射信号(<600nm),该段波长信号存在蓝绿区的细胞自发荧光干扰,并且穿透深度有限,不利于生物成像。而具有长波发射性能的荧光探针,可以消除细胞内蓝绿区自发荧光的影响,从而降低背景噪音,提高信噪比。
因此,研发出一种具有长波发射性能的粘度荧光探针,并应用于细胞内粘度检测的研究具有重大的意义。
发明内容
本发明要解决的技术问题是提供一种检测粘度的荧光探针,具有长波发射性能,并具有较大的Stokes位移特性,使吸收和发射光谱之间重叠小,可避免能量转移而导致的荧光效率降低,提高检测灵敏度。
为了解决上述问题,本发明的技术方案如下:
一种检测粘度的荧光探针,具有如下式(Ⅰ)所示的结构:
式(Ⅰ)。
本发明还提供一种检测粘度的荧光探针的制备方法,包括如下步骤:
步骤S1,以1,1,2-三甲基-1H-苯并(E)吲哚和碘乙烷反应制备得到3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚;
步骤S2,将N-[(3-(苯胺基亚甲基)-2-氯-1-环己烯-1-基)亚甲基]苯胺盐酸盐与步骤S1所得的3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚在碱性条件下反应,经柱层析提纯之后得到纯品;
步骤S3,将步骤S2所得纯品与苯并噻唑-2-乙腈在碱性条件下反应,经提纯得到荧光探针分子P-1纯品。
进一步地,一种检测粘度的荧光探针的制备方法,包括如下步骤:
步骤S1,称取1g(4.8mmol)的1,1,2-三甲基-1H-苯并(E)吲哚,加入463μL(5.8mmol,1.2equiv)的碘乙烷于10mL的无水乙腈中,85℃下回流12h,反应结束冷却至室温,抽滤并用无水乙醚洗涤得到3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚;
合成路线为:
步骤S2,取0.36g(1mmol)的N-[(3-(苯胺基亚甲基)-2-氯-1-环己烯-1-基)亚甲基]苯胺盐酸盐,0.24g(1mmol)的3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚,无水乙酸钠81mg(1mmol),溶于15mL超干乙醇中,设置油浴温度80℃,回流8h,反应结束后,冷却至室温,用磷酸盐缓冲溶液中和,二氯甲烷萃取得到有机相,然后用无水硫酸镁干燥,浓缩后利用硅胶柱层析分离,得到纯品;
合成线路为:
步骤S3,取步骤S2的反应产物331mg(0.7mmol)、苯并噻唑-2-乙腈122mg(0.7mmol)、无水乙酸钠63mg(0.76mmol,1.1equiv),溶于5mL超干乙醇中,设置油浴温度85℃,回流6h,反应结束后,冷却至室温,浓缩后利用柱层析分离,得到荧光探针分子P-1纯品。
合成线路为:
进一步地,步骤S2中,柱层析分离工艺中,展开剂为甲醇和二氯甲烷的混合溶液,且甲醇与二氯甲烷的体积比为1:200。
进一步地,步骤S3中,柱层析分离工艺中,展开剂为二氯甲烷。
本发明还提供一种检测粘度的荧光探针在溶液体系或生物体系中粘度测定的应用。
进一步地,单光子荧光测定粘度时,激发波长为650nm,荧光发射峰的峰值在780nm处,随着粘度的增大,荧光强度增强,且粘度与荧光强度比I/I0的对数值呈线性关系,从而根据荧光强度比I/I0的对数值测定粘度。。
与现有技术相比,本发明提供的检测粘度的荧光探针及其制备方法和应用,有益效果在于:
一、本发明提供的检测粘度的荧光探针,具有很大的共轭结构,从而使该探针分子可以发出较长波长的光,在该探针分子的激发波长下激发可以发出较强的荧光,并且随着粘度的增大,荧光发射强度明显增大,粘度和荧光发射强度之间显示很好的线性关系。本发明的荧光探针,荧光测定的激发波长为650nm,荧光发射峰的峰值在780nm处,斯托克位移达到了130nm,斯托克位移变化大,使吸收和发射光谱之间重叠小,可避免能量转移而导致的荧光效率降低,提高检测灵敏度。
二、本发明提供的检测粘度的荧光探针,是一种长波发射的荧光探针,用于生物体系成像的过程中可以有较强的穿透深度,还可以有效的消除细胞自荧光的干扰,对细胞的毒性低,在生物分子检测领域具有广阔的应用前景。
三、本发明提供的检测粘度的荧光探针,是一种简单,快速,灵敏的细胞粘度检测试剂,能实现溶液体系或生物体系粘度的快速检测。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为荧光探针制备方法中化合物1的核磁氢谱图;
图2为荧光探针制备方法中化合物2的核磁氢谱图;
图3为本发明提供的荧光探针P-1的核磁氢谱图;
图4为荧光探针P-1在不同溶剂中的紫外吸收光谱图;
图5为荧光探针P-1在不同溶剂中的荧光发射光谱图;
图6为荧光探针P-1在不同粘度的溶液中的单光子荧光发射图谱;
图7为荧光探针P-1在不同粘度的溶液中荧光强度比I/I0的对数值与溶液粘度的线性关系图;
图8为荧光探针P-1在纯乙醇溶液中的紫外吸收图和在含有80%甘油的乙醇溶液的荧光发射图谱;
图9为荧光探针P-1在纯乙醇溶液中和含有80%甘油的乙醇溶液中的连续被激发一个小时后的荧光变化情况;
图10为荧光探针P-1对Hela细胞的CCK-8细胞毒性检测图。
具体实施方式
为了使本技术领域的人员更好地理解本发明实施例中的技术方案,并使本发明的上述目的、特征和优点能够更加明显易懂,下面对本发明的具体实施方式作进一步的说明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应该被视为在本文中具体公开。
实施例1
3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚的合成
称取1g(4.8mmol)的1,1,2-三甲基-1H-苯并(E)吲哚,加入463μL(5.8mmol,1.2equiv)的碘乙烷于10mL的无水乙腈中,85℃下回流12个小时,反应结束冷却至室温,抽滤并用无水乙醚洗涤得到化合物1,化合物1为淡紫色固体0.95g,产率83.2%。化合物1为3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚。
请参阅图1,为荧光探针制备方法中化合物1的核磁氢谱图。核磁测试的结果如下:1H NMR(400MHz,CDCl3)δ8.24–7.57(m,6H),4.84(d,J=6.2Hz,2H),3.22(s,3H),1.86(s,7H),1.68(s,3H)。
实施例2
荧光探针的合成
取0.36g(1mmol)的N-[(3-(苯胺基亚甲基)-2-氯-1-环己烯-1-基)亚甲基]苯胺盐酸盐,0.24g(1mmol)的3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚,无水乙酸钠81mg(1mmol),溶于15mL超干乙醇中,设置油浴温度80℃,回流8h,反应结束后,冷却至室温,用磷酸盐缓冲溶液中和,二氯甲烷多次萃取得到有机相(紫红色),然后用无水硫酸镁干燥,浓缩后利用硅胶柱层析分离,展开剂为甲醇/二氯甲烷(体积比为1:200),得绿色固体化合物2 61.8mg,产率13%。
请结合参阅图2,为荧光探针制备方法中化合物2的核磁氢谱图。核磁测试的结果如下:1H NMR(400MHz,DMSO)δ8.74(s,1H),8.08(d,J=8.6Hz,1H),7.88(t,J=7.8Hz,2H),7.73(d,J=12.1Hz,1H),7.48(t,J=7.3Hz,1H),7.42–7.34(m,3H),7.32–7.25(m,1H),7.24–7.13(m,3H),5.76(s,1H),5.66(d,J=12.3Hz,1H),3.95(d,J=5.1Hz,2H),2.68(dt,J=10.9,5.7Hz,4H),1.89(s,6H),1.83–1.75(m,2H),1.22(s,3H)。
取化合物2 331mg(0.7mmol),苯并噻唑-2-乙腈122mg(0.7mmol),无水乙酸钠63mg(0.76mmol,1.1equiv),溶于5mL超干乙醇中,设置油浴温度85℃,回流6h,反应结束后,冷却至室温,浓缩后利用柱层析分离,展开剂为二氯甲烷,得蓝色固体化合物198mg,产率54%,即荧光探针分子P-1。
请结合参阅图3,为本发明提供的荧光探针P-1的核磁氢谱图。核磁测试的结果如下:1H NMR(400MHz,DMSO)δ8.55(s,1H),8.20(d,J=8.6Hz,1H),8.14(d,J=7.8Hz,1H),8.04(d,J=8.0Hz,1H),8.02–7.96(m,3H),7.63–7.44(m,5H),7.40(t,J=7.5Hz,1H),5.86(d,J=13.1Hz,1H),4.10(d,J=6.6Hz,2H),3.05(t,J=5.7Hz,2H),2.74–2.67(m,2H),1.98–1.87(m,8H),1.29(t,J=6.7Hz,3H)。
根据荧光探针的核磁氢谱图,分析可知,荧光探针分子P-1的结构如式(I)
所示:
式(I)。
实施例3
为了探究荧光探针P-1对不同溶剂极性的反应情况,在浓度为1×10-5mol/L的条件下分别测试了荧光探针P-1在二氯甲烷(CH2Cl2),甲醇(MeOH),乙醇(EtOH),乙腈(Acetonitrile),N,N-二甲基甲酰胺(DMF),二甲基亚砜(DMSO)6种不同溶剂中的UV-Vis吸收光谱和荧光发射光谱的变化。
请结合参阅图4,为荧光探针P-1在不同溶剂中的紫外吸收光谱图。由图4可知,不同溶剂中的紫外的吸收峰在640~670之间,因此选择了650nm作为激发波长,以10×10cm作为狭缝宽度,在950V电压下,同样以浓度为1×10-5mol/L分别测试了荧光探针在6种不同溶剂中的荧光发射情况。请结合参阅图5,为荧光探针P-1在不同溶剂中的荧光发射光谱图。
实施例4
由于乙醇既有较低的毒性,且具有良好的性能,因此选择乙醇作为溶剂,通过调节乙醇和甘油的比例来测试化合物在不同粘度下的荧光性质。取实施例2制备的荧光探针P-1溶于DMSO(二甲基亚砜)中,制成10-3mol/L的储备液。从储备液中取出10μL加入到5mL的离心管当中,用不同比例的乙醇(EtOH)和甘油(Gly)稀释至1mL,即在1×10-5mol/L条件下测量其荧光性质。以10×10cm作为狭缝宽度,650nm作为激发波长,在950V电压下,以浓度为1×10- 5mol/L分别测试了荧光探针P-1在纯乙醇当中,EtOH:Gly=9:1(v:v),EtOH:Gly=8:2(v:v),EtOH:Gly=7:3(v:v),EtOH:Gly=6:4(v:v),EtOH:Gly=5:5(v:v),EtOH:Gly=4:6(v:v),EtOH:Gly=3:7(v:v),EtOH:Gly=2:8(v:v)中的荧光发射。请结合参阅图6,为荧光探针P-1在不同粘度的溶液中的单光子荧光发射图谱,图6中横坐标为波长,纵坐标为荧光强度。由图6可见,在纯乙醇溶液到甘油含量80%之间,随着溶液粘度的增大,荧光强度都出现逐渐增大的趋势,且最大发射波长为780nm处荧光强度最强。
请结合参阅图7,为荧光探针P-1在不同粘度的溶液中荧光强度比I/I0的对数值与溶液粘度的线性关系图。由图7可以看出,荧光探针P-1的荧光强度比(I/I0)的对数与溶液粘度(不同比例的甘油溶液)有良好的线性拟合关系,线性拟合R2=0.9791。
实施例5
为了探究荧光探针是否具有优异的光稳定性能,分别对荧光探针浓度为1×10- 5mol/L的纯乙醇溶液和含有80%甘油的DMSO溶液,在激发波长为650nm下连续照射1小时。请结合参阅图8和图9,其中图8为荧光探针P-1在纯乙醇溶液中的紫外吸收图和在含有80%甘油的乙醇溶液的荧光发射图谱;图9为荧光探针P-1在纯乙醇溶液中和含有80%甘油的乙醇溶液中的连续被激发一个小时后的荧光变化情况。由图8和图8可知,连续照射一小时后,荧光强度前后变化不大,说明荧光探针P-1具有比较好的光稳定性,且对比得出最大Stoke位移为130nm,斯托克位移变化大,使吸收和发射光谱之间重叠小,可避免能量转移而导致的荧光效率降低,提高检测灵敏度。
实施例6
荧光探针P-1对Hela细胞的CCK-8细胞毒性检测
处于生长对数期的Hela细胞以每孔104个接种于96孔板,放入细胞培养箱(37℃,5%CO2)中贴壁生长24h。将细胞培养基吸除,加入用培养基稀释的荧光探针P-1(浓度分别为5,10,15,20,30μM)与HeLa细胞共孵育24h,弃去培养基,每孔加入100μL用培养基稀释的10%的CCK-8溶液继续孵育2h。用酶标仪(450nm)记录每孔的吸光度值。按下式计算细胞存活率:
其中A0、A1、A2分别为空白组(纯培养基,无探针)、实验组(细胞用探针处理)、对照组(细胞无处理)的吸光度值。
请结合参阅图10,为荧光探针P-1对Hela细胞的CCK-8细胞毒性检测图。由图10可知,随着探针浓度从5μM增加至30μM,细胞存活率无明显降低,仍能达到90%,说明探针P-1基本无毒性,是可能用于细胞粘度检测的。
与现有技术相比,本发明提供的检测粘度的荧光探针及其制备方法和应用,有益效果在于:
一、本发明提供的检测粘度的荧光探针,具有很大的共轭结构,从而使该探针分子可以发出较长波长的光,在该探针分子的激发波长下激发可以发出较强的荧光,并且随着粘度的增大,荧光发射强度明显增大,粘度和荧光发射强度之间显示很好的线性关系。本发明的荧光探针,荧光测定的激发波长为650nm,荧光发射峰的峰值在780nm处,斯托克位移达到了130nm,斯托克位移变化大,使吸收和发射光谱之间重叠小,可避免能量转移而导致的荧光效率降低,提高检测灵敏度。
二、本发明提供的检测粘度的荧光探针,是一种长波发射的荧光探针,用于生物体系成像的过程中可以有较强的穿透深度,还可以有效的消除细胞自荧光的干扰,对细胞的毒性低,在生物分子检测领域具有广阔的应用前景。
三、本发明提供的检测粘度的荧光探针,是一种简单,快速,灵敏的细胞粘度检测试剂,能实现溶液体系或生物体系粘度的快速检测。
以上对本发明的实施方式作出详细说明,但本发明不局限于所描述的实施方式。对本领域的技术人员而言,在不脱离本发明的原理和精神的情况下对这些实施例进行的多种变化、修改、替换和变型均仍落入在本发明的保护范围之内。
Claims (4)
2.根据权利要求1所述的检测粘度的荧光探针的制备方法,其特征在于,包括如下步骤:
步骤S1,称取1g浓度为4.8mmol的1,1,2-三甲基-1H-苯并(E)吲哚,加入463μL浓度为5.8mmol、当量equiv为1.2的碘乙烷于10mL的无水乙腈中,85℃下回流12h,反应结束冷却至室温,抽滤并用无水乙醚洗涤得到3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚;
步骤S2,取0.36g浓度为1mmol的N-[(3-(苯胺基亚甲基)-2-氯-1-环己烯-1-基)亚甲基]苯胺盐酸盐,0.24g浓度为1mmol的3-乙基-1,1,2-三甲基-1H-苯并(E)吲哚,浓度为1mmol的无水乙酸钠81mg,溶于15mL超干乙醇中,设置油浴温度80℃,回流8h,反应结束后,冷却至室温,用磷酸盐缓冲溶液中和,二氯甲烷萃取得到有机相,然后用无水硫酸镁干燥,浓缩后利用硅胶柱层析分离,得到纯品;
步骤S3,取浓度为0.7mmol的步骤S2的反应产物331mg、浓度为0.7mmol的苯并噻唑-2-乙腈122mg、浓度为0.76mmol,当量equiv为1.1的无水乙酸钠63mg,溶于5mL超干乙醇中,设置油浴温度85℃,回流6h,反应结束后,冷却至室温,浓缩后利用柱层析分离,得到荧光探针分子P-1纯品。
3.根据权利要求2所述的检测粘度的荧光探针的制备方法,其特征在于,步骤S2中,柱层析分离工艺中,展开剂为甲醇和二氯甲烷的混合溶液,且甲醇与二氯甲烷的体积比为1:200。
4.根据权利要求2所述的检测粘度的荧光探针的制备方法,其特征在于,步骤S3中,柱层析分离工艺中,展开剂为二氯甲烷。
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