CN113698928A - 碳点及其制备方法和在制备靶向线粒体的荧光探针中的应用 - Google Patents
碳点及其制备方法和在制备靶向线粒体的荧光探针中的应用 Download PDFInfo
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Abstract
本发明公开了一种碳点及其制备方法和在制备靶向线粒体的荧光探针中的应用,该碳点的制备方法,包括如下步骤:分别称取0.1~1g酒石酸,0.1~1g二乙氨基酚以备用;将称量好的酒石酸和二乙氨基酚放入超纯水中,搅拌混匀;将上述混匀的溶液装入反应釜中,并将反应釜在160~200℃条件下加热6~48小时后,将温度降为室温;将反应获得的棕色溶液装入透析袋中,并将其放入超纯水中透析,并每隔一段时间换水一次以除去杂质,最终获得酒红色碳点溶液。本发明利用水热方法一步制备的碳点具有高的荧光量子产率和稳定的荧光性质,且无毒性。细胞成像实验表明本发明制备的碳点可以作为线粒体荧光探针,并在疾病诊断、细胞成像以及分子检测等领域有潜在的应用。
Description
技术领域
本发明属于荧光碳点技术领域,尤其涉及一种碳点及其制备方法和在制备靶向线粒体的荧光探针中的应用。
背景技术
线粒体是哺乳动物细胞的重要细胞器,是细胞的动力站。除了提供细胞能量外,它们还与许多重要的细胞代谢过程有关,如细胞生长、坏死和凋亡。有趣的是,线粒体在这些生物过程中表现出包括裂变、融合和运输在内的动态特性。线粒体的这种动态变化对维持其形态和功能起着基础性作用。因此,可视化和监测活细胞内线粒体的动力学具有重要意义。到目前为止,各种探针已被应用于线粒体成像,包括荧光蛋白和有机小分子。然而,这些荧光探针仍有一些局限性。例如,荧光蛋白总是需要高效的蛋白表达和合适的抗体。对于有机小分子荧光染料,其具有制备方法复杂、抗光漂白性低和细胞毒性高。由于碳点具有制备方法简单、无毒性、抗光漂白等优点,逐渐成为替代荧光蛋白和有机小分子的候选材料。尽管如此,目前制备的大多数碳点不具有靶向线粒体的性质。
发明内容
针对现有技术的不足,本发明利用水热方法一步制备出一种高纯度且具有高荧光量子产率的碳点,并验证出其可以作为荧光探针靶向活细胞线粒体。
本发明具体是通过以下技术方案来实现的:
本发明第一方面提供一种碳点的制备方法,包括如下步骤:
步骤1:分别称取0.1~1g酒石酸,0.1~1g二乙氨基酚以备用;
步骤2:将称量好的酒石酸和二乙氨基酚放入超纯水中,搅拌混匀;
步骤3:将上述混匀的溶液装入反应釜中,并将反应釜在160~200℃条件下加热6~48小时后,将温度降为室温;
步骤4:将反应获得的棕色溶液装入透析袋中,并将其放入超纯水中透析,并每隔一段时间换水一次以除去杂质,最终获得酒红色碳点溶液。
作为本发明的进一步说明,步骤1中,所述酒石酸的称取量为0.523g,所述二乙氨基酚的称取量为0.476g。
作为本发明的进一步说明,步骤2中,超纯水用量为30mL,水电阻率为18.4MΩ·cm-1。
作为本发明的进一步说明,步骤3中,所述反应釜采用50mL的聚四氟乙烯反应釜。
作为本发明的进一步说明,步骤3中,所述反应釜的反应条件具体为:将所述反应釜置于烘箱中恒温180℃加热24小时后,将温度降为25℃。
作为本发明的进一步说明,步骤4中,所述透析袋为500Da的透析袋,超纯水用量为2L,且每次换水除杂时间间隔为4小时。
本发明第二方面提供一种荧光碳点,所述荧光碳点由上述的制备方法制得。
作为本发明的进一步说明,所述荧光碳点中的化学键以C-C/C=C、C-O/C-N和C=O为主。
作为本发明的进一步说明,所述荧光碳点的最佳激发波长和发射波长分别为513nm和535nm,且在365nm的紫外灯下呈酒红色。
本发明还提供了上述的荧光碳点在制备能够靶向活细胞线粒体的荧光探针中的应用。
与现有技术相比,本发明具有以下有益的技术效果:
本发明利用水热方法一步制备的碳点具有高的荧光量子产率和稳定的荧光性质,且无毒性。细胞成像实验表明本发明制备的碳点可以作为线粒体荧光探针,并在疾病诊断、细胞成像以及分子检测等领域有潜在的应用。
附图说明
图1为本发明制备的碳点的透射电镜图;
图2为本发明制备的碳点的粒径分布图;
图3为本发明制备的碳点的红外图谱;
图4为本发明制备的碳点的X射线光电子能的全谱谱图;
图5为本发明制备的碳点中碳元素的X射线光电子能的窄谱谱图;
图6为本发明制备的碳点溶液的紫外可见吸收光谱;
图7为本发明制备的碳点溶液的最佳激发(左边第一条)和发射光谱;
图8为本发明制备的碳点溶液在不同激发波长下的3D光谱;
图9为本发明制备的碳点溶液的荧光量子产率;
图10为本发明制备的碳点溶液在不同离子下的荧光强度变化;
图11为本发明制备的碳点溶液在不同pH值下的荧光强度变化;
图12为本发明制备的碳点溶液在不同离子强度下的荧光强度变化;
图13为本发明制备的碳点的细胞毒性图;
图14为本发明制备的碳点的共定位双染图。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和具体实施例对本发明进行详细描述。需要说明的是,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
实施例1:
提供一种碳点的制备方法,包括如下步骤:
步骤1:分别称取0.523g酒石酸,0.476g二乙氨基酚以备用。
步骤2:将称量好的酒石酸和二乙氨基酚放入装有30mL超纯水(水电阻率为18.4MΩ·cm-1)的烧杯中,搅拌混匀。
步骤3:将上述混匀的溶液装入50mL的聚四氟乙烯反应釜中,并将反应釜至于烘箱中恒温180℃加热24小时后,将温度降为25℃。
步骤4:将反应获得的棕色溶液装入500Da的透析袋中,并将其放入装有2L超纯水的烧杯中透析,并每隔4小时换水一次以除去杂质,最终获得酒红色碳点溶液。
实施例2:
提供一种碳点的制备方法,包括如下步骤:
步骤1:分别称取0.1g酒石酸,0.1g二乙氨基酚以备用。
步骤2:将称量好的酒石酸和二乙氨基酚放入装有30mL超纯水(水电阻率为18.4MΩ·cm-1)的烧杯中,搅拌混匀。
步骤3:将上述混匀的溶液装入50mL的聚四氟乙烯反应釜中,并将反应釜至于烘箱中恒温160℃加热6小时后,将温度降为25℃。
步骤4:将反应获得的棕色溶液装入500Da的透析袋中,并将其放入装有2L超纯水的烧杯中透析,并每隔4小时换水一次以除去杂质,最终获得酒红色碳点溶液。
实施例3:
提供一种碳点的制备方法,包括如下步骤:
步骤1:分别称取1g酒石酸,1g二乙氨基酚以备用。
步骤2:将称量好的酒石酸和二乙氨基酚放入装有30mL超纯水(水电阻率为18.4MΩ·cm-1)的烧杯中,搅拌混匀。
步骤3:将上述混匀的溶液装入50mL的聚四氟乙烯反应釜中,并将反应釜至于烘箱中恒温200℃加热48小时后,将温度降为25℃。
步骤4:将反应获得的棕色溶液装入500Da的透析袋中,并将其放入装有2L超纯水的烧杯中透析,并每隔4小时换水一次以除去杂质,最终获得酒红色碳点溶液。
对实施例1制备的碳点进行表征,具体表征结果如下:
1、通过透射电镜对上述实施例1制备的碳点进行表征,可知其粒径大约为~4nm(如图1)。
2、利用动态光散射粒度仪对碳点溶液进测试可知,制备的碳点粒径基本为~4.5nm(如图2),这与透射电镜获得的结果相当。
3、制备的碳点通过红外测试表征可知(如图3),该碳点含有多种化学键和基团,即–OH、–NH2和C–N。
4、从制备碳点的X射线光电子能谱可知,碳点含有C、O和N三种元素,且元素含量分别为62.5%、32.6%和4.9%(如图4)。由碳点中C元素的X射线光电子能谱可知,碳点中的化学键以C-C/C=C、C-O/C-N、和C=O为主(如图5)。
5、对碳点水溶液品荧光性质的研究发现,制备的碳点溶液在274、386和513nm处有三个吸收峰,274nm的吸收对应了C=C的π-π*跃迁,386和513nm处的吸收对应的为C=O/C–O/C–N的n-π*跃迁(如图6)。此外,碳点水溶液的最佳激发波长和发射波长分别为513nm和535nm(如图7),且在365nm的紫外灯下呈酒红色。对其在不同激发波长(300~600nm)下的荧光光谱的研究发现,碳点的发射峰位置会随着激发峰的改变而改变(如图8)。对碳点荧光量子产率的测试,可知其在水溶液的荧光量子产率高达24.5%(如图9)。
6、对碳点荧光稳定性的研究可以发现,测试了在不同阴阳离子下、不同pH值及在不同离子强度下的碳点的荧光强度。如图10,碳点在不同阴阳离子的作用下,其荧光强度基本保持不变。说明生物体内的不同离子对碳点荧光没有影响。图11显示了不同pH值对碳点荧光强度的影响。结果表明碳点的荧光强度变化不随着PH的变化成线性变化且碳点的荧光强度无明显改变。接下来,测试了不同离子强度(即加入不同浓度的NaCl的)对碳点荧光强度的影响。结果发现碳点的荧光强度无明显的变化(图12)。对碳点的细胞毒性研究表明,当碳点浓度为300μg/mL时,细胞的存货率大约为90%,说明制备的碳点基本无毒(如图13)。
将制备的碳点作为荧光探针,与人食管鳞癌细胞(KYSE150)共孵育,利用共定位的方法,确定了该碳点作为荧光探针可以靶向活细胞的线粒体。
综上所述,本发明利用水热方法一步制备的碳点具有高的荧光量子产率和稳定的荧光性质,且无毒性。细胞成像实验表明制备的碳点可以作为线粒体荧光探针,并在疾病诊断、细胞成像以及分子检测等领域有潜在的应用。
最后应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或等同替换,而不脱离本发明技术方案的精神和范围。
Claims (10)
1.一种碳点的制备方法,其特征在于,包括如下步骤:
步骤1:分别称取0.1~1g酒石酸,0.1~1g二乙氨基酚以备用;
步骤2:将称量好的酒石酸和二乙氨基酚放入超纯水中,搅拌混匀;
步骤3:将上述混匀的溶液装入反应釜中,并将反应釜在160~200℃条件下加热6~48小时后,将温度降为室温;
步骤4:将反应获得的棕色溶液装入透析袋中,并将其放入超纯水中透析,并每隔一段时间换水一次以除去杂质,最终获得酒红色碳点溶液。
2.根据权利要求1所述的碳点的制备方法,其特征在于,步骤1中,所述酒石酸的称取量为0.523g,所述二乙氨基酚的称取量为0.476g。
3.根据权利要求1所述的碳点的制备方法,其特征在于,步骤2中,超纯水用量为30mL,水电阻率为18.4MΩ·cm-1。
4.根据权利要求1所述的碳点的制备方法,其特征在于,步骤3中,所述反应釜采用50mL的聚四氟乙烯反应釜。
5.根据权利要求1所述的碳点的制备方法,其特征在于,步骤3中,所述反应釜的反应条件具体为:将所述反应釜置于烘箱中恒温180℃加热24小时后,将温度降为25℃。
6.根据权利要求1所述的碳点的制备方法,其特征在于,步骤4中,所述透析袋为500Da的透析袋,超纯水用量为2L,且每次换水除杂时间间隔为4小时。
7.一种荧光碳点,其特征在于,所述荧光碳点由权利要求1-6中任一项所述的制备方法制得。
8.根据权利要求7所述的荧光碳点,其特征在于,所述荧光碳点中的化学键以C-C/C=C、C-O/C-N和C=O为主。
9.根据权利要求7所述的荧光碳点,其特征在于,所述荧光碳点的最佳激发波长和发射波长分别为513nm和535nm,且在365nm的紫外灯下呈酒红色。
10.权利要求7所述的荧光碳点在制备能够靶向活细胞线粒体的荧光探针中的应用。
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