CN113845906A - 一种氮掺杂碳量子点荧光探针的合成及应用 - Google Patents
一种氮掺杂碳量子点荧光探针的合成及应用 Download PDFInfo
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Abstract
本发明公开了一种氮掺杂碳量子点的合成、及在Fe3+和PPi检测方面的应用;以柠檬酸和缩二脲为碳源和氮源,利用水热法合成制备了表面含有丰富官能团的氮掺杂碳量子点(N‑CDs),N‑CDs通过荧光猝灭效应实现了对Fe3+的高选择性和高灵敏度的检测;经Fe3+猝灭的N‑CDs荧光可通过焦磷酸盐(PPi)作用后恢复,使得N‑CDs/Fe3+传感器可用于PPi的高灵敏检测;Fe3+和PPi的线性检测范围分别为0‑200和0‑150μM,检测限(LOD)为2.71和1.12μM;N‑CDs在实际自来水样品中进行了Fe3+检测,取得了令人满意的结果;更重要的是,N‑CDs具有优异的光学特性,低毒性使其可用于Hela细胞和斑马鱼中的Fe3+和PPi的成像研究。
Description
技术领域
本发明属于碳纳米材料的制备、离子检测及生物成像技术领域,具体涉及一种碳量子点的合成方法、及在Fe3+和PPi检测中的应用。
背景技术
碳量子点是继富勒烯、碳纳米管和石墨烯之后最引人注目的碳纳米材料,因其具有优异的光学性能,易修饰,低毒等优点而得到研究者的广泛关注。碳量子点的原料来源广泛,制备成本较低,具有规模化生产的潜力,在医学成像、环境监测、化学分析、催化、能源等众多领域都具有较好的应用前景。
传统的半导体量子点往往含有铅、镉等金属离子,因而毒性大,对环境危害大,难以在生物体系内应用。碳量子点作为一种新型的碳纳米材料,在性质上与各种金属量子点类似,但制备过程不涉及重金属的使用,原料来源广泛且廉价,日常食物如土豆、西红柿等均可作为碳源。此外,碳量子点表面丰富的羟基、羧基等亲水性官能团保证其在水中具有良好的溶解性,能更好的应用于生物体系内。
铁(Fe3+)是动植物必需的微量元素之一,在环境和临床医学中发挥着重要作用。细胞内正常Fe3+浓度通常维持在0.3±0.1mM,缺乏Fe3+会导致缺铁性贫血、糖尿病、呼吸问题。Fe3+增强了自由基的产生,从而导致了肝脏和肾脏损伤以及心力衰竭。此外过量的Fe3+积累同样会造成严重的疾病,如帕金森症、阿尔茨海默症、癌症等。同时环境中Fe3+的含量是监测水污染的重要指标之一。
阴离子在整个生物系统中无处不在,并在广泛的化学和生物过程中发挥着基本作用。焦磷酸盐(PPi),作为阴离子的一种,它是生物能量循环和DNA合成的一部分,也是一些结晶反应的抑制剂。PPi被认为是软骨钙病或焦磷酸钙二水合晶体沉积疾病的潜在生物标志物。迄今为止,大多数的PPi的检测方法都是基于有机小分子探针。但由于其毒性、不良的生物相容性以及光学性能的缺陷导致在生物体系中应用受阻。
一直以来,研究人员致力于Fe3+和PPi检测方法的开发,先后发展了电感耦合等离子体光谱、液相色谱和原子吸收光谱等一系列方法。然而,仪器操作复杂、检测耗时、成本高等问题依然存在,阻碍了其规模化应用。与其他检测方法相比,荧光分析技术具有较低的细胞毒性,同时其作为一种非入侵的检测手段,因具有灵敏度高、响应迅速、选择性好、无损伤成像等优点而成为研究热点。因此,开发一种能够同时检测生物和环境样品中的Fe3+和PPi的荧光探针具有重要意义。
发明内容
为解决目前可用于同时检测Fe3+及PPi的碳量子点荧光探针缺乏的问题,本发明提供了一种碳量子点荧光探针及其制备方法,以及在实际水样中的应用、细胞及斑马鱼成像中的应用。
为达到上述目的,本发明的技术方案如下:
本发明提供一种氮掺杂碳量子点荧光探针的合成方法,合成方法包括以下步骤:
(1)将柠檬酸与缩二脲溶于双蒸水中,超声30分钟,得到混合液;
(2)将上述混合液转移至聚四氟乙烯内衬的高压反应釜中,进行反应;
(3)将反应得到的黄色溶液冷却,离心,用0.22μm的滤膜过滤后再用透析袋透析,得到纯化后液体;
(4)将纯化后液体在真空条件下冻干得到黄色固体粉末,即为N掺杂碳量子点,将N掺杂碳量子点配置成1.0mg/mL的N-CDs溶液,储存在4℃的冰箱中备用。
作为本发明进一步地改进,步骤(1)中,所述柠檬酸与缩二脲的质量比为1:0.3~1:0.8,优选地,柠檬酸与缩二脲的质量比为1:0.49;所述柠檬酸与双蒸水的质量体积比为1:23.78~1:25(g/ml)。
作为本发明进一步地改进,步骤(2)中,所述反应的温度为180-210℃,反应时间为4~6小时,优选地,反应温度为200℃;反应时间为5小时。
作为本发明进一步地改进,步骤(3)中,所述冷却的温度为-40~-60℃,离心的转速为8000~10000rpm,离心的时间为15~30分钟。
作为本发明进一步地改进,步骤(3)中,所述透析袋的截留分子量为500-1000。
本发明还提供了一种N掺杂碳量子点荧光探针,通过上述N掺杂碳量子点荧光探针的合成方法制备得到,所述N掺杂碳量子点的粒径为4.28±0.5nm;所述N掺杂碳量子点的晶格条纹间距为0.21nm,与石墨的(100)面相似。
本发明还提供了一种氮掺杂碳量子点荧光探针在实际水样中检测Fe3+的应用。
本发明还提供了一种氮掺杂碳量子点荧光探针在细胞成像中的应用。
本发明还提供了一种氮掺杂碳量子点荧光探针在斑马鱼成像中的应用。
本发明的有益效果为:
(1)本发明中所用的氮掺杂试剂为缩二脲,相比之前的其他氮掺杂试剂,缩二脲在提高碳量子点荧光性能的同时,针对目标检测物Fe3+及PPi更具有靶向性。
(2)本发明碳量子点可以应用在自来水中的Fe3+的检测。
(3)本发明碳量子点可以实现在HeLa细胞中对Fe3+及PPi进行成像。
(4)本发明碳量子点可以实现在斑马鱼中对Fe3+及PPi进行成像。
附图说明
图1为本发明实施例1中所述氮掺杂碳量子点N-CDs的合成过程图;
图2为本发明N-CDs的透射电镜TEM图及粒径分布直方图;
图3为本发明N-CDs的傅里叶红外光谱图;
图4为本发明N-CDs的X射线光电子能谱图;
图5为本发明N-CDs对不同浓度Fe3+的荧光图;
图6为本发明N-CDs的不同浓度PPi的荧光图;
图7为本发明N-CDs在HeLa细胞中对Fe3+及PPi的成像图;
图8为本发明N-CDs在斑马鱼中对Fe3+及PPi的成像图。
具体实施方式
下面结合附图和具体实施方式,进一步阐明本发明,应理解下述具体实施方式仅用于说明本发明而不用于限制本发明的范围。
实施例1
一种氮掺杂碳量子点N-CDs的制备,合成过程如图1所示,具体步骤如下:
(1)称取0.6304g柠檬酸与0.3092g缩二脲,溶于15mL双蒸水中,超声30分钟;
(2)将上述混合物溶液转移至100mL聚四氟乙烯内衬的高压反应釜中,将反应釜置于200℃的环境中反应5小时;
(3)将反应所得到的黄色溶液冷却至室温,然后离心25分钟,除去大颗粒,再用0.22μm的滤膜过滤,最后再用透析袋透析24小时;
(4)最后将上述纯化后的液体在-50℃的真空条件下冻干得到氮掺杂碳量子点;
图2为氮掺杂碳量子点N-CDs的透射电镜TEM图及粒径分布直方图,在1.72到7.57nm的范围内可以观察到球形纳米粒子,平均尺寸为4.28±0.5nm。在HRTEM图像中观察到间距为0.21nm的清晰晶格条纹(图2插图),这与石墨的(100)平面中的晶格极为相似,表明该氮掺杂碳量子点具有优异的结晶度。
图3为氮掺杂碳量子点N-CDs的红外光谱图,从图中可以看出,在3000~3500cm-1处的特征吸收峰为OH/NH伸缩振动,1629cm-1处的特征吸收峰属于C=O伸缩振动,特征吸收峰为OH/NH伸缩振动,在1390cm-1对应于C-N的伸缩振动。此外,1101cm-1处的峰可归因于C-OH的伸缩振动。由此表明该碳量子点表面含有羟基、羧基、羰基和氨基,表明氮掺杂碳量子点制备成功。这些官能团不仅提高了碳量子点的亲水性和水溶液的稳定性,而且可以为金属离子提供作用位点。
图4为氮掺杂碳量子点N-CDs的X射线光电子能谱图(XPS),通过XPS进一步研究氮掺杂碳量子点的元素组成。如图4所示,可以观察到三个典型的峰(284.52eV、399.52eV、532.24eV),分别属于C1s、N1s和O1s。该碳量子点对应的元素含量为:C1s占61.73%,N1s占10.72%,O1s占27.55%。
实施例2
实施例1制备的氮掺杂碳量子点N-CDs用于Fe3+的检测,如图5所示,随着Fe3+浓度的增加(0、50、100、200μM),碳量子点的荧光强度逐渐下降,在0-30μM范围内,Fe3+浓度与碳量子点荧光强度呈现线性关系,线性回归方程为Y=-0.0021[Fe3+]+0.98407,相关系数R2=0.99291,检测限(LOD)为2.71μM。
实施例3
在实施例2体系中加入PPi,氮掺杂碳量子点N-CDs用于PPi的检测,如图6所示,随着PPi浓度的增加(0、20、80、150μM),荧光逐渐恢复,PPi浓度继续增加至200μM,荧光无明显变化,最终大约能恢复至猝灭前荧光强度的80%。在0-12μM范围内,PPi浓度与荧光强度呈现线性关系,线性回归方程为Y=0.01125[PPi]+1.00756,相关系数R2=0.99767,检测限(LOD)为1.12μM。
实施例4
实施例1中制备的氮掺杂碳量子N-CDs点在实际样品中的应用,利用标准加入法测定自来水中的Fe3+。结果如表1所示,其回收率在88.97%-105.43%之间,RSD在0.12%-1.06%之间,该方法证实了该碳量子点用于Fe3+检测的可行性,因此有望用于实际样品的检测。
表1
实施例5
氮掺杂碳量子点N-CDs在HeLa细胞中对Fe3+及PPi的成像研究。
为了证明氮掺杂碳量子点在生物系统的实际应用,在共聚焦荧光显微镜下对细胞进行了生物荧光成像实验。将HeLa细胞接到培养皿中在37℃条件下培养24h,HeLa细胞分成四组,与氮掺杂碳量子点N-CDs(10mM)溶液一起孵育30分钟,然后用不同浓度的Fe3+(0、50、100和200μM)进一步处理30分钟。使用激光共聚焦显微镜获得的图像如图7(a1-h1为蓝色通道,a2-h2为明场)所示,仅对N-CDs观察到亮蓝色荧光,随着Fe3+浓度从50μM增加到200μM,由于稳定的N-CDs-Fe3+复合物的形成,蓝色通道的荧光缓慢下降。随后,同样的实验过程被用于PPi的成像,随着PPi浓度的增加,由于Fe3+与PPi的结合远大于N-CDs,N-CDs从N-CDs-Fe3+复合物中释放出来,荧光逐渐增强。证明合成的氮掺杂碳量子点N-CDs可以实现活细胞内对Fe3+及PPi的成像。
实施例6
氮掺杂碳量子点N-CDs在斑马鱼中对Fe3+及PPi的成像研究。
基于活细胞中N-CDs成像的满意结果,我们进一步探索其在体内的应用。选择5天龄的斑马鱼作为成像模型。制备7组斑马鱼样品。斑马鱼用氮掺杂碳量子点N-CDs(10mM)预处理30分钟,然后在E3培养基中用0、50、100或200μM Fe3+和20、80、150μM PPi孵育1小时。然后通过激光共聚焦显微镜成像,激发波长为405nm。结果与Hela细胞相似(图8,a1-h1为蓝色通道,a2-h2为明场)。与N-CDs共培养的斑马鱼的成像在405nm激光脉冲下显示蓝色荧光。随着Fe3+的加入,蓝色通道荧光减弱,而PPi的加入,荧光恢复,表明N-CDs可作为荧光探针对Fe3+和PPi进行体内成像。
需要说明的是,以上内容仅仅说明了本发明的技术思想,不能以此限定本发明的保护范围,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰均落入本发明权利要求书的保护范围之内。
Claims (9)
1.一种氮掺杂碳量子点荧光探针的合成方法,其特征在于,合成方法包括以下步骤:
(1)将柠檬酸与缩二脲溶于双蒸水中,超声30分钟,得到混合液;
(2)将上述混合液转移至聚四氟乙烯内衬的高压反应釜中,进行反应;
(3)将反应得到的黄色溶液冷却,离心,用0.22μm的滤膜过滤后再用透析袋透析,得到纯化后液体;
(4)将纯化后液体在真空条件下冻干得到黄色固体粉末,即为N掺杂碳量子点,将N掺杂碳量子点配置成1.0mg/mL的N-CDs溶液,储存在4℃的冰箱中备用。
2.根据权利要求1所述的一种氮掺杂碳量子点荧光探针的合成方法,其特征在于,步骤(1)中,所述柠檬酸与缩二脲的质量比为1:0.3~1:0.8;所述柠檬酸与双蒸水的质量体积比为1:23.78~1:25。
3.根据权利要求1所述的一种氮掺杂碳量子点荧光探针的合成方法,其特征在于,步骤(2)中,所述反应的温度为180~210℃,反应时间为4~6小时。
4.根据权利要求1所述的一种N掺杂碳量子点荧光探针的合成方法,其特征在于,步骤(3)中,所述冷却的温度为-40~-60℃,离心的转速为8000~10000rpm,离心的时间为15~30分钟。
5.根据权利要求1所述的一种氮掺杂碳量子点荧光探针的合成方法,其特征在于,步骤(3)中,所述透析袋的截留分子量为500-1000。
6.一种氮掺杂碳量子点荧光探针,其特征在于,通过权利要求1-5任一项所述的N掺杂碳量子点荧光探针的合成方法制备得到,所述N掺杂碳量子点的粒径为4.28±0.5nm。
7.一种如权利要求6所述的氮掺杂碳量子点荧光探针在实际水样中检测Fe3+的应用。
8.一种如权利要求6所述的氮掺杂碳量子点荧光探针在细胞成像中的应用。
9.一种如权利要求6所述的氮掺杂碳量子点荧光探针在斑马鱼成像中的应用。
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