CN116239101A - 具有近红外荧光及超氧化物歧化酶活性碳点纳米酶及其制备方法和应用 - Google Patents
具有近红外荧光及超氧化物歧化酶活性碳点纳米酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶及其制备方法和应用。所述制备方法利用磺酸基与酰氯的反应,在碳纤维粉与强酸共反应得到的原料碳点的基础上,修饰具有近红外荧光性质的小分子,获得同时具有近红外荧光及超氧化物歧化酶活性的碳点纳米酶。所述制备方法简单、产物具有近红外荧光性质及良好的超氧化物歧化酶活性,在细胞成像、氧化损伤保护、抗老化、抗炎治疗中具有广阔的应用前景。
Description
技术领域
本发明涉及新材料领域,特别涉及一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶及其制备方法和应用。
背景技术
纳米酶是一类具有类酶活性的纳米材料。目前纳米酶的催化类型涉及三大类,分别是氧化还原酶类、水解酶类、裂合酶类。其中,具有氧化还原酶活性的纳米酶,对活性氧簇(Reactive oxygen species,ROS)具有重要的调控作用。目前所报道的纳米酶大多数为金属氧化物(如氧化铁、氧化铜、氧化铈等)或贵金属(金、银、钯、铂等)类纳米材料,合成过程较为复杂、产率低不利于大批量生产,且稳定性不足,在生物医学应用时难以降解,而且可能释放金属离子而产生生物毒性,因此存在潜在的生物安全性问题。所以,发展毒性低、生物相容性好、酶活性高且可调的新型纳米酶具有十分重要的意义。
碳点,作为一种零维碳纳米材料合成方法简单、成本低廉、且具有独特的荧光性质。碳点的粒径小、比表面积大、导电性优异,可以作为电子供体和电子受体,有望可成为一类理想的可用于均相催化的碳纳米酶。目前的制备方法获得的碳点纳米酶,主要用于模拟天然的过氧化物酶(POD)活性,极大限制了其应用。此外,常见碳点的荧光范围难以到达近红外区间,难以应用于活体医学影像中,因此,需要采用新的制备方法和分离手段,获得具有新的酶催化能力,同时兼具良好近红外荧光性质的碳点纳米酶。
发明内容
针对现有技术中的缺陷,本发明提出了一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶及其制备方法和应用。所述制备方法利用磺酸基与酰氯的反应,在碳纤维粉与强酸共反应得到的原料碳点的基础上,修饰上具有近红外荧光性质的小分子,获得同时具有近红外荧光及超氧化物歧化酶活性的碳点纳米酶。
本发明提供一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶的制备方法,包括如下步骤:
(1)碳纤维粉与浓硝酸和浓硫酸反应得到原料碳点,所述原料碳点具有超氧化物歧化酶活性;
(2)利用磺酸基与酰氯的反应,实现对步骤(1)中所述原料碳点的进一步修饰,基于小分子的荧光,获得具有近红外荧光特性的碳点,所述具有近红外荧光特性的碳点仍然保持超氧化物歧化酶活性;所述小分子具有近红外荧光性质;
(3)分离步骤(2)的反应产物,同时收集产物,纯化得到所述具有近红外荧光及超氧化物歧化酶活性碳点纳米酶。
进一步的,所述步骤(1)中将碳纤维粉与浓硝酸、浓硫酸均匀混合,加热沸腾,冷凝回流反应后冷却;冷却溶液调节pH为5~6;过滤后浓缩,透析后收集冻干,得到所述原料碳点。
进一步的,所述调节pH步骤使用NaHCO3固体。
进一步的,所述步骤(2)中称取步骤(1)制备得到的原料碳点粉末加入无水乙腈分散,再加入二氯亚砜,加热或回流后,蒸干溶剂,得到酰氯化碳点;所述酰氯化碳点加水分散后,过滤,将磺化Cy5.5-伯胺溶于无水N,N-二甲基甲酰胺后加入所述酰氯化碳点水溶液中,室温搅拌反应过夜;旋转蒸发反应产物后得到具有超氧化物歧化酶活性的近红外荧光碳点。
进一步的,所述酰氯化碳点和磺化Cy5.5-伯胺的质量比为5~20:1。
进一步的,所述步骤(3)的分离步骤采用琼脂糖凝胶电泳;所述纯化步骤采用透析的方法。
进一步的,所述琼脂糖凝胶电泳使用1%琼脂糖凝胶,电压为135V。
本发明还提供一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶,由所述的制备方法制备得到。
本发明还提供所述的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶在细胞成像、氧化损伤保护、抗老化、抗炎治疗中的应用。
综上,与现有技术相比,本发明达到了以下技术效果:
本发明制备碳点的方法简便,利用磺酸基与酰氯的作用,借助染料分子赋予碳点近红外荧光的优良特性,同时保持碳点超氧化物歧化酶活性,可高效制备得到同时具有近红外荧光和超氧化物歧化酶活性特性的碳点纳米酶,制备方法具有普适性,制备得到的具有近红外荧光和超氧化物歧化酶活性特性的碳点纳米酶在氧化损伤保护、抗老化、抗炎治疗中具有广阔的应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,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相关的附图。
图1为本发明的方法中利用凝胶电泳进行分离的分离结果图。
图2为本发明的方法合成碳点纳米酶的紫外吸收表征图。
图3为本发明的方法合成碳点纳米酶的荧光光谱图。
图4为本发明的原料碳点SOD-Cdot和具有近红外荧光的Cy5.5-SOD-Cdot的超氧化物歧化酶活性检测图。
图5为本发明的方法制备得到的碳点纳米酶用于细胞成像的结果。
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方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
本发明利用磺酸基与酰氯的反应,在碳纤维粉与强酸共反应得到的原料碳点的基础上,修饰上具有近红外荧光性质的小分子,获得同时具有近红外荧光及超氧化物歧化酶活性的碳点纳米酶。通过高普适性方法制备得到同时具有近红外荧光和超氧化物歧化酶活性的碳纳米材料,制备方法简单、材料具有近红外荧光性质及良好的超氧化物歧化酶活性,在细胞成像、氧化损伤保护、抗老化、抗炎治疗等实际应用中具有广阔的前景。
本发明的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶的制备方法,包括以下步骤:
(1)碳纤维粉与浓硝酸和浓硫酸反应得到原料碳点,所述原料碳点具有超氧化物歧化酶活性;
(2)利用磺酸基与酰氯的反应,实现对步骤(1)中所述原料碳点的进一步修饰,基于小分子的荧光,获得具有近红外荧光特性的碳点,所述具有近红外荧光特性的碳点仍然保持超氧化物歧化酶活性;所述小分子具有近红外荧光性质。
(3)利用凝胶电泳法或透析法分离步骤(2)的反应产物,优选为琼脂糖凝胶电泳,比透析用时短,节约时间;同时用凝胶电泳法收集产物,纯化得到所述具有近红外荧光及超氧化物歧化酶活性碳点纳米酶。
除非特别说明,本发明采用的试剂、方法和设备为本领域常规试剂、方法和设备。
实施例1
本发明的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶的制备方法,具体包括如下步骤:
(1)将碳纤维粉与浓硝酸、浓硫酸按照质量:体积:体积=1:50:25的比例均匀混合,加热沸腾,保持冷凝回流反应2h后冷却。冷却溶液中加入NaHCO3固体调节pH为5~6之间。0.22μm滤膜过滤后浓缩到适量体积,转入3.5kDa透析袋中,超纯水里透析5天后收集冻干,得到原料碳点,命名为SOD-Cdot。
(2)冻干粉末进行酰氯化反应,具体步骤为:称取5mg原料碳点粉末加入10mL无水乙腈分散,再加入1-2mL二氯亚砜,加热或回流一定时间后,蒸干所有溶剂,得到酰氯化碳点。酰氯化碳点加水分散后,过0.22μm滤膜,再按照酰氯化碳点:磺化Cy5.5-伯胺质量比为10:1的比例(也可为5:1、20:1等任意比例),将磺化Cy5.5-伯胺溶于无水N,N-二甲基甲酰胺(DMF)后加入酰氯化碳点水溶液中,室温搅拌反应过夜。旋转蒸发反应产物后得到具有超氧化物歧化酶活性的近红外碳点。
(3)分离纯化:配制1%琼脂糖凝胶,135V,30min电泳分离,即可将未反应酰氯化碳点、未反应磺化Cy5.5-伯胺、产物近红外碳点分离开。如图1所示,图1为本方发中利用凝胶电泳进行分离的分离结果图,其中1为产物Cy5.5-SOD-Cdot;2和3均为未结合染料Cy5.5;4为对照原料碳点SOD-Cdot。说明通过电泳的方法再利用电泳收集近红外碳点组分,超纯水透析1天后冻干,得到纯度较高的具有超氧化物歧化酶活性的近红外碳点纳米酶,命名为Cy5.5-SOD-Cdot。
实施例2
对实施例1制备得到的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶进行紫外吸收光谱和荧光光谱的表征。结果如图2和图3所示。图2为本发明的方法合成碳点纳米酶的紫外吸收表征图。图3为本方法合成碳点纳米酶的荧光光谱图。SOD-Cdot代表对照的原料碳点,Cy5.5-SOD-Cdot代表本发明的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶。由图中结果可知,本发明合成的碳点纳米酶具有近红外荧光的优良特性。
实施例3
对实施例1制备的碳点纳米酶进行超氧化物歧化酶活性检测,酶活检测法使用超氧化物歧化酶检测试剂盒为Dojindo S311-10;所用样品的终浓度为0-20μg/mL。结果如图4所示,SOD-Cdot代表对照的原料碳点,Cy5.5-SOD-Cdot代表本发明的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶。检测结果可知SOD-Cdot的超氧化物歧化酶活性为27126U/mg,Cy5.5-SOD-Cdot的超氧化物歧化酶活性可高达7180U/mg。说明本发明制备的碳点纳米酶具有超高的超氧化物歧化酶活性。
综合实施例2和实施例3的结果,说明了本发明的制备方法能够成功制备得到同时具有近红外荧光的优良特性和超高的超氧化物歧化酶活性的碳点纳米酶。
实施例4
为了验证本发明制备得到的碳点纳米酶是否可以应用于细胞成像,将一定量的Cy5.5-SOD-Cdot加入巨噬细胞RAW264.7中,培养过夜,洗去培养基后用荧光显微镜观察。结果如图5所示,从左到右分别为明场、荧光场、叠加;标尺为100μm,其中荧光场中激发滤光片为620/60nm,发射滤光片为700/75nm。结果可见,有红色荧光显示在巨噬细胞RAW264.7内,说明本发明的Cy5.5-SOD-Cdot碳点纳米酶可进入细胞并应用于细胞成像。
综合以上实施例,本发明公开了一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶及其制备方法和应用。所述制备方法利用磺酸基与酰氯的反应,在碳纤维粉与强酸共反应得到的原料碳点的基础上,修饰上具有近红外荧光性质的小分子,获得同时具有近红外荧光及超氧化物歧化酶活性的碳点纳米酶。所述制备方法简单,产物具有近红外荧光性质及良好的超氧化物歧化酶活性,在细胞成像、氧化损伤保护、抗老化、抗炎治疗等实际应用中具有广阔的前景。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶的制备方法,其特征在于,包括如下步骤:
(1)碳纤维粉与浓硝酸和浓硫酸反应得到原料碳点,所述原料碳点具有超氧化物歧化酶活性;
(2)利用磺酸基与酰氯的反应,实现对步骤(1)中所述原料碳点的进一步修饰,基于小分子的荧光,获得具有近红外荧光特性的碳点,所述具有近红外荧光特性的碳点仍然保持超氧化物歧化酶活性;所述小分子具有近红外荧光性质;
(3)分离步骤(2)的反应产物,同时收集产物,纯化得到所述具有近红外荧光及超氧化物歧化酶活性碳点纳米酶。
2.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)中将碳纤维粉与浓硝酸、浓硫酸均匀混合,加热沸腾,冷凝回流反应后冷却;冷却溶液调节pH为5~6;过滤后浓缩,透析后收集冻干,得到所述原料碳点。
3.根据权利要求2所述的制备方法,其特征在于,所述调节pH步骤使用NaHCO3固体。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤(2)中称取步骤(1)制备得到的原料碳点粉末加入无水乙腈分散,再加入二氯亚砜,加热或回流后,蒸干溶剂,得到酰氯化碳点;所述酰氯化碳点加水分散后,过滤,将磺化Cy5.5-伯胺溶于无水N,N-二甲基甲酰胺后加入所述酰氯化碳点水溶液中,室温搅拌反应过夜;旋转蒸发反应产物后得到具有超氧化物歧化酶活性的近红外荧光碳点。
5.根据权利要求4所述的制备方法,其特征在于,所述酰氯化碳点和磺化Cy5.5-伯胺的质量比为5~20:1。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤(3)的分离步骤采用琼脂糖凝胶电泳;所述纯化步骤采用透析的方法。
7.根据权利要求6所述的制备方法,其特征在于,所述琼脂糖凝胶电泳使用1%琼脂糖凝胶,电压为135V。
8.一种具有近红外荧光及超氧化物歧化酶活性碳点纳米酶,其特征在于,由权利要求1~7任一项所述的制备方法制备得到。
9.权利要求8所述的具有近红外荧光及超氧化物歧化酶活性碳点纳米酶在细胞成像、氧化损伤保护、抗老化、抗炎治疗中的应用。
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