CN112604684B - 一种金碳复合纳米颗粒模拟酶及其制备方法 - Google Patents
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Abstract
本发明公开了一种树枝状金碳复合纳米颗粒模拟酶的制备。其纳米颗粒形貌呈树枝状结构,纳米颗粒组成Au/C原子比为1.07:84.64,平均粒径为68±15 nm。将柠檬酸溶于水后加入乙二胺搅拌均匀,升温至200℃,恒温反应5小时,将得到的碳溶液加水稀释并超声分散,随后向碳的水溶液中加入氯金酸和AA溶液(氯金酸与AA的摩尔比为1:5),30℃下静置一个小时后,反应结束后离心分离,洗涤得到金碳复合纳米颗粒,分散到水中得到得到金碳纳米粒子模拟酶溶液。所得树枝状金碳复合纳米颗粒模拟酶在酸性环境下具有很好的类酶活,作为一种新颖模拟酶在免疫分析、生物检测及临床诊断等领域都有潜在的应用价值。
Description
技术领域
本发明属于催化材料和分析化学领域,具体涉及一种金碳复合纳米颗粒模拟酶及其制备方法。
背景技术
酶是催化反应的生物分子之一,具有催化的高选择性和加速化学反应的特点。然而,天然酶的固有缺点如制备与纯化成本较高,稳定性低,存储寿命短,此外还严重依赖于温度,pH,反应环境等限制着天然酶的发展。为了克服这些局限性,投入大量精力来开发高度稳定,催化可调控,低成本且易于合成,结构涵盖从分子领域到无机纳米材料的酶模拟物。纳米酶是具有两种独特特性的酶模拟物,它兼具纳米材料的特性和天然酶的催化活性,因此,纳米酶材料和结构已广泛用于医药,化工,食品安全,农业和环境管理等领域。
近年来,随着纳米科技的兴起和迅速发展,纳米材料表现出许多与块体材料显著不同的新奇性质。自首次报道合成具有类过氧化物酶的四氧化三铁(Fe3O4)磁性纳米粒子(NPs)并应用于酶联免疫分析以来,越来越多的表现出优异的类酶活性并基于纳米材料的酶模拟物相继被报道。例如贵金属NPs,金属氧化物NPs,金属硫化物NPs,碳基纳米材料和其他一些纳米复合材料等。
此外,我们课题组还报道了Au@Pt、AuPt、AgPd、AgPt等双金属纳米颗粒具有类过氧化物酶、类氧化酶、类过氧化氢酶等特征,并开发了它们在生物检测中的应用。这些纳米材料合成方法简单,催化过程可控可调节,对环境的依赖性较低。他们优异的类酶催化活性的可能归因于大小与天然酶相当,具有很好分散度和高比表面积是几个关键因素,对后续的纳米酶材料的设计提供了新的思路。
当金属材料的尺寸减小到纳米级尺寸,所得的含纳米粒子变为最有效的催化剂之一,并已被广泛用于催化。我们认为不论是金属自身还是作为金属与金属的复合结构,具有的类酶活性源于自身独特的属性。
发明内容
本发明目的是提供一种树枝状金碳复合纳米颗粒模拟酶,它具有光调控类氧化物酶活性的特征,可用作氧化物模拟酶。
为达到上述目的,采用技术方案如下:
金碳复合纳米颗粒模拟酶,其纳米颗粒形貌呈树枝状结构,纳米颗粒组成Au/C的原子比为1.07: 84.64,平均粒径为68 ± 15 nm。
上述金碳复合纳米颗粒模拟酶的制备方法,包括以下步骤:
1)将柠檬酸溶于中,随后加入乙二胺搅拌均匀,升温至200℃,恒温反应5小时;
2)将得到的碳溶液加水稀释并超声分散,随后向碳的水溶液中加入氯金酸和AA溶液(氯金酸与AA的摩尔比为1:5);
3)30℃下静置一个小时后,反应结束后离心分离,洗涤得到金碳复合纳米颗粒,分散到水中得到金碳纳米粒子模拟酶溶液。
按上述方案,碳的水溶液的稀释比例为5mL的水中加入400微升的纯碳溶液。
按上述方案,所用原料氯金酸溶液与AA溶液的物质的量之比为1:5。
按上述方案,离心转速为8000-9000 rpm/min,时长5-10min。
本发明树枝状金碳复合纳米颗粒模拟酶作为氧化酶可以在没有H2O2存在的情况下可与3,3’,5,5’-四甲基联苯胺(TMB)发生显色反应,将TMB氧化为蓝色,证实金碳复合纳米颗粒模拟酶具有类氧化物酶活性的特征,可用作氧化物模拟酶。
本发明相对于现有技术,有益效果如下:
本专利申请中设计金属-碳这一结构纳米颗粒,突破了金颗粒自身不具有类酶活性的属性,与碳的复合形式,也增加了催化过程中的电子转移,在可见光范围内提供广泛的光学响应结合出色的热化学性能稳定性,将催化过程变得可控可调节。
两步合成了金碳复合纳米颗粒模拟酶溶液,合成方法与操作相对简单、高效、重复性高,所用试剂无毒,反应中产生的产物和副产物也对环境友好。
所得金碳复合纳米颗粒模拟酶溶液在不存在过氧化氢的情况下对有机底物3,3’,5,5’-四甲基联苯胺具有光激发催化氧化活性,表现出光调控的类氧化酶活性特征。
附图说明
图1是实施例1中金碳复合纳米颗粒的透射电镜照片;
图2是不同金含量的金碳复合纳米颗粒与金颗粒、碳溶液等不同纳米颗粒在光照作用下催化TMB的氧化反应的活性对比图;
图3 是不同金含量的金碳复合纳米颗粒与金颗粒、碳溶液等不同纳米颗粒在不同光源下催化TMB 的氧化反应的活性对比图;
附图4是金碳复合纳米颗粒模拟酶在非小细胞肺癌细胞上抗肿瘤实验用酶联仪测定细胞的吸光度对比图。
具体实施方式
以下实施例进一步阐释本发明的技术方案,但不作为对本发明保护范围的限制。
实施例1
金碳复合纳米颗粒模拟酶的制备:
将1.05克柠檬酸溶解于10mL的去离子水中,磁力搅拌15分钟后加入0.335mL乙二胺继续搅拌15分钟后,将混合溶液转移至50mL聚四氟乙烯反应釜中200℃反应5小时。将所得到的棕黑色溶液12000 rpm/min高速离心,去除大颗粒物。
将得到的碳溶液加水稀释并超声分散,随后向碳的水溶液中加入氯金酸和AA溶液(氯金酸与AA的摩尔比为1:5),30℃下静置一个小时后,反应结束后将得到的溶液8000rpm/min高速离心,两次洗涤得到金碳复合纳米颗粒,分散到水中得到金碳纳米粒子模拟酶溶液。
附图1为本实施例所得金碳复合纳米结构的透射电子显微镜图。金碳复合纳米颗粒的形貌为树枝状结构。金碳复合纳米颗粒的尺寸粒径为68 ± 15nm。
金碳合金纳米颗粒模拟酶溶液的类酶活性的比色测定步骤如下:
类氧化酶特征测试:取5.7ml 去离子水,依次向其中加入60µl 20mM 3,3’,5,5’-四甲基联苯胺(TMB)和500µl 1.8mM金碳复合纳米颗粒模拟酶溶液(实施例1制得的纳米酶颗粒分散于2mL的去离子水中所得到的溶液),然后将上述溶液混合均匀;室温(25℃)下放置在带有420截止滤光片的光源下10-25分钟后,即可观察到溶液从无色变为蓝色,这些表明金碳复合纳米颗粒模拟酶对水溶液中的溶解氧氧化底物3,3’,5,5’-四甲基联苯胺有很高的催化活性,表明本发明的金碳复合纳米颗粒模拟酶具有类似氧化酶的特征,可作为氧化酶模拟酶。
附图2分别是不同金含量的金碳复合纳米颗粒与金颗粒、碳溶液等不同纳米颗粒催化TMB的氧化反应的活性对比图。从图中可以看出,与单独的金颗粒与碳溶液相比相比,金碳复合纳米颗粒催化TMB 的效果最为显著。不同的金含量的金碳纳米复合模拟酶表现出不同的催化活性,在催化TMB的实验中,且随着金含量从0.6mM到1.8mM的增加,类氧化物酶表现越为优异,但是金的含量达到1.8mM后,类氧化物酶的活性会有下降的趋势。
附图3分别是不同金含量的金碳复合纳米颗粒与金颗粒、碳溶液等不同纳米颗粒在不同光源下催化TMB 的氧化反应的活性对比图。浓度相同的溶液在三种不同的光源下进行TMB催化实验,与520带通,700带通光源相比,在420截止光源下可以达到最优的催化效果。
附图4是金碳复合纳米颗粒模拟酶在非小细胞肺癌细胞上抗肿瘤实验,在96孔板上先进行细胞培养,再将金碳复合纳米颗粒均匀涂布在细胞上,培养四个小时后,用660激光照射,待进行12小时后,用酶联仪测定细胞的吸光度。
实施例2
将1.05克柠檬酸溶解于10mL的去离子水中,磁力搅拌15分钟后加入0.335mL乙二胺继续搅拌15分钟后,将混合溶液转移至50mL聚四氟乙烯反应釜中200℃反应5小时。将所得到的棕黑色溶液12000 rpm/min高速离心,去除大颗粒物。
将得到的碳溶液加水稀释并超声分散,随后向碳的水溶液中加入氯金酸和AA溶液(氯金酸与AA的摩尔比为1:10),30℃下静置一个小时后,反应结束后将得到的溶液9000rpm/min高速离心,两次洗涤得到金碳复合纳米颗粒,分散到水中得到金碳纳米粒子模拟酶溶液。
实施例3
将1.05克柠檬酸溶解于10mL的去离子水中,磁力搅拌15分钟后加入0.335mL乙二胺继续搅拌15分钟后,将混合溶液转移至50mL聚四氟乙烯反应釜中200℃反应5小时。将所得到的棕黑色溶液12000 rpm/min高速离心,去除大颗粒物。
将得到的碳溶液加水稀释并超声分散,随后向碳的水溶液中加入氯金酸和AA溶液(氯金酸与AA的摩尔比为1:7),30℃下静置一个小时后,反应结束后将得到的溶液8500rpm/min高速离心,两次洗涤得到金碳复合纳米颗粒,分散到水中得到金碳纳米粒子模拟酶溶液。
由上可知,本发明制备的树枝状金碳复合纳米颗粒溶液,具有类似氧化酶的催化功能,可作为一种新颖的氧化物模拟酶。
Claims (4)
1.一种金碳复合纳米颗粒模拟酶,其特征在于:所述金碳复合纳米颗粒模拟酶具有光激发氧化活性;纳米颗粒形貌呈树枝状结构,纳米颗粒组成Au/C的原子比为84.64:1.07,平均粒径为68±15nm,所述金碳复合纳米颗粒模拟酶的制备方法,包括以下步骤:
1)将柠檬酸溶于去离子水中,随后加入乙二胺搅拌均匀,升温至200℃,恒温反应5小时,将上述反应所得棕黑色溶液在12000rpm/min高速离心,去除大颗粒物,得到所需碳溶液;
2)将得到的碳溶液加水稀释并超声分散,随后向碳的水溶液中加入氯金酸和AA溶液;
3)30℃下静置一个小时后,反应结束后离心分离,洗涤得到金碳复合纳米颗粒,分散到水中得到金碳复合纳米颗粒模拟酶溶液;
其中碳的水溶液的稀释比例为5mL的水中加入400微升的纯碳溶液,氯金酸溶液与AA溶液的物质的量之比为1:5-10。
2.如权利要求1所述金碳复合纳米颗粒模拟酶,其特征在于:步骤3)中离心转速为8000-9000rpm/min,时长5-10min。
3.如权利要求1所述金碳复合纳米颗粒模拟酶作为光激发氧化物模拟酶应用。
4.一种利用权利要求1所述金碳复合纳米颗粒模拟酶在非小细胞肺癌细胞上抗肿瘤实验的方法,包括以下步骤:
在96孔板上先进行细胞培养,再将金碳复合纳米颗粒模拟酶均匀涂布在细胞上,培养四个小时后,用660激光照射,待进行12小时后,用酶联仪测定细胞的吸光度。
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