CN115615983A - 一种钼掺杂碳点纳米酶及其制备方法和应用 - Google Patents
一种钼掺杂碳点纳米酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种钼掺杂碳点纳米酶及其制备方法和应用,所述钼掺杂碳点纳米酶是以钼酸铵和抗坏血酸为原料,通过一步水热法制备得到,制备过程简便快速,原料来源广泛。钼掺杂碳点纳米酶表现出了高的类过氧化物酶活性,对H2O2和TMB均表现出较高的亲和力。通过Russell机理,钼掺杂碳点纳米酶可以在酸性条件下催化过氧化氢产生单线态氧,基于青霉素类抗生素可以和单线态氧发生反应,抑制其对TMB的氧化,从而实现显色信号的变化。因此,碳点纳米酶可以通过比色法用于青霉素类抗生素的分析检测。
Description
技术领域
本发明涉及碳点纳米材料,具体涉及一种钼掺杂碳点纳米酶及其制备方法,以及该碳点纳米酶作为探针用于青霉素类抗生素的灵敏检测。
背景技术
青霉素类抗生素是一类高效、低毒、临床应用广泛的抗生素。它的成功研制大大增强了人类抵抗细菌性感染的能力,它的出现开创了用抗生素治疗疾病的新纪元。然而抗生素的滥用也给人类健康与环境带来很多问题,在养殖业中滥用青霉素类抗生素,可能会引起肉、禽、蛋、奶等食品中抗生素残留,对人类健康产生潜在的威胁。而且水产养殖业中抗生素投放到水中仅有小部分为生物所利用,未被食用及食用后的排泄物长期存在于水体中。这些残留的抗生素不仅影响水体质量,而且对水蚤和微生物种群有不良作用。因此,建立快速有效的检测青霉素类抗生素的方法具有重要意义。
酶是极其重要的生物催化剂,具有底物特异性和优异的催化效率。然而由于容易变性和降解,催化效率有限,产物分离和纯化成本高,回收和再利用困难,天然蛋白酶的实际应用受大极大的限制。纳米酶是一种具有类酶活性的新型的功能性纳米材料。与天然酶相比,纳米酶具有催化效率高,活性稳定,活性易调节(调节纳米尺寸,表面修饰),可以规模化制备和成本低等优点,这使纳米酶在医学、化工、食品、农业和环境等领域得到广泛应用。
基于金属的纳米酶在电催化,传感和燃料电池领域脱颖而出。而碳点作为一种新兴的纳米材料,以其良好的生物相容性、低细胞毒性、优异的光物理性能、廉价的前驱体和简便的制备方法被广泛开发。将金属掺杂到碳点中构建纳米酶材料可以解决纳米酶聚集和高成本的问题。更重要的是,由于碳点良好的生物相容性,这种金属掺杂的碳点纳米酶具有极好的生物安全性。因此,通过简便的合成方法一步构建金属掺杂碳点纳米酶并开发其应用具有重要意义。
发明内容
为解决上述技术问题,本发明的目的在于提供一种性质优异的钼掺杂碳点纳米酶(Mo-CDs)及其制备方法和应用;所述制备方法简便,原料来源广泛;所制备的Mo-CDs可用于青霉素类抗生素的灵敏检测。
本发明提供的一种钼掺杂碳点纳米酶的制备方法,包括如下步骤:
1)、将0.20-1.20g四水合钼酸铵和0.23-1.40g抗坏血酸置于烧杯中,加入适量超纯水并超声至全部溶解;将混合物转移至聚四氟乙烯反应釜中并置于烘箱内,180-220℃反应9-15h,得到黄色碳点溶液;
2)、将反应釜自然冷却至室温,8000-12000rpm离心10-30min后取上清液,用0.22μm滤膜过滤,透析8-24h后得到黄色碳点纳米酶溶液;
3)、将上述钼掺杂碳点纳米酶溶液冷冻干燥后得到黄色的钼掺杂碳点纳米酶固体粉末。
上述方法制备的钼掺杂碳点纳米酶性质稳定,具有良好的水溶性和分散性,通过TEM表征可看出其形貌为单分散准球形颗粒,通过XPS表征可以看出Mo元素成功掺杂进碳点。所述碳点纳米酶制备方法简便快速,只需一步反应即可得到目标碳点,无需繁琐的纯化过程。该钼掺杂碳点纳米酶作为一种类过氧化物酶在酸性条件下,可以催化过氧化氢产生过氧自由基,并通过Russell机理自发发生重组生成单线态氧。相比于天然过氧化物酶——辣根过氧化酶,碳点纳米酶对底物H2O2和3,3’5,5’-四甲基联苯胺(TMB)均表现出更高的亲和力。米氏常数Km分别为0.1764mM和0.2446mM。基于青霉素类抗生素可以和单线态氧发生反应,抑制其对TMB的氧化,从而实现显色信号的变化。因此,碳点纳米酶可以通过比色法用于青霉素类抗生素的分析检测。以氨苄青霉素为例,该方法在氨苄青霉素(AMP)浓度为0.05μg/mL-100μg/mL范围内呈现出良好的线性相关性,ΔA652=0.00471[AMP]+0.00581,氨苄青霉素的最低检出限为12ng/ml。
本发明提供了一种用钼掺杂碳点纳米酶检测青霉素类抗生素的方法,具体检测步骤为:
1)、配置浓度为10mg/mL、pH=4的权利要求2所述钼掺杂碳点纳米酶溶液;
2)、配置浓度为50mM的H2O2溶液;浓度为25mM的3,3’5,5’-四甲基联苯胺(TMB)溶液;
3)、分别配置浓度为0,0.001,0.002,0.005,0.01,0.05,0.1,0.5,1,5,10,15,20,25,30,50mg/mL的青霉素类抗生素的标准储备溶液;
4)、将20μL 10mg/mL、pH=4的钼掺杂碳点纳米酶溶液,20μL不同浓度的青霉素类抗生素的标准储备溶液,40μL 25mM TMB溶液,20μL 50mM、pH=4的H2O2溶液依次加入1900μLpH=4的醋酸-醋酸钠缓冲溶液,放入40℃恒温器,反应10min,记录652nm处的紫外吸光谱变化,根据青霉素类抗生素的浓度和相对紫外吸收峰变化值之间的关系建立检测青霉素类抗生素的标准曲线,并计算检出限和线性范围;
5)、定量检测:测定待测样品与0.1mg/mL碳点纳米酶,0.5mM TMB溶液以及0.5mMH2O2放入40℃恒温器,反应10min后在652nm处的紫外吸光强度,通过步骤4)中获得的标准曲线,得到待测样品中青霉素类抗生素的含量。
本发明具有以下有益技术效果:
(1)本发明提供的钼掺杂碳点纳米酶通过一步水热法制备所得,制备过程简便快速,原料来源广泛,无需繁琐的纯化过程,合成的碳点纳米酶具有良好的生物相容性、低细胞毒性、优异的光物理性能。
(2)本发明提供的钼掺杂碳点纳米酶是一种类过氧化物酶,具有高活性,高底物特异性和高的催化稳定性,解决了常规金属纳米酶聚集和高成本的问题,并对H2O2和TMB均表现出良好的亲和力。
(3)本发明提供的钼掺杂碳点纳米酶在酸性条件下催化过氧化氢可有效生成单线态氧。基于活性氧对青霉素类抗生素的清除作用,碳点纳米酶可用于青霉素类抗生素的分析检测。本发明提供的检测青霉素类抗生素的比色分析法具有良好的选择性和灵敏度。
附图说明
图1为实施例1制备的碳点纳米酶的透射电镜图(TEM);
图2为实施例1制备的碳点纳米酶的红外光谱图;
图3为实施例1制备的碳点纳米酶的X射线光电子能谱图(XPS);
图4为不同反应体系(a)碳点纳米酶+TMB+H2O2,(b)碳点纳米酶+H2O2,(c)碳点纳米酶+TMB,(d)TMB+H2O2,(e)碳点纳米酶,(f)H2O2,(g)TMB的紫外吸收光谱;
图5为实施例1制备的碳点纳米酶检测青霉素类抗生素的选择性对比图,图中抗生素包含氨苄青霉素(AMP),青霉素G钠(PG),青霉素V钾(PVK),阿莫西林(AMX),盐酸环丙沙星(NA),诺氟沙星(NFX),左氧氟沙星(LVLX),红霉素(ERY),土霉素(OTC),链霉素(SM),甲硝唑(MTZ),奥硝唑(ONZ),替硝唑(TNZ);
图6为实施例1制备的碳点纳米酶与不同浓度氨苄青霉素反应后的紫外吸光变化图(A)及其在0.05 -100μg/mL范围内的线性关系图(B);
图7为实施例1制备的碳点纳米酶与不同浓度氨苄青霉素反应后不同显色的试纸条;
图8为颜色识别软件上对应的颜色信息亮度L和饱和度S(L/S)与氨苄青霉素浓度的关系图。
具体实施方式
下面结合实施例对本发明做详细说明,实施例给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1
碳点纳米酶的制备方法:
1)、分别取0.61g四水合钼酸铵和0.70g抗坏血酸置于烧杯中,用20mL超纯水超声至全部溶解。将反应混合物转移至水热反应釜中,置于烘箱内,200℃条件下反应12h,得到黄色碳点溶液;
2)、取出水热反应釜,自然冷却至室温,10000rpm离心20min后取上清液,用0.22μm滤膜过滤,透析12h(MW:500D)后得到黄色碳点纳米酶溶液;
3)、将上述碳点纳米酶水溶液冷冻干燥后得到黄色的碳点纳米酶固体状粉末。
实施例2
将实施例1制备的碳点纳米酶进行TEM扫描,红外吸收光谱和XPS能谱表征(见图1-3),结果表明碳点纳米酶平均粒径在3.49nm左右,呈均匀的球形分散性分布,主要含有C、H、O、N、Mo五种元素。
实施例3
配置实施例1制备的钼掺杂碳点纳米酶溶液(浓度为0.1mg/mL,醋酸—醋酸钠缓冲溶液pH=4),与H2O2(0.5mM)和TMB(0.5mM),40℃反应10min后,扫描紫外吸收光谱,在652nm出现了显著的吸收峰(图4),表明碳点纳米酶具有较高的类过氧化物酶活性,可以催化氧化TMB变为蓝色产物oxTMB。
实施例4
取20μL 10mg/mL的钼掺杂碳点纳米酶溶液,20μL 50mg/mL的各种抗生素标准液,40μL 25mM TMB溶液,20μL 50mM的H2O2溶液依次加入1900μL pH=4的醋酸-醋酸钠缓存溶液,放入40℃恒温器,反应10min,记录652nm处的紫外吸光度变化。如图5所示,与其他种类抗生素相比,有青霉素类抗生素存在时,652nm处的紫外吸光度明显降低,表明钼掺杂碳点纳米酶在酸性条件下催化H2O2产生的活性氧可以与青霉素类抗生素发生,因此可以将该反应体系用于青霉素类抗生素的分析检测。
实施例5
将20μL 10mg/mL的钼掺杂碳点纳米酶溶液,20μL不同浓度的氨苄青霉素溶液,40μL 25mM TMB溶液,20μL 50mM的H2O2溶液依次加入1900μL pH=4的醋酸-醋酸钠缓冲溶液中,放入40℃恒温器反应10min,记录652nm处的紫外吸光度变化值(图6A)。根据青霉素类抗生素的浓度和吸光度变化值之间的关系,建立检测青霉素类抗生素的标准曲线,并计算检出限和线性范围。该方法在0.05μg/mL—100μg/mL范围内呈现出良好的线性关系,ΔA652=0.00471[AMP]+0.00581,氨苄青霉素的最低检出限为12ng/mL(图6B)。
实施例6
建立用于青霉素类抗生素检测的试纸条。以氨苄青霉素为例,首先将滤纸切成1cm*3cm条形。将Mo-CDs,TMB和H2O2溶液均匀地喷洒在滤纸表面,10分钟后多孔滤纸呈现蓝色。随后将不同浓度的氨苄青霉素滴加在滤纸上,待反应10分钟后通过智能手机摄像头拍摄试纸条的颜色(图7)。最后通过智能手机上颜色识别软件分析图像,将其转换为相应的HSL值,其中H、L和S分别表示色调、亮度和饱和度。以L和S的比值(L/S)为纵坐标,以氨苄青霉素浓度为横坐标,建立定量检测的线性关系L/S=0.0585C+0.1810(R2=0.992)(图8)。
Claims (7)
1.一种钼掺杂碳点纳米酶的制备方法,包括如下步骤:
1)、将0.20-1.20g四水合钼酸铵和0.23-1.40g抗坏血酸置于烧杯中,加入适量超纯水并超声至全部溶解;将混合物转移至聚四氟乙烯反应釜中并置于烘箱内,180-220℃反应9-15h,得到黄色碳点溶液;
2)、将反应釜自然冷却至室温,8000-12000rpm离心10-30min后取上清液,用0.22μm滤膜过滤,透析8-24h后得到黄色碳点纳米酶溶液;
3)、将上述钼掺杂碳点纳米酶溶液冷冻干燥后得到黄色的钼掺杂碳点纳米酶固体粉末。
2.如权利要求1所述方法制备的钼掺杂碳点纳米酶。
3.如权利要求2所述的钼掺杂碳点纳米酶作为检测青霉素类抗生素试剂的应用。
4.如权利要求3所述的钼掺杂碳点纳米酶在检测青霉素类抗生素中的应用,所述的青霉素类抗生素为氨苄青霉素、青霉素V钾、青霉素G钠或阿莫西林。
5.一种钼掺杂碳点纳米酶检测青霉素类抗生素的方法,其特征在于步骤为:
1)、配置浓度为10mg/mL、pH=4的权利要求2所述钼掺杂碳点纳米酶溶液;
2)、配置浓度为50mM的H2O2溶液;浓度为25mM的3,3’5,5’-四甲基联苯胺(TMB)溶液;
3)、分别配置浓度为0,0.001,0.002,0.005,0.01,0.05,0.1,0.5,1,5,10,15,20,25,30,50mg/mL的青霉素类抗生素的标准储备溶液;
4)、将20μL 10mg/mL、pH=4的钼掺杂碳点纳米酶溶液,20μL不同浓度的青霉素类抗生素的标准储备溶液,40μL 25mM TMB溶液,20μL 50mM、pH=4的H2O2溶液依次加入1900μL pH=4的醋酸-醋酸钠缓冲溶液,放入40℃恒温器,反应10min,记录652nm处的紫外吸光谱变化,根据青霉素类抗生素的浓度和相对紫外吸收峰变化值之间的关系建立检测青霉素类抗生素的标准曲线,并计算检出限和线性范围;
5)、定量检测:测定待测样品与0.1mg/mL碳点纳米酶,0.5mM TMB溶液以及0.5mM H2O2放入40℃恒温器,反应10min后在652nm处的紫外吸光强度,通过步骤4)中获得的标准曲线,得到待测样品中青霉素类抗生素的含量。
6.一种青霉素类抗生素检测试纸条,其特征在于含有如权利要求2所述的钼掺杂碳点纳米酶。
7.如权利要求6所述的试纸条在青霉素类抗生素检测中的应用。
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