CN111439783B - 一种钒酸银其制备方法及作为类过氧化物酶检测尿酸的应用 - Google Patents

一种钒酸银其制备方法及作为类过氧化物酶检测尿酸的应用 Download PDF

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CN111439783B
CN111439783B CN202010153953.7A CN202010153953A CN111439783B CN 111439783 B CN111439783 B CN 111439783B CN 202010153953 A CN202010153953 A CN 202010153953A CN 111439783 B CN111439783 B CN 111439783B
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董雯佳
郑来宝
庄筱红
相振波
楼永良
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Abstract

本发明公开了一种钒酸银其制备方法及作为类过氧化物酶检测尿酸的应用，其分子式为Ag2V4O11。本发明的优点和有益效果是：第一次证明了所合成的Ag2V4O11纳米带具有类过氧化物酶活性，与单纯的采用贵金属作为模拟酶检测相比降低了成本。

Description

一种钒酸银其制备方法及作为类过氧化物酶检测尿酸的应用
技术领域
本发明涉及生物检测技术领域和复合氧化物领域，具体是指钒酸银其制备方法及作为类过氧化物酶检测尿酸的应用。
背景技术
过氧化物酶(Horseradish peroxidase，简称HRP)是由微生物或植物所产生的一类氧化还原酶，是以过氧化氢为电子受体催化底物氧化的酶。HRP是免疫酶标技术中最为常用的工具酶之一，常用于标记抗体。HRP是一类具有高度生物催化作用的蛋白质，因此，其结构易受环境变化的影响，从而导致酶失活。除此之外，HRP存在难提纯故成本高、不易保存和不利于抗体和抗原结合等缺点。这些缺点同样限制了它的大范围应用。
现有技术中，具有类过氧化物酶的材料(即模拟酶)主要可以分为以下四类：1)铁基纳米材料(BiFeO3(，CoFe2O4，MnFe2O4，FeS，FeSe等)；2)非铁金属(CeO2，MnO2，CuO(Chenet al.，2012b)，Co3O4，V2O5，CuS，CdS等)；3)贵金属纳米材料(纳米金，纳米铂，Ag3PO4纳米晶体等)；4)碳基纳米材料(碳纳米管，氧化石墨烯等)。这其中，贵金属纳米材料因为其独特的理化性质及良好的催化特性吸引了人们更多的关注。然而贵金属纳米材料自身同样存在原料价格较高，纳米材料易团聚及难以回收等缺点，这些缺点都限制了其进一步应用。
含有不同比例的银、钒、氧的复合氧化物统称为钒酸银，根据银、钒、氧比例的不同，钒酸银具有多种不同的组成与结构。钒酸银材料是一类广泛应用于光学、电学和催化等方面的功能材料，目前的研究报道主要是关于AgVO3、Ag3VO4、Ag2V4O11、Ag1.2V3O8及AgxV2O5。例如中国专利公开号为107376911A公开了一种α-钒酸银作为模拟酶的应用，该技术方案所涉及的钒酸银为AgVO3。钒酸银由于其独特的电子、光子和磁学特性，已经成为近年来的研究热点。钒酸银具有独特的晶体结构，其作为模拟酶在生物检测等领域的应用潜能尚未开发。
本申请所新合成的钒酸银为Ag2V4O11，该结构还未见报道也为应用于模拟酶在生物检测领域应用。
发明内容
为解决现有技术存在的问题和不足，本发明的目的是提供一种钒酸银其制备方法及作为类过氧化物酶检测尿酸的应用，本发明第一次证明了所合成的Ag2V4O11纳米带具有类过氧化物酶活性，与单纯的采用贵金属作为模拟酶检测相比降低了成本。
为实现上述目的，本发明的第一个方面是提供一种钒酸银，其分子式为Ag2V4O11。
本发明的第二个方面是提供一种钒酸银的制备方法，其特征在于包括以下步骤：
(1)将0.002mol NH4VO3和0.001mol AgNO3在磁力搅拌的条件下分别溶解在50和20mL去离子水中；
(2)将(1)中所得AgNO3溶液在磁力搅拌的条件下逐滴加入到NH4VO3溶液中；
(3)用1mol/L HNO3溶液调节(2)所得溶液的pH至3；
(4)将反应溶液继续在室温下搅拌1h和熟化4h；
(5)将反应溶液转移到80mL水热釜中，150℃水热24h；
(6)反应溶液冷却至室温后，真空抽滤获得产物，并将产物用去离子水清洗多次后，放入烘箱中70℃干燥12h，即得产物分子式为Ag2V4O11的钒酸银。
本发明的第三个方面是一种如所述的钒酸银用于类过氧化物酶的应用。
本发明还提供一种如所述的钒酸银作为类过氧化物酶用作尿酸的的检测方法，
(a)10μL 5.0mg/mL尿酸酶与待测样品加入到200μL pH＝8.5的100mM PBS溶液中，然后将上述溶液在37℃水浴保温30分钟；
(b)分别加入10μL 1mg mL-1的Ag2V4O11悬浊液，160μL 8mM TMB溶液和1620μL100mM PBS缓冲溶液，该缓冲溶液pH＝5.0，上述溶液30℃水
热7min；
(c)通过检测反应溶液652nm处吸光度，进而计算溶液中尿酸浓度。
本发明的优点和有益效果是：第一次证明了所合成的Ag2V4O11纳米带具有类过氧化物酶活性，与单纯的采用贵金属作为模拟酶检测相比降低了成本。
具体详细见实施例实验数据。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，根据这些附图获得其他的附图仍属于本发明的范畴。
图1所制备Ag2V4O11纳米带的X射线衍射(XRD)谱图；
图2所制备Ag2V4O11纳米带的扫描电镜图片和元素分布图；
图3不同反应体系在652nm处光吸收特性和反应溶液的变化情况比较图；
图4本发明应用例1的检测图，其中A图为尿酸检测响应值曲线；B图为尿酸检测的线性区间曲线。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
实施例
Ag2V4O11纳米带的制备如图1所示，包括以下步骤：
(1)将0.002mol NH4VO3和0.001mol AgNO3在磁力搅拌的条件下分别溶解在50和20mL去离子水中；(2)将(1)中所得AgNO3溶液在磁力搅拌的条件下逐滴加入到NH4VO3溶液中；(3)用1mol/L HNO3溶液调节(2)所得溶液的pH至3；(4)将反应溶液继续在室温下搅拌1h和熟化4h；(5)将反应溶液转移到80mL水热釜中，150℃水热24h；(6)反应溶液冷却至室温后，真空抽滤获得产物，并将产物用去离子水清洗多次后，放入烘箱中70℃干燥12h。
Ag2V4O11纳米带的表征
使用X射线衍射分析所合成物质的组成进行分析，所合成物质衍射谱图与单斜相的Ag2V4O11(JCPDS Card No.49-0166)高度吻合，证明合成的物质为Ag2V4O11。
利用扫描电镜对合成的Ag2V4O11纳米带的形貌和元素组成进行分析。从图2中可以看出所合成的Ag2V4O11微观形貌为宽度20-40nm的纳米带状结构。元素分布显示，所合成物质由Ag、V、O三种元素组成。
通过催化过氧化物酶典型催化底物H2O2与3,3',5,5'-四甲基联苯胺(TMB，无色)生成氧化态的TMB(蓝色)来检测所制备Ag2V4O11纳米带是否具有类过氧化物酶的催化活性。从图3插图中，可以观察到不同体系的溶液颜色变化：(a)H2O2+Ag2V4O11纳米带体系，溶液无色透明；(b)TMB+Ag2V4O11纳米带体系，溶液无色透明；(c)TMB+H2O2体系，溶液呈非常淡的淡蓝色；(d)H2O2+TMB+Ag2V4O11纳米带体系，溶液呈现较明显的蓝色。图3是三种反应体系对应的紫外可见吸收光谱图，体系a和b在652nm几乎没有吸收峰，体系c有较小的吸收峰，这可能与浓度较高的H2O2有关，较高浓度的H2O2氧化TMB生成氧化态的TMB，而体系d(H2O2+TMB+Ag2V4O11纳米带体系)在652nm处有较明显的吸收峰且峰形较好，说明Ag2V4O11纳米带催化氧化TMB生成氧化态TMB，具有类过氧化物酶活性。
应用例1
尿酸在尿酸酶的催化下，生成H2O2，Ag2V4O11纳米带模拟过氧化物酶催化尿酸分解生成的H2O2和与催化催化底物TMB反应生成蓝色产物氧化态TMB，达到间接检测尿酸的目的。根据尿酸浓度不同反应液显色程度不同而建立了一个比色检测尿酸的方法，利用氧化态TMB在652nm下的吸光度与UA浓度成一定比例，作出一条标准曲线。图4B是利用652nm下的吸光度与尿酸浓度作的线性标准曲线。线性范围为1-110μM，方法的检出限为0.35μM。线性方程为A652 nm＝0.10155+0.01018CUA，标准偏差R2＝0.9996。
应用例2
表1不同血清样品中尿酸检测结果与比较
Figure GDA0003970404720000051
收集普通人(样品1-3)和痛风病人(4和5)的血清用此方法进行检测。所有样品进行检测前离心取上清液，然后用pH＝7.4的PBS稀释十倍。实验结果显示，此方法尿酸检出结果与医院检测结果十分接近，可用于血清中尿酸含量的检测。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。

Claims (1)

1.一种钒酸银作为类过氧化物酶用于尿酸的非诊断目的的检测方法，其特征在于：
所述的钒酸银的制备方法包括以下步骤：
(1)将0.002mol NH4VO3和0.001mol AgNO3在磁力搅拌的条件下分别溶解在50和20mL去离子水中；
(2)将(1)中所得AgNO3溶液在磁力搅拌的条件下逐滴加入到NH4VO3溶液中；
(3)用1mol/L HNO3溶液调节(2)所得溶液的pH至3；
(4)将反应溶液继续在室温下搅拌1h和熟化4h；
(5)将反应溶液转移到80mL水热釜中，150℃水热24h；
(6)反应溶液冷却至室温后，真空抽滤获得产物，并将产物用去离子水清洗多次后，放入烘箱中70℃干燥12h，即得产物分子式为Ag2V4O11的钒酸银；
所述的检测方法包括以下步骤：
(a)10μL 5.0mg/mL尿酸酶与待测样品加入到200μL pH＝8.5的100mMPBS溶液中，然后将上述溶液在37℃水浴保温30分钟；
(b)分别加入10μL 1mg·mL-1的Ag2V4O11悬浊液，160μL 8mM TMB溶液和1620μL 100mMPBS缓冲溶液，该缓冲溶液pH＝5.0，上述溶液30℃水热7min；
(c)通过检测反应溶液652nm处吸光度，进而计算溶液中尿酸浓度。
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