CN108872217A - 二氧化铱纳米酶的合成与应用 - Google Patents
二氧化铱纳米酶的合成与应用 Download PDFInfo
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Abstract
本发明涉及纳米催化、分析化学领域，具体包括二氧化铱（IrO2）纳米酶的合成与应用。IrO2纳米酶在酸性pH下具有过氧化物酶的活性，可催化过氧化氢和有机显色剂发生显色反应。本发明利用制备得到的IrO2纳米酶与肌氨酸氧化酶偶联来实现肌氨酸的定量分析。该发明可用于食品发酵、生物医药、化工、环境、生物技术等领域的定量分析和催化剂等方面。
Description
技术领域
本发明涉及分析化学领域，具体包括二氧化铱（IrO2）纳米酶的合成其作为仿酶材料的应用。
背景技术
纳米酶是一类既有纳米材料的独特性能，又有催化功能的模拟酶。纳米酶克服了天然酶的许多缺点，如价格昂贵、易失活和储存条件要求苛刻等，对生物传感、免疫分析、癌症诊断和治疗等领域产生了巨大影响。
到目前为止，多种无机纳米材料，都表现出仿过氧化物酶、仿氧化酶、仿过氧化氢酶或仿超氧化物歧化酶等性能。其中，仿过氧化物酶活性指纳米材料具有同过氧化物酶一样作用的以H2O2为电子受体催化底物氧化的酶。
已报道的具有仿过氧化物酶活性的无机纳米材料包括Fe3O4、γ-Fe2O3、FePO4、FePt、CuO、Cu3(PO4)2、CuS、Cu、CuInS2、Co3O4、CoFe、CoFe2O4、CeO2、CePO4、Gd、MnO2、MnSe、ZnO、BiFeO3、MoS2 、WC、VO2、V2O3、Ir、Pd-Ir、Au、Pt、Au/CuS、Bi/Au、Ag/Pt、Ag/Pd、Ag/Au、Ag/Pt、Au/Pd、Si-dots、V2O5。值得注意的是，并不是所有金属氧化物都具有仿过氧化物酶活性。二氧化铱尚未报道具有仿酶活性。
贵金属及其氧化物纳米颗粒对一些化学反应有着极高的催化活性。IrO2具有许多重要应用，包括作为超级电容和电致变色材料，神经元间刺激电极，用于pH感测和成像的超微电极以及用于氧和氯反应的催化剂。IrO2纳米颗粒在较宽的pH范围内对水分解析氧反应表现出较高的电催化活性。IrO2纳米颗粒近年来也被用作酶的电子传导基质，使其可用于生物传感器。但我们目前尚未发现IrO2具有仿酶活性。
肌氨酸（Sarcosine），又名N‒甲基甘氨酸，是胆碱自然代谢为甘氨酸过程中的一个非编码氨基酸中间体，可以由氯乙酸与甲胺反应制得，也可以由甘氨酸通过N-甲基转移酶作用生成。肌氨酸有甜味，溶于水，存在于人体的肌肉及其他一些组织中，生理状态下正常人体血清中的肌氨酸含量是1.59±1.08 nmol/L。肌氨酸可以从饮食摄入的胆碱或蛋氨酸代谢而来，但很快在体内转换为甘氨酸。甘氨酸作为结构性氨基酸，在人体生理过程中发挥着重要作用，是谷胱甘肽、肌酸、嘌呤和丝氨酸等活细胞必需成分的代谢来源。肌氨酸可激活前列腺癌细胞，并且其在尿液中被检测到意味着恶性前列腺癌的发生。肌氨酸成为前列腺癌进展和转移时显著增加的一个代谢产物，能够在尿液中被检测到，因而被确定为前列腺癌的一个特异性指标。因此肌氨酸的检测在治疗前列腺癌中具有重要作用。常用的肌氨酸的检测方法有电致化学发光分析、质谱法、荧光检测分析。目前，尚未报道基于纳米酶的肌氨酸检测方法。肌氨酸氧化酶（Sarcosine oxidase，简称SOX，EC.1.5.3.1）属于黄素蛋白氧化酶类,以FAD为辅因子,可催化肌氨酸中N‒甲基的氧化还原，是检测血清或尿液中肌酐含量的关键酶之一，广泛应用于医疗诊断领域。它可催化肌氨酸降解为甘氨酸、甲醛和过氧化氢。作为一种重要的诊断酶制剂，SOX被广泛应用于人体血清中肌酐水平的检测，用来判断肾功能的健康程度。
综上所述，至今为止，基于纳米酶的肌氨酸检测分析鲜有报道，尚未报道IrO2纳米酶的合成及其应用。
发明内容
本发明的目的是确定IrO2纳米酶的合成及其作为仿酶材料的应用。
为实现上述目的，本发明采用的技术方案为：
一种纳米酶材料及其应用，所述IrO2纳米酶具有仿过氧化物酶活性。
优选是，所述仿过氧化物酶材料IrO2纳米酶合成过程为：
(1)将K2IrCl6加入到柠檬酸氢钠溶液中，用NaOH调pH至7.5；
(2)回流30 min，室温冷却后，再调pH至7.5，继续回流，重复该过程直至pH恒定7.5；
(3)将溶液通过氧气鼓泡进一步回流2小时，即得IrO2纳米酶。
进一步的优选，所述仿过氧化物酶材料IrO2纳米酶可作为仿过氧化物酶催化剂，用于基于仿过氧化物酶活性的应用。
更优选是，所述仿过氧化物酶材料IrO2纳米酶在酸性条件下作为具有过氧化物酶活性的催化剂，用于对肌氨酸进行定性/定量检测。具体的肌氨酸检测方法：
(1)包含一定浓度的肌氨酸、肌氨酸氧化酶的磷酸盐缓冲液（pH 8.0, 10 mmol/L）在37℃下反应30分钟；
(2)将上述溶液离心，取上清液10 µL，加入权利要求1-3中所述IrO2纳米酶、有机显色剂、醋酸盐缓冲液（pH 3.5, 100 mmol/L），继续在37 ℃下反应30分钟；
(3)观察溶液颜色变化来实现定性检测；
(4)利用酶标仪检测有机显色剂氧化物的相应吸光值来实现定量检测。
所述检测肌氨酸时加入有机显色剂；
所述有机显色剂为2, 2’-联氮-双（3-乙基苯并噻唑啉-6-磺酸）二胺盐（ABTS）或3,3’,5, 5’-四甲基联苯胺（TMB）。
本发明的效果是：
1.本发明合成了具有过氧化物酶活性的IrO2纳米酶，并且最适pH在3.5左右，在酸性条件下（pH 3.5−6.0）表现出仿过氧化物酶活性。
2.本发明将IrO2纳米酶纳米粒子应用于肌氨酸的定性/定量分析，实现了在酸性pH条件下（pH 3.5−6.0）利用分光光度法对肌氨酸的定性/定量检测。
3.本发明提供在酸性pH条件下发挥仿酶活性的材料，应用于人体血清中肌酐水平的检测，用来判断肾功能的健康程度，广泛应用于医疗诊断领域。
附图说明
图1为本发明实施例提供的IrO2纳米酶的透射电子显微镜图；
图2为本发明实施例提供的IrO2纳米酶仿酶活性效果图；
图3为本发明实施例提供的IrO2纳米酶仿酶活性的pH优化效果图；
图4为本发明实施例提供的H2O2的定量检测标准工作曲线；
图5为本发明实施例提供的肌氨酸的检测示意图；
图6为本发明实施例提供的肌氨酸的定性检测照片；
图7为本发明实施例提供的肌氨酸的定量检测标准工作曲线。
具体实施方式
为了更清楚更深入地说明本发明的内容，下面将进一步列举一些实施例，但本发明不局限于所列举的实施例。下列实施例中具体实验条件或方法如未注明，均按本领域的常规条件或方法进行。
实施例1
仿过氧化物酶材料IrO2纳米酶的制备：
将30 mg K2IrCl6加入到50 mL柠檬酸氢钠(3.8 mol/L)溶液中。使用NaOH（0.25 mol/L）溶液将所得溶液pH调至7.5，然后在恒定搅拌下回流30分钟。此后，将其在室温下冷却，然后进行pH调节，搅拌和回流，重复该过程直到获得恒定的pH 7.5。为了获得IrO2纳米酶的悬浮液，将溶液通过氧气鼓泡进一步回流2小时，得IrO2纳米酶。图1为通过透射电子显微镜观察到的IrO2纳米酶的TEM成像。
实施例2
IrO2纳米酶的仿酶活性验证：
纳米酶实验体系a：催化反应体系为包含H2O2（0.5 mmol/L）、上述实施例获得的IrO2纳米酶（70 µg/mL）、有机显色剂TMB（0.5 mmol/L）的醋酸盐缓冲液（pH 3.5, 100 mmol/L）。在室温（25 ℃）下反应30分钟后，利用酶标仪检测其300−800 nm内的吸光值。
另做两个对照试验：其中一个对照实验b的催化反应体系中不加IrO2纳米酶，在与上述实验体系同样条件下反应30分钟后检测吸光值；另一份对照实验c的催化反应体系为IrO2纳米酶（70 µg/mL）在醋酸盐缓冲液（pH 3.5, 100 mmol/L）中，在与上述实验体系同样条件下静置30分钟后检测吸光值。
如图2所示，实验体系a显示出明显的峰，说明IrO2纳米酶在pH 4.0处具有明显的仿过氧化物酶的活性；对照试验b在650 nm附近无明显的峰，说明若无IrO2纳米酶作催化剂将无明显反应；对照试验c在650 nm附近无明显的峰，说明实验体系a的峰不是IrO2纳米酶自身的响应峰。
实施例3
IrO2纳米酶仿酶活性的pH优化：
催化反应体系为包含H2O2（0.5 mmol/L）、IrO2纳米酶（70 µg/mL）、有机显色剂TMB（0.5mmol/L）的不同pH的缓冲液（pH 1.0−2.0，甘氨酸-盐酸缓冲液；pH 3.0−6.0，醋酸-醋酸钠缓冲液；pH 6.5−8.0，磷酸盐缓冲液；pH 9.0−10.0，Tris-盐酸缓冲液；pH 11.0−12.0，碳酸氢钠-氢氧化钠缓冲液）。在室温（25 ℃）下反应30分钟后，利用酶标仪检测其650 nm内的吸光值。如图3所示，IrO2纳米酶在酸性的pH下（pH 3.5−6.0）表现出过氧化物酶的活性，并且最适pH为3.5左右。
实施例4
H2O2的定量检测：
催化反应体系为包含不同浓度的H2O2（0.01−3 mmol/L）、IrO2纳米酶（70 µg/mL）、有机显色剂TMB（0.5 mmol/L）的醋酸盐缓冲液（pH 3.5, 100 mmol/L）。在室温（25 ℃）下反应10分钟后，利用酶标仪检测其300−800 nm内的吸光值。由650 nm处吸光值扣除空白对照后绘制出H2O2标准工作曲线。如图4所示，线性范围0−1.5 mmol/L，线性方程为y=0.47x+0.15 (R2=0.997)。
实施例5
肌氨酸的定性检测：
由于肌氨酸在肌氨酸氧化酶催化下生成甘氨酸、H2O2和HCHO，具有仿过氧化物酶活性的IrO2纳米酶可在H2O2存在下氧化TMB显色，从而实现肌氨酸的定量检测。图5为肌氨酸的检测示意图。催化反应体系为包含不同浓度的肌氨酸（0−100 µg/mL）、肌氨酸氧化酶（300 µg/mL）的磷酸盐缓冲液（pH 8.0，10 mmol/L）。在37 ℃下反应30分钟后，将上述溶液离心，取上清液10 µL，加入IrO2纳米酶（70 µg/mL）、有机显色剂TMB（0.5 mmol/L），醋酸盐缓冲液（pH3.5，100 mmol/L）。在37 ℃下反应10 min，如图6所示，观察颜色变化。
实施例6
肌氨酸的定量检测：
催化反应体系为包含不同浓度的肌氨酸（0−100 µg/mL）、肌氨酸氧化酶（300 µg/mL）的磷酸盐缓冲液（pH 8.0，10 mmol/L）。在37 ℃下反应30分钟后，将上述溶液离心，取上清液10 µL，加入IrO2 纳米酶（70 µg/mL）、有机显色剂TMB（0.5 mmol/L），醋酸盐缓冲液（pH3.5, 100 mmol/L），反应30分钟后，利用酶标仪检测其650 nm处的吸光值并绘制出肌氨酸标准工作曲线。如图7所示，线性范围0−60 µg/mL，线性方程为y=0.008x+0.012 (R2=0.995)。
Claims (5)
1.一种二氧化铱（IrO2）纳米酶，其特征在于：所述IrO2纳米酶具有仿过氧化物酶活性。
2.根据权利要求1所述的二氧化铱（IrO2）纳米酶，其特征在于，合成步骤如下：
(1)将K2IrCl6加入到柠檬酸氢钠溶液中，用NaOH调pH至7.5；
(2)回流30 min，室温冷却后，再调pH至7.5，继续回流，重复该过程直至pH恒定在7.5；
(3)将溶液通过氧气鼓泡进一步回流2小时，即得IrO2纳米酶。
3.一种基于IrO2纳米酶的应用，其特征在于：权利要求1、2中所述的IrO2纳米酶可作为仿过氧化物酶催化剂，用于基于仿过氧化物酶活性的应用。
4.一种基于IrO2纳米酶的肌氨酸定性/定量分析应用，其特征在于定性/定量分析应用的步骤如下：
(1)包含一定浓度的肌氨酸、肌氨酸氧化酶的磷酸盐缓冲液（pH 8.0, 10 mmol/L）在37℃下反应30分钟；
(2)将上述溶液离心，取上清液10 µL，加入权利要求1-3中所述IrO2纳米酶、有机显色剂、醋酸盐缓冲液（pH 3.5，100 mmol/L），继续在37 ℃下反应30分钟；
(3)观察溶液颜色变化来实现定性检测；
(4)利用酶标仪检测有机显色剂氧化物的相应吸光值来实现定量检测。
5.根据权利要求4所述的有机显色剂为3, 3,5, 5’-四甲基联苯胺（TMB）或2, 2’-联氮-双（3-乙基苯并噻唑啉-6-磺酸）二胺盐（ABTS）。
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