基于铜离子(Ⅱ)类过氧化物酶性质可视化比色法检测过氧化氢  

A Visual Colorimetric Method for Hydrogen Peroxide Detection Based on the Peroxidase-Like Properties of Cu(Ⅱ)

作　　者：马纪 黄国霞[1,2] 李军生 阎柳娟[1,2] 张倩 MA Ji;HUANG Guo-xia;LI Jun-sheng;YAN Liu-juan;ZHANG Qian(Guangxi Key Laboratory of Green Processing of Sugar Resources,College of Biological and Chemical Engineering,Guangxi University of Science and Technology,Liuzhou 545006,China;Guangxi Liuzhou Luosifen Research Center of Engineering Technology,Liuzhou 545006,China)

机构地区：[1]广西糖资源绿色加工重点实验室/生物与化学工程学院,广西科技大学,广西柳州545006 [2]广西柳州螺蛳粉工程技术研究中心,广西柳州545006

出　　处：《光谱学与光谱分析》2022年第9期2795-2799,共5页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金面上基金项目(21966008);广西自然科学基金面上基金项目(2020GXNSFAA159021);广西高校中青年教师基础能力提升项目(2020KY08006);广西糖资源绿色加工重点实验室基金项目(GXTZY202002);广西科技大学博士基金项目(校科博20z26)资助。

摘　　要：过氧化氢(H_(2)O_(2))在食品工业、环境监测分析、燃料电池、临床诊断等领域得到十分广泛的应用。过氧化氢不仅是严重疾病的生物标志物,也是重要的食品添加剂。无论从食品安全还是人类健康考虑,对过氧化氢构建简便、快速、灵敏的检测方法具有重要意义。比色法因具有易操作、成本低及检测结果可视化等优点被广泛关注。比色法普遍使用的酶是辣根过氧化物酶(HRP)。但天然酶存在易失活、生产成本高及稳定性差等不足。纳米酶克服了HRP易失活的缺点。然而部分纳米酶合成复杂、需要表征、水溶性差及催化活性低等。与HRP和纳米酶相比,具有类过氧化物酶性质的铜离子(Ⅱ),不仅具有灵敏度高的特点,而且不需要复杂的合成、易获得、易储存、不需修饰可直接使用、操作简单及分析成本低廉等优势。铜离子(Ⅱ)类酶能够通过催化过氧化氢生成羟基自由基,氧化3,3’,5,5’-四甲基联苯胺(TMB)而产生氧化态TMB,使溶液的颜色由无色逐渐转变为蓝色,从而产生光信号,这些光信号可以肉眼识别和通过紫外分光光度计检测。基于上述原理构建过氧化氢的快速比色传感并应用于银耳样品的检测。实验考察了体系中过氧化氢浓度、pH值、温度等因素对吸光度的影响。选择缓冲溶液pH值为3.0,温度40℃,TMB和铜离子(Ⅱ)浓度分别为6.0×10^(-4)和8.0×10^(-3)mol·L^(-1),反应时间为20 min。在最优条件下,过氧化氢浓度与体系的吸光度值呈良好的线性关系,线性范围是0.08~40μmol·L^(-1),检测限为0.14μmol·L^(-1)。该法可检测银耳中过氧化氢的浓度,加标回收率在97.10%~107.08%之间,相对标准偏差(RSD)小于5%。该研究无需特殊检测设备条件下实现过氧化氢的简单、快速、成本低廉且灵敏的可视化检测,有利于过氧化氢的定量检测在食品领域和临床上的快速推广。Hydrogen peroxide(H_(2)O_(2))is widely used in the food industry,environmental monitoring and analysis,fuel cell,clinical diagnosis and other fields.Hydrogen peroxide is not only a biomarker of severe diseases but also an important food additive.In terms of food safety and human health,it is of great significance to construct a simple,rapid and sensitive detection method.Colorimetric method is widely concerned because of its advantages such as easy operation,low cost and visualization of detection results.The enzyme commonly used in colorimetric method is horseradish peroxidase(HRP).However,natural enzymes are easy to deactivate,have high production costs and have poor stability.Nanoenzyme overcomes the disadvantage of HRP’s easy inactivation.However,some nanoenzymes are complex in synthesis,need to be characterized,and have poor water solubility and have low catalytic activity.Compared with HRP and nanoenzyme,copper ion(Ⅱ)with peroxidase-like properties has the characteristics of high sensitivity and does not need complex synthesis,is easy to obtain,easy to store,without modification can be directly used,simple operation and low analysis cost,etc.Copper ion(Ⅱ)can catalyze hydrogen peroxide to generate hydroxyl radicals and oxidize 3,3’,5,5’-tetramethyl benzidine(TMB)to produce the oxidation state TMB,which makes the color in the solution gradually change from colorless to blue,thus producing light signals that can be recognized by the naked eye and detected by ultraviolet spectrophotometer.Based on the above principles,a fast colorimetry sensor based on hydrogen peroxide was constructed and applied to detect tremella samples.The effects of hydrogen peroxide concentration,pH value,temperature and other factors on the absorbance were investigated.The pH value of the buffer solution was 3.0,the temperature was 40℃,the concentration of TMB and Cu(Ⅱ)was 6.0×10^(-4)and 4.0×10^(-3)mol·L^(-1),respectively,and the reaction time was 20 min.Under the optimal conditions,the concentration of hydrogen peroxid

关 键 词：铜离子(Ⅱ) 过氧化物模拟酶 比色法 过氧化氢 

分 类 号：O657.3[理学—分析化学]