锰单原子纳米酶比色法检测碱性磷酸酶活性  

Colorimetric Detection of Alkaline Phosphatase Activity Based on Manganese Single Atom Nanozyme

作　　者：王波 张续东 康革 刘芳宁 赵丹 陈传霞 逯一中 WANG Bo;ZHANG Xu-Dong;KANG Ge;LIU Fang-Ning;ZHAO Dan;CHEN Chuan-Xia;LU Yi-Zhong(School of Materials Science and Engineering,University of Jinan,Jinan 250022,China;Department of Environmental Engineering and Chemistry,Luoyang Institute of Science and Technology,Luoyang 471023,China)

机构地区：[1]济南大学材料科学与工程学院,济南250022 [2]洛阳理工学院环境工程与化学学院,洛阳471023

出　　处：《分析化学》2022年第1期54-63,共10页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21902061,22104046,21904048,22172063);山东省自然科学基金项目(Nos.ZR2020QB033,ZR2019YQ10)资助。

摘　　要：利用溶剂热法,合成了具有类过氧化物酶活性的锰单原子纳米酶,用于碱性磷酸酶(ALP)活性的检测。以甲酰胺为碳源和氮源,MnCl_(2)为锰源,经180℃反应12 h,获得富含单分散Mn-N4位点的锰、氮共掺杂碳材料(f-MnNC)。通过透射电子显微镜、X-射线衍射仪、X-射线光电子能谱、红外光谱等对f-MnNC的结构和形貌进行了表征,利用紫外-可见吸收光谱对f-MnNC的类过氧化物酶活性进行了研究。具有类过氧化物酶活性的f-MnNC可催化H_(2)O_(2)将无色的3,3′,5,5′-四甲基联苯胺(TMB)氧化为蓝色的氧化态TMB(oxTMB)。考察了f-MnNC催化TMB-H_(2)O_(2)显色反应条件对此过氧化物酶活性的影响,并进行了稳态动力学分析。结果表明,f-MnNC的酶催化反应遵循典型的Michaelis-Menten动力学模型。利用抗坏血酸磷酸酯(AA2P)为底物,ALP催化AA2P水解产生抗坏血酸(AA),AA具有还原性,可通过还原oxTMB使得体系蓝色变浅,吸光度降低。信号降低程度与ALP活性有关,据此可对ALP活性进行定量检测。本方法检测ALP活性的线性范围为0.1~10 mU/mL,检出限(S/N=3)为0.059 mU/mL。其它生物体系中常见蛋白和生物酶对ALP检测无明显干扰,表明本方法检测ALP活性具有良好的选择性。将本方法用于血清中ALP活性的检测,结果令人满意。Manganese(Mn)single atom nanozyme with peroxidase-like activity was synthesized through a solvothermal method and used for the detection of alkaline phosphatase(ALP)activity.By using formamide as the carbon and nitrogen source and MnCl_(2)as the Mn source,formamide converted Mn and nitroren co-doped carbon(f-MnNC)could be obtained after reacting at 180℃for 12 h.The structure and morphology were characterized by transmission electron microscopy,X-ray photoelectron spectroscopy,X-ray diffractometer,etc.,and the peroxidase-like activity was investigated mainly using UV-visible absorption spectroscopy.In the presence of H_(2)O_(2),f-MnNC could catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine(TMB)into blue oxidized TMB(oxTMB),with a characteristic absorption peak centered at 652 nm.ALP could catalyze the hydrolysis of L-ascorbic acid-2-phosphate(AA2P)to produce ascorbic acid(AA),which then reduced oxTMB to TMB,resulting in faded color and decreased absorbance.The degree of colorimetric signal change was related to ALP activity,and thus ALP activity could be quantitatively detected by employing AA2P as the substrate.In the activity range of 0.1-10 mU/mL,there was a good linear relationship between the absorbance at 652 nm and the ALP activity,and the detection limit(S/N=3)was 0.059 mU/mL.Other proteins and enzymes had no obvious interference with ALP detection,indicating good selectivity for ALP activity detection.The method was successfully applied to the determination of ALP activity in serum with satisfactory results.

关 键 词：锰单原子纳米酶 比色 碱性磷酸酶 抗坏血酸 还原性 

分 类 号：O657.3[理学—分析化学] R313[理学—化学]