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
王波;张续东;康革;刘芳宁;赵丹;陈传霞;逯一中;
摘要(Abstract)：
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-N_4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_2O_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_2O_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。
关键词(KeyWords)： 锰单原子纳米酶;比色;碱性磷酸酶;抗坏血酸;还原性
基金项目(Foundation): 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~~
作者(Authors): 王波;张续东;康革;刘芳宁;赵丹;陈传霞;逯一中;
DOI: 10.19756/j.issn.0253-3820.210812
参考文献(References)：
- [1] MILLáN J L.Purinerg.Signal.,2006,2(2):335.
- [2] FERNANDEZ N J,KIDNEY B A.Vet.Clin.Path.,2007,36(3):223-233.
- [3] GOGGINS S,NAZ C,MARSH B J,FROST C G.Chem.Commun.,2015,51(3):561-564.
- [4] KREUZER M P,O′SULLIVAN C K,GUILBAULT G G.Anal.Chim.Acta,1999,393(1):95-102.
- [5] BLUM J S,LI R H,MIKOS A G,BARRY M A.J.Cell Biochem.,2001,80(4):532-537.
- [6] HASEGAWA T,SUGITA M,TAKATANI K,MATSUURA H,UMEMURA T,HARAGUCHI H.Bull.Chem.Soc.Jpn.,2006,79(8):1211-1214.
- [7] RUAN C M,WANG W,GU B H.Anal.Chem.,2006,78(10):3379-3384.
- [8] WEI H,CHEN C G,HAN B Y,WANG E K.Anal.Chem.,2008,80(18):7051-7055.
- [9] CHEN C X,ZHAO D,JIANG Y Y,NI P J,ZHANG C H,WANG B,YANG F,LU Y Z,SUN J.Anal.Chem.,2019,91(23):15017-15024.
- [10] ZHAO W,CHIUMAN W,LAM J C,BROOK M A,LI Y.Chem.Commun.,2007,43(36):3729-3731.
- [11] SUN J,ZHAO J,BAO X,WANG Q,YANG X.Anal.Chem.,2018,90(10):6339-6345.
- [12] CHEN C X,ZHANG G L,NI P J,JIANG Y Y,LU Y Z,LU Z L.Microchim.Acta,2019,186(6):348.
- [13] XIANYU Y L,WANG Z,JIANG X Y.ACS Nano,2014,8(12):12741-12747.
- [14] CHEN C X,YUAN Q,NI P J,JIANG Y Y,ZHAO Z L,LU Y Z.Analyst,2018,143(16):3821-3828.
- [15] MA J L,YIN B C,WU X,YE B C.Anal.Chem.,2016,88(18):9219-9225.
- [16] CHEN C X,ZHAO J H,LU Y Z,SUN J,YANG X R.Anal.Chem.,2018,90(5):3505-3511.
- [17] KIM T I,KIM H,CHOI Y,KIM Y.Chem.Commun.,2011,47(35):9825-9827.
- [18] CHEN C X,ZHAO D,WANG B,NI P J,JIANG Y Y,ZHANG C H,YANG F,LU Y Z,SUN J.Anal.Chem.,2020,92(6):4639-4646.
- [19] SUN J,HU T,CHEN C X,ZHAO D,YANG F,YANG X R.Anal.Chem.,2016,88(19):9789-9795.
- [20] ZHAO D,LI J,PENG C Y,ZHU S Y,SUN J,YANG X R.Anal.Chem.,2019,91(4):2978-2984.
- [21] MALASHIKHINA N,GARAI-IBABE G,PAVLOV V.Anal.Chem.,2013,85(14):6866-6870.
- [22] BABSON A L,GREELEY S J,COLEMAN C M,PHILLIPS G E.Clin.Chem.,1966,12(8):482-490.
- [23] CHEN Xi,CHEN Jin,ZHANG Hui-Yan,WANG Fu-Bing,WANG Fang-Fang,JI Xing-Hu,HE Zhi-Ke.Chin.J.Anal.Chem.,2016,44(4):591-596.陈熙，陈锦，张慧妍，汪付兵，王方方，吉邢虎，何治柯.分析化学，2016,44(4):591-596.
- [24] YANG X,GAO Z.Chem.Commun.,2015,51(32):6928-6931.
- [25] WANG C,GAO J,CAO Y,TAN H.Anal.Chim.Acta,2018,1004:74-81.
- [26] SONG H,YE K,PENG Y,WANG L,NIU X.J.Mater.Chem.B,2019,7(38):5834-5841.
- [27] TIAN F,ZHOU J,MA J,LIU S,JIAO B,HE Y.Microchim.Acta,2019,186(7):408.
- [28] JIAO L,YAN H,WU Y,GU W,ZHU C,DU D,LIN Y.Angew.Chem.,Int.Ed.,2020,59(7):2565-2576.
- [29] HAN L,ZHANG H,CHEN D,LI F.Adv.Funct.Mater.,2018,28(17):1800018.
- [30] ZHU Wei-Ran,HAO Nan,YANG Xiao-Di,WANG Kun.Chin.J.Anal.Chem.,2020,48(6):727-732.朱巍然，郝楠，杨小弟，王坤.分析化学，2020,48(6):727-732.
- [31] ZHANG G,JIA Y,ZHANG C,XIONG X,SUN K,CHEN R,CHEN W,KUANG Y,ZHENG L,TANG H,LIU W,LIU J,SUN X,LIN W F,DAI H.Energy Environ.Sci.,2019,12(4):1317-1325.
- [32] GONG Z,YANG B,LIN H,TANG Y,TANG Z,ZHANG J,ZHANG H,LI Y,XIE Y,LI Q,CHI L.ACS Nano,2016,10(4):4228-4235.
- [33] CHEN Z,MITCHELL S,VOROBYEVA E,LEARY R K,HAUERT R,FURNIVAL T,RAMASSE Q M,THOMAS J M,MIDGLEY P A,DONTSOVA D,ANTONIETTI M,POGODIN S,LóPEZ N,PéREZ-RAMíREZ J.Adv.Funct.Mater.,2017,27(8):1605785.
- [34] JIANG B,DUAN D,GAO L,ZHOU M,FAN K,TANG Y,XI J,BI Y,TONG Z,GAO G F,XIE N,TANG A,NIE G,LIANG M,YAN X.Nat.Protoc.,2018,13(7):1506-1520.
- [35] GAO L,ZHUANG J,NIE L,ZHANG J,ZHANG Y,GU N,WANG T,FENG J,YANG D,PERRETT S,YAN X.Nat.Nanotechnol.,2007,2(9):577-583.
- [36] QIAO F,CHEN L,LI X,LI L,AI S.Sens.Actuators,B,2014,193:255-262.
- [37] XU W,JIAO L,YAN H,WU Y,CHEN L,GU W,DU D,LIN Y,ZHU C.ACS Appl.Mater.Interfaces,2019,11(25):22096-22101.
- [38] WU Y,WU J,JIAO L,XU W,WANG H,WEI X,GU W,REN G,ZHANG N,ZHANG Q,HUANG L,GU L,ZHU C.Anal.Chem.,2020,92(4):3373-3379.
- [39] GE C,WU R,CHONG Y,FANG G,JIANG X,PAN Y,CHEN C,YIN J J.Adv.Funct.Mater.,2018,28(28):1801484.
- [40] CHEN C X,LIU W D,NI P J,JIANG Y Y,ZHANG C H,WANG B,LI J K,CAO B Q,LU Y Z,CHEN W.ACS Appl.Mater.Interfaces,2019,11(50):47564-47570.
- [41] LIU W D,CHU L,ZHANG C H,NI P J,JIANG Y Y,WANG B,LU Y Z,CHEN C X.Chem.Eng.J.,2021,415:128876.
- [42] JIANG X,WANG X,LIN A,WEI H.Anal.Chem.,2021,93(14):5954-5962.
- [43] WANG J W,NI P J,CHEN C X,JIANG Y Y,ZHANG C H,WANG B,CAO B Q,LU Y Z.Microchim.Acta,2020,187(2):115.
- [44] ZHANG Q,YU Y,YUN X,LUO B,JIANG H,CHEN C,WANG S,MIN D.ACS Appl.Nano Mater.,2020,3(6):5212-5219.
- [45] HAYAT A,GONCA B,ANDREESCU S.Biosens.Bioelectron.,2014,56:334-339.
- [46] CHEN Q,LI S,LIU Y,ZHANG X,TANG Y,CHAI H,HUANG Y.Sens.Actuators,B,2020,305:127511.
- [47] XIE X,WANG Y,ZHOU X,CHEN J,WANG M,SU X.Analyst,2021,146(3):896-903.