Microchimica Acta

, 186:408 | Cite as

MnO2 nanosheets as oxidase mimics for colorimetric detection of alkaline phosphatase activity

  • Fengyu Tian
  • Jing Zhou
  • Jing Ma
  • Siyuan Liu
  • Bining Jiao
  • Yue HeEmail author
Original Paper


A sensitive colorimetric method is described for the determination of the activity of alkaline phosphatase (ALP). It is based on the regulation of the oxidase-mimicking activity of MnO2 nanosheets. In the absence of ALP, MnO2 nanosheets are capable of catalyzing the oxidation of the colorless substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen to form a blue oxidized product (TMB Ox) with an absorption peak at 652 nm. In the presence of ALP and its substrate ascorbic acid-2-phosphate, the latter is hydrolyzed to form ascorbic acid (AA). AA triggers the decomposition of MnO2 nanosheets by reducing MnO2 to Mn2+, thereby weakening the enzyme mimicking activity of the MnO2 nanosheets and causing a drop in absorbance. The drop in absorbance at 652 nm is related to the ALP activity in the range from 0.05–10 m-units per mL (mU·mL−1), and the detection limit is 0.05 mU·mL−1. The method was applied to the determination of ALP in spiked calf serum samples and gave satisfactory results.

Graphical abstract

Schematic presentation of a facile and sensitive colorimetric method for detecting the activity of alkaline phosphatase (ALP) based on enzymatic regulation of the oxidase-mimicking activity of MnO2 nanosheets.


Enzyme activity assay Visual detection Nanozyme Ascorbic acid Ascorbic acid-2-phosphate 3,3′,5,5′-Tetramethylbenzidine 



The authors thank the National Natural Science Foundation of China (No. 21405125), the Natural Science Foundation Project of CQ (No. cstc2018jscx-msybX0263) and the Fundamental Research Funds for the Central Universities (No. XDJK2019C026) for kindly support. In addition, the authors also thank China Agriculture Research System (No. CARS-27), National Risk Assessment Program for Agricultural Products Quality and Safety (No. GJFP2019003 and GJFP2019013) for financial support.

Compliance with ethical standards

The author(s) declare that they have no competing interests.

All procedures performed in studies involving animals were in accordance with the ethical standards of Chinese laws and guidelines (GKFCZ2001545).

Supplementary material

604_2019_3519_MOESM1_ESM.doc (2.1 mb)
ESM 1 (DOC 2162 kb)


  1. 1.
    Millan JL (2006) Alkaline phosphatases : structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes. Purinergic Signal 2(2):335–341CrossRefGoogle Scholar
  2. 2.
    Ma JL, Yin BC, Wu X, Ye BC (2016) Copper-mediated DNA-Scaffolded silver nanocluster on-off switch for detection of pyrophosphate and alkaline phosphatase. Anal Chem 88(18):9219–9225CrossRefGoogle Scholar
  3. 3.
    Wang C, Gao J, Cao Y, Tan H (2018) Colorimetric logic gate for alkaline phosphatase based on copper (II)-based metal-organic frameworks with peroxidase-like activity. Anal Chim Acta 1004:74–81CrossRefGoogle Scholar
  4. 4.
    He Y, Jiao B (2017) Determination of the activity of alkaline phosphatase based on the use of ssDNA-templated fluorescent silver nanoclusters and on enzyme-triggered silver reduction. Microchim Acta 184(10):4167–4173CrossRefGoogle Scholar
  5. 5.
    Liu X, Fan N, Wu L, Wu C, Zhou Y, Li P, Tang B (2018) Lighting up alkaline phosphatase in drug-induced liver injury using a new chemiluminescence resonance energy transfer nanoprobe. Chem Commun (Camb) 54(88):12479–12482CrossRefGoogle Scholar
  6. 6.
    Hu Q, Zhou B, Dang P, Li L, Kong J, Zhang X (2017) Facile colorimetric assay of alkaline phosphatase activity using Fe (II)-phenanthroline reporter. Anal Chim Acta 950:170–177CrossRefGoogle Scholar
  7. 7.
    Zhang Z, Chen Z, Wang S, Cheng F, Chen L (2015) Iodine-mediated etching of gold Nanorods for Plasmonic ELISA based on colorimetric detection of alkaline phosphatase. ACS Appl Mater Inter 7(50):27639–27645CrossRefGoogle Scholar
  8. 8.
    Song H, Wang H, Li X, Peng Y, Pan J, Niu X (2018) Sensitive and selective colorimetric detection of alkaline phosphatase activity based on phosphate anion-quenched oxidase-mimicking activity of Ce (IV) ions. Anal Chim Acta 1044:154–161CrossRefGoogle Scholar
  9. 9.
    Wu S, Duan N, Qiu Y, Li J, Wang Z (2017) Colorimetric aptasensor for the detection of salmonella enterica serovar typhimurium using ZnFe2O4-reduced graphene oxide nanostructures as an effective peroxidase mimetics. Int J Food Microbiol 261:42–48CrossRefGoogle Scholar
  10. 10.
    Zhao Z, Fan H, Zhou G, Bai H, Liang H, Wang R, Zhang X, Tan W (2014) Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet-aptamer nanoprobe. J Am Chem Soc 136(32):11220–11223CrossRefGoogle Scholar
  11. 11.
    Han L, Zhang H, Chen D, Li F (2018) Protein-directed metal oxide Nanoflakes with tandem enzyme-like characteristics: colorimetric glucose sensing based on one-pot enzyme-free Cascade catalysis. Adv Funct Mater 28(17):1800018CrossRefGoogle Scholar
  12. 12.
    He L, Lu Y, Wang F, Jing W, Chen Y, Liu Y (2018) Colorimetric sensing of silver ions based on glutathione-mediated MnO2 nanosheets. Sensor Actuat B-Chem 254:468–474CrossRefGoogle Scholar
  13. 13.
    Xiao T, Sun J, Zhao J, Wang S, Liu G, Yang X (2018) FRET effect between fluorescent Polydopamine nanoparticles and MnO2 Nanosheets and its application for sensitive sensing of alkaline phosphatase. ACS Appl Mater Inter 10(7):6560–6569CrossRefGoogle Scholar
  14. 14.
    Ge J, Cai R, Chen X, Wu Q, Zhang L, Jiang Y, Cui C, Wan S, Tan W (2019) Facile approach to prepare HSA-templated MnO2 nanosheets as oxidase mimic for colorimetric detection of glutathione. Talanta 195:40–45CrossRefGoogle Scholar
  15. 15.
    Yan X, Song Y, Zhu C, Li H, Du D, Su X, Lin Y (2018) MnO2 Nanosheet-carbon dots sensing platform for sensitive detection of organophosphorus pesticides. Anal Chem 90(4):2618–2624CrossRefGoogle Scholar
  16. 16.
    Ge J, Xing K, Geng X, Hu YL, Shen XP, Zhang L, Li ZH (2018) Human serum albumin templated MnO2 nanosheets are oxidase mimics for colorimetric determination of hydrogen peroxide and for enzymatic determination of glucose. Mikrochim Acta 185(12):559CrossRefGoogle Scholar
  17. 17.
    Liu J, Meng L, Fei Z, Dyson PJ, Zhang L (2018) On the origin of the synergy between the Pt nanoparticles and MnO2 nanosheets in wonton-like 3D nanozyme oxidase mimics. Biosens Bioelectron 121:159–165CrossRefGoogle Scholar
  18. 18.
    Sun Y, Tan H, Li Y (2018) A colorimetric assay for acetylcholinesterase activity and inhibitor screening based on the thiocholine-induced inhibition of the oxidative power of MnO2 nanosheets on 3,3′,5,5′-tetramethylbenzidine. Mikrochim Acta 185(10):446CrossRefGoogle Scholar
  19. 19.
    He Y, Wang C, Zhao Q, Zhang Y, Chen A, Pang J, Fang Q, Cui Y, Jiao B (2017) Facile and sensitive fluorescence sensing of alkaline phosphatase activity using NMM/G-quadruplex. Talanta 172:171–175CrossRefGoogle Scholar
  20. 20.
    Liu J, Meng L, Fei Z, Dyson PJ, Jing X, Liu X (2017) MnO2 nanosheets as an artificial enzyme to mimic oxidase for rapid and sensitive detection of glutathione. Biosens Bioelectron 90:69–74CrossRefGoogle Scholar
  21. 21.
    Wei H, Wang E (2013) Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem Soc Rev 42(14):6060–6093CrossRefGoogle Scholar
  22. 22.
    Darabdhara G, Boruah PK, Das MR (2018) Colorimetric determination of glucose in solution and via the use of a paper strip by exploiting the peroxidase and oxidase mimicking activity of bimetallic Cu-Pd nanoparticles deposited on reduced graphene oxide, graphitic carbon nitride, or MoS2 nanosheets. Microchim Acta 186(1)Google Scholar
  23. 23.
    Wang C, Tang G, Tan H (2018) Colorimetric determination of mercury (II) via the inhibition by ssDNA of the oxidase-like activity of a mixed valence state cerium-based metal-organic framework. Mikrochim Acta 185(10):475CrossRefGoogle Scholar
  24. 24.
    Yan X, Song Y, Wu X, Zhu C, Su X, Du D, Lin Y (2017) Oxidase-mimicking activity of ultrathin MnO2 nanosheets in colorimetric assay of acetylcholinesterase activity. Nanoscale 9(6):2317–2323CrossRefGoogle Scholar
  25. 25.
    Na W, Li N, Xingguang S (2018) Enzymatic growth of single-layer MnO2 nanosheets in situ: application to detect alkaline phosphatase and ascorbic acid in the presence of sulfanilic acid functionalized graphene quantum dots. Sensor Actuat B-Chem 274:172–179CrossRefGoogle Scholar
  26. 26.
    He L, Wang F, Chen Y, Liu Y (2018) Rapid and sensitive colorimetric detection of ascorbic acid in food based on the intrinsic oxidase-like activity of MnO2 nanosheets. Luminescence 33(1):145–152CrossRefGoogle Scholar
  27. 27.
    Hu Q, He M, Mei Y, Feng W, Jing S, Kong J, Zhang X (2017) Sensitive and selective colorimetric assay of alkaline phosphatase activity with Cu (II)-phenanthroline complex. Talanta 163:146–152CrossRefGoogle Scholar
  28. 28.
    Han X, Han M, Ma L, Qu F, Kong RM, Qu F (2019) Self-assembled gold nanoclusters for fluorescence turn-on and colorimetric dual-readout detection of alkaline phosphatase activity via DCIP-mediated fluorescence resonance energy transfer. Talanta 194:55–62CrossRefGoogle Scholar
  29. 29.
    Gao Z, Deng K, Wang XD, Miro M, Tang D (2014) High-resolution colorimetric assay for rapid visual readout of phosphatase activity based on gold/silver core/shell nanorod. ACS Appl Mater Inter 6(20):18243–18250CrossRefGoogle Scholar
  30. 30.
    Lin C, Zheng H, Sun M, Guo Y, Luo F, Guo L, Qiu B, Lin Z, Chen G (2018) Highly sensitive colorimetric aptasensor for ochratoxin a detection based on enzyme-encapsulated liposome. Anal Chim Acta 1002:90–96CrossRefGoogle Scholar
  31. 31.
    Zhou CH, Zi QJ, Wang J, Zhao WY, Cao Q (2018) Determination of alkaline phosphatase activity and of carcinoembryonic antigen by using a multicolor liquid crystal biosensor based on the controlled growth of silver nanoparticles. Mikrochim Acta 186(1):25CrossRefGoogle Scholar
  32. 32.
    Yang J, Zheng L, Wang Y, Li W, Zhang J, Gu J, Fu Y (2016) Guanine-rich DNA-based peroxidase mimetics for colorimetric assays of alkaline phosphatase. Biosens Bioelectron 77:549–556CrossRefGoogle Scholar
  33. 33.
    Tang Z, Zhang H, Ma C, Gu P, Zhang G, Wu K, Chen M, Wang K (2018) Colorimetric determination of the activity of alkaline phosphatase based on the use of cu (II)-modulated G-quadruplex-based DNAzymes. Mikrochim Acta 185(2):109CrossRefGoogle Scholar
  34. 34.
    Wu T, Hou W, Ma Z, Liu M, Liu X, Zhang Y, Yao S (2019) Colorimetric determination of ascorbic acid and the activity of alkaline phosphatase based on the inhibition of the peroxidase-like activity of citric acid-capped Prussian blue nanocubes. Microchim Acta 186(2):123CrossRefGoogle Scholar
  35. 35.
    Liu SG, Han L, Li N, Xiao N, Ju YJ, Li NB, Luo HQ (2018) A fluorescence and colorimetric dual-mode assay of alkaline phosphatase activity via destroying oxidase-like CoOOH nanoflakes. J Mater Chem B 6(18):2843–2850CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Quality & Safety Risk Assessment for Citrus Products (Chongqing), Ministry of Agriculture, Citrus Research InstituteSouthwest UniversityChongqingPeople’s Republic of China
  2. 2.National Citrus Engineering Research CenterChongqingPeople’s Republic of China
  3. 3.College of Food ScienceSouthwest UniversityChongqingPeople’s Republic of China

Personalised recommendations