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Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes

Nature Protocolsvolume 13pages15061520 (2018) | Download Citation


Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biological detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe3O4 nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase–like catalytic activities. Given the complex interdependence of the physicochemical properties and catalytic characteristics of nanozymes, it is important to establish a standard by which the catalytic activities and kinetics of various nanozymes can be quantitatively compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addition, we describe the detailed experimental procedures for a typical nanozyme strip–based biological detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic standard. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h.

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  1. 1.

    Gao, L. et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2, 577–583 (2007).

  2. 2.

    Kotov, N. A. Inorganic nanoparticles as protein mimics. Science 330, 188–189 (2010).

  3. 3.

    Fu, S. et al. Structural effect of Fe3O4 nanoparticles on peroxidase-like activity for cancer therapy. Colloids Surface B 154, 239 (2017).

  4. 4.

    Dong, J. et al. Co3O4 nanoparticles with multi-enzyme activities and their application in immunohistochemical assay. ACS Appl. Mater. Interfaces 6, 1959 (2014).

  5. 5.

    Jia, H. et al. Peroxidase-like activity of the Co3O4 nanoparticles used for biodetection and evaluation of antioxidant behavior. Nanoscale 8, 5938 (2016).

  6. 6.

    Mu, J., Zhang, L., Zhao, G. & Wang, Y. The crystal plane effect on the peroxidase-like catalytic properties of Co3O4 nanomaterials. Phys. Chem. Chem. Phys. 16, 15709–15716 (2014).

  7. 7.

    Guo, Y. et al. Fabrication of Ag-Cu2O/reduced graphene oxide nanocomposites as SERS substrates for in situ monitoring of peroxidase-like catalytic reaction and biosensing. ACS Appl. Mater. Interfaces 9, 19074–19081 (2017).

  8. 8.

    Dutta, A. K. et al. Synthesis of FeS and FeSe nanoparticles from a single source precursor: a study of their photocatalytic activity, peroxidase-like behavior, and electrochemical sensing of H2O2. ACS Appl. Mater. Interfaces 4, 1919–1927 (2012).

  9. 9.

    Dai, Z., Liu, S., Bao, J. & Ju, H. Nanostructured FeS as a mimic peroxidase for biocatalysis and biosensing. Chem. Eur. J. 15, 4321–4326 (2009).

  10. 10.

    Celardo, I., Pedersen, J. Z., Traversa, E. & Ghibelli, L. Pharmacological potential of cerium oxide nanoparticles. Nanoscale 3, 1411–1420 (2011).

  11. 11.

    Zhao, H., Dong, Y., Jiang, P., Wang, G. & Zhang, J. Highly dispersed CeO2 on TiO2 nanotube: a synergistic nanocomposite with superior peroxidase-like activity. ACS Appl. Mater. Interfaces 7, 6451–6461 (2015).

  12. 12.

    Shi, W., Zhang, X., He, S. & Huang, Y. CoFe2O4 magnetic nanoparticles as a peroxidase mimic mediated chemiluminescence for hydrogen peroxide and glucose. Chem. Commun. 47, 10785–10787 (2011).

  13. 13.

    Fan, Y. & Huang, Y. The effective peroxidase-like activity of chitosan-functionalized CoFe2 O4 nanoparticles for chemiluminescence sensing of hydrogen peroxide and glucose. Analyst 137, 1225–1231 (2012).

  14. 14.

    Luo, W. et al. Ultrasensitive fluorometric determination of hydrogen peroxide and glucose by using multiferroic BiFeO3 nanoparticles as a catalyst. Talanta 81, 901–907 (2010).

  15. 15.

    Vernekar, A. A., Das, T., Ghosh, S. & Mugesh, G. A remarkably efficient MnFe2O4‐based oxidase nanozyme. Chem. Asian J. 11, 72–76 (2016).

  16. 16.

    Peng, Y. et al. Size-and shape-dependent peroxidase-like catalytic activity of MnFe2O4 nanoparticles and their applications in highly efficient colorimetric detection of target cancer cells. Dalton Trans. 44, 12871–12877 (2015).

  17. 17.

    Liu, Q. et al. 5,10,15,20-Tetrakis (4-carboxyl phenyl) porphyrin–CdS nanocomposites with intrinsic peroxidase-like activity for glucose colorimetric detection. Mater. Sci. Eng. C 42, 177–184 (2014).

  18. 18.

    Garai-Ibabe, G., Möller, M., Saa, L., Grinyte, R. & Pavlov, V. Peroxidase-mimicking DNAzyme modulated growth of CdS nanocrystalline structures in situ through redox reaction: application to development of genosensors and aptasensors. Anal. Chem. 86, 10059–10064 (2014).

  19. 19.

    Jiang, L. et al. An ultrasensitive electrochemical aptasensor for thrombin based on the triplex-amplification of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme and horseradish peroxidase decorated FeTe nanorods. Analyst 138, 1497–1503 (2013).

  20. 20.

    Liu, W. et al. Paper-based colorimetric immunosensor for visual detection of carcinoembryonic antigen based on the high peroxidase-like catalytic performance of ZnFe2O4–-multiwalled carbon nanotubes. Analyst 139, 251–258 (2014).

  21. 21.

    Zhao, M. et al. Controlled synthesis of spinel ZnFe2O4 decorated ZnO heterostructures as peroxidase mimetics for enhanced colorimetric biosensing. Chem. Commun. 69, 7656–7658 (2013).

  22. 22.

    Zhou, Y. et al. Enzyme-mimetic effects of gold@platinum nanorods on the antioxidant activity of ascorbic acid. Nanoscale 5, 1583–1591 (2013).

  23. 23.

    Vineh, M. B., Saboury, A. A., Poostchi, A. A., Rashid, A. M. & Parivar, K. Stability and activity improvement of horseradish peroxidase by covalent immobilization on functionalized reduced graphene oxide and biodegradation of high phenol concentration. Int. J. Biol. Macromol. 17, 32776–32779 (2017).

  24. 24.

    Wang, S. et al. Mimicking horseradish peroxidase and NADH peroxidase by heterogeneous Cu2+-modified graphene oxide nanoparticles. Nano Lett. 17, 2043–2048 (2017).

  25. 25.

    Voeikov, V. L. & Yablonskaya, O. I. Stabilizing effects of hydrated fullerenes C60 in a wide range of concentrations on luciferase, alkaline phosphatase, and peroxidase in vitro. Electromagn. Biol. Med. 34, 160–166 (2015).

  26. 26.

    Wang, H. et al. Platinum nanocatalysts loaded on graphene oxide-dispersed carbon nanotubes with greatly enhanced peroxidase-like catalysis and electrocatalysis activities. Nanoscale 6, 8107–8116 (2014).

  27. 27.

    He, W. et al. Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays. Biomaterials 32, 1139–1147 (2011).

  28. 28.

    Zheng, X. et al. Catalytic gold nanoparticles for nanoplasmonic detection of DNA hybridization. Angew. Chem. Int. Ed. Engl. 123, 12200–12204 (2011).

  29. 29.

    Zhang, X., He, S., Chen, Z. & Huang, Y. CoFe2O4 nanoparticles as oxidase mimic-mediated chemiluminescence of aqueous luminol for sulfite in white wines. J. Agric. Food Chem. 61, 840–847 (2013).

  30. 30.

    Su, L. et al. Colorimetric detection of urine glucose based ZnFe2O4 magnetic nanoparticles. Anal. Chem. 84, 5753–5758 (2012).

  31. 31.

    Liu, X. et al. BSA-templated MnO2 nanoparticles as both peroxidase and oxidase mimics. Analyst 137, 4552 (2012).

  32. 32.

    Yan, X. et al. Oxidase-mimicking activity of ultrathin MnO2 nanosheets in colorimetric assay of acetylcholinesterase activity. Nanoscale 9, 2317 (2017).

  33. 33.

    Liu, J. et al. MnO2 nanosheets as an artificial enzyme to mimic oxidase for rapid and sensitive detection of glutathione. Biosens. Bioelectron. 90, 69 (2016).

  34. 34.

    Asati, A., Santra, S., Kaittanis, C., Nath, S. & Perez, J. M. Oxidase-like activity of polymer-coated cerium oxide nanoparticles. Angew. Chem. Int. Ed. Engl. 48, 2308 (2009).

  35. 35.

    Asati, A., Kaittanis, C., Santra, S. & Perez, J. M. The pH-tunable oxidase-like activity of cerium oxide nanoparticles achieves sensitive fluorigenic detection of cancer biomarkers at neutral pH. Anal. Chem. 83, 2547–2553 (2011).

  36. 36.

    Natalio, F. et al. Vanadium pentoxide nanoparticles mimic vanadium haloperoxidases and thwart biofilm formation. Nat. Nanotechnol. 7, 530–535 (2012).

  37. 37.

    Paras, J. et al. Helium‐based cold atmospheric plasma‐induced reactive oxygen species‐mediated apoptotic pathway attenuated by platinum nanoparticles. J. Cell. Mol. Med. 20, 1737–1748 (2016).

  38. 38.

    Clark, A., Zhu, A., Kai, S. & Petty, H. R. Cerium oxide and platinum nanoparticles protect cells from oxidant-mediated apoptosis. J. Nanopart. Res. 13, 5547–5555 (2011).

  39. 39.

    Rasouli, V. J., Taghi, M. M., Sarami, F. M. & Mahvash, J. Polyhydroxylated fullerene nanoparticles attenuate brain infarction and oxidative stress in rat model of ischemic stroke. EXCLI J. 15, 378–390 (2016).

  40. 40.

    Hu, Z. et al. Photodynamic anticancer activities of water-soluble C 60 derivatives and their biological consequences in a HeLa cell line. Chem. Biol. Interact. 195, 86–94 (2012).

  41. 41.

    Heckert, E. G., Karakoti, A. S., Seal, S. & Self, W. T. The role of cerium redox state in the SOD mimetic activity of nanoceria. Biomaterials 29, 2705–2709 (2008).

  42. 42.

    Chen, J., Patil, S., Seal, S. & Mcginnis, J. F. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nat. Nanotechnol. 1, 142–150 (2006).

  43. 43.

    Silva, G. A. Nanomedicine: seeing the benefits of ceria. Nat. Nanotechnol. 1, 92–94 (2006).

  44. 44.

    Wei, H. & Wang, E. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev. 42, 6060–6093 (2013).

  45. 45.

    Liang, M. et al. Fe3O4 magnetic nanoparticle peroxidase mimetic-based colorimetric assay for the rapid detection of organophosphorus pesticide and nerve agent. Anal. Chem. 85, 308–312 (2012).

  46. 46.

    Zhuang, J. et al. Ex vivo detection of iron oxide magnetic nanoparticles in mice using their intrinsic peroxidase-mimicking activity. Mol. Pharm. 9, 1983–1989 (2012).

  47. 47.

    Ghadi, A., Mahjoub, S., Tabandeh, F. & Talebnia, F. Synthesis and optimization of chitosan nanoparticles: potential applications in nanomedicine and biomedical engineering. Caspian J. Intern. Med. 5, 156 (2014).

  48. 48.

    Fan, K., Cao, C. & Pan, Y. et al. Magnetoferritin nanoparticles for targeting and visualizing tumour tissues. Nat. Nanotechnol. 7, 459–464 (2012).

  49. 49.

    Brynskikh, A. M. et al. Macrophage delivery of therapeutic nanozymes in a murine model of parkinson’s disease. Nanomedicine 5, 379–396 (2010).

  50. 50.

    Duan, D. et al. Nanozyme-strip for rapid local diagnosis of Ebola. Biosens. Bioelectron. 74, 134–141 (2015).

  51. 51.

    Sharif, A. et al. Soft template synthesis of super paramagnetic Fe3O4 nanoparticles a novel technique. J. Inorg. Organomet. Polym. Mater. 19, 355–360 (2009).

  52. 52.

    Bi, Y. et al. Genesis, evolution and prevalence of H5N6 avian influenza viruses in China. Cell Host Microbe 20, 810–821 (2016).

  53. 53.

    World Organisation for Animal Health. Avian influenza (infection with avian influenza viruses). OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Chapter 2.3.4. (2015).

  54. 54.

    World Health Organization. WHO Manual on Animal Influenza Diagnosis and Surveillance. (2002).

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This work was supported by the National Key R&D Program of China (2017YFA0205501), the National Natural Science Foundation of China (81722024, 81571728 and 31530026), the Key Research Program of Frontier Sciences (QYZDY-SSW-SMC013), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA09030306 and XDPB0304), the Youth Innovation Promotion Association (2014078) and the Sanming Project of Medicine in Shenzhen (SZSM201612031).

Author information

Author notes

  1. These authors contributed equally: Bing Jiang, Demin Duan, and Lizeng Gao.


  1. Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    • Bing Jiang
    • , Demin Duan
    • , Mengjie Zhou
    • , Kelong Fan
    • , Minmin Liang
    •  & Xiyun Yan
  2. School of Medicine, Yangzhou University, Yangzhou, China

    • Lizeng Gao
    • , Yan Tang
    •  & Juqun Xi
  3. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing, China

    • Yuhai Bi
    • , Zhou Tong
    •  & George Fu Gao
  4. Institute of Translation Medicine, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, China

    • Ni Xie
    • , Aifa Tang
    •  & Guohui Nie


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M.L. conceived and designed the experiments. B.J., D.D. and L.G. performed the experiments. M.L., B.J., D.D., L.G. and X.Y. reviewed, analyzed and interpreted the data. Y.B., Z.T. and G.F.G. provided the inactivated H1N1 virus. M.L. wrote the paper. B.J., D.D., L.G., M.Z., K.F., Y.T., J.X., Y.B., Z.T., G.F.G., N.X., A.T., G.N., M.L. and X.Y. discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Minmin Liang or Xiyun Yan.

Integrated supplementary information

  1. Supplementary Figure 1 Standardization of the peroxidase-like activity of Fe3O4 NPs with different surface modifications.

    (a) The catalytic activity and (b) specific activity of Fe3O4 NPs capped with SiO2, polyethylene glycol (PEG) or dextran. SA indicates specific activities. Error bars shown represent the standard error derived from three repeated measurements

  2. Supplementary Figure 2 Size-dependent peroxidase-like activity of Fe3O4 nanozymes.

    (a) TEM images of Fe3O4 NPs with three different sizes. (b) The catalytic activity (left) and specific activity (right) of Fe3O4 NPs of three different sizes. The smaller NPs show higher catalytic activity under the same conditions. SA indicates specific activities. Error bars shown represent the standard error derived from three repeated measurements

  3. Supplementary Figure 3 Standardization of the peroxidase-like activity of commercial Fe3O4 NPs.

    (a) The catalytic activity and (b) specific activity of commercial Fe3O4 NPs of two different sizes. The smaller NPs show higher catalytic activity under the same conditions. SA indicates specific activities. Error bars shown represent the standard error derived from three repeated measurements

  4. Supplementary Figure 4 Effect of temperature on the catalytic activity of carbon, Fe3O4 and Au peroxidase nanozymes and HRP.

    Error bars shown represent the standard error derived from three repeated measurements

  5. Supplementary Figure 5 Standardization of HRP catalytic activity.

    (a) Reaction-time curve of TMB colorimetric reaction catalyzed by HRP. (b) The specific activity (SA) of HRP was calculated to be 504 U/mg protein using the nanozyme activity standardization method described herein. Error bars shown represent the standard error derived from three repeated measurements

  6. Supplementary Figure 6 The catalytic activity of nanozymes is from the NPs instead of the dissolved ions in acidic reaction solution.

    The Fe3O4, carbon and Au NPs were respectively incubated in pH 3.6 reaction solution for 400 s (the time taken for activity measurement), and then removed by centrifugation. The activity of the leaching solution was compared with that of the collected NPs

  7. Supplementary Figure 7 Characterization of the structural integrity of NPs.

    DLS analysis of Fe3O4, carbon and Au NPs before (a) and after (b) incubation under the standard reaction solution (pH 3.6 NaAc–HAc buffer)

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–7

  2. Combined Supplementary Information

    Supplementary Methods and Supplementary Data 1

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