Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

Nature Protocols volume 13, pages 1506–1520 (2018)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Standardization of nanozyme peroxidase-like catalytic activity.
Fig. 2: Characterization of the catalytic kinetics of peroxidase nanozymes and natural (HRP).
Fig. 3: Peroxidase nanozyme-based strip method for detecting H1N1 virus with the standardized nanozyme activity units as guide.

Similar content being viewed by others

References

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  PubMed  Google Scholar 

  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).

    PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf (2015).

  54. World Health Organization. WHO Manual on Animal Influenza Diagnosis and Surveillance. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf. (2002).

Download references

Acknowledgements

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.

Authors and Affiliations

  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

Authors
  1. Bing Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Demin Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lizeng Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mengjie Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kelong Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yan Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Juqun Xi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yuhai Bi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhou Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. George Fu Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ni Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Aifa Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Guohui Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Minmin Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Xiyun Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Minmin Liang or Xiyun Yan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Intrinsic peroxidase-like activity of ferromagnetic nanoparticles: https://doi.org/10.1038/nnano.2007.260

Magnetoferritin nanoparticles for targeting and visualizing tumour tissues: https://doi.org/10.1038/nnano.2012.90

Nanozyme-strip for rapid local diagnosis of Ebola: https://doi.org/10.1016/j.bios.2015.05.025

Integrated supplementary information

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

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

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

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

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

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

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

Supplementary Text and Figures

Supplementary Figures 1–7

Combined Supplementary Information

Supplementary Methods and Supplementary Data 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jiang, B., Duan, D., Gao, L. et al. Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes. Nat Protoc 13, 1506–1520 (2018). https://doi.org/10.1038/s41596-018-0001-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-018-0001-1

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research