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

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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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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.


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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

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.

Correspondence to Minmin Liang or Xiyun Yan.

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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)

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Supplementary Figures 1–7

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Supplementary Methods and Supplementary Data 1

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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) doi:10.1038/s41596-018-0001-1

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