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Stable isotope labeling of metal/metal oxide nanomaterials for environmental and biological tracing


Engineered nanomaterials (NMs) are often compositionally indistinguishable from their natural counterparts, and thus their tracking in the environment or within the biota requires the development of appropriate labeling tools. Stable isotope labeling has become a well-established such tool, developed to assign ‘ownership’ or a ‘source’ to engineered NMs, enabling their tracing and quantification, especially in complex environments. A particular methodological challenge for stable isotope labeling is to ensure that the label is traceable in a range of environmental or biological scenarios but does not induce modification of the properties of the NM or lose its signal, thus retaining realism and relevance. This protocol describes a strategy for stable isotope labeling of several widely used metal and metal oxide NMs, namely ZnO, CuO, Ag, and TiO2, using isotopically enriched precursors, namely 67Zn or 68Zn metal, 65CuCl2, 107Ag or 109Ag metal, and 47TiO2 powder. A complete synthesis requires 1–8 d, depending on the type of NM, the precursors used, and the synthesis methods adopted. The physicochemical properties of the labeled particles are determined by optical, diffraction, and spectroscopic techniques for quality control. The procedures for tracing the labels in aquatic (snail and mussel) and terrestrial (earthworm) organisms and for monitoring the environmental transformation of labeled silver (Ag) NMs are also described. We envision that this labeling strategy will be adopted by industry to facilitate applications such as nanosafety assessments before NMs enter the market and environment, as well as for product authentication and tracking.

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Fig. 1: Schematic of the stable isotope labeling of NMs.
Fig. 2
Fig. 3: Comparative particle sizing of labeled versus unlabeled NMs.
Fig. 4: Enhanced detection sensitivity for NMs provided by stable isotope labeling.
Fig. 5: Transformation of 107Ag NMs and 109AgNO3 in an aqueous system.

Data availability

The data that support the plots within this paper are available from the corresponding author upon reasonable request.


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This work was supported by Marie Skłodowska-Curie Individual Fellowships (NanoLabels 750455 to P.Z.; NanoBBB 798505 to Z.G.) under the European Union’s Horizon 2020 research program. Financial support from MHRD-IMPRINT funds is appreciated. Partial funding from EU H2020 project ACEnano (grant agreement no. 2016-720952) is also acknowledged.

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Authors and Affiliations



P.Z., S.M., and Z.G. wrote the paper. E.V.-J. and M.R. designed part of the experiments and revised the paper. S.M. performed part of the experiments.

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Correspondence to Peng Zhang or Eugenia Valsami-Jones.

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Key references using this protocol

Dybowska, A.D. et al. Environ. Pollut. 159, 266–273 (2011):

Yu, S., Yin, Y., Zhou, X., Dong, L. & Liu, J. Environ. Sci. Nano 3, 883–893 (2016):

Misra, S.K. et al. Environ. Sci. Technol. 46, 1216–1222 (2012):

Laycock, A. et al. Environ. Sci. Nano 1, 271–283 (2014):

Bourgeault, A. et al. Environ. Sci. Technol. 49, 2451–2459 (2015):

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Zhang, P., Misra, S., Guo, Z. et al. Stable isotope labeling of metal/metal oxide nanomaterials for environmental and biological tracing. Nat Protoc 14, 2878–2899 (2019).

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