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Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging

Abstract

The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody–silica–UCNP conjugate takes 3 d, the streptavidin–poly(ethylene glycol)–UCNP conjugate takes 2–3 weeks, upconversion-linked immunosorbent assay takes 2–4 d and immunocytochemistry takes 8–10 h. The procedures can be performed after standard laboratory training in nanomaterials research.

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Fig. 1: Two methods for surface modification and bionconjugation of UCNPs.
Fig. 2: Purification of protein-conjugated UCNPs and analysis of aggregation.
Fig. 3: Workflow of UCNPs synthesis for highly sensitive assays and imaging applications.
Fig. 4: Schemes of two ULISA procedures for the detection of the cancer marker PSA.
Fig. 5: Scheme of immunocytochemical labeling of HER2.
Fig. 6: Apparatus for UCNP synthesis.
Fig. 7: Upconversion detection instruments.
Fig. 8: TEM images and emission spectra of UCNPs obtained by a seed-mediated synthesis.
Fig. 9: Purification of streptavidin–PEG–UCNPs by sucrose gradient centrifugation and aggregation analysis.
Fig. 10: ULISA of PSA.
Fig. 11: ICC based on streptavidin–PEG–neridronate–UCNP labels.

Data availability

This article presents representative data that support the procedures. Additional data are available in the supporting research papers (gel electrophoresis74, digital immunoassays34,41 and ICC36). Primary data underlying the figures shown in this protocol are available upon a reasonable request from the corresponding authors. Source data are provided with this paper.

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Acknowledgements

H.H.G. acknowledges support from the German Research Foundation (DFG: GO 1968/ 7–1 (Heisenberg Program) and GO 1968/6–1). A.H. and F.F. acknowledge institutional support grant RVO 68081715. A.H., Z.F., and P.S. acknowledge grant 21-03156S from the Czech Science Foundation. Z.F. and P.S. acknowledge the support of the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR) under the projects INTER-ACTION (LTAB19011) and CEITEC 2020 (LQ1601). D.H. acknowledges grant 21-04420S from the Czech Science Foundation. We thank P. Bouchal and P. Bouchalová for providing cell slides, N. Velychkivska for NMR measurements and V. Vykoukal for taking TEM images.

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Contributions

A.H., Z.F., M.J.M., U.K. and J.C.B. contributed with experiments, A.H., Z.F., M.J.M., U.K., J.C.B. and H.H.G. wrote the manuscript, and P.S., F.F., D.H. and H.H.G. supervised.

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Correspondence to Antonín Hlaváček, Zdeněk Farka or Hans H. Gorris.

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Competing interests

M.J.M. was a graduate student in the Gorris group when he completed the work described in the paper and is now employed by Lumito, a company that is pursuing applications of UCNPs in IHC.

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Nature Protocols thanks Jiajia Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Farka, Z. et al. Nanoscale 12, 8303–8313 (2020): https://doi.org/10.1039/c9nr10568a

Mickert, M. J. et al. Anal. Chem. 91, 9435–9441 (2019): https://doi.org/10.1021/acs.analchem.9b02872

Hlaváček, A. et al. Anal. Chem. 91, 1241–1246 (2019): https://doi.org/10.1021/acs.analchem.8b04488

Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Figs. 1–8.

Reporting Summary

Supplementary Table 2

Computation of chemicals for coating UCNPs with a carboxylated silica shell

Source data

Source Data Fig. 10

Unprocessed micrographs

Source Data Fig. 11

Unprocessed micrograph

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Hlaváček, A., Farka, Z., Mickert, M.J. et al. Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging. Nat Protoc 17, 1028–1072 (2022). https://doi.org/10.1038/s41596-021-00670-7

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  • DOI: https://doi.org/10.1038/s41596-021-00670-7

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