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Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays

Abstract

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6–15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4–7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.

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Fig. 1: Overview of the scPISA procedure for the determination of low-copy-number proteins in single living cells.
Fig. 2: Fabrication of extraction microprobes.
Fig. 3: Fabrication of plasmonic nanotags.
Fig. 4: Single-cell manipulation via a three-dimensional manipulator.
Fig. 5: Raman spectrum acquisition and analysis.
Fig. 6: Examples of anticipated results of detection of survivin in single living cells by the scPISA.

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Data availability

The detailed raw data for Fig. 6 and their interpretation for the low-copy-number protein detection in single living cells by scPISA are available in the original publication24. Source data are provided with this paper.

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Acknowledgements

This work is supported by Key Scientific Instrumentation Grant (21627810) from the National Natural Science Foundation of China, and Excellent Research Program of Nanjing University (ZYJH004).

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J.L., R.Y.W. and Z.L. developed the protocol. J.L., H.H. and Z.L. wrote the paper. All authors have discussed the results and approved the final version of the manuscript.

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Correspondence to Zhen Liu.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Jakub Dostálek, Ugur Tamer 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

Liu, J. et al. Angew. Chem. Int. Ed. 55, 13215–13218 (2016): https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201608237

Liu, J. et al. Chem. Sci. 9, 7241–7246 (2018): https://pubs.rsc.org/en/content/articlepdf/2018/sc/c8sc02533a

Wen, Y. R. et al. Anal. Chem. 92, 12498–12508 (2020): https://pubs.acs.org/doi/10.1021/acs.analchem.0c02344

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Source Data Fig. 5

Raw data for Fig. 5e and 5f.

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Liu, J., He, H., Xie, D. et al. Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays. Nat Protoc 16, 3522–3546 (2021). https://doi.org/10.1038/s41596-021-00547-9

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