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  • Brief Communication
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Multiplexed direct detection of barcoded protein reporters on a nanopore array


Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.

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Fig. 1: NTERs.
Fig. 2: Classification and multiplexed detection of NTER expression levels with a MinION.

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Data availability

Data are available upon request and on can be found on

Code availability

Codes are available upon request and can be found on Custom MinION MinKNOW runscripts can also be obtained from Oxford Nanopore Technologies upon request.


  1. Ghim, C. M., Lee, S. K., Takayama, S. & Mitchell, R. J. The art of reporter proteins in science: past, present and future applications. BMB Rep. 43, 451–460 (2010).

    Article  CAS  Google Scholar 

  2. Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci 42, 111–129 (2017).

    Article  CAS  Google Scholar 

  3. Martin, L., Che, A. & Endy, D. Gemini, a bifunctional enzymatic and fluorescent reporter of gene expression. PLoS ONE 4, e7569 (2009).

    Article  Google Scholar 

  4. Parrello, D., Mustin, C., Brie, D., Miron, S. & Billard, P. Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach. PLoS ONE 10, e0122848 (2015).

    Article  Google Scholar 

  5. Shimo, T., Tachibana, K. & Obika, S. Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening. PLoS ONE 13, e0197373 (2018).

    Article  Google Scholar 

  6. Wroblewska, A. et al. Protein barcodes enable high-dimensional single-cell CRISPR screens. Cell 175, 1141–1155 (2018).

    Article  CAS  Google Scholar 

  7. He, W., Yuan, S., Zhong, W. H., Siddikee, M. A. & Dai, C. C. Application of genetically engineered microbial whole-cell biosensors for combined chemosensing. Appl. Microbiol. Biotechnol. 100, 1109–1119 (2016).

    Article  CAS  Google Scholar 

  8. Nielsen, A. A. K. et al. Genetic circuit design automation. Science 352, aac7341 (2016).

    Article  Google Scholar 

  9. Shi, W., Friedman, A. K. & Baker, L. A. Nanopore sensing. Anal. Chem. 89, 157–188 (2017).

    Article  CAS  Google Scholar 

  10. Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016).

    Article  Google Scholar 

  11. Garalde, D. R. et al. Highly parallel direct RNA sequencing on an array of nanopores. Nat. Methods 15, 201–206 (2018).

    Article  CAS  Google Scholar 

  12. Nivala, J., Marks, D. B. & Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat. Biotechnol. 31, 247–250 (2013).

    Article  CAS  Google Scholar 

  13. Nivala, J., Mulroney, L., Li, G., Schreiber, J. & Akeson, M. Discrimination among protein variants using an unfoldase-coupled nanopore. ACS Nano 8, 12365–12375 (2014).

    Article  CAS  Google Scholar 

  14. Yim, H. H. & Villarejo, M. osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli. J. Bacteriol. 174, 3637–3644 (1992).

    Article  CAS  Google Scholar 

  15. Kotzsch, A. et al. A secretory system for bacterial production of high-profile protein targets. Protein Sci. 20, 597–609 (2011).

    Article  CAS  Google Scholar 

  16. Goyal, P. et al. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature 516, 250–253 (2014).

    Article  CAS  Google Scholar 

  17. Taylor, S. S. et al. PKA: a portrait of protein kinase dynamics.Biochim. Biophys. Acta Proteins Proteom. 1697, 259–269 (2004).

    Article  CAS  Google Scholar 

  18. Román, R. et al. Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide. J. Biotechnol. 239, 57–60 (2016).

    Article  Google Scholar 

  19. Peroutka, R. J., Elshourbagy, N., Piech, T. & Butt, T. R. Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A 2. Protein Sci. 17, 1586–1595 (2008).

    Article  CAS  Google Scholar 

  20. Gorochowski, T. E. et al. Genetic circuit characterization and debugging using RNA‐seq. Mol. Syst. Biol. 13, 952 (2017).

    Article  Google Scholar 

  21. Gach, P. C. et al. A droplet microfluidic platform for automating genetic engineering. ACS Synth. Biol. 5, 426–433 (2016).

    Article  CAS  Google Scholar 

  22. Chao, R., Mishra, S., Si, T. & Zhao, H. Engineering biological systems using automated biofoundries. Metab. Eng. 42, 98–108 (2017).

    Article  Google Scholar 

  23. Madison, A. C. et al. Scalable device for automated microbial electroporation in a digital micro fluidic platform. ACS Synth. Biol. 6, 1701–1709 (2017).

    Article  CAS  Google Scholar 

  24. Chen, Z. & Elowitz, E. B. Programmable protein circuit design. Cell 184, 2284–2301 (2021).

    Article  CAS  Google Scholar 

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We thank additional members of the Molecular Information Systems Lab for helpful discussion and feedback on this work. The OsmY expression plasmid was generously provided by C. Bryan and L. Carter (Institute for Protein Design, University of Washington). We also thank A. Heron and R. Gutierrez (Oxford Nanopore Technologies) for providing the configurable MinION run script and discussions on its use, and M. Jain (UCSC) for a custom Matlab script that facilitated visualization of the raw MinION data. This work was supported in part by NSF EAGER Award no. 1841188 and NSF CCF Award no. 2006864 to L.C. and J.N., an NIH/NCI Cancer Center Support Grant (no. P30 CA015704) Pilot Award and NSF Award 2021552 to J.N. and a sponsored research agreement from Oxford Nanopore Technologies.

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Authors and Affiliations



N.C., K.Z., A.N. and N.B. performed wet laboratory experiments. K.Z. and K.D. developed the data analysis pipeline and performed computational analyses. Z.S. implemented the machine learning approach. N.B., K.S., L.C. and J.N. supervised the project. J.N. conceived and directed the project. All authors contributed to writing and editing of the manuscript.

Corresponding author

Correspondence to Jeff Nivala.

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Competing interests

A provisional patent has been filed by the University of Washington covering aspects of this work (Patent Application no. 17/283,007). K.S. is an employee of Microsoft. J.N. is a consultant to Oxford Nanopore Technologies. The remaining authors declare no competing interests.

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Peer review information Nature Biotechnology thanks Yi-Tao Long, Meni Wanunu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Cardozo, N., Zhang, K., Doroschak, K. et al. Multiplexed direct detection of barcoded protein reporters on a nanopore array. Nat Biotechnol 40, 42–46 (2022).

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