Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Ensemble multicolour FRET model enables barcoding at extreme FRET levels

Abstract

Quantitative models of Förster resonance energy transfer (FRET)—pioneered by Förster—define our understanding of FRET and underpin its widespread use. However, multicolour FRET (mFRET), which arises between multiple, stochastically distributed fluorophores, lacks a mechanistic model and remains intractable. mFRET notably arises in fluorescently barcoded microparticles, resulting in a complex, non-orthogonal fluorescence response that impedes their encoding and decoding. Here, we introduce an ensemble mFRET (emFRET) model, and apply it to guide barcoding into regimes with extreme FRET. We further introduce a facile, proportional multicolour labelling method using oligonucleotides as homogeneous linkers. A total of 580 barcodes were rapidly designed and validated using four dyes—with FRET efficiencies reaching 76%—and used for multiplexed immunoassays with cytometric readout and fully automated decoding. The emFRET model helps to expand the barcoding capacity of barcoded microparticles using common organic dyes and will benefit other applications subject to stochastic mFRET.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Spectrally overlapping classifier dyes and the impact of mFRET on the fluorescence response of barcoded microparticles.
Fig. 2: One-pot DNA-assisted microparticle labelling conserves dye proportions.
Fig. 3: Schematic representation of the emFRET model and experimental validation of the MFM.
Fig. 4: In silico design and experimental verification of four-colour barcodes with extreme emFRET.
Fig. 5: Multicolour fluorescence model enables automated decoding.
Fig. 6: Screening of binding specificities for a 35-plex sandwich immunoassay.

Similar content being viewed by others

References

  1. Nolan, J. P. & Sklar, La Suspension array technology: evolution of the flat-array paradigm. Trends Biotechnol. 20, 9–12 (2002).

    Article  CAS  Google Scholar 

  2. Wilson, R., Cossins, A. R. & Spiller, D. G. Encoded microcarriers for high-throughput multiplexed detection. Angew. Chem. Int. Ed. 45, 6104–6117 (2006).

    Article  CAS  Google Scholar 

  3. Fulton, R. J., McDade, R. L., Smith, P. L., Kienker, L. J. & Kettman, J. R. Advanced multiplex analysis with the FlowMetrix system. Clin. Chem. 43, 1749–1756 (1997).

    CAS  Google Scholar 

  4. Wu, W. et al. Antibody array analysis with label-based detection and resolution of protein size. Mol. Cell. Proteomics 8, 245–257 (2009).

    Article  CAS  Google Scholar 

  5. Wang, L. & Tan, W. Multicolor FRET silica nanoparticles by single wavelength excitation. Nano. Lett. 6, 84–88 (2006).

    Article  CAS  Google Scholar 

  6. Vaidya, S. V., Couzis, A. & Maldarelli, C. Reduction in aggregation and energy transfer of quantum dots incorporated in polystyrene beads by kinetic entrapment due to cross-linking during polymerization. Langmuir 31, 3167–3179 (2015).

    Article  CAS  Google Scholar 

  7. Clapp, A. R., Medintz, I. L. & Mattoussi, H. Förster resonance energy transfer investigations using quantum-dot fluorophores. ChemPhysChem 7, 47–57 (2006).

    Article  CAS  Google Scholar 

  8. Wagh, A. et al. Polymeric nanoparticles with sequential and multiple FRET cascade mechanisms for multicolor and multiplexed imaging. Small 9, 2129–2139 (2013).

    Article  CAS  Google Scholar 

  9. Stuchlý, J. et al. An automated analysis of highly complex flow cytometry-based proteomic data. Cytom. A 81, 120–129 (2012).

    Article  Google Scholar 

  10. Han, M., Gao, X., Su, J. Z. & Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat. Biotechnol. 19, 631–635 (2001).

    Article  CAS  Google Scholar 

  11. Wang, G. et al. Highly efficient preparation of multiscaled quantum dot barcodes for multiplexed hepatitis B detection. ACS Nano 7, 471–481 (2013).

    Article  CAS  Google Scholar 

  12. Ferguson, J. A., Steemers, F. J. & Walt, D. R. High-density fiber-optic DNA random microsphere array. Anal. Chem. 72, 5618–5624 (2000).

    Article  CAS  Google Scholar 

  13. Förster, T. Experimentelle und theoretische Untersuchung des zwischengmolekularen übergangs von Elektronenanregungsenergie. Naturforsch. A Astrophys. Phys. Phys. Chem. 4a, 321–327 (1949).

    Google Scholar 

  14. Wolber, P. & Hudson, B. An analytic solution to the Förster energy transfer problem in two dimensions. Biophys. J. 28, 197–210 (1979).

    Article  CAS  Google Scholar 

  15. Watrob, H. M., Pan, C. P. & Barkley, M. D. Two-step FRET as a structural tool. J. Am. Chem. Soc. 125, 7336–7343 (2003).

    Article  CAS  Google Scholar 

  16. Fabian, A., Horvath, G., Vamosi, G., Vereb, G. & Szollosi, J. TripleFRET measurements in flow cytometry. Cytom. A 83, 375–385 (2013).

    Article  Google Scholar 

  17. Stein, I. H., Steinhauer, C. & Tinnefeld, P. Single-molecule four-color FRET visualizes energy-transfer paths on DNA origami. J. Am. Chem. Soc. 133, 4193–4195 (2011).

    Article  CAS  Google Scholar 

  18. Lee, J. et al. Single-molecule four-color FRET. Angew. Chem. Int. Ed. 49, 9922–9925 (2010).

    Article  CAS  Google Scholar 

  19. Uphoff, S. et al. Monitoring multiple distances within a single molecule using switchable FRET. Nat. Methods 7, 831–836 (2010).

    Article  CAS  Google Scholar 

  20. Bunt, G. & Wouters, F. S. FRET from single to multiplexed signaling events. Biophys. Rev. 9, 119–129 (2017).

    Article  Google Scholar 

  21. Buckhout-White, S. et al. Assembling programmable FRET-based photonic networks using designer DNA scaffolds. Nat. Commun. 5, 5615–5630 (2014).

    Article  CAS  Google Scholar 

  22. Spillmann, C. M. et al. Extending FRET cascades on linear DNA photonic wires. Chem. Commun. 50, 7246–7249 (2014).

    Article  CAS  Google Scholar 

  23. Raicu, V. Efficiency of resonance energy transfer in homo-oligomeric complexes of proteins. J. Biol. Phys. 33, 109–127 (2007).

    Article  CAS  Google Scholar 

  24. Liu, J. & Lu, Y. FRET study of a trifluorophore-labelled DNAzyme. J. Am. Chem. Soc. 124, 15208–15216 (2002).

    Article  CAS  Google Scholar 

  25. Shapiro, H. M. Practical Flow Cytometry (Wiley, Hoboken, NJ, 2003).

    Book  Google Scholar 

  26. Mátyus, L. Fluorescence resonance energy transfer measurements on cell surfaces. A spectroscopic tool for determining protein interactions. J. Photochem. Photobiol. 12, 323–337 (1992).

    Article  Google Scholar 

  27. Berney, C. & Danuser, G. FRET or no FRET: a quantitative comparison. Biophys. J. 84, 3992–4010 (2003).

    Article  CAS  Google Scholar 

  28. Corry, B., Jayatilaka, D. & Rigby, P. A flexible approach to the calculation of resonance energy transfer efficiency between multiple donors and acceptors in complex geometries. Biophys. J. 89, 3822–3836 (2005).

    Article  CAS  Google Scholar 

  29. Koppel, D. E., Fleming, P. J. & Strittmatter, P. Intramembrane positions of membrane-bound chromophores determined by excitation energy transfer. Biochemistry 18, 5450–5457 (1979).

    Article  CAS  Google Scholar 

  30. Nguyen, H. Q. et al. Programmable microfluidic synthesis of over one thousand uniquely identifiable spectral codes. Adv. Opt. Mater. 5, 1600548 (2016).

    Article  Google Scholar 

  31. Lee, J. et al. Universal process-inert encoding architecture for polymer microparticles. Nat. Mater. 13, 524–529 (2014).

    Article  CAS  Google Scholar 

  32. Fielding, A. H. Cluster and Classification Techniques for the Biosciences Vol. 53 (Cambridge Univ. Press, Cambridge, 2007).

  33. King, L. E. et al. Ligand binding assay critical reagents and their stability: recommendations and best practices from the Global Bioanalysis Consortium Harmonization Team. AAPS J. 16, 504–515 (2014).

    Article  CAS  Google Scholar 

  34. Uhlen, M. et al. A proposal for validation of antibodies. Nat. Methods 13, 823–827 (2016).

    Article  CAS  Google Scholar 

  35. Pla-Roca, M. et al. Antibody colocalization microarray: a scalable technology for multiplex protein analysis in complex samples. Mol. Cell. Proteomics 11, https://doi.org/10.1074/mcp.M111.011460 (2012).

    Article  Google Scholar 

  36. Juncker, D., Bergeron, S., Laforte, V. & Li, H. Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing. Curr. Opin. Chem. Biol. 18, 29–37 (2014).

    Article  CAS  Google Scholar 

  37. Schweitzer, B. et al. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359–365 (2002).

    Article  CAS  Google Scholar 

  38. Gonzalez, R. M. et al. Development and validation of sandwich ELISA microarrays with minimal assay interference. J. Proteome Res. 7, 2406–2414 (2008).

    Article  CAS  Google Scholar 

  39. Hardin, B. E. et al. Increased light harvesting in dye-sensitized solar cells with energy relay dyes. Nat. Photon. 3, 406–411 (2009).

    Article  CAS  Google Scholar 

  40. Lakowicz, J. R. Principles of Fluorescence Spectroscopy (Springer, New York, NY, 2006).

    Book  Google Scholar 

  41. Förster, T. in Modern Quantum Chemistry Part III: Action of Light and Organic Crystals (ed. Sinanglu, O.) 93–137 (Academic, New York, NY, 1965)..

  42. Dempster, A., Laird, N. & Donald, R. Maximum likelihood from incomplete data via the EM algorithm. J. R. Statist. Soc. Ser. B 39, 1–38 (1977).

    Google Scholar 

Download references

Acknowledgements

The authors thank T. Gervais for discussions, and J. Munzar for proofreading our manuscript. The authors thank NSERC and FQRNT for funding. M.D. acknowledges the NSERC-CREATE ISS programme for support. The flow cytometry work was performed at two McGill core flow facilities, namely the Microbiology and Immunology (MIMM) department and the Life Science Complex, which is supported by funding from the Canadian Foundation for Innovation.

Author information

Authors and Affiliations

Authors

Contributions

M.D. and D.J. developed the approach. M.D. and A.N. conceived the experiments. M.D. developed the models and performed the experiments. M.K. and M.D. developed the decoding algorithm. M.D and D.J. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to David Juncker.

Ethics declarations

Competing interests

D.J. and M.D. are inventors on a provisional US patent application 62/568,998 that covers some of the aspects reported here and was filed by McGill University on 6 October 2017. M.D. and D.J. are founders and shareholders of nplex biosciences inc.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–14, Supplementary Tables 1–2, Supplementary Notes and Supplementary References

Supplementary Table 3

Designed barcodes

Supplementary Table 4

35-plex sandwich assay: barcodes and reagents

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dagher, M., Kleinman, M., Ng, A. et al. Ensemble multicolour FRET model enables barcoding at extreme FRET levels. Nature Nanotech 13, 925–932 (2018). https://doi.org/10.1038/s41565-018-0205-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-018-0205-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing