Ensemble multicolour FRET model enables barcoding at extreme FRET levels

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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.

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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.


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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

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.

Correspondence to David Juncker.

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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.

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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

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

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