Ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays

Abstract

The detection and quantification of low-abundance molecular biomarkers in biological samples is challenging. Here, we show that a plasmonic nanoscale construct serving as an ‘add-on’ label for a broad range of bioassays improves their signal-to-noise ratio and dynamic range without altering their workflow and readout devices. The plasmonic construct consists of a bovine serum albumin scaffold with approximately 210 IRDye 800CW fluorophores (with a fluorescence intensity approximately 6,700-fold that of a single 800CW fluorophore), a polymer-coated gold nanorod acting as a plasmonic antenna and biotin as a high-affinity biorecognition element. Its emission wavelength can be tuned over the visible and near-infrared spectral regions by modifying its size, shape and composition. It improves the limit of detection in fluorescence-linked immunosorbent assays by up to 4,750-fold and is compatible with multiplexed bead-based immunoassays, immunomicroarrays, flow cytometry and immunocytochemistry methods, and it shortens overall assay times (to 20 min) and lowers sample volumes, as shown for the detection of a pro-inflammatory cytokine in mouse interstitial fluid and of urinary biomarkers in patient samples.

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Fig. 1: Plasmonic-fluor synthesis and material characterization.
Fig. 2: Plasmon-enhanced fluorescence and colloidal stability of plasmonic-fluors.
Fig. 3: p-FLISA and multiplexed bead-based immunoassay.
Fig. 4: Plasmonic-fluor-enhanced high-throughput proteome profiler array.
Fig. 5: Plasmonic-fluor-enhanced ICC and flow cytometry.
Fig. 6: Flow cytometry analysis of BMDC maturation maker probed by conventional fluor (680LT) and plasmonic-fluor–680LT.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Raw imaging data (collected and analysed via the software indicated in the Reporting Summary) are available from figshare with the identifier https://doi.org/10.6084/m9.figshare.11888055.

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Acknowledgements

We acknowledge support from National Science Foundation (award nos. CBET-1512043 and CBET-1900277), National Institutes of Health (R01DE027098 and R01CA141521), Center for Multiple Myeloma Nanotherapy (U54 CA199092) and a grant from the Barnes-Jewish Hospital Research Foundation (no. 3706). We thank K. Naegle for providing access to a LI-COR Odyssey CLx scanner, L. Setton for the flow cytometer, J. Rudra and T. Pietka for Luminex readers, and the Nano Research Facility (NRF) and Institute of Materials Science and Engineering (IMSE) at Washington University for providing access to electron microscopy facilities. We also thank G. Genin for inspiring discussions and suggestions.

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Contributions

S.S. and J.L. designed the project and experiments. J.L. synthesized plasmonic-fluors. A.S. and J.L. designed and performed the flow cytometry experiments of SK-BR-3 and BMDCs. R.G. performed the AFM and TEM characterization. Z.W. performed the immunocytochemistry experiment of SK-BR-3 cells and B.X. did the confocal imaging of the cells. P.R., S.C. and H.G.D. performed the SEM characterizations. R.T. performed the fluorescence lifetime measurements and analysed the data. S.A. helped to design the lifetime measurement experiments and analysed the data. J.J.M. helped to design the kidney disease-related experiments and provided the kidney disease patient samples. S.S. and J.L. wrote the paper. All authors reviewed and commented on the manuscript.

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Correspondence to Srikanth Singamaneni.

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Competing interests

J.L., J.J.M. and S.S. are inventors on a provisional patent related to this technology. The technology has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC, which is developing plasmonic-fluor products. J.L., J.J.M. and S.S. are co-founders/shareholders of Auragent Bioscience LLC. These potential conflicts of interest have been disclosed and are being managed by Washington University in St. Louis. The remaining authors declare no competing interests.

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Luan, J., Seth, A., Gupta, R. et al. Ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays. Nat Biomed Eng 4, 518–530 (2020). https://doi.org/10.1038/s41551-020-0547-4

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