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


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


  1. 1.

    Cohen, L. & Walt, D. R. Highly sensitive and multiplexed protein measurements. Chem. Rev. 119, 293–321 (2018).

    PubMed  Google Scholar 

  2. 2.

    Hanash, S. M., Pitteri, S. J. & Faca, V. M. Mining the plasma proteome for cancer biomarkers. Nature 452, 571–579 (2008).

    CAS  PubMed  Google Scholar 

  3. 3.

    Shaw, L. M., Korecka, M., Clark, C. M., Lee, V. M.-Y. & Trojanowski, J. Q. Biomarkers of neurodegeneration for diagnosis and monitoring therapeutics. Nat. Rev. Drug Discov. 6, 295–303 (2007).

    CAS  PubMed  Google Scholar 

  4. 4.

    Blennow, K. & Zetterberg, H. Understanding biomarkers of neurodegeneration: ultrasensitive detection techniques pave the way for mechanistic understanding. Nat. Med. 21, 217–219 (2015).

    CAS  PubMed  Google Scholar 

  5. 5.

    Savage, M. J. et al. A sensitive aβ oligomer assay discriminates Alzheimer’s and aged control cerebrospinal fluid. J. Neurosci. 34, 2884–2897 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Westermann, D., Neumann, J. T., Sörensen, N. A. & Blankenberg, S. High-sensitivity assays for troponin in patients with cardiac disease. Nat. Rev. Cardiol. 14, 472–483 (2017).

    CAS  PubMed  Google Scholar 

  7. 7.

    Rissin, D. M. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 28, 595–599 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Tabakman, S. M. et al. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range. Nat. Commun. 2, 466 (2011).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Zhang, B. et al. Diagnosis of Zika virus infection on a nanotechnology platform. Nat. Med. 23, 548–550 (2017).

    CAS  PubMed  Google Scholar 

  10. 10.

    Zhang, B., Kumar, R. B., Dai, H. & Feldman, B. J. A plasmonic chip for biomarker discovery and diagnosis of type 1 diabetes. Nat. Med. 20, 948–953 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Zhang, B. et al. Plasmonic micro-beads for fluorescence enhanced, multiplexed protein detection with flow cytometry. Chem. Sci. 5, 4070–4075 (2014).

    CAS  Google Scholar 

  12. 12.

    Steward, M. W. & Lew, A. M. The importance of antibody affinity in the performance of immunoassays for antibody. J. Immunol. Methods 78, 173–190 (1985).

    CAS  PubMed  Google Scholar 

  13. 13.

    Roth, S. et al. Photobleaching: improving the sensitivity of fluorescence‐based immunoassays by photobleaching the autofluorescence of magnetic beads (Small 3/2019). Small 15, 1970016 (2019).

    Google Scholar 

  14. 14.

    Zhang, P. et al. Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip. Nat. Biomed. Eng. 3, 438–451 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Espina, V. et al. Protein microarray detection strategies: focus on direct detection technologies. J. Immunol. Methods 290, 121–133 (2004).

    CAS  PubMed  Google Scholar 

  16. 16.

    Chen, Z. et al. Protein microarrays with carbon nanotubes as multicolor Raman labels. Nat. Biotechnol. 26, 1285–1292 (2008).

    CAS  PubMed  Google Scholar 

  17. 17.

    Luan, J. et al. Add-on plasmonic patch as a universal fluorescence enhancer. Light. Sci. Appl. 7, 29 (2018).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Reisch, A. et al. Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nat. Commun. 5, 4089 (2014).

    CAS  PubMed  Google Scholar 

  19. 19.

    Hu, J. et al. Sensitive and quantitative detection of C-reaction protein based on immunofluorescent nanospheres coupled with lateral flow test strip. Anal. Chem. 88, 6577–6584 (2016).

    CAS  PubMed  Google Scholar 

  20. 20.

    Huang, L. et al. Brilliant pitaya‐type silica colloids with central–radial and high‐density quantum dots incorporation for ultrasensitive fluorescence immunoassays. Adv. Funct. Mater. 28, 1705380 (2018).

    Google Scholar 

  21. 21.

    Reisch, A. & Klymchenko, A. S. Fluorescent polymer nanoparticles based on dyes: seeking brighter tools for bioimaging. Small 12, 1968–1992 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Shulov, I. et al. Fluorinated counterion-enhanced emission of rhodamine aggregates: ultrabright nanoparticles for bioimaging and light-harvesting. Nanoscale 7, 18198–18210 (2015).

    CAS  PubMed  Google Scholar 

  23. 23.

    Melnychuk, N. & Klymchenko, A. S. DNA-functionalized dye-loaded polymeric nanoparticles: ultrabright FRET platform for amplified detection of nucleic acids. J. Am. Chem. Soc. 140, 10856–10865 (2018).

    CAS  PubMed  Google Scholar 

  24. 24.

    Xu, Z. et al. Broad‐spectrum tunable photoluminescent nanomaterials constructed from a modular light‐harvesting platform based on macrocyclic amphiphiles. Adv. Mater. 28, 7666–7671 (2016).

    CAS  PubMed  Google Scholar 

  25. 25.

    Kasha, M., Rawls, H. & El-Bayoumi, M. A. The exciton model in molecular spectroscopy. Pure Appl. Chem. 11, 371–392 (1965).

    CAS  Google Scholar 

  26. 26.

    Méallet-Renault, R. et al. Fluorescent nanoparticles as selective Cu (II) sensors. Photochem. Photobiol. Sci. 5, 300–310 (2006).

    PubMed  Google Scholar 

  27. 27.

    Trofymchuk, K., Reisch, A., Shulov, I., Mély, Y. & Klymchenko, A. S. Tuning the color and photostability of perylene diimides inside polymer nanoparticles: towards biodegradable substitutes of quantum dots. Nanoscale 6, 12934–12942 (2014).

    CAS  PubMed  Google Scholar 

  28. 28.

    Trofymchuk, K. et al. Giant light-harvesting nanoantenna for single-molecule detection in ambient light. Nat. Photon. 11, 657–663 (2017).

    CAS  Google Scholar 

  29. 29.

    Holmberg, A. et al. The biotin–streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26, 501–510 (2005).

    CAS  PubMed  Google Scholar 

  30. 30.

    Chen, H., Shao, L., Li, Q. & Wang, J. Gold nanorods and their plasmonic properties. Chem. Soc. Rev. 42, 2679–2724 (2013).

    CAS  PubMed  Google Scholar 

  31. 31.

    Huang, X., Neretina, S. & El‐Sayed, M. A. Gold nanorods: from synthesis and properties to biological and biomedical applications. Adv. Mater. 21, 4880–4910 (2009).

    CAS  PubMed  Google Scholar 

  32. 32.

    Dreaden, E. C., Alkilany, A. M., Huang, X., Murphy, C. J. & El-Sayed, M. A. The golden age: gold nanoparticles for biomedicine. Chem. Soc. Rev. 41, 2740–2779 (2012).

    CAS  PubMed  Google Scholar 

  33. 33.

    Ge, S., Kojio, K., Takahara, A. & Kajiyama, T. Bovine serum albumin adsorption onto immobilized organotrichlorosilane surface: influence of the phase separation on protein adsorption patterns. J. Biomater. Sci. Polym. Ed. 9, 131–150 (1998).

    CAS  PubMed  Google Scholar 

  34. 34.

    Ashitate, Y., Tanaka, E., Stockdale, A., Choi, H. S. & Frangioni, J. V. Near-infrared fluorescence imaging of thoracic duct anatomy and function in open surgery and video-assisted thoracic surgery. J. Thorac. Cardiovasc. Surg. 142, 31–38 (2011).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Liu, C. et al. Fluorescence‐converging carbon nanodots‐hybridized silica nanosphere. Small 12, 4702–4706 (2016).

    CAS  PubMed  Google Scholar 

  36. 36.

    Flauraud, V. et al. In-plane plasmonic antenna arrays with surface nanogaps for giant fluorescence enhancement. Nano Lett. 17, 1703–1710 (2017).

    CAS  PubMed  Google Scholar 

  37. 37.

    Tam, F., Goodrich, G. P., Johnson, B. R. & Halas, N. J. Plasmonic enhancement of molecular fluorescence. Nano Lett. 7, 496–501 (2007).

    CAS  PubMed  Google Scholar 

  38. 38.

    Kinkhabwala, A. et al. Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna. Nat. Photon. 3, 654–657 (2009).

    CAS  Google Scholar 

  39. 39.

    Cai, Y. et al. Photoluminescence of gold nanorods: Purcell effect enhanced emission from hot carriers. ACS Nano 12, 976–985 (2018).

    CAS  PubMed  Google Scholar 

  40. 40.

    Liu, B. et al. High performance, multiplexed lung cancer biomarker detection on a plasmonic gold chip. Adv. Funct. Mater. 26, 7994–8002 (2016).

    CAS  Google Scholar 

  41. 41.

    Mayer, K. M. & Hafner, J. H. Localized surface plasmon resonance sensors. Chem. Rev. 111, 3828–3857 (2011).

    CAS  PubMed  Google Scholar 

  42. 42.

    Haes, A. J., Chang, L., Klein, W. L. & Van Duyne, R. P. Detection of a biomarker for Alzheimer’s disease from synthetic and clinical samples using a nanoscale optical biosensor. J. Am. Chem. Soc. 127, 2264–2271 (2005).

    CAS  PubMed  Google Scholar 

  43. 43.

    Bardhan, R., Grady, N. K., Cole, J. R., Joshi, A. & Halas, N. J. Fluorescence enhancement by Au nanostructures: nanoshells and nanorods. ACS Nano 3, 744–752 (2009).

    CAS  PubMed  Google Scholar 

  44. 44.

    Khatua, S. et al. Resonant plasmonic enhancement of single-molecule fluorescence by individual gold nanorods. ACS Nano 8, 4440–4449 (2014).

    CAS  PubMed  Google Scholar 

  45. 45.

    Anger, P., Bharadwaj, P. & Novotny, L. Enhancement and quenching of single-molecule fluorescence. Phys. Rev. Lett. 96, 113002 (2006).

    PubMed  Google Scholar 

  46. 46.

    Tian, L. et al. Gold nanocages with built-in artificial antibodies for label-free plasmonic biosensing. J. Mater. Chem. B 2, 167–170 (2014).

    CAS  PubMed  Google Scholar 

  47. 47.

    Abadeer, N. S., Brennan, M. R., Wilson, W. L. & Murphy, C. J. Distance and plasmon wavelength dependent fluorescence of molecules bound to silica-coated gold nanorods. ACS Nano 8, 8392–8406 (2014).

    CAS  PubMed  Google Scholar 

  48. 48.

    Mishra, H., Mali, B. L., Karolin, J., Dragan, A. I. & Geddes, C. D. Experimental and theoretical study of the distance dependence of metal-enhanced fluorescence, phosphorescence and delayed fluorescence in a single system. Phys. Chem. Chem. Phys. 15, 19538–19544 (2013).

    CAS  PubMed  Google Scholar 

  49. 49.

    Yan, B. et al. Engineered SERS substrates with multiscale signal enhancement: nanoparticle cluster arrays. ACS Nano 3, 1190–1202 (2009).

    CAS  PubMed  Google Scholar 

  50. 50.

    Pierre, M. C. S. & Haes, A. J. Purification implications on SERS activity of silica coated gold nanospheres. Anal. Chem. 84, 7906–7911 (2012).

    CAS  PubMed  Google Scholar 

  51. 51.

    Bardhan, R., Grady, N. K. & Halas, N. J. Nanoscale control of near‐infrared fluorescence enhancement using Au Nanoshells. Small 4, 1716–1722 (2008).

    CAS  PubMed  Google Scholar 

  52. 52.

    Thompson, D. K., Huffman, K. M., Kraus, W. E. & Kraus, V. B. Critical appraisal of four IL-6 immunoassays. PLoS ONE 7, e30659 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Samant, P. P. & Prausnitz, M. R. Mechanisms of sampling interstitial fluid from skin using a microneedle patch. Proc. Natl Acad. Sci. USA 115, 4583–4588 (2018).

    PubMed  Google Scholar 

  54. 54.

    Mishra, J. et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 365, 1231–1238 (2005).

    CAS  PubMed  Google Scholar 

  55. 55.

    Han, W. et al. Urinary biomarkers in the early diagnosis of acute kidney injury. Kidney Int. 73, 863–869 (2008).

    CAS  PubMed  Google Scholar 

  56. 56.

    Munier, M. et al. Physicochemical factors affecting the stability of two pigments: R-phycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum. Food Chem. 150, 400–407 (2014).

    CAS  PubMed  Google Scholar 

  57. 57.

    Yuan, H., Khatua, S., Zijlstra, P., Yorulmaz, M. & Orrit, M. Thousand‐fold enhancement of single‐molecule fluorescence near a single gold nanorod. Angew. Chem. Int. Ed. 52, 1217–1221 (2013).

    CAS  Google Scholar 

  58. 58.

    Ma, Y. et al. Au@ Ag core−shell nanocubes with finely tuned and well-controlled sizes, shell thicknesses, and optical properties. ACS Nano 4, 6725–6734 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Liu, K.-K., Tadepalli, S., Tian, L. & Singamaneni, S. Size-dependent surface enhanced Raman scattering activity of plasmonic nanorattles. Chem. Mater. 27, 5261–5270 (2015).

    CAS  Google Scholar 

  60. 60.

    Dixit, C. K. & Aguirre, G. R. Protein microarrays with novel microfluidic methods: current advances. Microarrays 3, 180–202 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Jain, P. K., Lee, K. S., El-Sayed, I. H. & El-Sayed, M. A. Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine. J. Phys. Chem. B 110, 7238–7248 (2006).

    CAS  PubMed  Google Scholar 

  62. 62.

    De La Rica, R. & Stevens, M. M. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nat. Nanotechnol. 7, 821–824 (2012).

    CAS  PubMed  Google Scholar 

  63. 63.

    Deng, W., Xie, F., Baltar, H. T. & Goldys, E. M. Metal-enhanced fluorescence in the life sciences: here, now and beyond. Phys. Chem. Chem. Phys. 15, 15695–15708 (2013).

    CAS  PubMed  Google Scholar 

  64. 64.

    Lee, K.-S. & El-Sayed, M. A. Dependence of the enhanced optical scattering efficiency relative to that of absorption for gold metal nanorods on aspect ratio, size, end-cap shape, and medium refractive index. J. Phys. Chem. B 109, 20331–20338 (2005).

    CAS  PubMed  Google Scholar 

  65. 65.

    Gole, A. & Murphy, C. J. Azide-derivatized gold nanorods: functional materials for “click” chemistry. Langmuir 24, 266–272 (2008).

    CAS  PubMed  Google Scholar 

  66. 66.

    Tebbe, M., Kuttner, C., Männel, M., Fery, A. & Chanana, M. Colloidally stable and surfactant-free protein-coated gold nanorods in biological media. ACS Appl. Mater. Interfaces 7, 5984–5991 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Nedrebø, T., Reed, R. K., Jonsson, R., Berg, A. & Wiig, H. Differential cytokine response in interstitial fluid in skin and serum during experimental inflammation in rats. J. Physiol. 556, 193–202 (2004).

    PubMed  PubMed Central  Google Scholar 

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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.

Author information




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).

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