Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye

Journal name:
Nature Nanotechnology
Year published:
Published online


In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living1, 2. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources3, 4, 5, 6, 7, 8, 9, 10, 11. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10−18 g ml−1. p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.

At a glance


  1. Schematic representation of the sandwich ELISA format used here and two possible signal generation mechanisms.
    Figure 1: Schematic representation of the sandwich ELISA format used here and two possible signal generation mechanisms.

    In sandwich ELISA the target molecule is anchored to the substrate by capture antibodies and recognized by primary antibodies. In the present work, the enzyme is linked to the immunocomplex through interactions between enzyme-decorated streptavidin and biotinylated secondary antibodies. a, In conventional colorimetric ELISA, enzymatic biocatalysis generates a coloured compound. b, In plasmonic ELISA the biocatalytic cycle of the enzyme generates coloured nanoparticle solutions of characteristic tonality (S, substrate; P, product; NP, nanoparticle).

  2. Generation of coloured solutions for detection with the naked eye.
    Figure 2: Generation of coloured solutions for detection with the naked eye.

    a, In the presence of hydrogen peroxide, gold ions are reduced. b, High concentrations of hydrogen peroxide favour the formation of non-aggregated, spherical nanoparticles that give rise to a red solution. c, When the concentration of hydrogen peroxide decreases, for example due to the biocatalytic action of the enzyme catalase, aggregates of nanoparticles are formed and this turns the solution blue.

  3. Generation of nanoparticle solutions with different colours depends on the concentration of hydrogen peroxide.
    Figure 3: Generation of nanoparticle solutions with different colours depends on the concentration of hydrogen peroxide.

    Different concentrations of hydrogen peroxide were added to a solution containing gold ions (0.1 mM) in MES buffer (1 mM, pH 6.5). a, Photograph showing the generation of nanoparticle solutions with different colours and intensities after 15 min. The tonality of the solution changes from red to blue between 120 and 100 µM. b, Ultraviolet–visible spectra for different hydrogen peroxide concentrations. The localized surface plasmon resonance peak redshifts when the concentration of hydrogen peroxide is 100 µM or lower. c,d, Transmission electron microscopy (TEM) images of nanoparticles grown with hydrogen peroxide at concentrations of 100 µM (c) and 120 µM (d). Scale bars = 50 nm (c) and 100 nm (d). e, Graph showing that the absorbance of the solutions at 550 nm varies with concentration of hydrogen peroxide.

  4. Naked-eye detection of serum proteins with plasmonic ELISA.
    Figure 4: Naked-eye detection of serum proteins with plasmonic ELISA.

    a, PSA. b, p24. PSA and p24 can be detected by the generation of blue nanoparticle solutions. The signal (−ΔA550) is expressed as the decrease in absorbance with respect to the blank monitored at 550 nm. Blue curves were obtained by spiking PSA into bovine serum or p24 in female serum. Red curves were obtained by spiking the unrelated protein BSA. Error bars indicate the standard deviation of three independent measurements.

  5. Detection of p24 in sera from donors with different viral loads.
    Figure 5: Detection of p24 in sera from donors with different viral loads.

    The order in the photograph is directly related to the order in the table. a, HIV-infected samples with a high viral load yield blue-coloured nanoparticle solutions. b, HIV-infected patients with a viral load undetectable with the nucleic acid-based test yield blue-coloured nanoparticle solutions. c, Non-infected HIV-negative (HIV-ve) donors yield red-coloured nanoparticle solutions.


  1. Patton, J. C., Coovadia, A. H., Meyers, T. M. & Sherman, G. G. Evaluation of the ultrasensitive human immunodeficiency virus type (HIV-1) p24 antigen assay performed on dried blood spots for diagnosis of HIV-1 infection in infants. Clin. Vaccine Immunol. 15, 388391 (2008).
  2. Tang, S. & Hewlett, I. Nanoparticle-based immunoassays for sensitive and early detection of HIV-1 capsid (p24) antigen. J. Infect. Dis. 201, S59S64 (2010).
  3. Rodriguez-Lorenzo, L., de la Rica, R., Alvarez-Puebla, R., Liz-Marzan, L. M. & Stevens, M. M. Plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth. Nature Mater. 11, 604607 (2012).
  4. Nam, J. M., Thaxton, C. S. & Mirkin, C. A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 18841886 (2003).
  5. Rissin, D. M. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnol. 28, 595599 (2010).
  6. Tabakman, M. N. et al. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range. Nature Commun. 2, 466 (2011).
  7. Laromaine, A., Koh, L. L., Murugesan, M., Ulijn, R. V. & Stevens, M. M. Protease-triggered dispersion of nanoparticle assemblies. J. Am. Chem. Soc. 129, 41564157 (2007).
  8. De la Rica, R., Baldi, A., Fernandez-Sanchez, C. & Matsui, H. Single-cell pathogen detection with a reverse-phase immunoassay on impedimetric transducers. Anal. Chem. 81, 77327736 (2009).
  9. De la Rica, R., Fratila, R. M., Szarpak, A., Huskens, J. & Velders, A. H. Multivalent nanoparticle networks as ultrasensitive enzyme sensors. Angew. Chem. Int. Ed. 50, 57035706 (2011).
  10. Rodriguez-Lorenzo, L. et al. Zeptomol detection through controlled ultrasensitive surface-enhanced Raman scattering. J. Am. Chem. Soc. 131, 46164618 (2009).
  11. Chen, S., Svedendahl, M., van Duyne, R. P. & Käll, M. Plasmon-enhanced colorimetric ELISA with single molecule sensitivity. Nano Lett. 11, 18261830 (2011).
  12. Kowalczyk, B., Walker, D. A., Soh, S. & Grzybowski, B. A. Nanoparticle supracrystals and layered supracrystals as chemical amplifiers. Angew. Chem. Int. Ed. 49, 57375741 (2009).
  13. Aili, D., Selegard, R., Baltzer, L., Enander, K. & Liedberg, B. Colorimetric protein sensing by controlled assembly of gold nanoparticles functionalized with synthetic receptors. Small 5, 24452452 (2009).
  14. Qu, W., Liu, Y., Liu, D., Wang, Z. & Jiang, X. Copper-mediated amplification allows readout of immunoassays by the naked eye. Angew. Chem. Int. Ed. 50, 34423445 (2011).
  15. Engelbrekt, C. et al. Green synthesis of gold nanoparticles with starch–glucose and application in bioelectrochemistry. J. Mater. Chem. 19, 78397847 (2009).
  16. Thaxton, C. S. et al. Nanoparticle-based bio-barcode assay redefines ‘undetectable’ PSA and biochemical recurrence after radical prostatectomy. Proc. Natl Acad. Sci. USA 106, 1843718442 (2009).
  17. De la Rica, R. & Matsui, H. Urease as a nanoreactor for growing crystalline ZnO nanoshells at room temperature. Angew. Chem. Int. Ed. 47, 54155417 (2008).
  18. Pejoux, C., de la Rica, R. & Matsui, H. Biomimetic crystallization of sulfide semiconductor nanoparticles in aqueous solution. Small 6, 9991002 (2010).
  19. De la Rica, R., Fabijanic, K. I., Baldi, A. & Matsui, H. Biomimetic crystallization nanolithography: simultaneous nanopatterning and crystallization. Angew. Chem. Int. Ed. 49, 14471450 (2010).
  20. Fan, R. et al. Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nature Biotechnol. 26, 13731378 (2008).

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  1. Department of Materials, Department of Bioengineering and Institute for Biomedical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK

    • Roberto de la Rica &
    • Molly M. Stevens


R.R. elaborated the concept, designed and performed experiments, and wrote the paper. M.M.S. supervised the project, participated in scientific discussions, and revised the paper.

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The authors declare no competing financial interests.

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