Skip to main content

Thank you for visiting 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:

A plasmonic ‘antenna-in-box’ platform for enhanced single-molecule analysis at micromolar concentrations


Single-molecule fluorescence techniques1,2,3 are key for a number of applications, including DNA sequencing4,5, molecular and cell biology6,7 and early diagnosis8. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur2. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes9,10,11,12,13. Although these nanoantennas enhance fluorescence brightness14,15,16,17,18,19,20, large background signals20,21,22 and/or unspecific binding to the metallic surface23,24,25 have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel ‘antenna-in-box’ platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.

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

Access options

Buy this article

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

Figure 1: Antenna-in-box platform for single-molecule analysis at high concentrations.
Figure 2: Enhanced single-molecule analysis with an antenna-in-box.
Figure 3: Fluorescence enhancement and volume reduction as a function of nanoantenna gap size.
Figure 4: Applicability of the antenna-in-box to detect and discriminate individual biomolecules at 10 µM concentrations.

Similar content being viewed by others


  1. Lu, H. P., Xun, L. & Xie, X. S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998).

    Article  CAS  Google Scholar 

  2. Craighead, H. G. Future lab-on-a-chip technologies for interrogating individual molecules. Nature 442, 387–393 (2006).

    Article  CAS  Google Scholar 

  3. Zander, C., Enderlein, J. & Keller, R. A. Single-Molecule Detection in Solution—Methods and Applications (Wiley, 2002).

    Book  Google Scholar 

  4. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  Google Scholar 

  5. Uemura, S. et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010).

    Article  CAS  Google Scholar 

  6. Bacia, K., Kim, S. A. & Schwille, P. Fluorescence cross-correlation spectroscopy in living cells. Nature Methods 3, 83–89 (2006).

    Article  CAS  Google Scholar 

  7. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

    Article  CAS  Google Scholar 

  8. Pitschke, M., Prior, R., Haupt, M. & Riesner, D. Detection of single amyloid β-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy. Nature Med. 4, 832–834 (1998).

    Article  CAS  Google Scholar 

  9. Levene, M. J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).

    Article  CAS  Google Scholar 

  10. Novotny, L. & van Hulst, N. Antennas for light. Nature Photon. 5, 83–90 (2011).

    Article  CAS  Google Scholar 

  11. Biagioni, P., Huang, J. S. & Hecht, B. Nanoantennas for visible and infrared radiation. Rep. Prog. Phys. 75, 024402 (2012).

    Article  Google Scholar 

  12. Schuller, J. A. et al. Plasmonics for extreme light concentration and manipulation. Nature Mater. 9, 193–204 (2010).

    Article  CAS  Google Scholar 

  13. Zijlstra, P., Paulo, P. M. R. & Orrit, M. Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod. Nature Nanotech. 7, 379–382 (2012).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  15. Kühn, S., Håkanson, U., Rogobete, L. & Sandoghdar, V. Enhancement of single-molecule fluorescence using a gold nanoparticle as an optical nanoantenna. Phys. Rev. Lett. 97, 017402 (2006).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Aouani, H. et al. Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations. Nano Lett. 11, 637–644 (2011).

    Article  CAS  Google Scholar 

  18. Bermúdez Ureña, E. et al. Excitation enhancement of a quantum dot coupled to a plasmonic antenna. Adv. Mater. 24, OP314–OP320 (2012).

    Google Scholar 

  19. Busson, M. P., Rolly, B., Stout, B., Bonod, N. & Bidault, S. Accelerated single photon emission from dye molecule-driven nanoantennas assembled on DNA. Nature Commun. 3, 962 (2012).

    Article  Google Scholar 

  20. Acuna, G. P. et al. Fluorescence enhancement at docking sites of DNA-directed self-assembled nanoantennas. Science 338, 506–510 (2012).

    Article  CAS  Google Scholar 

  21. Choudhury, S. D., Ray, K. & Lakowicz, J. R. Silver nanostructures for fluorescence correlation spectroscopy: reduced volumes and increased signal intensities. J. Phys. Chem. Lett. 3, 2915–2919 (2012).

    Article  Google Scholar 

  22. Lu, G. W. et al. Plasmonic near-field in the vicinity of a single gold nanoparticle investigated with fluorescence correlation spectroscopy. Nanoscale 4, 3359–3364 (2012).

    Article  CAS  Google Scholar 

  23. Estrada, L. C., Aramendia, P. F. & Martinez, O. E. 10000 times volume reduction for fluorescence correlation spectroscopy using nano-antennas. Opt. Express 16, 20597–20602 (2008).

    Article  CAS  Google Scholar 

  24. Kinkhabwala, A. A., Yu, Z. F., Fan, S. H., & Moerner, W. E. Fluorescence correlation spectroscopy at high concentrations using gold bowtie nanoantennas. Chem. Phys. 406, 3–8 (2012).

    Article  CAS  Google Scholar 

  25. 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. 125, 1255–1259 (2013).

    Article  Google Scholar 

  26. Rigneault, H. et al. Enhancement of single-molecule fluorescence detection in subwavelength apertures. Phys. Rev. Lett. 95, 117401 (2005).

    Article  Google Scholar 

Download references


The research leading to these results received funding from the European Commission's Seventh Framework Programme (FP7-ICT-2011-7) under grant agreements 288263 (NanoVista), ERC StG 278242 (ExtendFRET) and ERC AdG (NanoAntennas), the Spanish Ministry of Science and Innovation and the Agence Nationale de la Recherche under grant ANR-10-INBS-04-01 (France Bio Imaging). The authors thank A. Brisson for providing the Annexin sample.

Author information

Authors and Affiliations



J.W., H.R., N.F.v.H. and M.G-P. conceived and designed the experiments. D.P., J.W. and S.B.M. performed the experiments and analysed the data. M.M. and T.S.v.Z. fabricated the antennas. J.W., M.G-P. and N.F.v.H. wrote the manuscript.

Corresponding author

Correspondence to Jérôme Wenger.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Information (PDF 4288 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Punj, D., Mivelle, M., Moparthi, S. et al. A plasmonic ‘antenna-in-box’ platform for enhanced single-molecule analysis at micromolar concentrations. Nature Nanotech 8, 512–516 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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