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

Abstract

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.

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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.

References

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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 

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Acknowledgements

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.

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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.

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Correspondence to Jérôme Wenger.

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The authors declare no competing financial interests.

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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). https://doi.org/10.1038/nnano.2013.98

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  • DOI: https://doi.org/10.1038/nnano.2013.98

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