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BioNanoScience

, Volume 3, Issue 1, pp 30–36 | Cite as

Imaging of Protein Secretion from a Single Cell Using Plasmonic Substrates

  • Henryk Szmacinski
  • Vladimir Toshchakov
  • Wenji Piao
  • Joseph R. Lakowicz
Article

Abstract

Detecting, imaging, and monitoring cell function on a single-cell basis is very important in the field of immunology research where many molecules are secreted from cells in response to external stimuli including immunization. Here, we introduce substrates with plasmonic nanoparticles and fluorescence microscopy as promising imaging methods for studies on molecular processes controlling cell behavior, particularly secretion of cytokines. We developed a unique composition of silver and silica layers of plasmonic nanostructures which resulted in fluorescence enhancement of more than 200-fold for the ensemble of molecules in the immunoassay. For the proof of concept demonstration, we used primary mouse macrophages and imaged tumor necrosis alpha secretion after stimulation of the cells with lipopolysaccharide. We demonstrate that metal-enhanced fluorescence assay provides imaging capability detection of cytokine secretion from a single cell without extensive biochemical procedures as required with standard methods. In addition, it is demonstrated that cell viability can be controlled during secretion.

Keywords

Plasmonic nanostructures Silver nanoparticles Metal-enhanced fluorescence Microphage cells Cell assays Tumor necrosis factor alpha 

Notes

Acknowledgments

This research was supported by NIH grants R21CA147975, HG005090, HG002655, and AI0082299.

References

  1. 1.
    Henry, A. I., Bingham, J. M., Ringe, E., Marks, L. D., Schatz, G. C., Van Duyne, R. P. (2011). Correlated structure and optical property studies of plasmonic nanoparticles. Journal of Physical Chemistry C, 115, 9291–9305.CrossRefGoogle Scholar
  2. 2.
    Luk’yanchuk, B., Zheludev, N. I., Maier, S. A., Halas, N. J., Nordlander, P., Giessen, H., Chong, C. T. (2010). The Fano resonance in plasmonic nanostructures and metamaterials. Nature Materials, 9, 707–715.CrossRefGoogle Scholar
  3. 3.
    Stiles, P. L., Dieringer, J. A., Shah, N. C., Van Duyne, R. P. (2008). Surface-enhanced Raman spectroscopy. Annual Review of Analytical Chemistry, 1, 601–626.CrossRefGoogle Scholar
  4. 4.
    Stewart, M. E., Anderson, C. R., Thompson, L. B., Maria, J., Grey, S. K., Rogers, J. A., Nuzzo, R. G. (2008). Nanostructured plasmonic sensors. Chemical Review, 108, 494–521.CrossRefGoogle Scholar
  5. 5.
    Lakowicz, J. R., Ray, K., Chowdhury, M., Szmacinski, H., Fu, Y., Zhang, J., Nowaczyk, K. (2008). Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy. Analyst, 133, 1308–1346.CrossRefGoogle Scholar
  6. 6.
    Fort, E., & Grésillon, S. (2008). Surface enhanced fluorescence. Journal of Physics D: Applied Physics, 41, 1–31.CrossRefGoogle Scholar
  7. 7.
    Hao, Q., Qiu, T., Chu, P. K. (2012). Surface—enhanced cellular fluorescence imaging. Progress in Surface Science, 87, 23–45.CrossRefGoogle Scholar
  8. 8.
    Ming, T., Chen, H., Jiang, R., Li, Q., Wang, J. (2011). Plasmon-controlled fluorescence: beyond the intensity enhancement. Journal of Physical Chemistry Letters, 3, 191–201.CrossRefGoogle Scholar
  9. 9.
    Arnáiz, B., Martinez-Ávila, O., Falcon-Perez, J., Penadés, S. (2012). Cellular uptake of gold nanoparticles bearing HIV gp120 oligomannosides. Bioconjugate Chemistry, 23, 814–825.CrossRefGoogle Scholar
  10. 10.
    Saha, A., Basiruddin, S. K., Sarkar, R., Pradhan, N., Jana, N. R. (2009). Functionalized plasmonic-fluorescent nanoparticles for imaging and detection. Journal of Physical Chemistry C, 113, 18492–18498.CrossRefGoogle Scholar
  11. 11.
    Zhang, J., Fu, Y., Mei, Y., Jiang, F., Lakowicz, J. R. (2010). Fluorescent metal nanoshell probe to detect single miRNA in lung cancer cell. Analytical Chemistry, 82, 4464–4471.CrossRefGoogle Scholar
  12. 12.
    Zhang, J., Fu, Y., Liang, D., Shao, R. Y., Lakowicz, J. R. (2009). Fluorescent avidin-bound silver particle: a strategy for single target molecule detection on a cell membrane. Analytical Chemistry, 81, 883–889.CrossRefGoogle Scholar
  13. 13.
    Huang, C.-S., George, S., Lu, M., Chaudhery, V., Tan, R., Zangar, R. C., Cunningham, B. T. (2011). Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays. Analytical Chemistry, 83, 1425–1430.CrossRefGoogle Scholar
  14. 14.
    Chan, L. L., Gosangari, S. L., Watkin, K. L., Cunningham, B. T. (2007). A label-free photonic crystal biosensor imaging method for detection of cancer cell cytotoxicity and proliferation. Apoptosis, 12, 1061–1068.CrossRefGoogle Scholar
  15. 15.
    Ballarini, M., Frascella, F., Enrico, E., Mandracci, P., De Leo, N., Michelotti, F., Giorgis, F., Descrovi, E. (2012). Bloch surface waves-controlled fluorescence emission: coupling into nanometer-sized polymeric waveguides. Applied Physics Letters, 100, 063305.CrossRefGoogle Scholar
  16. 16.
    Le Moal, E., Fort, E., Lévêque-Fort, S., Cordelières, F. P., Fontaine-Aupart, M. P., Ricolleau, C. (2007). Enhanced fluorescence cell imaging with metal-coated slides. Biophysical Journal, 92, 2150–2161.CrossRefGoogle Scholar
  17. 17.
    Rice, J. M., Stern, L. J., Guignon, E. F., Lawrence, D. A., Lynes, M. A. (2012). Antigen-specific T cell phenotyping microarrays using grating coupled surface plasmon resonance imaging and surface plasmon coupled emission. Biosensors and Bioelectronics, 31, 264–269.CrossRefGoogle Scholar
  18. 18.
    Thorpe, R., Wadhwa, M., Page, C., Mire-Sluis, A. (1999). Biological characterization and assay of cytokines and growth factors. Development in Biological Standarization, 97, 61–71.Google Scholar
  19. 19.
    Moody, M. D., Van Arsdell, S. W., Murphy, K. P., Orencole, S. F., Burns, C. (2001). Array-based ELISA for high-throughput anlysis of human cytokines. Biotechniques, 31(1), 186–196.Google Scholar
  20. 20.
    Becher, B., Blain, M., Giacomini, P. S., Antel, J. P. (1999). Inhibition of Th1 polarization by soluble TNF receptor is dependent on antigen-presenting cell-derived IL-12. Journal of Immunology, 162, 684–688.Google Scholar
  21. 21.
    Ewen, C., & Baca-Estrada, M. E. (2001). Evaluation of interleukin-4 concentration by ELISA is influenced by the consumption of IL-4 by cultured cells. Journal of Interferon Research, 21, 39–43.CrossRefGoogle Scholar
  22. 22.
    Barnes, A. (1998). Measurement of serum cytokines. Lancet, 352, 324–325.CrossRefGoogle Scholar
  23. 23.
    Dinter, A., & Berger, E. G. (1998). Golgi-disturbing agents. Histochemistry and Cell Biology, 109, 571–590.CrossRefGoogle Scholar
  24. 24.
    Asemissen, A. M., Nagorsen, D., Keilholz, U., Letsch, A., Schmittel, A., Thiel, E., Scheibenbogen, C. (2001). Flow cytometric determination of intracellular or secreted IFNγ for the quantification of antigen reactive T cells. Journal of Immunological Methods, 251, 101–108.CrossRefGoogle Scholar
  25. 25.
    Tanguay, S., Killion, J. (1994). Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine and Cytokine Research, 13, 259–263.Google Scholar
  26. 26.
    Boulet, S., Ndongala, M. I., Peretz, Y., Boisvert, M. P., Boulassel, M. R., Tremblay, C., Routy, J. P., Sekaly, R. P., Bernard, N. F. (2007). A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-γ HIV-specific immune responses. Journal of Immunological Methods, 320, 18–29.CrossRefGoogle Scholar
  27. 27.
    Karulin, A. Y., Hesse, M. D., Tary-Lehmann, M., Lehmann, P. V. (2000). Single-cytokine producing CD4 memory cells predominate in type 1 and type 2 immunity. Journal of Immunology, 164, 1862–1872.Google Scholar
  28. 28.
    Gazagne, A., Claret, E., Wijdenes, J., Yssel, H., Bousquet, F., Levy, E., Vielh, P., Scotte, F., Le Goupil, T., Fridman, W. H., Tartour, E. (2003). A Fluorospot assay to detect single T-lymphocytes simultaneously producing multiple cytokines. Journal of Immunological Methods, 283, 91–98.CrossRefGoogle Scholar
  29. 29.
    Chen, D. S., Soen, Y., Stuge, T. B., Lee, P. P., Webere, J. S., Brown, P. O., Davis, M. M. (2005). Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray. PLoS Medicine, 2(10), e265. doi: 10.1371/journal.pmed.0020265.CrossRefGoogle Scholar
  30. 30.
    Shirai, A., Holmes, K., Klinman, D. M. (1993). Detection and quantitation of cells secreting IL-6 under physiological conditions in BALB/c mice. Journal of Immunology, 150, 793–799.Google Scholar
  31. 31.
    Szmacinski, H., Badugu, R., Lakowicz, J. R. (2010). Fabrication and characterization of planar plasmonic substrates with high fluorescence enhancement. Journal of Physical Chemistry C, 114, 21142–21149.CrossRefGoogle Scholar
  32. 32.
    Szmacinski, H., Bagugu, R., Mahdavi, F., Blair, S., Lakowicz, J. R. (2012). Large fluorescence enhancements of fluorophore ensembles with multilayer plasmonic substrates: comparison of theory and experimental results. Journal of Physical Chemistry C, 116, 21563–21571.CrossRefGoogle Scholar
  33. 33.
    Szmacinski, H., Murtaza, Z., Lakowicz, J. R. (2010). Time-resolved method for one-step immunoassays using plasmonic nanostructures. Journal of Physical Chemistry C, 114, 7236–7241.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Henryk Szmacinski
    • 1
  • Vladimir Toshchakov
    • 2
  • Wenji Piao
    • 2
  • Joseph R. Lakowicz
    • 1
  1. 1.Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, School of MedicineUniversity of MarylandBaltimoreUSA
  2. 2.Department of Microbiology and Immunology, School of MedicineUniversity of MarylandBaltimoreUSA

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