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.

Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties


Molecular optical imaging has shown promise in visualizing molecular biomarkers with subcellular resolution both noninvasively and in real-time. Here, we use gold nanoparticles as optical probes to provide meaningful signal in the presence of targeted biomarkers. We present a novel conjugation technique to control the binding orientation of antibodies on the surface of gold nanoparticles to maximize antibody functionality. Briefly, a heterobifunctional linker, hydrazide-polyethylene glycol-dithiol, is used to directionally attach the Fc, or nonbinding region of the antibody, to the gold nanoparticle surface. The conjugation strategy allows for multiplexing various glycosylated antibodies on a single nanoparticle. We present a method to prepare multifunctional nanoparticles by incorporating targeting and delivery moieties on the same nanoparticle that addresses the challenge of imaging intracellular biomarkers. The time estimate for the entire protocol is 6 h.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


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

Figure 1
Figure 2: Gold nanoparticle synthesis.
Figure 3: TEM image and UV-Vis spectrum of 18-nm gold nanoparticles.
Figure 4: The results show that the surface of 18-nm gold nanoparticles was functionalized with nearly the same ratio of antibodies present in the initial reaction solution.
Figure 5: Appearance of solutions at each step in the conjugation process.
Figure 6: UV-Vis spectra of bare 18-nm gold nanoparticles (blue line), and the same particles after conjugation with antibodies (green line).
Figure 7: Standard pyrene-actin polymerization assay shows similar polymerization rates with and without the presence of gold nanoparticle-based contrast agents.
Figure 8: Darkfield transmittance images of fibroblasts labeled with gold nanoparticle contrast agents targeted to actin.


  1. Alivisatos, P. The use of nanocrystals in biological detection. Nat. Biotechnol. 22, 47–52 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Aaron, J. et al. Plasmon resonance coupling of metal nanoparticles for molecular imaging of carcinogenesis in vivo. J. Biomed. Opt. 12, 034007 (2007).

    Article  PubMed  Google Scholar 

  3. Kumar, S., Harrison, N., Richards-Kortum, R. & Sokolov, K. Plasmonic nanosensors for imaging intracellular biomarkers in live cells. Nano Lett. 7, 1338–1343 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Sokolov, K. et al. Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Res. 63, 1999–2004 (2003).

    CAS  PubMed  Google Scholar 

  5. El-Sayed, I.H., Huang, X. & El-Sayed, M.A. Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles. Cancer Lett. 239, 129–135 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Huang, X., El-Sayed, I.H., Qian, W. & El-Sayed, M.A. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 128, 2115–2120 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Loo, C. et al. Nanoshell-enabled photonics-based imaging and therapy of cancer. Technol. Cancer Res. Treat. 3, 33–40 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Han, M.S., Lytton-Jean, A.K.R., Oh, B.-K., Heo, J. & Mirkin, C.A. Colorimetric screening of DNA-binding molecules with gold nanoparticle probes. Angew. Chem. Int. Ed. Engl. 45, 1807–1810 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Liu, G.L. et al. A nanoplasmonic molecular ruler for measuring nuclease activity and DNA footprinting. Nat. Nanotechnol. 1, 47–52 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Soennichsen, C., Reinhard, B.M., Liphardt, J. & Alivisatos, A.P. A molecular ruler based on plasmon coupling of single gold and silver nanoparticles. Nat. Biotechnol. 23, 741–745 (2005).

    Article  CAS  Google Scholar 

  11. Kumar, S. & Richards-Kortum, R. Optical molecular imaging agents for cancer diagnostics and therapeutics. Nanomed. 1, 23–30 (2006).

    Article  CAS  Google Scholar 

  12. El-Sayed, I.H., Huang, X. & El-Sayed, M.A. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano Lett. 5, 829–834 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Loo, C. et al. Gold nanoshell bioconjugates for molecular imaging in living cells. Opt. Lett. 30, 1012–1014 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Pathak, S., Davidson, M.C. & Silva, G.A. Characterization of the functional binding properties of antibody conjugated quantum dots. Nano Lett. 7, 1839–1845 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Aaron, J.S. et al. Increased optical contrast in imaging of epidermal growth factor receptor using magnetically actuated hybrid gold/iron oxide nanoparticles. Opt. Express 14, 12930–12943 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Spangler, B.D. & Tyler, B.J. Capture agents for a quartz crystal microbalance-continuous flow biosensor: functionalized self-assembled monolayers on gold. Anal. Chim. Acta 399, 51–62 (1999).

    Article  CAS  Google Scholar 

  17. Won, J. et al. A magnetic nanoprobe technology for detecting molecular interactions in live cells. Science 309, 121–125 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Mallidi, S., Larson, T., Aaron, J., Sokolov, K. & Emelianov, S. Molecular specific optoacoustic imaging with plasmonic nanoparticles. Opt. Express 15, 6583–6588 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Larson, T.A., Bankson, J., Aaron, J. & Sokolov, K. Hybrid plasmonic magnetic nanoparticles as molecular specific agents for MRI/optical imaging and photothermal therapy of cancer cells. Nanotechnology 18, 325101 (2007).

    Article  Google Scholar 

  20. Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature 241, 20–22 (1973).

    CAS  Google Scholar 

  21. Cooper, J.A., Walker, S.B. & Pollard, T.D. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J. Muscle Res. Cell Motil. 4, 253–262 (1983).

    Article  CAS  PubMed  Google Scholar 

  22. Kouyama, T. & Mihashi, K. Fluorimetry Study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur. J. Chem. 114, 33–38 (1981).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to K Sokolov.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kumar, S., Aaron, J. & Sokolov, K. Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nat Protoc 3, 314–320 (2008).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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