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

Nature Protocols volume 3, pages 314–320 (2008)Cite this article

Abstract

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.

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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.

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of Texas at Austin, Austin, 78712, Texas, USA

    S Kumar, J Aaron & K Sokolov

  2. Department of Biomedical Engineering, MD Anderson Cancer Center, Houston, 77030, Texas, USA

    K Sokolov

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  1. S Kumar
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Correspondence to K Sokolov.

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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). https://doi.org/10.1038/nprot.2008.1

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