Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates

Bioconjugate Chemistry
Rachel KozlowskiR Brian Dyer

Abstract

Functional enzyme-nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein-gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV-vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determ...Continue Reading

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Citations

Jan 21, 2021·Proceedings of the National Academy of Sciences of the United States of America·Rachel KozlowskiR Brian Dyer
May 4, 2021·Journal of the American Chemical Society·Alexandra V RamseyMatthew B Francis
Apr 13, 2019·The Journal of Physical Chemistry Letters·Anup GhoshLev Chuntonov

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