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Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.

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 ? 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 determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein-AuNP bioconjugates, particularly enzyme activity.

SUBMITTER: Kozlowski R

PROVIDER: S-EPMC6093776 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.

Kozlowski Rachel R Ragupathi Ashwin A Dyer R Brian RB

Bioconjugate chemistry 20180726 8

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 coverage ...[more]

PMID: 30004227

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Project description:Gold nanoparticles (AuNPs) bound with biomolecules have emerged as suitable biosensors exploiting unique surface chemistries and optical properties. Many efforts have focused on antibody bioconjugation to AuNPs resulting in a sensitive bioconjugate to detect specific types of bacteria. Unfortunately, bacteria thrive under various harsh environments, and an understanding of bioconjugate stability is needed. Here, we show a method for optimizing Listeria monocytogenes polyclonal antibodies bioconjugation mechanisms to AuNPs via covalent binding at different pH values, from 2 to 11, and 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid, NaOH, HCl conditions. By fitting Lorentz curves to the amide I and II regions, we analyze the stability of the antibody secondary structure. This shows an increase in the apparent breakdown of the antibody secondary structure during bioconjugation as pH decreases from 7.9 to 2. We find variable adsorption efficiency, measured as the percentage of antibody adsorbed to the AuNP surface, from 17 to 27% as pH increases from 2 to 6 before decreasing to 8 and 13% at pH 7.9 and 11, respectively. Transmission electron microscopy (TEM) analysis reveals discrepancies between size and morphological changes due to the corona layer assembly from antibody binding to single nanoparticles versus aggregation or cluster self-assembly into large aggregates. The corona layer formation size increases from 3.9 to 5.1 nm from pH 2 to 6, at pH 7.9, there is incomplete corona formation, whereas at pH 11, there is a corona layer formed of 6.4 nm. These results indicate that the covalent binding process was more efficient at lower pH values; however, aggregation and deactivation of the antibodies were observed. We demonstrate that optimum bioconjugation condition was determined at pH 6 and MES buffer-type by indicators of covalent bonding and stability of the antibody secondary structure using Fourier transform-infrared, the morphological characteristics and corona layer formation using TEM, and low wavelength shifts of ultraviolet-visible after bioconjugation.

Dataset Information

Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.

Publications

Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.

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