Novel detection strategies that exploit the unique properties of gold nanoparticles (AuNPs) hold great promise for the advancement of detection technologies that mitigate biological threats and aid in therapeutic development. Central to these emerging techniques is the immobilization of selective recognition elements such as antibodies onto the AuNP surface. Optimization of the loading density and proper orientation of immobilized antibodies is critical to maximize the analytical performance. Recent studies have demonstrated that proteins with cysteine residues adsorb to gold nanoparticles with strong binding affinity. Moreover, localized regions of charge are responsible for the orientation of the immobilized protein. Here, we measure the adsorption dynamics of anti-horseradish peroxidase antibody (anti-HRP) on gold nanoparticles using nanoparticle tracking analysis (NTA). Solution pH is used to alter the charge distribution on the antibody to investigate the impact on adsorption. NTA facilitates in situ measurement of antibody adsorption on AuNP by measuring the increase in hydrodynamic diameter (D<sub>h</sub>) of the AuNPs as a function of antibody concentration. Adsorption isotherms are modeled by the Hill-modified Langmuir equation to determine the adsorption affinity, protein layer thickness, and binding cooperativity at each pH. Our data show a monolayer of antibody is formed at saturation at each pH; however, the increase in D<sub>h </sub>at monolayer coverage is pH-dependent, suggesting differences in antibody orientation. Differences in antibody orientation were further confirmed by an enzyme-mediated assay to quantify the amount of antibody available for antigen binding. Ultimately, these studies describe, in part, our efforts to elucidate the effect of charge on antibody adsorption onto AuNP to maximize antigen-binding activity of antibody-AuNP conjugates and enhance the performance of biosensing technologies.

Impact of pH on the orientation of antibody adsorbed onto gold nanoparticles

2019 Spring National Meeting

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