pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles

Novel detection strategies that exploit the unique properties of gold nanoparticles (AuNPs) hold great promise for the advancement of diagnostic testing. Fundamental to many of these nanoparticle-enabled techniques is the immobilization of antibodies onto the AuNP surface to afford selective binding to target species. Orientation and loading density of the immobilized antibodies govern Fab accessibility and are critical to the analytical performance. Here, we use pH to systematically control the surface charge distribution on an antibody and investigate the impact of protein charge on adsorption to AuNPs. Nanoparticle tracking analysis (NTA) is used to measure the adsorption dynamics of anti-horseradish peroxidase antibody (anti-HRP) onto AuNPs at different pHs. NTA enables in situ measurement of antibody adsorption on AuNP by measuring the increase in hydrodynamic diameter (DH) of the AuNPs as a function of antibody concentration. The adsorption affinity, protein layer thickness, and binding cooperativity at each pH are extracted from the best fit of the adsorption isotherms to the Hill-modified Langmuir equation. Our data show a monolayer of antibody is formed at saturation at pHs 7.5, 8.0, and 8.5, and no difference in anti-HRP-AuNP binding constants is observed in this pH range (Kd ∼11 nM). However, the increase in DH of the AuNPs with adsorbed protein at monolayer coverage is pH-dependent, measuring 13.2 ± 1.1 nm, 9.8 ± 1.0 nm, and 7.4 ± 0.6 nm for pHs 7.5, 8.0, and 8.5, respectively. Moreover, results of an enzyme-mediated assay reveal the antigen-binding capacity of the immobilized anti-HRP antibody is 33 ± 2%, 23 ± 7%, and 18 ± 2% when adsorbed at pHs 7.5, 8.0, and 8.5, respectively. Our data confirm that antibody charge can be altered with pH to modulate and optimize antibody orientation on AuNP. These studies describe our continued efforts to establish design criteria to prepare conjugates with maximum antigen-binding activity that will enhance the performance of biofunctional nanomaterials