pH-dependent streptavidin conjugation on non-spherical gold nanoparticles: Electrokinetic characterization and biosensing performance

Abstract

Gold nanoparticles (AuNPs) have garnered significant attention in biomedical applications due to their tunable optical and surface properties. This study investigates the effect of pH on the adsorption behavior, electrokinetic characteristics, and biofunctional performance of non-spherical, cetyltrimethylammonium chloride (CTAC)-capped AuNPs functionalized with streptavidin (SA). Hexagonal AuNPs with an average diameter of 75 ± 12 nm were synthesized via a modified multi-step growth method and conjugated with SA at pH 5, 7, and 9. Micro-BCA analysis revealed that protein adsorption increased under acidic conditions (523 ± 55, 447 ± 45, and 298 ± 33 molecules per particle at pH 5, 7, and 9, respectively). Electrophoretic mobility measurements revealed a pH-dependent surface charge shift, with plain CTAC-coated AuNPs exhibiting positive Ue values, which reversed to negative values after streptavidin binding. An isoelectric point was observed near pH 5.5–6, and mobility plateaus at high ionic strength indicated the presence of a hydrodynamically permeable layer. Duval-Ohshima model fitting demonstrated a consistent softness parameter (λ ≈ 0.5 nm) corresponding to a uniform hydrated streptavidin layer. Donnan (ψDON) and surface (ψ0) potentials also shifted from positive for CTAC-AuNPs to moderately negative values after conjugation. Despite greater adsorption at pH 5, the resulting conjugates showed reduced functional performance, likely due to partial protein deformation and disordered multilayer accumulation. In contrast, conjugates prepared at pH 7 retained structural integrity, exhibited stable negative mobility without a distinct isoelectric point, and achieved the strongest biotin-BSA binding and lateral-flow assay signals. These findings highlight a critical balance between adsorption strength and bioactivity and demonstrate the utility of the Duval-Ohshima model as a quantitative framework for analyzing soft, biomolecule-coated nanostructures.