Elucidating Antibody Conjugation and Orientation Dynamics on Phenylalanine-Functionalized Gold Nanoparticles: The Role of Lipid Coating and Other Physiological Conditions.

Gold nanoparticles (AuNPs) functionalized with antibodies offer significant potential to advance biomedical applications due to their unique optical properties and the specificity of antibody-antigen interactions. A critical aspect of optimizing these AuNP-based systems is the effective adsorption of antibodies on the nanoparticle surface. Recent research has focused on developing new strategies to enhance antibody loading and orientation, with the aim of improving antibody activity. However, the lack of robust analytical methods for accurately quantifying the activity of conjugated antibodies and comparing immobilization strategies remains a significant challenge. Herein, for the first time, we describe the effect of DPPC and DOPC lipid coatings on the interaction of phenylalanine functionalized gold nanoparticles (AuPhe NPs) with immunoglobulin G (IgG) antibody under varying pH (∼6, 7.4, and 9) and buffer systems (HEPES and phosphate). Using several techniques, we reveal the superior performance of lipid-coated AuPhe NPs, particularly those coated with DPPC, compared to native AuPhe NPs in terms of stability, antigen-binding activity, and antibody orientation. Between the two different buffer systems, antibody adsorption on AuPhe NPs is significantly higher in the zwitterionic buffer (HEPES) compared to the negatively charged phosphate buffer. Furthermore, at lower pH, native AuPhe NPs and DOPC-coated AuPhe NPs undergo aggregation, and DPPC-coated AuPhe NPs remain stable. Considering the vital role of lipid coatings under varying physiological conditions, we propose that lipid-coated AuPhe NPs serve as robust platforms for diverse biomedical applications, ensuring enhanced stability and efficiency in antibody-mediated processes.