Abstract: When nanoprobes are applied for detection in complex biological matrices such as serum, a protein corona is prone to form on their surface, which may mask the recognition molecules and thus lead to probe inactivation. To investigate the effects of different antibody immobilization strategies on antibody bioactivity and anti-interference performance, gold nanoparticles were used as the carrier in this study to systematically compare three immobilization methods for rabbit anti-mouse IgG: direct physical adsorption, covalent conjugation via mercaptopropionic acid, and site-directed immobilization mediated by staphylococcal protein A. We comprehensively adopted nanoparticle tracking analysis, BCA protein quantitation and dot blot assay to quantitatively evaluate the antibody loading capacity per nanoparticle and the accessible binding orientation of antibodies for each method. Furthermore, a serum-mimicking environment was constructed to analyze the masking effect of protein corona on antibody exposure and the actual target antigen capture capability of the nanoprobes. The experimental results demonstrated that the physical adsorption method via direct co-incubation exhibited the optimal performance in antibody loading capacity and binding orientation optimization. Moreover, this method maintained the highest antibody accessibility and antigen-binding capacity after incubation in serum, and its overall performance was significantly superior to those of the two chemical modification methods. This study reveals that the simple and direct physical adsorption method possesses unique advantages in the preparation of nanoprobes for detection in complex biological samples, which provides novel experimental evidence and practical strategies for optimizing nanoprobe design and improving the reliability of in vitro diagnostics.

Key words: Gold nanoprobes, Protein corona, Antibody immobilization strategies, Antibody orientation, Serum detection