Abstract Background Bovine serum albumin (BSA) serves as a blocking agent in in-vitro diagnostic (IVD) applications, with fatty acid stabilized (FAS) and fatty acid free (FAF) BSA as common options. The presence of fatty acids and pH influence the conformation of BSA. While previous studies have explored the individual effects of fatty acids or pH on BSA blocking, limited data exists on their combined impact. This study employs atomic force microscopy (AFM) to investigate how pH and fatty acids jointly affect BSA adsorption onto silica, a surface relevant within diagnostics. Methods AFM, a sensitive microscopic technique, provides topographic images of surfaces with superior resolution compared to optical microscopy. We conducted experiments to assess the adsorption of 1% FAS and FAF BSA on a silica surface at pH 5.2 and pH 7. Evaluation of the coatings involved scratching to assess overall thickness, and roughness, indicating thickness variability. Results Results revealed that both fatty acids and pH play significant roles in shaping the BSA coating characteristics on silica surfaces. In three out of four experimental conditions, the average thickness of the BSA coating ranged between 3-4 nm, except for FAS BSA at pH 7, which exhibited a thicker average coating measuring 5.6 nm. The pH impacted the roughness of the BSA coating. Coatings prepared at pH 5.2 displayed smoother surfaces compared to those at pH 7. Utilizing the root mean square average height variability analysis, there is 95% confidence that the FAF and FAS BSA surfaces at pH 5.2 had a height range between 2.4-4.3 nm and 2.0-4.9 nm, respectively, indicating complete coverage of the silica surface without much height variation. In contrast, the FAF and FAS BSA coatings at pH 7 exhibited a wider range of heights, with 95% confidence intervals spanning from -1.5-8.6 nm and 0.0-11.2 nm, respectively. This variability suggests potential issues including incomplete BSA coverage at the lower range, and steric hindrance at the higher end, highlighting the importance of pH in optimizing BSA coatings for various applications. Conclusions Silica is commonly used in IVD assays, with BSA frequently employed to coat and passivate the surface. Our AFM experiments highlight the impact of pH and fatty acid presence on coating thickness and uniformity. Notably, FAS BSA at pH 7 exhibited the thickest coating; however, its increased roughness may pose challenges in IVD applications due to steric hindrance blocking antibody-antigen interactions. Alternatively, FAF BSA at pH 7 displayed an adequate thickness but also considerable roughness, suggesting potential insufficiencies in surface coverage and passivation. FAS and FAF BSA coatings at pH 5.2 showed relatively consistent thickness, indicating a more uniform surface which may be ideal for IVD coating applications. These AFM experiments were conducted on pure silica surfaces, which may differ from surfaces engineered for IVD. Additionally, both FAF and FAS BSA grades are proven to be effective in IVD applications. Further research employing AFM and other biophysical techniques is warranted to deepen our understanding of pH and fatty acid effects on BSA-surface interactions, aiming to enhance blocking strategies in IVD.
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Dec 2, 2024
Feilian Chen + 3
Just Published
Open Access