Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

Ultrathin protein coatings such as BSA are widely used to impart antifouling and stealth properties to flat and nanoparticle surfaces. Despite broad use, many commercial BSA variants exist that differ by purification method (cold ethanol fractionation, heat-shock fractionation, or combinations) and by the presence or absence of fatty acid stabilizers, with little guidance on which type is optimal for antifouling. Prior work typically evaluated a single BSA type or isolated parameters (e.g., ionic strength, heating) rather than the dominant variability source—the purification route and fatty acid content. This study asks which BSA variants yield superior antifouling coatings and why, by systematically comparing conformational stability, adsorption behavior, and coating performance of fatted versus defatted BSA prepared by different fractionation methods.

The authors note extensive use of BSA for surface passivation in assays (ELISA, blots, immunohistochemistry, PCR) and in biosensing and nanomedicine. Prior studies compared BSA adsorption on different substrates or examined specific factors (ionic strength, heating) but generally used a single BSA source. Reports highlight variability among commercial BSAs and the need for predictors of protein adsorption at nano-bio interfaces. However, comparative evaluation of BSA types arising from different purification methods and fatty acid content had not been addressed, motivating the present systematic comparison.

Six commercial BSA types from Sigma-Aldrich were selected based on fractionation route and fatty acid content: fatted BSA obtained by (1) cold ethanol fractionation (A2153; BSA 1), (2) heat-shock fractionation (A3059; BSA 2), and (3) cold ethanol followed by heat-shock (A7638; BSA 3). Corresponding defatted (fatty acid-free) versions were A6003 (BSA 4), A7030 (BSA 5), and A0281 (BSA 6). Defatting involved activated charcoal treatment per manufacturer; ≤0.01% fatty acids were confirmed by gas chromatography, supported by IR spectroscopy.

• Solution and sample conditions: Tris buffer (10 mM, pH 7.5) with 150 mM NaCl; protein concentrations included 100 µM for adsorption assays, 2.5 µM for CD, and 451 µM for DLS aggregation assays. Where applicable, caprylic acid (octanoic acid) doping was performed at a 10:1 molar ratio (caprylic acid:BSA) at pH 7.5 to convert defatted into fatted-like BSA.
• Conformational stability in solution: Dynamic light scattering (DLS) measured hydrodynamic diameter and thermal aggregation onset with stepwise temperature increases (25–75 °C, 5 °C increments). Circular dichroism (CD) spectroscopy quantified secondary structure and α-helicity across temperatures using [θ]222 and a standard helicity equation.
• Adsorption on flat silica: Quartz crystal microbalance with dissipation (QCM-D) tracked real-time adsorption kinetics (Δf) and viscoelasticity (ΔD) on silica-coated sensors at 25 °C. 100 µM BSA was flowed for 30 min followed by buffer wash; Δfmax and |Δfmax/ΔDmax| were analyzed to infer adsorption amount and denaturation.
• Adsorption on nanostructured silica: Localized surface plasmon resonance (LSPR) on silica-coated gold nanodisk arrays quantified wavelength shifts (Δλ) during adsorption and after rinse; Δλmax and the initial rate (dΔλ/dt)max were used to assess uptake and adsorption-induced spreading/denaturation.
• Adsorption-induced structural changes: ATR-FTIR measured amide I bands for BSA in solution and after adsorption/washing on a ZnSe ATR crystal; Gaussian deconvolution yielded fractional α-helix, random coil, etc., to quantify denaturation.
• Antifouling performance on surfaces: QCM-D biofouling assay on silica surfaces. After forming BSA coatings (100 µM, 60 min flow, 40 min wash), undiluted FBS (100%) was flowed for 80 min, followed by a wash. Passivation efficiency was calculated as 1 − (ΔFFBS−BSA / ΔFControl).
• Blocking performance on hydrophobic membranes: Western blot on nitrocellulose membranes pre-exposed to human serum. Membranes were blocked with fatted (BSA 1) or defatted (BSA 5) 3% BSA in TBST; nonspecific band intensities quantified by chemiluminescence imaging and ImageJ analysis.
• Antifouling performance on nanoparticles: 100-nm silica nanoparticles were coated by incubating with 1 mg/mL BSA at 37 °C for 2 h, washed by centrifugation, and incubated in normal human serum (NHS). Complement activation was quantified by ELISA for SC5b-9; protection (%) was calculated relative to uncoated nanoparticles (positive control) and NHS alone (negative control).
• Statistical analysis: One-way or two-way ANOVA with appropriate multiple comparisons and unpaired t-tests (GraphPad Prism v8).

• Solution conformational stability: All BSA types were ~8 nm at 25–55 °C by DLS. Defatted BSAs (4–6) began aggregating at lower temperatures (size ~20 nm at 60 °C; ~45 nm at 65 °C), while fatted BSAs (1–3) exhibited later onset (BSA 1 at ~65 °C, BSA 2 at ~70 °C, BSA 3 at ~75 °C), indicating greater stability with fatty acids. CD showed ~60–63% α-helix at 25 °C for all; at 65 °C, defatted <50% vs fatted >51% helicity on average.
• Adsorption amount and viscoelasticity on silica (QCM-D): Defatted BSAs showed larger Δfmax (~35–45 Hz) vs fatted (~15–35 Hz), indicating greater uptake. |Δfmax/ΔDmax| and F–D analysis indicated more denaturation (lower dissipation per mass) for defatted vs greater viscoelasticity for fatted, consistent with higher stability of fatted.
• Adsorption on nanostructured silica (LSPR): Defatted BSAs had higher Δλmax (~1.2 ± 0.2 nm) vs fatted (~0.6 ± 0.2 nm), corroborating higher uptake and tighter packing. Initial adsorption rate (dΔλ/dt)max was higher for defatted (~0.6–0.7 nm min⁻1) vs fatted (~0.2–0.3 nm min⁻1), indicating greater adsorption-induced spreading/denaturation.
• Adsorption-induced structural changes (ATR-FTIR): In solution, fatted and defatted helicities were ~63% and 60%, respectively. After adsorption and washing, fatted decreased to ~53% (≈10% net loss) and defatted to ~45% (≈15% net loss). Random coil fractions increased by ~9% (fatted) and ~12% (defatted), indicating greater surface-induced denaturation for defatted.
• Antifouling on flat surfaces (serum biofouling): Defatted BSAs outperformed fatted; defatted BSA 5 and 6 achieved ~90% passivation efficiency, while fatted BSA 1 and 3 were <40%.
• Blocking on nitrocellulose membranes: Defatted BSA blocking reduced nonspecific band intensities compared to fatted, consistent with denser coverage.
• Antifouling on nanoparticles (complement activation): Defatted BSA coatings inhibited >60% SC5b-9 generation, whereas fatted inhibited ~40% or less, indicating better protection against complement activation with defatted coatings.
• Mechanism: Fatty acids insert into BSA hydrophobic pockets, stabilizing structure and increasing negative surface charge via carboxylate headgroups. This yields less adsorption-induced denaturation (larger molecular footprint) and enhanced protein–protein electrostatic repulsion, reducing packing density and coating performance. Removing fatty acids lowers conformational stability and charge repulsion, enabling greater spreading and tighter adlayer packing, thus superior antifouling.
• Control validation: Doping defatted BSA with caprylic acid converted behavior to fatted-like, increasing conformational stability and reducing adsorption uptake and denaturation.

The study directly addresses which BSA variants provide optimal antifouling coatings by linking purification-dependent fatty acid content to conformational stability, adsorption behavior, and performance. Defatted BSAs, being less conformationally stable and less negatively charged, undergo greater surface-induced denaturation and reduced inter-protein repulsion, enabling higher adsorption uptake and tighter packing on both flat and nanostructured silica and on silica nanoparticles. Consequently, defatted coatings exhibit markedly better resistance to serum biofouling and more effectively suppress nanoparticle-induced complement activation. Reintroducing fatty acids (caprylic acid) restores higher stability and reduces adsorption/denaturation, confirming causality. These mechanistic insights provide a clear rationale for selecting defatted BSA for antifouling applications and highlight how bound small-molecule ligands modulate protein adsorption and coating properties.

Fatty acid-free (defatted) BSA consistently forms denser, more effective antifouling coatings than fatty acid-stabilized (fatted) BSA across purification routes. Mechanistically, reduced conformational stability and lower charge repulsion in defatted BSA promote greater adsorption-induced spreading and higher packing density, enhancing surface passivation and mitigating complement activation on nanoparticles. The work offers practical guidance to preferentially use defatted BSA for antifouling coatings and suggests broader applicability to other fatty acid-binding proteins (e.g., human serum albumin) and diverse nanoparticle systems. Future studies can extend these insights to different surface chemistries, particle shapes and sizes, and explore how controlled ligand binding tunes protein adsorption behavior for optimized biointerfaces.