Temperature-Induced Denaturation of BSA Protein Molecules for Improved Surface Passivation Coatings

Abstract

Bovine serum albumin (BSA) is the most widely used protein for surface passivation applications, although it has relatively weak, nonsticky interactions with hydrophilic surfaces such as silica-based materials. Herein, we report a simple and versatile method to increase the stickiness of BSA protein molecules adsorbing onto silica surfaces, resulting in up to a 10-fold improvement in blocking efficiency against serum biofouling. Circular dichroism spectroscopy, dynamic light scattering, and nanoparticle tracking analysis showed that temperature-induced denaturation of BSA proteins in bulk solution resulted in irreversible unfolding and protein oligomerization, thereby converting weakly adhesive protein monomers into a more adhesive oligomeric form. The heat-treated, denatured BSA oligomers remained stable after cooling. Room-temperature quartz crystal microbalance-dissipation and localized surface plasmon resonance experiments revealed that denatured BSA oligomers adsorbed more quickly and in larger mass quantities onto silica surfaces than native BSA monomers. We also determined that the larger surface contact area of denatured BSA oligomers is an important factor contributing to their more adhesive character. Importantly, denatured BSA oligomers were a superior passivating agent to inhibit biofouling on silica surfaces and also improved Western blot application performance. Taken together, the findings demonstrate how temperature-induced denaturation of BSA protein molecules can lead to improved protein-based coatings for surface passivation applications.

Quantitative assessment of bovine serum albumin proteins for blocking applications

“…Across the temperature range of 25 to 55 °C, the DLS data showed that all six BSA proteins have ~8-nm diameter, which agrees well with the expected size of BSA monomers 25,26,37,38 (Fig. 2b and Supplementary Fig.…”

“…Thermally induced oligomerization of BSA molecules is triggered by irreversible conformational changes in the protein structure once there is a critical degree of protein unfolding and corresponding loss of α-helical character. The CD spectroscopy data showed that all six BSA proteins exhibited around 60-63% α-helical character at 25 °C, followed by a progressive decrease in α-helical character with increasing temperature 25,26,39,40 (Fig. 2c and Supplementary Figs.…”

“…However, there is essentially no discussion in the scientific literature about what type(s) of BSA reagents are useful to pick for blocking applications, especially from a mechanistic perspective. Most blocking-related studies that investigate mechanistic details have only evaluated a single type of BSA and compare BSA adsorption and surface-induced conformational changes on different surfaces 23,24 or investigate the effects of ionic strength 25 or BSA pretreatment 26 . However, the comparison of BSA proteins obtained through different purification methods remains unexplored despite being the greatest source of variability in BSA reagent options on the market.…”

Amyloid‐Like Protein Aggregates: A New Class of Bioinspired Materials Merging an Interfacial Anchor with Antifouling

“…BSA is the most abundant protein in plasma, and its function is to maintain the osmotic pressure of the blood, to provide pH buffering, and to transport many small molecules, such as calcium, bilirubin, and some drug molecules. [ 25–29 ] BSA is also well known to resist the nonspecific adsorption of proteins, [ 30,31 ] so it is often used as a passivating agent for immunofluorescence and other related experiments to prevent nonspecific adsorption of other protein molecules. [ 32 ] In the present work, the phase transition is triggered by the reduction of the disulfide bond of BSA via tris(2‐carboxyethyl)phosphine (TCEP).…”

Microbial degradation of organic pollutants in groundwater related to underground coal gasification

“…When temperature was above 40°C, the degradation efficiencies of phenol dropped rapidly due to the possible denaturation of enzyme protein. 31 The pH favourable for the growth of JC and WX bacteria was in range of 6-8, and the degradation efficiency of phenol reached the maximum at pH = 7 as shown in Figure 4B. It was also observed that the degradation efficiency of phenol by WX bacteria was higher than that by JC bacteria in weak acid or weak alkali solutions, but significantly decreased and became far lower than that by JC bacteria at pH = 9.…”