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ABSTRACT: Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins behaved similarly on addition of the alkaline-surfactant cleaning solution, in that platinum and gold exhibited an increase, while tungsten, titanium, and stainless steel showed a decrease in weight. According to dissipation measurements with the QCM-D, the adsorbed layer for platinum and gold was rigid, while that for the tungsten, titanium, and stainless steel was much more flexible. The removal efficiency of adsorbed-protein by alkaline solution of SDS depended on the water content of the adsorbed layers for W, Ti, and SS, while for Pt and Au, it depended on secondary structural content. When protein adsorption was high (Pt, Au), protein-protein interactions and protein-surface interactions were dominant and the removal of protein layers was limited. Water content of the adsorbed protein layer was the determining factor for how efficiently the layer was removed by alkaline SDS when protein adsorption was low. Hence, protein-protein and protein-surface interactions were minimal and protein structure was less perturbed in comparison with those for high protein adsorption. Secondary structural content determined the efficient removal of adsorbed protein for high adsorbed amount.
Langmuir 03/2011; 27(5):1830-6. · 4.19 Impact Factor
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ABSTRACT: Combining a wide range of protein adsorption experiments (three globular proteins on eight well-defined homogeneous surfaces) with Monte Carlo simulations of lattice proteins at different concentrations and on surfaces of varying "polarity", we explore the extent and rheological behavior of adsorbed proteins as a function of substrate polarity, "on" rate constants (k(a)) and steric parameters (|A(1)|) from the random sequential adsorption model, and demonstrate a folding to unfolding transition upon adsorption. We show that model globular proteins (hen egg lysozyme, ribonuclease A, and insulin dimer) behave similarly with respect to adsorption. Experimentally, above a substrate wettability cos theta > 0.4 (where theta is the sessile contact angle of water on a substrate in air), the adsorbed mass, rigidity, and k(a) of the proteins are diminished, while the steric factor |A(1)| is increased, suggesting a lower packing density. To analyze these results, we have invoked computer simulations. We show that changing surface polarity has two profound effects. First, the amount adsorbed increases as the surfaces become more apolar. Further, the proteins become less stable as their adsorbed amount increased because they gain a large number of interprotein and protein-surface interactions. Finally, apolar surfaces served to reduce the unfolding free energy barriers, further facilitating the reorganizing of proteins on these surfaces. Thus, increasing the nonpolar nature of the surfaces resulted in a more rigid adsorbed layer, in good agreement with the experiments.
Langmuir 07/2010; 26(13):10803-11. · 4.19 Impact Factor
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ABSTRACT: We offer a novel process to render hydrophobic surfaces resistant to relatively small proteins during adsorption. This was accomplished by self-assembly of a well-known natural osmolyte, trimethylamine oxide (TMAO), a small amphiphilic molecule, on a hydrophobic alkanethiol surface. Measurements of lysozyme (LYS) adsorption on several homogeneous substrates formed from functionalized alkanethiol self-assembled monolayers (SAMs) in the presence and absence of TMAO, and direct interaction energy between the protein and functionalized surfaces, demonstrate the protein-resistant properties of a noncovalently adsorbed self-assembled TMAO layer. Molecular dynamics simulations clearly show that TMAO molecules concentrate near the CH(3)-SAM surface and are preferentially excluded from LYS. Interestingly, TMAO molecules adsorb strongly on a hydrophobic CH(3)-SAM surface, but a trade-off between hydrogen bonding with water, and hydrophobic interactions with the underlying substrate results in a nonintuitive orientation of TMAO molecules at the interface. Additionally, hydrophobic interactions, usually responsible for nonspecific adsorption of proteins, are weakly affected by TMAO. In addition to TMAO, other osmolytes (sucrose, taurine, and betaine) and a larger homologue of TMAO (N,N-dimethylheptylamine-N-oxide) were tested for protein resistance and only N,N-dimethylheptylamine-N-oxide exhibited resistance similar to TMAO. The principle of osmolyte exclusion from the protein backbone is responsible for the protein-resistant property of the surface. We speculate that this novel process of surface modification may have wide applications due to its simplicity, low cost, regenerability, and flexibility.
Langmuir 03/2010; 26(12):9695-702. · 4.19 Impact Factor
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ABSTRACT: The stability of tethered globular proteins under denaturing conditions was interrogated with a hydrophobic surface, since conventional structural methods like circular dichroism (CD) and fluorescence or infrared spectroscopy could not be used because of the presence of an opaque solid substrate and extremely low surface concentrations. For free protein in solution, CD spectra gave well-known unfolding denaturing curves for lysozyme (LYS) and ribonuclease A (RNase A). The unfolding process for covalently tethered LYS and RNase A was followed, with multimolecular force spectroscopy (using an atomic force microscope in force-mode), via the adhesion energy between a functionalized self-assembled monolayer (CH(3)-SAM) probe and the protein molecules covalently bound to a carboxylic SAM on a gold-coated glass coverslip. The adhesion energy passed through a maximum for the tethered proteins during excursions with temperature or chemical denaturants. The initial rise in adhesion energy on increasing the temperature or GuHCl concentration was due to increasing exposure of the unfolded hydrophobic core of the proteins to the CH(3)-SAM tip, while the decrease in adhesion energy at high temperature or large concentrations of denaturant is attributed to interprotein association with nearest neighbors. Attempts to recover their folded state upon cooling (or reducing GuHCl concentration) were unsuccessful. Also, dilution of surface-tethered LYS reduced the aggregation with nearest neighbors about 6-fold. These results are in qualitative agreement with Monte Carlo simulations on a simple two-letter lattice protein model, especially for low concentrations of grafted proteins.
Langmuir 04/2009; 25(9):4998-5005. · 4.19 Impact Factor
