Selective adsorption of protein molecules on phase-separated sapphire surfaces

Site-selective adsorption of protein molecules was found on sapphire surfaces that exhibit a phase separation into two domains: weakly charged hydrophobic domain and negatively charged hydrophilic one. Ferritin and bovine serum albumin molecules, which are negatively charged in a buffer solution, are adsorbed to the hydrophobic domains. Avidin molecules, which are positively charged, are adsorbed to the other domain. Fibrinogen molecules, which consist of both negative and positive modules, are adsorbed to the whole sapphire surface. Hemoglobin molecules, whose net charge is almost zero, are also adsorbed to the whole surfaces. These results indicate that electrostatic double layer interaction is the primary origin of the observed selectivity. Dependence of protein adsorption or desorption behaviors on the pH value can also be interpreted by the proposed model.     

Competitive adsorption of model charged proteins: the effect of total charge and charge distribution

The adsorption of mixtures of charged proteins on charged surfaces is studied using a molecular theory. The theory explicitly treats each of the molecular species in the system. The mixtures treated in this work are composed by two types of proteins, dissociated monovalent salt and solvent. The intermolecular and surface interactions include electrostatic, van der Waals and excluded volume. The theory is more general than the Poisson-Boltzmann approach since the size and shape of all the molecular components are explicitly treated. The studies presented in this work concentrate on the differences in competitive adsorption when the proteins in the mixtures differ in their total charge or in the spatial distribution of the charges within the proteins. In the cases of mixtures that differ in the number of charges it is found, as expected, that the particles with the larger charge adsorb in excess. The ratio of adsorbed proteins can vary by 3-5 orders of magnitude by varying the bulk salt concentration from 1 to 100 mM. This is the result of an increase on the adsorption of the proteins with larger charge and an even stronger decrease on the adsorption of the less charged particles. The simple model systems studied provide guidelines on how to separate charge ladder proteins and proteins with different charge distributions. In the case of proteins with the same total charge but different charge distribution, it is found that the partition of the proteins depends upon the bulk composition. However, in general the particles with the highest localized charge tend to adsorb more on the surfaces. The proteins are adsorbed in one or more layers. The structure of the second adsorbed layer is determined mostly by the bulk properties of the solution. In all cases it is found that in the range of salt concentrations studied the number of adsorbed ions from the salt is very large. This is due to competitive adsorption with the proteins and their very low bulk concentration compared to the salt. The limitations of the theory and directions for improvement of the approach as well as the model for the proteins are discussed.     

Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy

Electrostatic interactions between negatively charged polymer surfaces and factor XII (FXII), a blood coagulation factor, were investigated by sum frequency generation (SFG) vibrational spectroscopy, supplemented by several analytical techniques including attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance (QCM), ζ-potential measurement, and chromogenic assay. A series of sulfonated polystyrenes (sPS) with different sulfonation levels were synthesized as model surfaces with different surface charge densities. SFG spectra collected from FXII adsorbed onto PS and sPS surfaces with different surface charge densities showed remarkable differences in spectral features and especially in spectral intensity. Chromogenic assay experiments showed that highly charged sPS surfaces induced FXII autoactivation. ATR-FTIR and QCM results indicated that adsorption amounts on the PS and sPS surfaces were similar even though the surface charge densities were different. No significant conformational change was observed from FXII adsorbed onto surfaces studied. Using theoretical calculations, the possible contribution from the third-order nonlinear optical effect induced by the surface electric field was evaluated, and it was found to be unable to yield the SFG signal enhancement observed. Therefore it was concluded that the adsorbed FXII orientation and ordering were the main reasons for the remarkable SFG amide I signal increase on sPS surfaces. These investigations indicate that negatively charged surfaces facilitate or induce FXII autoactivation on the molecular level by imposing specific orientation and ordering on the adsorbed protein molecules. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Adsorption of the protein bovine serum albumin in a planar poly(acrylic acid) brush layer as measured by optical reflectometry

The adsorption of bovine serum albumin (BSA) in a planar poly(acrylic acid) (PAA) brush layer has been studied by fixed-angle optical reflectometry. The influence of polymer length, grafting density, and salt concentration is studied as a function of pH. The results are compared with predictions of an analytical polyelectrolyte brush model, which incorporates charge regulation and excluded volume interactions. A maximum in adsorption is found near the point of zero charge (pzc) of the protein. At the maximum, BSA accumulates in a PAA brush to at least 30 vol %. Substantial adsorption continues above the pzc, that is, in the pH range where a net negatively charged protein adsorbs into a negatively charged brush layer, up to a critical pH value. This critical pH value decreases with increasing ionic strength. The adsorbed amount increases strongly with both increasing PAA chain length and increasing grafting density. Experimental data compare well with the analytical model without having to include a nonhomogeneous charge distribution on the protein surface. Instead, charge regulation, which implies that the protein adjusts its charge due to the negative electrostatic potential in the brush, plays an important role in the interpretation of the adsorbed amounts. Together with nonelectrostatic interactions, it explains the significant protein adsorption above the pzc.     

Strong resistance of a thin crystalline layer of balanced charged groups to protein adsorption

Resistance of mixed self-assembled monolayers (SAMs) with various counter-charged terminal groups of different valence and protonation/deprotonation states to nonspecific protein adsorption is investigated. It is demonstrated that excellent nonfouling surfaces can be readily constructed from mixed positively and negatively charged components of equal valence in a wide range of thiol solution compositions. Furthermore, the lattice structure of one of the mixed SAM systems studied is revealed by atomic force microscopy (AFM) to be (5.2 +/- 0.2 A x 5.2 +/- 0.2 A)60 degrees . Results indicate that the packing structure of mixed charged SAMs is determined by strong charge-charge interactions of the terminal groups rather than S-Au and chain-chain interactions. This work provides direct evidence that conformational flexibility is not required for protein resistance of a surface and even a single compact layer of charged groups of balanced charge with a crystalline structure can resist nonspecific protein adsorption, suggesting that tightly bound water molecules on the topmost part of the mixed SAMs play a dominant role in surface resistance to nonspecific protein adsorption.     

Probing interactions between aggrecan and mica surface by the atomic force microscopy

Aggrecan is a bottlebrush shaped macromolecule found in the extracellular matrix of cartilage. The negatively charged glycosaminoglycan (GAG) chains attached to its protein backbone give aggrecan molecules a high charge density, which is essential for exerting high osmotic swelling pressure and resisting compression under external load. In solution, aggrecan assemblies are insensitive to the presence of calcium ions, and show distinct osmotic pressure versus concentration regimes. The aim of this study is to investigate the effect of ionic environment on the structure of aggrecan molecules adsorbed onto well‐controlled mica surfaces. The conformation of the aggrecan was visualized using Atomic Force Microscopy. On positively charged APS mica the GAG chains of the aggrecan molecules are distinguishable, and their average dimensions are practically unaffected by the presence of salt ions. With increasing aggrecan concentration they form clusters, and at higher concentrations they form a continuous monolayer of conforming molecules. On negatively charged mica, the extent of aggrecan adsorption varies with salt composition. Understanding aggrecan adsorption onto a charged surface provides insight into its interactions with bone and implant surfaces in the biological milieu. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2010     

Monte Carlo simulations of antibody adsorption and orientation on charged surfaces

Monte Carlo simulations were performed to study the adsorption and orientation of antibodies on charged surfaces based on both colloidal and all-atom models. The colloidal model antibody consists of 12 connected beads representing the 12 domains of an antibody molecule. The structure of the all-atom antibody model was taken from the protein databank. The effects of the surface charge sign and density, the solution pH and ionic strength on the adsorption and orientation of different colloidal model antibodies with different dipole moments were examined. Simulation results show that both the 12-bead and the all-atom models of the antibody, for which the dipole moment points from the Fc to (Fab)2 fragments, tend to have the desired "end-on" orientation on positively charged surfaces and undesired "head-on" orientation on negatively charged surfaces at high surface charge density and low solution ionic strength where electrostatic interactions dominate. At low surface charge density and high solution ionic strength where van der Waals interactions dominate, 12-bead model antibodies tend to have "lying-flat" orientation on surfaces. The orientation of adsorbed antibodies results from the compromise between electrostatic and van der Waals interactions. The dipole moment of an antibody is an important factor for antibody orientation on charged surfaces when electrostatic interactions dominate. This charge-driven protein orientation hypothesis was verified by our simulations results in this work. It was further confirmed by surface plasmon resonance biosensor and time-of-flight secondary ion mass spectrometry experiments reported elsewhere.     

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO(2) wafers was achieved under pH 11.6, 50mM CaCl(2), and at 70 degrees C. XG-ALG films presented mean thickness of (16+/-2)nm and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAlt adsorbed irreversibly onto XG-ALG forming homogenous monolayers approximately (4+/-1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue.     

Interactions between charged surfaces immersed in a polyelectrolyte solution

With grand canonical simulations invoking a configurationally weighted scheme, we have calculated interactions between charged surfaces immersed in a polyelectrolyte solution. In contrast to previous simulations of such systems, we have imposed full equilibrium conditions (i.e., we have included diffusive equilibrium with a bulk solution). This has a profound impact on the resulting interactions: even at modest surface charge densities, oppositely charged chains will, at sufficiently large separations, adsorb strongly enough to overcompensate for the nominal surface charge. This phenomenon, known as charge inversion, generates a double-layer repulsion and a free-energy barrier. Simpler canonical approaches, where the chains are assumed to neutralize the surfaces perfectly, will not capture this stabilizing barrier. The barrier height increases with the length of the polyions. Interestingly enough, the separation at which the repulsion becomes attractive is independent of chain length. The short chains here are unable to reach across from one surface to the other. We therefore conclude that the transition to an attractive regime is not provided by the formation of such "intersurface" bridges. With long chains and at large separations, charge inversion displays decaying oscillatory behavior (i.e., the apparent surface charge switches sign once again). This is due to polyion packing effects. We have also investigated responses to salt addition and changes in polyelectrolyte concentration. Our results are in qualitative and semiquantitative agreement with experimental findings, although it should be noted that our chains are comparatively short, and the experimental surface charge density is poorly established.     

Electrostatic interactions between amphoteric latex particles and proteins

The electrostatic interactions between amphoteric polymethyl methacrylate latex particles and proteins with different pI values were investigated. These latex particles possess a net positive charge at low pH, but they become negatively charged at high pH. The nature and degree of interactions between these polymer particles and proteins are primarily controlled by the electrostatic characteristics of the particles and proteins under the experimental conditions. The self-promoting adsorption process from the charge neutralization of latex particles by the proteins, which have the opposite net charge to that of the particles, leads to a rapid reduction in the zeta potential of the particles (in other words colloidal stability), and so strong flocculation occurs. On the other hand, the electrostatic repulsion forces between similarly charged latex particles and the proteins retard the adsorption of protein molecules onto the surfaces of the particles. Therefore, latex particles exhibit excellent colloidal stability over a wide range of protein concentrations. A transition from net negative charge to net positive charge, and vice versa (charge reversal), was observed when the particle surface charge density was not high enough to be predominant in the protein adsorption process.     

Interactions between charged surfaces with ionizable sites

A key factor controlling the interactions between surfaces in aqueous solutions is the surface charge density. Surfaces typically become charged though a titration process where surface groups can become ionized based on their dissociation constant and the pH of the solution. In this work, we use a Monte Carlo method to treat this process in a system with two planar surfaces with explicitly described ionizable sites in a salt solution. We focus on a system with a surface density of ionizable sites set to 4.8 nm(-2), corresponding to silica. We find that the surface charge density changes as the surfaces come close to contact due to interactions between the ionizable groups on each surface. In addition, we observe an attraction between the surfaces above a threshold surface charge, in good agreement with previous theoretical predictions based on uniformly charged surfaces. However, close to contact we find the force is significantly different than for the uniformly charged case.     

Phase behaviour of oppositely charged protein and surfactant mixtures: DOTAC-BLG-Water

The interactions between the negatively charged protein, β-lactoglobulin (BLG) and the cationic surfactant dodecyltrimethylammonium chloride (DOTAC) in water have been investigated by determining the phase equilibria of the ternary system within the concentration range of 20 wt.% of both protein and surfactant. Three main regions are formed—an isotropic solution phase, a white precipitation region and a blueish, isotropic, highly viscous gel phase. The protein solution can solubilize 1 mole surfactant, [DOTAC] per mole protein, [BLG] prior to precipitation. The protein-surfactant precipitate complex is neutral and consists of 8 [DOTAC]/[BLG]. The net charge of the protein in water at aqueous pH is −7 and this agrees with the determined composition. The pH is, however, decreased on addition of DOTAC, but this does not seem to affect the composition of the precipitate significantly. The amount of precipitate reaches a maximum at about 8 [DOTAC]/[BLG] and thereafter a plateau region occurs where no more precipitate seems to be formed. On further increasing the surfactant concentration the precipitate redissolves either into a solution phase directly or into a solution phase via a gel phase depending on the protein concentration. On decreasing salt concentration the ternary system shows similar phase behaviour, but the stability of the regions are different. It is also observed that oppositely charged protein-surfactant systems show similar phase behaviour irrespective of nature of the net charge on the protein.     

Image charges and dispersion forces in electric double layers: the dependence of wall-wall interactions on salt concentration and surface charge density

The interaction pressure between two planar charged walls is calculated for a range of conditions. The diffuse electric double layers between the two wall surfaces are treated with ion-wall dispersion forces and ionic image charge interactions taken into account. Both these interactions are due to dielectric discontinuities at the surfaces. Ion-ion and ion-image charge correlations are explicitly included. The ion-wall dispersion interactions can give rise to appreciable ion specific effects, which are particularly strong when the counterions to the surfaces are highly polarizable. The mechanisms of these effects are investigated, and their influence on the net interaction pressure between the walls is studied for a range of surface charge densities, strengths of the anion-wall dispersion interaction and bulk electrolyte concentrations. When the strength of the anion-wall dipersion interaction is increased, the pressure generally becomes less repulsive (or more attractive) for positive surfaces. The opposite happens in general for negative surfaces but to a much lesser extent. The effects are largest for large surface charge densities and high electrolyte concentrations. The image charge interactions give rise to a considerable depletion attraction between the walls for low surface charge densities.     

Selective protein adsorption on a phase-separated solvent-cast polymer blend

Polymer-based biomedical devices are growing increasingly sophisticated as compositions evolve toward copolymers and blends in order to satisfy complex design criteria. Such polymers afford opportunities for both micro- and macrophase separation at nano- and micro-length scales and raise questions concerning the role of heterogeneous surface morphology on protein adsorption. Adsorbed protein layers play a critical role in mediating the interaction of cells with polymer surfaces, and both understanding and controlling protein adsorption is assuming greater significance in the development of surfaces with enhanced physiological compatibility. Here we study the short-time adsorption of ferritin, a model protein highly resistant to denaturation and easily imaged in the transmission electron microscope (TEM), onto a phase-separated homopolymer blend of polycaprolactone (PCL) and a polycarbonate derived from desaminotyrosyl-tyrosine dodecyl ester (PDTD). At physiological pH, ferritin selectively adsorbs onto the PDTD phase at a surface density approximately three times greater than that on the PCL phase. By decreasing the pH below ferritin's isoelectric point so its average charge becomes positive, the selective adsorption disappears and the surface density of adsorbed ferritin becomes independent of the phase separation. We attribute the selectivity to the electrostatic repulsion between ferritin and hydrolytically charged PCL, both of which will have a net negative charge at physiological pH. To perform these experiments, we solvent-cast ultrathin polymer films onto dissolvable salt substrates, and we characterize the morphology by TEM imaging and quantitative spatially resolved electron energy-loss spectroscopy (EELS). We find that the film morphology depends strongly on such processing-related variables as the solvent evaporation rate and the nature of the surface in contact with the polymer film during casting. The adsorption of ferritin depends on whether the film is phase-separated as well as to which surface of the film the protein solution is exposed, and these findings suggest that seemingly small variations in polymer processing that influence both the bulk and surface morphology can have a profound effect on the short-time protein adsorption.     

Activity of an enzyme immobilized on polyelectrolyte multilayers

The immobilization of α-chymotrypsin on the surface of boron silicate glass microspheres is conducted via the technique of multilayer adsorption of polyelectrolytes. It is shown that the enzyme is adsorbed on both positively and negatively charged surfaces and its activity is partially preserved relative to that in solution. The activity of the enzyme depends on the number of polyectrolyte layers preliminarily adsorbed on glass microspheres and on the charge of the surface. The activity of α-chymotrypsin adsorbed on the negatively charged surface is four times higher than the activity of this enzyme adsorbed on a positively charged surface.     

Human serum albumin self-assembly on weak polyelectrolyte multilayer films structurally modified by pH changes

Adsorption of proteins onto film surfaces built up layer by layer from oppositely charged polyelectrolytes is a complex phenomenon, governed by electrostatic forces, hydrogen bonds, and hydrophobic interactions. The amounts of the interacting charges, however, both in polyelectrolytes and in proteins adsorbed on such films are a function of the pH of the solution. In addition, the number and the accessibility of free charges in proteins depend on the secondary structure of the protein. The subtle interplay of all these factors determines the adsorption of the proteins onto the polyelectrolyte film surfaces. We investigated the effect of these parameters for polyelectrolyte films built up from weak "protein-like" polyelectrolytes (i.e., polypeptides), poly(L-lysine) (PLL), and poly(glutamic acid) (PGA) and for the adsorption of human serum albumin (HSA) onto these films in the pH range 3.0-10.5. It was found that the buildup of the polyelectrolyte films is not a simple function of the pure charges of the individual polyelectrolytes, as estimated from their respective pKa values. The adsorption of HSA onto (PLL/PGA)n films depended strongly on the polyelectrolyte terminating the film. For PLL-terminated polyelectrolyte films, at low pH, repulsion, as expected, is limiting the adsorption of HSA (having net positive charge below pH 4.6) since PLL is also positively charged here. At high pH values, an unexpected HSA uptake was found on the PGA-ending films, even when both PGA and HSA were negatively charged. It is suggested that the higher surface rugosity and the decrease of the alpha-helix content at basic pH values (making accessible certain charged groups of the protein for interactions with the polyelectrolyte film) could explain this behavior.     

Molecular dynamics simulations of polyelectrolyte adsorption

We have performed molecular dynamics simulations of polyelectrolyte adsorption at oppositely charged surfaces from dilute polyelectrolyte solutions. In our simulations, polyelectrolytes were modeled by chains of charged Lennard-Jones particles with explicit counterions. We have studied the effects of the surface charge density, surface charge distribution, solvent quality for the polymer backbone, strength of the short-range interactions between polymers and substrates on the polymer surface coverage, and the thickness of the adsorbed layer. The polymer surface coverage monotonically increases with increasing surface charge density for almost all studied systems except for the system of hydrophilic polyelectrolytes adsorbing at hydrophilic surfaces. In this case the polymer surface coverage saturates at high surface charge densities. This is due to additional monomer-monomer repulsion between adsorbed polymer chains, which becomes important in dense polymeric layers. These interactions also preclude surface overcharging by hydrophilic polyelectrolytes at high surface charge densities. The thickness of the adsorbed layer shows monotonic dependence on the surface charge density for the systems of hydrophobic polyelectrolytes for both hydrophobic and hydrophilic surfaces. Thickness is a decreasing function of the surface charge density in the case of hydrophilic surfaces while it increases with the surface charge density for hydrophobic substrates. Qualitatively different behavior is observed for the thickness of the adsorbed layer of hydrophilic polyelectrolytes at hydrophilic surfaces. In this case, thickness first decreases with increasing surface charge density, then it begins to increase.     

THEORETICAL MODELING OF IONIC SURFACTANT ADSORPTION ON MINERAL OXIDE SURFACES

A model for the adsorption of ionic surfactants on oppositely charged solid surfaces of uniform charge density is developed. The model is based on the assumption that, on the solid surface, adsorbed surfactant monomers, monolayered and bilayered surfactant aggregates of different sizes and specifically adsorbing ions of added electrolyte constitute a mixture of hard discs. It means that only excluded area interactions between the surface discs are taken into account. To avoid a rapid two-dimensional condensation of the adsorbed surfactant the potential energy per molecule in the surface aggregates, which is a sum of chemical and electrostatic interactions, is assumed to decrease linearly with the increasing aggregate size. The electrostatic interactions of ionic species with the charged solid surface are described in terms of the Guy-Chapman theory of the double layer formation. The appropriate equations for adsorption isotherms of surfactant and electrolyte ions are derived and used to predict the experimental adsorption isotherms of DTAB on the precipitated silica at two different salt concentrations in the aqueous solution, On the basis of the obtained results the evolution of the adsorbed phase structure and the charge of silica particles with an increasing surface coverage is discussed.     

Theory of polyelectrolyte adsorption onto surfaces patterned with charge and topography

Mean-field theory is used to derive criteria for the adsorption of a weakly charged polyelectrolyte molecule from salt solution onto surfaces patterned with charge and topography. For flat surfaces patterned with periodic arrays of charged patches, the adsorbed layer thickness predicted using mean-field theory and that found by Brownian dynamics simulations are in quantitative agreement in the strong-adsorption regime, which corresponds to sufficiently small kappa or sufficiently large |sigma(eff)q|, where kappa is the inverse Debye screening length, sigma(eff) is an effective surface charge density, and q is the charge on each segment of the polyelectrolyte. Qualitative agreement is obtained in the weak-adsorption regime, and for the case where surfaces are patterned with both charge and topography. For uniformly charged, sinusoidally corrugated surfaces, the theory predicts that the critical temperature required for adsorption can be greater than or less than the corresponding value for a flat surface depending on the relative values of kappa and the corrugation wave number. If the surface charge is also allowed to vary sinusoidally, then adsorption is predicted to occur only when the topography crests have a surface charge opposite to that of the polyelectrolyte. Surfaces patterned with rectangular indentations having charged bottoms which are separated by flat charged plateaus are investigated as well. Adsorption is predicted to occur even when the net surface charge is zero, provided that the plateaus have a charge opposite to that of the polyelectrolyte. If the charge on the plateaus and polyelectrolyte is the same, adsorption may still occur if electrostatic attraction from the indentation bottoms is sufficiently strong.     

Adsorption and disruption of lipid bilayers by nanoscale protein aggregates

Nanoparticles taken into biological systems can have biological impacts through their interactions with cell membranes, accompanied by protein adsorption onto the nanoparticle surfaces, forming a so-called protein corona. Our current research aims to demonstrate that nanoscale protein aggregates behave like such nanoparticles with regard to the interaction with lipid membranes. In this study, the adsorption and disruption of the lipid membranes by protein aggregates were investigated using amyloid fibrils and nanoscale thermal aggregates of lysozyme. Both types of protein aggregates had disruptive effects on the negatively charged liposomes, similar to polycationic nanoparticles. Interestingly, adsorption of liposomes on the amyloid fibrils preceding disruption occurred even if the net charge of the liposome was zero, suggesting the importance of hydrophobic interactions in addition to electrostatic interactions. The results of the present study provide new insights into the biological impacts of nanoparticles in vivo.