On the stability of the polymer brushes formed by adsorption of ionomer complexes on hydrophilic and hydrophobic surfaces.

We have studied the effect of normal forces and shear forces on the stability and functionality of a polymer brush layer formed upon adsorption of polymeric micelles on hydrophilic and hydrophobic surfaces. The micelles consist of oppositely charged polyelectrolyte blocks (poly(acrylic acid) and poly(N-methyl 2-vinyl pyridinium iodide), and a neutral block (poly(vinyl alcohol)) or neutral grafts (poly(ethylene oxide)). The strength of the attachment of the micellar layers to various substrates was evaluated with Atomic Force Microscopy. Flow cell experiments allowed for the evaluation of long-term stability of coatings in lateral flow. Fixed angle optical reflectometry was used to quantify protein (BSA) adsorption on the micellar layers after their exposure to flow. The results show that adsorbed micellar layers are relatively weakly attached to hydrophobic surfaces and much stronger to hydrophilic surfaces, which has a significant impact on their stability. Adsorbed layers maintain their ability to suppress protein adsorption on hydrophilic surfaces but not on hydrophobic surfaces. Due to the relatively weak attachment to hydrophobic surfaces the structure of adsorbed layers may easily be disrupted by lateral forces, such that the complex coacervate-brush structure no longer exists.

Resolution of D- and L-glucoses by chiral N-octyl-beta-D-glycoside-Cu(II) complex adsorbed at the gas/liquid interface of small bubbles.

A new technique of the jet drop method (JDM) was applied to a chiral molecular discrimination of optically active D- or L-glucose (guest) by chiral N-octyl-beta-D-glycoside (ObetaDG)-Cu(II) complex (host) at the gas/liquid interface of small bubbles. The discrimination of glucoses as the guests is possible using ObetaDG adsorbed at the gas/liquid interface of bubbles where it acts as the host, either in the presence or the absence of Cu(II) ions. In order to make clear the host-guest interaction at the gas/liquid interface, the composition of 5000 top jet drops periodically collected onto a slide glass receiver was analyzed. The relative concentration (eta(i)-1) and the surface excess amount, Gamma(i)(0) of species i such as D- and L-glucoses, and ObetaDG were determined as a function of bubble size, d(b) and bulk concentration C(b). The partition coefficient, Pi(i)=Gamma(i)(0)/C(b) was also evaluated for each component. The adsorption of these materials either in the presence or absence of Cu(II) ions, was assigned to the Freundlich type, and the discrimination of D- and L-glucoses with ObetaDG was evaluated in terms of the Freundlich constant, k(i) and 1/n. The discrimination ability of ObetaDG was also evaluated by determining the equilibrium constants, K(c) of complex formations for the respective glucoses in the presence and absence of Cu(II) ions. It was found that L-glucose can form a more stable complex with ObetaDG-Cu(II).

Surface-modified nanoparticles as a new, versatile, and mechanically robust nonadhesive coating: suppression of protein adsorption and bacterial adhesion.

The synthesis of surface-modified silica nanoparticles, chemically grafted with acrylate and poly(ethylene glycol) (PEG) groups, and the ability of the resulting crosslinked coatings to inhibit protein adsorption and bacterial adhesion are explored. Water contact angles, nanoindentation, and atomic force microscopy were used to characterize the cross-linked coatings. Coatings showed a high degree of hydrophilicity combined with a remarkable hardness and stiffness in the dry state. Adsorption of the small protein lysozyme from buffer solution on coated silica wafers decreased significantly with increasing grafting density of the PEG groups on the nanoparticles and was completely inhibited at 0.6 chains nm(-2). Coatings significantly reduced adhesion of Staphylococcus epidermidis HBH 276 in a parallel plate flow chamber with respect to bare glass (>90%), whereas adhesion of Pseudomonas aeruginosa AK1 was only marginally affected by the presence of the coating (<15%). Passage of an air-bubble resulted in almost complete detachment (>93%) of both strains from coated glass, indicating that the adhesion strength between both bacterial strains and the coated surface was significantly reduced by the grafted PEG groups. These coatings thus provide a new method to prepare mechanically robust films with nonadhesive properties that will be extremely useful for the design of biocompatible surfaces in biomedical applications.

Reduction of protein adsorption to a solid surface by a coating composed of polymeric micelles with a glass-like core.

Adsorption studies by optical reflectometry show that complex coacervate core micelles (C3Ms) composed of poly([4-(2-amino-ethylthio)-butylene] hydrochloride)(49)-block-poly(ethylene oxide)(212) and poly([4-(2-carboxy-ethylthio)-butylene] sodium salt)(47)-block-poly(ethylene oxide)(212) adsorb in equal amounts to both silica and cross-linked 1,2-polybutadiene (PB). The C3Ms have an almost glass-like core and atomic force microscopy of a dried layer of adsorbed C3Ms shows densely packed flattened spheres on silica, which very probably are adsorbed C3Ms. Experiments were performed with different types of surfaces, solvents, and proteins; bare silica and cross-linked 1,2-PB, NaNO(3) and phosphate buffer, and lysozyme, bovine serum albumin, beta-lactoglobulin, and fibrinogen. On the hydrophilic surface the coating reduces protein adsorption >90% in 0.1 M phosphate buffer, whereas the reduction on the coated hydrophobic surface is much lower. Reduction is better in phosphate buffer than in NaNO(3), except for the positively charged lysozyme, where the effect is reversed.

Stability of globular proteins in H2O and D2O.

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion ("hard" globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability ("soft" globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O.

Interactive forces between co-aggregating and non-co-aggregating oral bacterial pairs.

The temporo-spatial development of plaque is governed by adhesive interactions between different co-aggregating bacterial strains and species. Physico-chemically, these interactions are due to attractive Lifshitz-Van der Waals and acid-base forces, and occur despite electrostatic repulsion and with a critical influence of temperature. The forces between co-aggregating and non-co-aggregating pairs have never been measured, however. The aim here, thus, is to investigate, by atomic force microscopy, whether there is a difference in interactive forces between co-aggregating and non-co-aggregating bacterial pairs at 10 degrees C, 22 degrees C, and 40 degrees C. Actinomyces naeslundii 147 was immobilized on poly-L-lysine-coated tipless AFM cantilevers, while streptococci were immobilized on poly-L-lysine-coated glass surfaces. Upon approach, a repulsive force was measured, regardless of whether a co-aggregating or non-co-aggregating pair was involved. However, upon retraction, the co-aggregating pair exhibited larger adhesive forces and energies than did the non-co-aggregating pair. Adhesive interactions between the co-aggregating pair were smallest at 40 degrees C.

BSA adsorption on bimodal PEO brushes.

BSA adsorption onto bimodal PEO brushes at a solid surface was measured using optical reflectometry. Bimodal brushes consist of long (N=770) and short (N=48) PEO chains and were prepared on PS surfaces, applying mixtures of PS(29)-PEO(48) and PS(37)-PEO(770) block copolymers and using the Langmuir-Blodgett technique. Pi-A isotherms of (mixtures of) the block copolymers were measured to establish the brush regime. The isotherms of PS(29)-PEO(48) show hysteresis between compression and expansion cycles, indicating aggregation of the PS(29)-PEO(48) upon compression. Mixtures of PS(29)-PEO(48) and PS(37)-PEO(770) demonstrate a similar hysteresis effect, which eventually vanishes when the ratio of PS(37)-PEO(770) to PS(29)-PEO(48) is increased. The adsorption of BSA was determined at brushes for which the grafting density of the long PEO chains was varied, while the total grafting density was kept constant. BSA adsorption onto monomodal PEO(48) and PEO(770) brushes was determined for comparison. The BSA adsorption behavior of the bimodal brushes is similar to the adsorption of BSA at PEO(770) monomodal brushes. The maximum of BSA adsorption at low grafting density of PEO(770) can be explained by ternary adsorption, implying an attraction between BSA and PEO. The contribution of primary adsorption to the total adsorbed amount is negligible.

Tethered polymer chains: surface chemistry and their impact on colloidal and surface properties.

In this review the grafting of polymer chains to solid supports or interfaces and the subsequent impact on colloidal properties is examined. We start by examining theoretical models for densely grafted polymers (brushes), experimental techniques for their preparation and the properties of the ensuing structures. Our aim is to present a broad overview of the state of the art in this field, rather than an in-depth study. In the second section the interactions of surfaces with tethered polymers with the surrounding environment and the impact on colloidal properties are considered. Various theoretical models for such interactions are discussed. We then review the properties of colloids with tethered polymer chains, interactions between planar brushes and nanocolloids, interactions between brushes and biocolloids and the impact of grafted polymers on wetting properties of surfaces, using the ideas presented in the first section. The review closes with an outlook to possible new directions of research.

Protein adsorption at polymer-grafted surfaces: comparison between a mixture of saliva proteins and some well-defined model proteins.

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1 h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, beta-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface.

Adsorption of IgG onto hydrophobic teflon. Differences between the F(ab) and F(c) domains.

The effect of differences in the degree of hydrophobicity of protein patches/fragments on the adsorption behaviour of the protein is investigated. The adsorption isotherm of a monoclonal mouse anti-human immunoglobulin G (isotype 2b) onto hydrophobic Teflon particles is measured using a depletion method. The adsorption-induced denaturation of the immunoglobulin as a function of the adsorbed amount is studied by differential scanning calorimetry, and the corresponding rearrangements in the secondary structure of the whole IgG molecule and its F(ab) and F(c) fragments are determined by circular dichroism spectroscopy. The effects of adsorption on the F(ab) and F(c) fragments in the intact IgG molecule occur independently. Adsorption of the whole IgG molecule leads to denaturation of the F(ab) fragments, whereas the F(c) fragment remains unperturbed; adsorption of the isolated fragments results in structural changes in both F(ab) and F(c). The surface hydrophobicity of the isolated fragments was studied by HPLC. These experiments support the hypothesis that differences in the degree of denaturation between F(ab) and F(c) are due to the higher degree of hydrophobicity of the F(ab) fragment. The adsorption-induced changes in the secondary structure are more prominent for the isolated fragments as compared to intact IgG. This is ascribed to the higher flexibility of the isolated fragment, as compared to the fragment in the whole molecule.

The Adsorption-Desorption Cycle. Reversibility of the BSA-Silica System.

The reversibility of the adsorption-desorption cycle was established by comparing the thermostability (determined by differential scanning calorimetry) and secondary structure (obtained by circular dichroism spectroscopy) of BSA before adsorption, adsorbed on, and exchanged from silica particles. Circular dichroism was also measured as a function of temperature at a given wavelength. Adsorbed BSA presents a higher thermostability and a lower alpha-helix content than the native protein while it regains its conformation when released from the surface back into the solution; the homomolecular exchange is reversible.The changes in ellipticity (at a given wavelength) as a function of the temperature show that the thermal denaturation of native, adsorbed, and exchanged BSA proceeds in two steps. For the dissolved protein, the first step up to 50 degrees C involves a slight change in the structure while in the 50-90 degrees C temperature range the actual unfolding takes place. For the adsorbed BSA, the first step proceeds up to 60 degrees C and includes some intermolecular association between the adsorbed protein molecules, which may be responsible for the increased thermostability. The unfolding occurs in the 60-90 degrees C range; it is less cooperative and involves a lower enthalpy change than the native protein. Adsorbed BSA presents the same secondary structure as that observed for dissolved BSA that has passed a heating-cooling cycle. Copyright 2001 Academic Press.

The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F(ab) and F(c) fragments.

The unfolding and further denaturation of IgG and its F(ab) and F(c) fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F(ab) and F(c) units was retained after fragmentation of the IgG. The F(ab) fragment denatured at approximately 61 degrees C and the F(c) fragment at 71 degrees C. The structural transitions observed in the whole IgG is the sum effect of those determined for the isolated F(ab) and F(c) fragments.

Adsorption of Immunoglobulin G on Core-Shell Latex Particles Precoated with Chaps.

The aim of this work is to investigate the adsorption behavior of a monoclonal antibody (immunoglobulin G, IgG) on latex particles, possessing reactive chloromethyl groups, precoated with 3-([3-cholamidopropyl]dimethylammonio-1-propanesulfonate (Chaps). The amount and reactivity of the surface chloromethyl groups were monitored by the nucleophilic attack of glycinate to the functional groups as a function of time at 22 and 36 degrees C. The extent of displacement of Chaps by IgG and the enthalpy of the process were determined under two different conditions of precoating the latex particles with Chaps, at 22 and 36 degrees C. The adsorption of IgG takes place in two steps; the first one involves physical interaction between IgG and the surface. This step is relatively fast (in the range of minutes) and independent of temperature. In the second step covalent bonding between the protein and the active surface groups occurs. This reaction is improved by raising the temperature because Chaps desorption, which exposes the reactive chloromethyl groups on the latex particles, is kinetically and thermodynamically favored at 36 degrees C and the covalent bonding of IgG is faster at 36 degrees C. Copyright 2000 Academic Press.

BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states.

The secondary structure and the thermostability of bovine serum albumin (BSA), before adsorption and after homomolecular displacement from silica and polystyrene particles, are studied by circular dichroism spectroscopy and differential scanning calorimetry. The structural perturbations induced by the hydrophilic silica surface are reversible, i.e. BSA completely regains the native structure and stability after being exchanged. On the other hand, the adsorption on, and subsequent desorption from, polystyrene particles causes irreversible changes in the stability and (secondary) structure of BSA. The exchanged proteins have a higher denaturation temperature and a lower enthalpy of denaturation than native BSA. The alpha-helix content is reduced while the beta-turn fraction is increased in the exchanged molecules. Both effects are more pronounced when the protein is displaced from less crowded sorbent surfaces. The irreversible surface-induced conformational change may be related to some aggregation of BSA molecules after being exposed to a hydrophobic surface.

The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein.

The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the F(ab) and F(c) fragments of the IgG molecule. The F(ab) fragment is most sensitive to heat treatment, whereas the F(c) fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate-determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.

ATR-FTIR Study of IgG Adsorbed on Different Silica Surfaces.

Thesecondary structure of adsorbed immunoglobulin G (IgG) on different silica surfaces (hydrophilic, hydrophobic, hydrophobic with preadsorbed triblock-copolymers consisting of a polypropylene oxide buoy and two polyethylene oxide chains dangling in the solution) is studied by ATR-FTIR. Some results for adsorbed bovine serum albumin (BSA) are also presented. The secondary structure of adsorbed IgG was quantified using second-derivative spectra for the input parameters of the curve-fitting analysis of the original spectra. The secondary structure of adsorbed IgG on a hydrophilic silica surface resembles that of IgG in solution (about 60% beta-sheet and almost no alpha-helix content). There is some loss in the helix content of BSA after adsorption on the hydrophilic surface, but this structural element is still the most important one in the adsorbed protein. The IR spectra of the adsorbed proteins on the hydrophobic silica surface can not be interpreted, probably because of a large contribution to the IR signal of water molecules that are exchanged against the proteins during adsorption. The presence of preadsorbed triblock-copolymers reduces the adsorbed amount and causes an effect on the adsorbed proteins similar to that exerted by ethylene glycol: a different type of beta-sheet structure in IgG and a more ordered alpha-helix structure in BSA are provoked. Copyright 1999 Academic Press.

Measurements of the wettability of protein-covered hydroxyapatite surfaces.

We developed a new method (dropping time method, DTM) to investigate the wettability of a surface of a protein layer adsorbed on glass plates in aqueous solution. However, the previous setup of DTM can only be utilized for optically transparent materials. In this study, we have extended the method to optically nontransparent materials such as hydroxyapatite plates. DTM is based on measuring the dropping time of a liquid film along a protein-covered surface when this surface is instantaneously vertically removed from the protein solution. The intensity of the reflected light beam depends on the presence of a liquid film on the surface. This allows to estimate the movement of the liquid film along the sorbent surface. Thus, the extended DTM can be used for determining the wettability of optically nontransparent solid plates. The adsorption behavior of four proteins (albumin, lysozyme, beta-lactoglobulin, ovalbumin) on a hydrophobic hydroxyapatite plate in water was studied by this method. When adsorbed from a protein solution of high concentration, the surfaces of adsorbed proteins, except ovalbumin, were fairly hydrophilic; this hydrophilicity was already attained at the initial stage of the adsorption process. The surface of ovalbumin on hydroxyapatite was more hydrophobic than those of the other proteins, and the hydrophilicity increased with the protein adsorption process. At low protein concentration, the hydrophilicity increased in the course of the adsorption process. The change in hydrophilicity with time depends on the kind of protein. Hen's egg lysozyme is more hydrophilic and the time to reach saturation is shorter than for the other proteins. The processes of increasing hydrophilicity of the surface of human serum albumin, beta-lactoglobulin and ovalbumin are similar. However, for beta-lactoglobulin hydrophobicity at adsorption saturation is stronger than for human serum albumin and ovalbumin. Thus, using DTM it is shown that the hydrophilicity of the surface of adsorbed protein on hydroxyapatite depends strongly on the kind of protein.

Interaction between fatty acid salts and elastin: kinetics, absorption equilibrium, and consequences for elasticity.

Elastin from bovine ligamentum nuchae is incubated in aqueous solutions of sodium salts of fatty acids (FAS). The FAS are laurate, myristate, and palmitate. Absorption of FAS in the elastin network is studied as a function of time, FAS concentration, and ionic strength. The consequences of this uptake for the elasticity of the elastin are studied by static and dynamic stress-strain measurements. Generally, distinction must be made between the initial time-dependent stage (I) and the final equilibrium stage (II). In I the initial rate of absorption follows a second-order binding mechanism, with the rate constant increasing with decreasing length of the FAS. In this regime, the elasticity modulus remains more or less unaffected. Especially in regime II the absorption of FAS is enhanced by a reduction in the cross-link density in the elastin network. This is ascribed to an osmotic pressure primarily caused by the concomitant uptake of low molecular weight ions in the elastin. The absorption equilibrium can be described by Langmuir theory. The absorption affinity increases with increasing hydrocarbon chain length of the FAS, indicating the contribution of hydrophobic interaction. Although the elasticity is not lost, the modulus is now reduced and a concomitant viscous component is developed.

Bovine serum albumin adsorption on titania surfaces and its relation to wettability aspects.

The adsorption of bovine serum albumin (BSA) from sodium chloride solution and Hanks' balanced salt solution (HBSS) onto TiO2-silicon surfaces is studied by reflectometry in stagnation point flow. The results are compared with those obtained by dynamic contact-angle (DCA) analysis of titanium substrates. The adsorption isotherms show that the adsorbed amount of protein always is lower in HBSS, that is, in the presence of calcium and phosphate ions. This may be related to the increase in surface hydrophilicity caused by these ions, as suggested by the authors in previous works. The rate of adsorption also is lower in HBSS solutions. Comparison of the initial adsorption rates with the rate of mass transfer to the surface reveals that in both solvents only a small fraction of the protein that arrives at the surface adsorbs onto it. Electrostatic and/or conformational effects can explain the energy barrier to adsorption. The DCA analysis of high concentration (4 mg/mL) protein solutions shows a strong reduction of the contact-angle hysteresis, both in HBSS and in NaCl solutions, which confirms that the immediate adsorption of the protein to the surface forms a stable, hydrophilic film.