Protein Adsorption Process Research Articles

Surface free energy and cell attachment onto ion-beam irradiated polymer surfaces

The paper reports evidence of the different cytocompatibility towards ion irradiated polymer surfaces. In particular, we studied the cell attachment, adhesion and spreading of normal human dermal fibroblast cells onto poly(hydroxymethylsiloxane) and poly(ethyleneterephthalate) surfaces modified by 50 keV Ar + beams. The cell response is discussed in connection with the radiation-induced changes of the polymers surface chemical structure and related surface free energy, investigated by means of X-ray photoelectron spectroscopy and static contact angle measurements. The biological response is interpreted in terms of the different modification trends of the Surface Free Energy components, and its relationship to the protein adsorption processes from culture medium or to the direct cell-surface interaction in a protein-free saline solution. The results point towards a critical role of the electron-donor character of the surfaces, which seems able to trigger the optimal cell response to the employed polymeric surfaces.

Two-Dimensional Crystallization of a Histidine-Tagged Protein on Monolayers of Fluidity-Enhanced Ni2+-Chelating Lipids

Protein two-dimensional (2D) crystallization on lipid monolayers is a powerful method for structure determination. This method has been extended using the specific and strong interaction between histidine residues (of an overexpressed protein) and Ni 2+ ions tethered at the headgroup of synthetic lipids. Understanding and then improving the process of adsorption and crystallization of proteins on a lipid monolayer are prerequisites for the production of large and well-ordered crystals of any soluble or membrane His-tagged proteins. These large high-quality arrays are necessary for structural studies at high resolution. We have investigated the steps of adsorption and 2D crystallization of His-HupR using three different lipids: (i) 2-(bis-carboxymethyl-amino)-6-[2-(1,3-di-O-oleyl-glyceroxy)-acetyl-amino] hexanoic acid nickel(II) (Ni-NTA-DOGA), which has been previously used, and two specifically designed Ni 2+ -chelating lipids, (ii) Ni-NTA-BB, which has two branched (B) alkyl chains and (iii) Ni-NTA-BF, a nonsymmetrical lipid with one branched (B) and one fluorinated (F) chain. These three lipids, when spread at the air-water interface, exhibit various fluidity properties. The adsorption and crystallization process have been monitored in situ and in real time using a variety of complementary techniques such as ellipsometry, shear rigidity measurements of the monolayer, and Brewster angle microscopy, and we have also developed X-ray reflectivity analysis to investigate the evolution of the electron density profile of the lipid-protein monolayer. Electron microscopy observations of the protein-lipid layers were also performed. We have found that the fluidity of the lipid monolayer has a marked influence on the rates of protein adsorption and crystallization of His-HupR. When Ni-NTA-BB is used to form the monolayer, it accelerates the process of protein adsorption and the protein crystallization is three times faster than when Ni-NTA-DOGA is used.

Protein Adsorption on Model Surfaces with Controlled Nanotopography and Chemistry

To evaluate the influence of substratum surface characteristics on protein adsorption processes, we have investigated the adsorption (adsorbed amount, supramolecular organization) of collagen on model substrata exhibiting controlled topography and surface chemistry. Substrata were prepared in two steps: (i) gold deposition onto silicon wafers (smooth substrata) and onto a support with nanoscale protrusions created by colloidal lithography (rough substrata); (ii) functionalization with CH3 (hydrophobic) and OH (hydrophilic) groups, using alkanethiol self-assembly. Atomic force microscopy (AFM) images were recorded under water, prior to and after collagen adsorption, and the images were analyzed quantitatively using two independent approaches. On smooth substrata, collagen formed a ∼6 nm thick, homogeneous layer with low roughness on hydrophilic surfaces, and a ∼20 nm thick layer exhibiting elongated aggregated structures on hydrophobic surfaces. Film thickness measurements (AFM) together with X-ray photoelectron spectroscopy (XPS) revealed larger adsorbed amounts on hydrophobic surfaces compared to hydrophilic ones. On rough substrata, the adsorbed amounts were similar to those found on smooth substrata; however, the collagen molecules no longer formed aggregated structures on the hydrophobic surfaces. It is concluded that while the adsorbed amount is only affected by the surface chemistry, the supramolecular organization of the adsorbed layer is controlled both by surface chemistry and topography. The approach presented here will have great value in biophysics for investigating bioadsorption and bioadhesion processes on substrata of defined surface properties.

High-resolution chiral separation using microfluidics-based membrane chromatography

A plastic microfluidic system, containing porous poly(vinylidene fluoride) (PVDF) membranes adsorbed with bovine serum albumin (BSA), is demonstrated for high resolution chiral separation of racemic tryptophan and thiopental mixtures. Microfluidic networks on poly(dimethylsiloxane) (PDMS) substrates are fabricated by capillary molding technique. This miniaturized chiral separation system consists of two layers of PVDF membranes which are sandwiched between two PDMS slabs containing microchannels facing the membranes. On-line adsorption of BSA onto the membranes is employed for the preparation of chiral stationary phase and the evaluation of solution conditions in an effort to achieve maximum protein adsorption. Variations in the mobile phase conditions, including solution pH and ammonium sulfate concentration, are studied for their effects on chiral separation. Based on the large surface area to volume ratio of porous membrane media, adsorbed BSA onto the PVDF membranes enables high resolution separation of racemic mixtures with sample consumption of sub-nanogram or less in the integrated microfluidic networks. In addition, the membrane pore diameter in the submicron range eliminates the constraints of diffusional mass-transfer resistance during protein adsorption and chiral chromatographic processes.

Mechanistic aspects of protein/material interactions probed by atomic force microscopy

Physicochemical studies on the mechanisms of protein adsorption onto solid material surfaces have been extensively performed so far, mainly based on the analysis of factors such as the equilibrium adsorbed amount (adsorption isotherms), time-dependent change of adsorbed amount (adsorption kinetics), and conformational change of adsorbed protein. However, direct understanding of the strength of the molecular interaction between protein and the material surface has not been established yet. For this issue, the force measurement techniques of an atomic force microscope (AFM) using a protein-modified probe tip are recently becoming powerful tools to analyze the actual interaction forces between protein and material surfaces. In this mini review, we discuss the characteristics and interpretation of the AFM force-versus-distance curves ( f– d curves) obtained with the protein-modified probe tip, and the relationship between the forces measured from the f– d curves and the driving forces in the natural process of protein adsorption. Relative degrees of each of the following contributions which determine the character of protein adsorption are clarified: (1) the intrinsic protein/material forces mediated by solvent, (2) the thermodynamic stability of protein/material adhesion interface, and (3) diffusion force of protein molecules. Within these driving forces, the latter two in particular are confirmed to play essential roles in determining the character of protein adsorption, based on the profiles of f– d curves.

Microcalorimetric studies on the interaction mechanism between proteins and hydrophobic solid surfaces in hydrophobic interaction chromatography: effects of salts, hydrophobicity of the sorbent, and structure of the protein.

This study examines the effects of different salts as well as the influence of the relative hydrophobicities of different sorbents on the adsorption processes of proteins in hydrophobic interaction chromatography (HIC). Comparative data acquired by the equilibrium binding analysis and by isothermal titration microcalorimetry (ITC) are presented. In particular, thermodynamic parameters, including the enthalpy changes, related to the interactions between several globular proteins and various Toyopearl 650 M sorbents under solvent conditions containing either 2.0 M ammonium sulfate or 2.0 M sodium sulfate at pH 7.0 and 298.15 K have been evaluated in terms of the molecular properties of these systems. The results reveal that the dependence of the free energy change, deltaGads, for protein adsorption to HIC sorbents on the salt composition can be mainly attributed to the enthalpy changes associated with protein and sorbent dehydration and hydrophobic interactions. Differences in binding mechanisms between the n-butyl- and phenyl-HIC sorbents were evident. In the latter case, the participation of pi-pi hydrophobic interactions leads to significant differences in the associated enthalpy and entropy changes. Furthermore, an increase in the hydrophobicity of either the sorbent or the protein resulted in more negative values for the free energy change, which arose mostly from dehydration processes. Entropic effects favoring HIC adsorption increased with an increase in the exposed nonpolar surface area of the protein. Consequently, an increased contribution from the entropy change to the respective change in free energy occurs when HIC sorbents or proteins of higher hydrophobicity are employed, with these larger entropy changes consistent with a change in the interaction mechanism from a binding event dominated by adsorption to a partitioning-like process. Data extracted from the ITC measurements also provided insight into the interaction mechanisms that occur between proteins and hydrophobic solid surfaces, yielding information that can be applied to the HIC purification of proteins according to the concept of critical hydrophobicity of the system and its thermodynamic consequences.

Study of human serum albumin-TiO 2 nanocrystalline electrodes interaction by impedance electrochemical spectroscopy

The adsorption of human serum albumin (HSA) onto nanocrystalline TiO 2 electrodes was studied by electrochemical impedance spectroscopy (EIS) in function of pH and electrode potential. The characterization and physico-chemical properties of the TiO 2 electrode were investigated by scanning electron microscopy (SEM), UV-photoelectron spectroscopy (UPS), cyclic voltammetry and capacitance measurements. The impedance response of the particulate TiO 2 electrode/protein interface was fitted using an equivalent circuit model to describe the adsorption process. The adsorbed protein layer, which is formed as soon as the protein is injected into the solution and becomes in contact with the electrode, was investigated as a function of electrode potential and solution pH. The measurements were performed under pseudo-steady-state and steady-state conditions, which gave information about the different states of the system. With the pseudo-steady state measurements, it was possible to determine two rate constants of the protein adsorption process, which correspond to two different states of the protein. The shortest one was associated with the first contact between the protein and the substrate and the second relaxation time, with the protein suffering an structural rearrangement due to the interaction with the TiO 2 electrode. It was detected that at sufficiently long times (approx. 1 h, where the system was under steady state conditions), a quasi-reversible protein adsorption mechanism was established. The measurements performed as a function of frequency under steady-state conditions, an equivalent circuit with a Warburg element gave the better fitting to data taken at −0.585 V closer to the oxide flat band potential and it was associated with protein diffusion. Experimental results obtained at only one frequency as a function of potential could be fitted to a model that takes into account non-specific and probable specific protein adsorption, which renders to be potential- and pH-dependent. Low capacity values were obtained in the whole potential range, which were measured in the presence and in the absence of the protein layer. The capacity dependence on potential and pH were associated with the generation of surface states on TiO 2. A surface state concentration of 4.1×10 18 cm −2 was obtained by relating the parallel capacitance with oxide surface states arising from the protein–oxide interaction.

Electrokinetic surface characterization of biomedical polymers — a survey

The application of streaming potential measurements in support of the development of blood compatible polymers is reviewed. Current knowledge about the relevance of interfacial charge of materials with respect to thrombogenicity of and coagulation activation by medical devices is discussed. Potentialities of streaming potential measurements for the characterization of biomedical polymers comprise the distinction between interfacial charge formation processes, the derivation of acid–base characteristics of surface functions, and the estimation of the relative contribution of known molecular surface sites to the interfacial charge density. Frequently, polymer surfaces consist of different types of surface sites. Beyond sites with Bronsted-acidic or -basic character the preferential adsorption of electrolyte ions onto indifferent surface sites may substantially contribute to the interfacial charge. Examples are given for the detection of acidic and basic properties of hemodialysis membranes of different shape made of poly(sulfone)/poly(vinylpyrrolidone) and cellulose derivates. For partially amine-modified cellulose flat membranes the percentage of basic surface sites is derived from the shift of the isoelectric point. Further, the use of streaming potential measurements is demonstrated in the optimization of surface modifications of cardiovascular implant materials by low pressure plasma treatments. The introduction of basic groups into the poly(tetrafluoroethylene) implant surfaces by ammonia plasma treatments manifests itself in the shift of the isoelectric point. A correlation between hydrophobicity and charge formation of the polymer–solution interface by preferential ion adsorption is confirmed for polymers without dissociating functions. This is based on the comparison of water contact angles for a series of poly(styrene) derivates of different hydrophobicity and the maximum zeta potential of these polymer films in potassium hydroxide solutions. Advanced applications of streaming potential measurements for the characterization of biomedical polymers are comprise the in situ characterization of protein adsorption processes onto polymer solids. This approach is demonstrated for the adsorption of the blood plasma proteins albumin and fibrinogen onto a fluorocarbon polymer substrate. The potentialities of a recently developed microslit electrokinetic setup are illustrated which permits to study simultaneously zeta potential and surface conductivity of flat polymer–liquid interfaces. Data gained by the microslit electrokinetic setup reveal the dramatic increase of the surface conductivity of a fluorocarbon polymer–solution interface caused by fibrinogen adsorption.

Wettability of proteins adsorbed on silica glasses treated with fluorosurfactants

Generally, the apolar/polar surface is probed by water-wetting, which is measured using a method such as the sessile liquid drop method. However, when one tries to measure the wetting of a surface where biological macromolecules are adsorbed, there is the problem of a change in conformation due to drying the surface; hence, using this method in situ information cannot be obtained. We have developed a new method that can be used to measure the wettability of the adsorbed protein surface without drying. This method, the dropping time method, which is based on measuring the dropping time of a film of liquid along a protein-covered surface when this surface is instantaneously vertically removed from the protein solution. The adsorption behavior of four proteins (albumin, lysozyme, β-lactoglobulin, ovalbumin) on the surface of silica glass that has been treated with various fluorosurfactants is studied using this method. At a high concentration of protein, the surfaces of adsorbed proteins of any kind are fairly hydrophilic on glass treated with all fluorosurfactants. At a lower concentration of protein, the hydrophilicity of the protein layer depends on the kind of fluorosurfactant and also on the protein adsorption process. The apolar glass surface becomes more hydrophilic with increasing dipping time in the protein solution. On the other hand, the hydrophilic glass surface shows a complex change in the hydrophilicity with elapsed time after dipping it into a solution of albumin or lysozyme, i.e., the hydrophilicity decreases in the early stage of the adsorption and then increases with proceeding adsorption.

Adsorption of β‐Lactoglobulin variants A and B to the air–water interface

Summary The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of β‐lactoglobulin (β‐lg) differ by only two amino acids (Asp‐64, Val‐118 in A, and Gly‐64, Ala‐118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of β‐lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer : monomer equilibrium in the bulk solution.

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, β–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, β–lactoglobulin and ovalbumin are similar. However, for β–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.

High-performance ion-exchange chromatography and adsorption of plasma proteins

Resolution and recovery are primary concerns in protein chromatography. Separations are often by size, ion exchange, or hydrophobic–hydrophilic properties of the support, eluent and protein. Adsorption of a protein to a synthetic surface plays an essential role in this complex process. In this study, we examined the adsorption properties of three representative plasma proteins (albumin, fibrinogen, and immunoglobulin G) on nonporous column materials containing either quaternary amine or sulfopropyl functional groups. The adsorption properties were studied at 37°C and pH 7.4. Salt gradients were used to examine the adsorption/desorption properties of each of the proteins on each type of surface. The salt concentrations at desorption were measured and compared to the protein isoelectric points. In addition, we examined protein recoveries as a function of desorption time. Our results suggest that protein recoveries depend not only on the protein, eluent and surface, but also the residence time and overall charge concentration during the initial adsorption process. Finally, we correlated the number of charge sites on a molecule with the width of a chromatographic band at half height. The data produced as a result of this study may be used to determine the actual unfolding time of a protein, given a certain set of conditions. The data may also help in understanding the chemistry and dynamics of the protein adsorption processes in ion-exchange chromatography as well as provide key structural information about the proteins.

Adsorption of proteins onto silica glass treated with fluorosurfactants and its wettability

we developed a new method which can measure wetting of the surface of adsorbed protein layers without drying. At a higher protein concentration, the surface of adsorbed protein layers on glass treated with fluorosurfactants is fairly hydrophilic. At a lower protein concentration, the surface hydrophilicity of the adsorbed protein layer depends on the kind of fluorosurfactants that are utilized for glass-treatments and also the protein adsorption process.

Protein-stabilized emulsions

The protein film is an essential element in protein-stabilized emulsions. Significant advances have been made in our understanding of the process of protein adsorption. Novel techniques and concepts have been applied to the determination of the structure and properties of the adsorbed layer.

Protein adsorption on solid surfaces

The research field of protein adsorption on surfaces appears to be as popular as ever. In the past year, several hundred published papers tackled problems ranging from fundamental aspects of protein surface interactions to applied problems of surface blood compatibility and protein surface immobilization. Although some parts of the protein adsorption process, such as kinetics and equilibrium interactions, can be accurately predicted, other aspects, such as the extent and the rate of protein conformational change, are still somewhat uncertain. The whole field is ripe for a comprehensive theory on protein adsorption.

Two-Dimensional Crystallization of Streptavidin Studied by Quantitative Brewster Angle Microscopy

Brewster angle microscopy (BAM) is extended to probe protein adsorption and aggregation processes at interfaces quantitatively. Since no p-polarized light is reflected from the plain air−water inte...

High-performance liquid chromatography of amino acids, peptides and proteins CXXXIX. Impact of operating parameters in large-scale chromatography of proteins

Large-scale chromatography has been playing an important role in downstream treatment processing in biotechnology. In order to improve the productivity, the throughput of the chromatographic equipment was often increased by increasing the flow-rate and/or by increasing the column sample loading. This paper reports the results of a study on the impact of these and other operating parameters in affinity and ion-exchange chromatographic columns when used for protein purification. A sectional model was developed to predict protein adsorption processes in a packed column. The formulations of this mathematical model are presented in the Appendix. The present study was carried out with computer simulation based on this model and using data obtained from laboratory-scale columns. This model can simulate both the adsorption and washing stages of the protein purification process for both porous and non-porous particles. The effects of changing operating parameters were simulated and contour plots were generated for the easy identification of these effects. It was shown that both flow-rate and column loading can have a considerable impact on the processing rate and the yield of the column. As for the column capacity utilization, the impact of changing flow-rate is not significant at column loading of less than 80% in the test case. It was suggest that the present investigation provides a systematic predictive strategy which will greatly reduce the need for expensive, labour-intensive and time-consuming experimental work during process scale-up.

High-performance liquid chromatography of amino acids, peptides and proteins CXXVI. Modelling of protein adsorption with non-porous and porous particles in a finite bath

Analytical solutions for a mathematical model describing dynamic adsorption processes of proteins onto non-porous adsorbent particles in a finite bath are presented. The model, based on the Langmuir adsorption isotherm, has been applied to experimental data obtained with affinity and ion-exchange adsorbents. The external film mass transfer resistance, as well as the rate of surface interaction between proteins and adsorbents, have been taken into account. The model has been extended to the case of adsorption onto porous particles by employing a linear driving force approximation for describing mass transfer in the pore fluid. This approach enables the derivation of an effective overall liquid phase mass transfer coefficient, permitting subsequent adaptation of the analytical solutions developed for non-porous particles. The evaluation of the effective liquid phase mass transfer coefficients is also described. Examples of a comparison between predicted and experimental dynamic adsorption curves for both dye-affinity and ion-exchange systems are presented. The application of the model for predicting the optimum operating conditions is discussed.

The role of protein charge in protein-lipid interactions. pH-Dependent changes of the electrophoretic mobility of liposomes through adsorption of water-soluble, globular proteins

The role of electrostatics in the adsorption process of proteins to preformed negatively-charged (phosphatidylcholine/phosphatidylglycerol) and neutral (phosphatidylcholine) liposomes was studied. The interaction was monitored at low ionic strength for a set of model proteins as a function of pH. The adsorption behavior of trypsin inhibitor (pI = 4.6), myoglobin (pI = 7.4), ribonuclease (pI = 9.6), and lysozyme (pI = 10.7) with preformed liposomes was investigated, along with changes in the electrophoretic mobility of liposomes through the adsorption of charged proteins. Mean protein charge was determined by acid/base titration. Significant adsorption of the proteins to negatively-charged liposomes was only found at pH values where the number of positive charge moieties exceeds the number of negative charge moieties on the protein by at least three charge units. Negligible adsorption to liposomes composed of zwitterionic lipids was observed in the pH range tested (4-9). The absolute value of the electrophoretic mobilities of negatively-charged, empty liposomes decreased after adsorption of positively-charged proteins. With increasing protein to phospholipid ratio, the drop in the electrophoretic mobility leveled off and reached a plateau; protein adsorption profiles showed a similar shape. Analysis of the data demonstrated that neutralization of the liposome charge due to the adsorption of the positively-charged proteins is the controlling factor in their adsorption. The plateau level reached depended on the type of protein and the pH of the incubation medium. This pH dependency could be ascribed to the mean positive charge of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarimetric optical-fibre sensor for biochemical measurements

The use of an optical-fibre polarimeter as a chemical sensor is demonstrated. The compound to be detected is allowed to adsorb onto a decladded 5 cm length of the fibre. The fibre is polarization maintaining with an elliptical fibre core and a D-shaped geometry. The overall retardation stability of this fibre polarimeter is ≈ 0.5 × 2π rad m −1 K −1. With this sensor adsorption processes of proteins can be followed on-line. The resulting relative phase retardations caused by the growth of a monolayer of antibodies (αhCG, αhSA) are ≅ 0.25 × 2π. For the much smaller protein hSA, this value is ≅ 0.1 × 2π.