The influence of electrostatic forces on protein adsorption

In this paper we investigate the importance of electrostatic double layer forces on the adsorption of human serum albumin by UV-ozone modified polystyrene. Electrostatic forces were measured between oxidized polystyrene surfaces and gold-coated atomic force microscope (AFM) probes in phosphate buffered saline (PBS) solutions. The variation in surface potential with surface oxygen concentration was measured. The observed force characteristics were found to agree with the theory of electrical double layer interaction under the assumption of constant potential. Chemically patterned polystyrene surfaces with adjacent 5 microm x 5 microm polar and non-polar domains have been studied by AFM before and after human serum albumin adsorption. A topographically flat surface is observed before protein adsorption indicating that the patterning process does not physically modify the surface. Friction force imaging clearly reveals the oxidation pattern with the polar domains being characterised by a higher relative friction compared to the non-polar, untreated domains. Far-field force imaging was performed on the patterned surface using the interleave AFM mode to produce two-dimensional plots of the distribution of electrostatic double-layer forces formed when the patterned polystyrene surfaces is immersed in PBS. Imaging of protein layers adsorbed onto the chemically patterned surfaces indicates that the electrostatic double-layer force was a significant driving force in the interaction of protein with the surface.     

The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes

In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of ～10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar membranes and charged proteins or biopolymers for encapsulation and delivery applications.     

The influence of temperature on the adsorption of mellitic acid onto goethite

The adsorption of mellitic acid (benzene-1,2,3,4,5,6-hexacarboxylic acid) onto goethite was investigated at five temperatures between 10 and 70 degrees C. Mellitic acid adsorption increased with increasing temperature below pH 7.5, but at higher pH the effect of increasing temperature was to reduce the amount adsorbed. Potentiometric titrations were conducted and adsorption isotherms were measured over the same temperature range, and the data obtained were used in conjunction with adsorption edge data to develop an Extended Constant Capacitance Surface Complexation Model of mellitic acid adsorption. A single set of reactions was used to model the adsorption for the three different experiment types at the five temperatures studied. The adsorption reactions proposed for mellitate ion (L(6-)) adsorption at the goethite surface (SOH) involved the formation of two outer-sphere complexes: SOH + L(6-) + 3H+ <==> [(SOH2)+ (LH2)(4-)]3-, 2SOH + L(6-) + 2H+ <==> [(SOH2)2(2+) (L)(6-)]4-. This mechanism is consistent with recent ATR-FTIR spectroscopic measurements of the mellitate-goethite system. Thermodynamic parameters calculated from the temperature dependence of the equilibrium constants for these reactions indicate that the adsorption of mellitic acid onto goethite is accompanied by a large entropy increase.     

Interactions of a hydrophobically modified polycation with zwitterionic lipid membranes

The interactions between synthetic polycations and phospholipid bilayers play an important role in some biophysical applications such as gene delivery or antibacterial usage. Despite extensive investigation into the nature of these interactions, their physical and molecular bases remain poorly understood. In this Article, we present the results of our studies on the impact of a hydrophobically modified strong polycation on the properties of a zwitterionic bilayer used as a model of the mammalian cellular membrane. The study was carried out using a set of complementary experimental methods and molecular dynamic (MD) simulations. A new polycation, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and its interactions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were examined using dynamic light scattering (DLS), zeta potential measurements, and cryo-transmission electron microscopy (cryo-TEM). Our results have shown that polymer 3 can efficiently associate with and insert into the POPC membrane. However, it does not change its lamellar structure, as was demonstrated by cryo-TEM. The influence of polymer 3 on the membrane functionality was studied by leakage experiments applying a fluorescence dye (calcein) encapsulated in the phospholipid vesicles. The MD simulations of model systems reveal that polymer 3 promotes formation of hydrophilic pores in the membrane, thus increasing considerably its permeability.     

Excess fibrinogen adsorption to monolayers of mixed lipids

Adsorption of fibrinogen to the monolayers of mixed lipids, dipalmitoyl phosphatidyl choline (DPPC) and eicosylamine (EA) was measured at a surface pressure of 20 mN/m by an in situ surface plasmon resonance technique. Pressure–area isotherms of DPPC + EA mixtures on water and buffer subphases indicated good lipid miscibility and some contraction of the monolayers at intermediate and higher surface pressures. Surface electric potential of the DPPC + EA monolayers showed excess values for intermediate DPPC:EA ratios. Fibrinogen adsorption and its adsorption rates from a dilute solution (0.03 mg/ml) were proportional to the fraction of EA in the monolayer indicating that protein binding was primarily driven by electrostatic interactions between positive EA charges in the monolayer and a net negative protein charge. At a higher protein concentration (0.06 mg/ml) both the fibrinogen adsorbed amount and its maximum adsorption rate showed excess values relative to the pure EA for 1:1, 2:1 and 3:1 DPPC + EA monolayers. This excess adsorption could be explained, in part, by the contraction of the monolayers with intermediate DPPC:EA ratios which resulted in an excess surface electric potential.     

The effect of cholesterol on the adsorption of phenyltin compounds onto phosphatidylcholine and sphingomyelin liposome membranes

Organometallic compounds are widely spread in the human environment sometimes, causing a substantial health risk. Their amphiphilic character enables them to intercalate and penetrate cell membranes, potentially affecting various vital cell functions. Compound adsorption onto the membrane depends on the compound properties, as well as on the membrane composition and state. When adsorbing onto the lipidic surface, phenyltins localize at areas where lipid bilayer organization is compatible with compound spatial requirements. The lipid bilayer is a dynamic and laterally nonuniform structure with complex local and global architecture correlated with a variety of cell functions. The selective binding of a toxic compound to selected membrane areas may, therefore, interfere with some types of cellular process. We present experimental results concerning phenyltin adsorption onto the lipid bilayer surface measured with the fluorescent probe fluorescein‐PE. Model lipid bilayers were formed from lipid mixtures mimicking various plasma membrane regions. The adsorption of Ph₃SnCl and P₂SnCl₂ onto the phosphatidylcholine–cholesterol bilayer was qualitatively different from sphingomyelin–cholesterol. The results presented indicate that phenyltins are likely to accumulate in areas containing phosphatidylcholine, outside of lipid rafts. Copyright © 2004 John Wiley & Sons, Ltd.     

A study of the adsorption of bile salts onto model lecithin membranes

Bile salts play a central role in the promotion of cytotoxicity or cytoprotection. In this study, we examined the interaction of different bile salts with egg lecithin vesicles using 31P NMR spectroscopy. The effects of taurochenodeoxycholate (TCDC or 3alpha,7alpha,-dihydroxy-5beta-cholanoyl taurine, of tauroursodeoxycholate (TUDC) or 3alpha,7beta,-dihydroxy-5beta-cholanoyl taurine) and of taurobetamuricholate (TbetaMC or 3alpha,6beta,7beta,-trihydroxy-5beta-cholanoyl taurine), at various bile salt/lecithin ratios, were evaluated. From the percent 31P present in vesicles, the micellar capacity of bile salts to dissolve lecithin was determined. TCDC was incorporated into vesicles for bile salt/lecithin molar ratios lower than 0.62 while for TUDC and TbetaMC, the critical ratios were 0.94 and 1.1, respectively. The 31P chemical shift change was markedly larger with TCDC than that found with TUDC and TbetaMC. In order to specify the low interactions observed between hydrophilic bile salts and lecithin, we determined the intermixed micellar/vesicular bile salt concentrations (IMVC) of bile salt/lecithin solutions using rapid ultrafiltration-centrifugation for TUDC and lecithin solubility measurements for TUDC, TbetaMC and TCDC. The low IMVC obtained indicate that even hydrophilic bile salts were bound mostly to the mixed aggregates. In conclusion, the low disturbance in the arrangement of lecithin induced by TUDC and TbetaMC appears to be due to the interfacial location of these bile salts. TCDC (7alpha OH) penetrates more deeply in the membrane than the 7beta hydroxylated bile salts that may partly explain the distinct damaging effects of these bile salts.     

Analysis of inorganic anions by electrostatic ion chromatography using zwitterionic/cationic mixed micelles as the stationary phase

The inability to separate fluoride, phosphate and sulfate by electrostatic ion chromatography (EIC) was overcome by using an ODS silica column coated with mixed zwitterionic-cationic surfactants as the stationary phase. The best results were obtained using the zwitterionic surfactant, 3-(N,N-dimethylmyristylammonium)-propanesulfonate (C19H41NO3S), and the cationic surfactant, myristyltrimethylammonium, CH3(CH2)13N+(CH3)3, in a 10:1 molar ratio in the column coating solution. With a dilute solution of sodium tetraborate as the eluent the model analyte anions were completely separated in the following elution order: F, HPO42-, SO42-, Cl-, NO2-, Br-, NO3-. The very early elution of phosphate and sulfate is most unusual and is unique to this system. Detection limits better than 1.1 x 10(-4) mM and linear calibration plots up to 7.0 mM were obtained with a suppressed conductivity system.     

Analysis of inorganic anions by electrostatic ion chromatography using zwitterionic/cationic mixed micelles as the stationary phase

The inability to separate fluoride, phosphate and sulfate by electrostatic ion chromatography (EIC) was overcome by using an ODS silica column coated with mixed zwitterionic-cationic surfactants as the stationary phase. The best results were obtained using the zwitterionic surfactant, 3-(N,N-dimethylmyristylammonium)-propanesulfonate (C₁₉H₄₁NO₃S), and the cationic surfactant, myristyltrimethylammonium, CH₃(CH₂)₁₃N⁺(CH₃)₃, in a 10:1 molar ratio in the column coating solution. With a dilute solution of sodium tetraborate as the eluent the model analyte anions were completely separated in the following elution order: F^–, HPO₄ ^2–, SO₄ ^2–, Cl^–, NO₂ ^–, Br^–, NO₃ ^–. The very early elution of phosphate and sulfate is most unusual and is unique to this system. Detection limits better than 1.1 × 10^–4 mM and linear calibration plots up to 7.0 mM were obtained with a suppressed conductivity system.     

The influence of anisotropic membrane inclusions on curvature elastic properties of lipid membranes

A membrane inclusion can be defined as a complex of protein or peptide and the surrounding significantly distorted lipids. We suggest a theoretical model that allows for the estimation of the influence of membrane inclusions on the curvature elastic properties of lipid membranes. Our treatment includes anisotropic inclusions whose energetics depends on their in-plane orientation within the membrane. On the basis of continuum elasticity theory, we calculate the inclusion-membrane interaction energy that reflects the protein or peptide-induced short-ranged elastic deformation of a bent lipid layer. A numerical estimate of the corresponding interaction constants indicates the ability of inclusions to sense membrane bending and to accumulate at regions of favorable curvature, matching the effective shape of the inclusions. Strongly anisotropic inclusions interact favorably with lipid layers that adopt saddlelike curvature; such structures may be stabilized energetically. We explore this possibility for the case of vesicle budding where we consider a shape sequence of closed, axisymmetric vesicles that form a (saddle-curvature adopting) membrane neck. It appears that not only isotropic but also strongly anisotropic inclusions can significantly contribute to the budding energetics, a finding that we discuss in terms of recent experiments.     

Large influence of cholesterol on solute partitioning into lipid membranes

Cholesterol plays an important role in maintaining the correct fluidity and rigidity of the plasma membrane of all animal cells, and hence, it is present in concentrations ranging from 20 to 50 mol %. Whereas the effect of cholesterol on such mechanical properties has been studied exhaustively over the last decades, the structural basis for cholesterol effects on membrane permeability is still unclear. Here we apply systematic molecular dynamics simulations to study the partitioning of solutes between water and membranes. We derive potentials of mean force for six different solutes permeating across 20 different lipid membranes containing one out of four types of phospholipids plus a cholesterol content varying from 0 to 50 mol %. Surprisingly, cholesterol decreases solute partitioning into the lipid tail region of the membranes much more strongly than expected from experiments on macroscopic membranes, suggesting that a laterally inhomogeneous cholesterol concentration and permeability may be required to explain experimental findings. The simulations indicate that the cost of breaking van der Waals interactions between the lipid tails of cholesterol-containing membranes account for the reduced partitioning rather than the surface area per phospholipid, which has been frequently suggested as a determinant for solute partitioning. The simulations further show that the partitioning is more sensitive to cholesterol (i) for larger solutes, (ii) in membranes with saturated as compared to membranes with unsaturated lipid tails, and (iii) in membranes with smaller lipid head groups.     

Properties of mixed lipid monolayers assembled on hydrophobic surfaces through vesicle adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.     

Modeling the influence of adsorbed DNA on the lateral pressure and tilt transition of a zwitterionic lipid monolayer

Certain lipid monolayers at the air-water interface undergo a second-order transition from a tilted to an untilted liquid-crystalline state of their lipid hydrocarbon chains at sufficiently large lateral pressure. Recent experimental observations demonstrate that in the presence of divalent cations DNA adsorbs onto a zwitterionic lipid monolayer and decreases the tilt transition pressure. Lowering of the tilt transition pressure indicates that the DNA condenses the lipid monolayer laterally. To rationalize this finding we analyze a theoretical model that combines a phenomenological Landau approach with an extension of the Poisson-Boltzmann model to zwitterionic lipids. Based on numerical calculations of the mean-field electrostatic free energy of a zwitterionic lipid monolayer-DNA complex in the presence of divalent cations, we analyze the thermodynamic equilibrium of DNA adsorption. We find that adsorbed DNA induces a 10% reduction of the electrostatic contribution to the lateral pressure exerted by the monolayer. This result implies a small but notable decrease in the tilt transition pressure. Additional mechanisms due to ion-ion correlations and headgroup reorientations are likely to further enhance this decrease.     

Electromagnetic-field-induced oscillations of the lipid domain structures in the mixed membranes.

The effect of external electromagnetic field (EMF) on a percolation structure formed during phase separation in the mixed phospholipid membranes was studied by computer simulation. Decay of the percolation structure under electromagnetic radiation was detected. It was shown that oscillation regime can be realized in this system: periodic alternation of formation and decay of the percolation cluster was observed under 10 kHz EMF. The decay of the lipid domain structure in the EMF results from anomalous increase of the permittivity of the continuous fluid lipid phase in the percolation threshold region. It is proposed that detected EMF effect can influence the signal and transport processes associated with percolation properties of biomembranes.     

Immunosensor interface based on physical and chemical immunoglobulin G adsorption onto mixed self-assembled monolayers

An immunosensor interface based on mixed hydrophobic self-assembled monolayers (SAMs) of methyl and carboxylic acid terminated thiols with covalently attached human Immunoglobulin G (hIgG), is investigated. The densely packed and organised SAMs were characterised by contact angle measurements and cyclic voltammetry. The effect of the non-ionic surfactant, Tween 20, in preventing nonspecific adsorption is addressed by ellipsometry during physical and covalent hIgG immobilization on pure and mixed SAMs, respectively. It is clearly demonstrated that nonspecific adsorption due to hydrophobic interactions of hIgG on methyl ended groups is totally inhibited, whereas electrostatic/hydrogen bonding interactions with the exposed carboxylic groups prevail in the presence of surfactant. Results of ellipsometry and Atomic Force Microscopy, reveal that the surface concentration of covalently immobilized hIgG is determined by the ratio of COOH / CH(3)-terminated thiols in SAM forming solution. Moreover, the ellipsometric data demonstrates that the ratio of bound anti-hIgG / hIgG depends on the density of hIgG on the surface and that the highest ratio is close to three. We also report the selectivity and high sensitivity achieved by chronoamperometry in the detection of adsorbed hIgG and the reaction with its antibody.     

Fabrication of alternating polycation and albumin multilayer coating onto stainless steel by electrostatic layer-by-layer adsorption

Multilayer films consisting of polyethylenimine (PEI) and albumin were successfully prepared on biomedical 316L stainless steel surface via electrostatic self-assembly of the PEI and albumin. The process of electrostatic self-assembly of PEI/albumin was monitored by125I radiolabeling, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The EIS data revealed that the multilayer coating was stable in Tris-HCl (pH 7.35) buffer solution for 21 days. 125I radiolabeling experiments indicated that less than 10% albumin was eluted by PBS in 45 days. Static platelet adhesion experiments indicated that the PEI/albumin deposited on stainless steel could resist platelet adhesion effectively. Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices.     

A theoretical study of micro-domain formation in mixed lipid membranes

The thermodynamic stability of micro-clusters in a membrane built-up by charged and uncharged lipid molecules is discussed. A simple variational function is proposed in order to describe the essential structure of such lipid domains. Solvent-screened electrostatic repulsion between the lipid ionic head groups, short-range forces between the lipid hydrophobic taisl and entropic effects are taken into account. The stability conditions as well as the composition and the size of the lipid micro-domains are calculated and expressed as a function of molecular parameters for the membrane and its environment (for example, short-range forces, surface charge density of the lipid bilayer, ion concentration of the electrolyte solution in contact with the lipid membrane and temperature). As an application, the effect of micro-domain formation on the number of adsorbed ions on a charged lipid membrane has been calculated.     

On the theory of polyelectrolyte adsorption : The effect on adsorption behavior of the electrostatic contribution to the adsorption free energy

A theory has been developed for the adsorption of polyelectrolytes on charged interfaces from an aqueous salt solution. This adsorption is determined by the electrical charge density of the polyelectrolyte, the adsorption energy, the salt concentration, the molecular weight, solubility, flexibility, and concentration of polymer. The theory relates these parameters to the properties of the adsorbed polymer layer, i.e., the amount of polymer adsorbed, the fraction of the adsorbent interface covered, the fraction of the segments actually adsorbed on the interface versus the fraction of the segments in the dangling loops, the final surface charge density, and the thickness of the adsorbed layer. As polyelectrolyte adsorption should resemble nonionic polymer adsorption at high ionic strength of the solution or low charge density on the polymer, this work is an extension of the nonionic polymer adsorption theory to polyelectrolyte adsorption. The following effects are taken into account: (a) the conformational change upon adsorption of a coil in solution into a sequence of adsorbed trains interconnected by loops dangling in solution; (b) the interactions of the adsorbed trains with the interface and with each other; (c) the interaction of the dangling loops with the solvent; (d) the change in surface charge density of the adsorbent due to adsorption of charged trains and the accompanying changes in the electrical double layer which contains “small” ions as well as charged loops; (e) the (induced) dipole interaction of the adsorbed trains with the charged adsorbent interface. The theory is worked out for low potentials (Debye—Hückel approximation); in Appendix B an outline of a more complete treatment is given. The predicted adsorption isotherms have the experimentally observed high-affinity character. A relation between the adsorption energy, the surface charge density on the adsorbent, the degree of dissociation of the polymer, and the salt concentration predicts the conditions under which no adsorption will occur. For adsorbent and polymer carrying the same type of charge (both positive or both negative) the adsorption is predicted to decrease with increased charge density on polymer or adsorbent and to increase with salt concentration. If adsorbent and polymer carry different type charges, the adsorption as a function of the degree of dissociation, α, goes through a maximum at a relatively low value of α and, depending on the adsorption energy, an increase in the salt concentration can then increase or decrease the adsorption. At finite polymer concentration in solution the number of adsorbed segments and the fraction of the interface covered practically do not change with an increase in polymer concentration, whereas the total number of polymer molecules adsorbed increases slightly, as does the average fraction of segments in loops. The experimental results for polyelectrolyte adsorption have been reviewed in general and, as far as data are available, the predictions of the theory seem to follow the experimentally observed trends quite closely, except for the thickness of the adsorbed layer. This thickness is systematically overestimated by the theory and two reasons for this are given. The theoretical model implies a not too low ionic strength of the solution. Extrapolation of results to solutions of very low ionic strength is not warranted.     

The influence of pressure-induced structural changes on the ionic conductivity of zwitterionic polymers

In this investigation, ionic conductivity measurements are performed on N-oxide polymers neutralized with NaOH. The polymer samples were conformed in disk shapes using an Instron machine considering various values of the pressure required to compact the samples. The dielectric properties of these systems were obtained by the a.c. spectroscopy technique. Results indicate a substantial variation in the ionic conductivity due to the pressure-induced structural changes on the morphology of these samples, as confirmed by WAXS studies. © 1994 John Wiley & Sons, Inc.