Zbigniew Adamczyk

Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements.

In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.

ROLE OF ELECTROSTATIC INTERACTIONS IN PARTICLE ADSORPTION

Abstract The role of the electrostatic double-layer interactions in adsorption of colloid particles at solid/liquid interface was reviewed. The phenomenological formulation of the governing PB equation was presented with the expressions for the pressure tensor enabling one to calculate forces, torques and interaction energies between particles in electrolyte solutions. Then, the limiting analytical results for an isolated double-layer (both spherical and planar) were discussed in relation to the effective surface potential concept. The range of validity of the approximate expression connecting the surface potential and the effective surface potential with surface charge for various electrolytes was estimated. The results for double-layer systems were next presented including the case of two planar double-layers and two dissimilar spherical particles. Limiting solutions for short and long distances as well as for low potentials (linear HHF model) were discussed. The approximate models for calculating interactions of spheres, i.e., the extended Derjaguin summation method and the linear superposition approach (LSA) were also introduced. The results stemming from these models were compared with the exact numerical solution obtained in bispherical coordinate system. Possibilities of describing interactions of nonspherical particles (e.g., spheroids) in terms of the Derjaguin and the equivalent sphere methods were pointed out. In further part of the review the role of these electrostatic interactions in adsorption of colloid particles was discussed. Theoretical predictions were presented enabling a quantitative determination of both the initial adsorption flux for low surface coverages and the surface blocking effects for larger surface coverages. Possibility of bilayer adsorption for dilute electrolytes was mentioned. The theoretical results concerning both the adsorption kinetics and structure formation were then confronted with experimental evidences obtained in the well-defined systems, e.g., the impinging-jet cells and the packed-bed columns of monodisperse spherical particles. The experiments proved that the initial adsorption flux was considerably increased in dilute electrolytes whereas the monolayer coverages were considerably decreased due to lateral interactions among particles. It was then concluded that the good agreement between experimental and theoretical data confirmed the thesis of an essential role of the electrostatic interactions in adsorption phenomena of colloid particles.

Application of the DLVO theory for particle deposition problems

Implications of the DLVO theory for problems associated with colloid particle adsorption and deposition at solid/liquid interfaces were reviewed. The electrostatic interactions between two planar double-layers described by the classical Poisson–Boltzmann (PB) equation were first discussed. Then, the approximate models for calculating interactions of curved interfaces (e.g. spheres) were exposed in some detail, inter alia the extended Derjaguin summation method and the linear superposition approach (LSA). The results stemming from these models were compared with the exact numerical solution for two dissimilar spheres (including the case of sphere/plane interactions) obtained in bispherical coordinate system. The electrostatic interaction energy was used in combination with dispersion interactions for constructing the DLVO energy profiles discussed next. The influence of surface roughness and charge heterogeneity on energy profiles was also discussed. It was demonstrated that in particle deposition problems the monotonically changing profiles determined by the electrostatic interactions played the most important role. In further part of the review the role of these electrostatic interactions in adsorption and deposition of colloid particles was discussed. The governing continuity equation was exposed incorporating the convective transport in the bulk and the specific force dominated transport at the surface. Approximate analytical models aimed at decoupling of these transfer steps were described. It was demonstrated that the surface boundary layer approximation (SFBLA) was the most useful one for describing the effect of electrostatic interaction at initial adsorption stages. A procedure of extending this model for non-linear adsorption regimes, governed by the steric barrier due to adsorbed particles, was also presented. The theoretical results were then confronted with experimental evidences obtained in the well-defined systems, e.g. the impinging-jet cells and the packed-bed columns of monodisperse spherical particles. The experiments proved that the initial adsorption flux of particles was considerably increased in dilute electrolytes due to attractive electrostatic interactions. This was found in a quantitative agreement with the convective diffusion theory. On the other hand, the rate of later adsorption stages was diminished by the electrostatic lateral interactions between adsorbed and adsorbing particles. Similarly, the experimental data obtained by various techniques (AFM, reflectometry, optical microscopy) demonstrated that these interactions reduced significantly the maximum monolayer coverages at low ionic strength. This behaviour was found in good agreement with theoretical MC-RSA simulation performed by using the DLVO energy profiles. The extensive experimental evidences seem, therefore, to support the thesis that the electrostatic interactions play an essential role in adsorption phenomena of colloid particles.

Structure and ordering in localized adsorption of particles

Abstract Localized, sequential adsorption of colloid particles interacting via screened Coulomb potential was analyzed theoretically and experimentally. The two-dimensional (2D) pair correlation functions were simulated by using the Monte Carlo technique for various surface concentrations θ and for various screening length parameters κα, characterizing the “softness” of the particle-particle interaction potential. For κα ⪢ 1 the hard-disk limiting behavior was confirmed (maximum surface concentration θmx = 55%). The θmx for soft disks, which is generally much smaller than the above value, was also determined as a function of the κα parameter. A distinctive tendency toward a short-range ordering, analogous to the 3D results obtaining previously for colloid suspensions, was found for surface concentration close to the θmx value. Theoretical predictions were tested by applying the direct experimental method based on microscope observations of particle adsorption. A monodisperse polystyrene suspension was used (particle size 0.90 μm) and the adsorbing surface was made of mica sheets. Experimental results proved to be in good agreement with Monte Carlo simulations illustrating well the tendency towards structurization (2D quasi-liquid phase formation) for surface concentrations close to the predicted θmx values.

Particle transfer to solid surfaces

Abstract Several theories for predicting deposition rates of flowing colloidal particles onto various collector surfaces are compared and discussed. Successful theories must include, besides hydrodynamic conditions in the vicinity of the collector and the energy of interaction between particles and the wall, a variety of other important factors affecting deposition such as particle detachment, aging of particle-collector bonds, masking, influence of the stability of the dispersion and surface heterogeneity and roughness. Several experimental procedures for measuring deposition rates are discussed and experimental data are compared with theoretical predictions. In the absence of energy barriers current theories predict deposition rates within 10–25% of their actual value. In their presence, discrepancies are usually much larger, mainly because theory neglects several of the above mentioned factors. Examples of dynamic simulation and Monte Carlo calculations suggest that these methods can incorporize the factors affecting deposition more readily than current theories.

Particle adsorption and deposition: role of electrostatic interactions

This work demonstrates how electrostatic interactions, described in terms of the classical DLVO theory, influence colloid particle deposition phenomena at solid/liquid interfaces. Electrostatic interactions governing particle adsorption in both non-polar and polar media (screened interactions) are discussed. Exact and approximate methods for calculating the interaction energy of spherical and non-spherical (anisotropic) particles are presented, including the Derjaguin method. Phenomenological transport equations governing particle deposition under the linear regime are discussed with the limiting analytical expressions for calculating initial flux. Non-linear adsorption regimes appearing for higher coverage of adsorbed particles are analysed. Various theoretical approaches are exposed, aimed at calculating blocking effects appearing due to the presence of adsorbed particles. The significant role of coupling between bulk transport and surface blocking is demonstrated. Experimental data obtained under well-defined transport conditions, such as diffusion and forced convection (impinging-jet cells), are reviewed. Various experimental techniques for detecting particles at interfaces are discussed, such as reflectometry, ellipsometry, streaming potential, atomic force microscopy, electron and optical microscopy, etc. The influence of ionic strength and flow rate on the initial particle deposition rate (limiting flux) is presented. The essential role of electrostatic interactions in particle deposition on heterogeneous surfaces is demonstrated. Experimental data pertinent to the high-coverage adsorption regime are also presented, especially the dependence of the maximum coverage of particles and proteins on the ionic strength. The influence of lateral electrostatic interactions on the structure of particle monolayers is elucidated, and the links between colloid and molecular systems are pointed out.

Fibrinogen Adsorption on Mica Studied by AFM and in Situ Streaming Potential Measurements

Adsorption of fibrinogen from aqueous solutions on mica was studied using AFM and in situ streaming potential measurements. In the first stage, bulk physicochemical properties of fibrinogen and the mica substrate were characterized for various ionic strength and pH. The zeta potential and number of uncompensated (electrokinetic) charges on the protein surfaces were determined from microelectrophoretic measurements. Analogously, using streaming potential measurements, the electrokinetic charge density of mica was determined for pH range 3-10 and the NaCl background electrolyte concentration of 10(-3) and 10(-2) M. Next, the kinetics of fibrinogen adsorption at pH 3.5 and 7.4 in the diffusion cell was studied using a direct AFM determination of the number of molecules per unit area of the mica substrate. Then, streaming potential measurements were performed to determine the apparent zeta potential of fibrinogen-covered mica for different pH and ionic strength in terms of its surface concentration. A quantitative interpretation of these streaming potential measurements was achieved in terms of the theoretical model postulating a side-on adsorption of fibrinogen molecules as discrete particles. On the basis of these results, the maximum coverage of fibrinogen Θ close to 0.29 was predicted, in accordance with previous theoretical predictions. It was also suggested that anomalous adsorption for pH 7.4, where fibrinogen and the mica substrate were both negatively charged, can be explained in terms of a heterogeneous charge distribution on fibrinogen molecules. It was estimated that the positive charge was 12 e (for NaCl concentration of 10(-2) M and pH 7.4) compared with the net charge of fibrinogen at this pH, equal to -21 e. Results obtained in this work proved that the coverage of fibrinogen can be quantitatively determined using the streaming potential method, especially for Θ < 0.2, where other experimental methods become less accurate.

Streaming potential studies of colloid, polyelectrolyte and protein deposition

Recent developments in the electrokinetic determination of particle, protein and polyelectrolyte monolayers at solid/electrolyte interfaces, are reviewed. Illustrative theoretical results characterizing particle transport to interfaces are presented, especially analytical formulae for the limiting flux under various deposition regimes and expressions for diffusion coefficients of various particle shapes. Then, blocking effects appearing for higher surface coverage of particles are characterized in terms of the random sequential adsorption model. These theoretical predictions are used for interpretation of experimental results obtained for colloid particles and proteins under convection and diffusion transport conditions. The kinetics of particle deposition and the structure of monolayers are analyzed quantitatively in terms of the generalized random sequential adsorption (RSA) model, considering the coupling of the bulk and surface transport steps. Experimental results are also discussed, showing the dependence of the jamming coverage of monolayers on the ionic strength of particle suspensions. In the next section, theoretical and experimental results pertaining to electrokinetics of particle covered surfaces are presented. Theoretical models are discussed, enabling a quantitative evaluation of the streaming current and the streaming potential as a function of particle coverage and their surface properties (zeta potential). Experimental data related to electrokinetic characteristics of particle monolayers, mostly streaming potential measurements, are presented and interpreted in terms of the above theoretical approaches. These results, obtained for model systems of monodisperse colloid particles are used as reference data for discussion of experiments performed for polyelectrolyte and protein covered surfaces. The utility of the electrokinetic measurements for a precise, in situ determination of particle and protein monolayers at various interfaces is pointed out.

Mechanisms of Fibrinogen Adsorption at Solid Substrates

Adsorption of fibrinogen, modeled as a linear chain of touching beads of various sizes, was theoretically studied using the random sequential adsorption (RSA) model. The adsorption process was assumed to consist of two steps: (i) formation of an irreversibly bound fibrinogen monolayer under the side-on orientation, which is independent of the bulk protein concentration and (ii) formation of the reversibly bound, end-on monolayer, whose coverage was dependent on the bulk concentration. Calculation based on the RSA model showed that the maximum surface concentration of the end-on (reversible) monolayer equals N(⊥∞) = 6.13 × 10(3) μm(-2) which is much larger than the previously found value for the side-on (irreversible) monolayer, equal to N(∞) = 2.27 × 10(3) μm(-2). Hence, the maximum surface concentration of fibrinogen in both orientations is determined to be 8.40 × 10(3) μm(-2) corresponding to the protein coverage of 5.70 mg m(-2) assuming 20% hydration. Additionally, the surface blocking function (ASF) was determined for the end-on fibrinogen adsorption, approximated for the entire range of coverage by the interpolating polynomial. For the coverage approaching the jamming limit, the surface blocking function (ASF) was shown to vanish proportionally to (θ(⊥∞) - θ(⊥))(2). These calculation allowed one to theoretically predict adsorption isotherms for the end-on regime of fibrinogen and adsorption kinetics under various transport conditions (diffusion and convection). Using these theoretical results, a quantitative interpretation of experimental data obtained by TIRF and ellipsometry was successfully performed. The equilibrium adsorption constant for the end-on adsorption regime was found to be 8.04 × 10(-3) m. On the basis of this value, the depth of the adsorption energy minimum, equal to -17.4 kT, was predicted, which corresponds to ΔG = -41.8 kJ mol(-1). This is in accordance with adsorption energy derived as the sum of the van der Waals and electrostatic interactions. Besides having significance for predicting fibrinogen adsorption, theoretical results derived in this work also have implications for basic science providing information on mechanisms of anisotropic protein molecule adsorption on heterogeneous surfaces.

Zeta potential of mica covered by colloid particles: a streaming potential study.

The streaming potential of mica covered by monodisperse latex particles was measured using the parallel-plate channel, four-electrode cell. The zeta potential of latex bearing amidine charged groups was regulated by the addition of NaCl (10(-4)-10(-2) M) and MgCl(2) (10(-4)-10(-2) M) at a constant pH 5.5 and by the change in pH (4-12) at 10(-2) M NaCl. The size of the latex particles, determined by dynamic light scattering, varied between 502 and 540 nm for the above electrolyte concentration range. Mica sheets have been covered with latex particles under diffusion transport conditions. The latex coverage was regulated by the bulk suspension concentration in the channel and the deposition time. The coverage was determined, with a relative precision of 2%, by the direct enumeration of particles by optical microscopy and AFM. The streaming potential of mica was then determined for a broad range of particle coverage 0 < theta < 0.5, the particle-to-substrate zeta potential ratio zeta(p)/zeta(i), and 8.8 < kappa a < 143 (thin double-layer limit). These experimental data confirmed that the streaming potential of covered surfaces is well reflected by the theoretical approach formulated in ref 32. It was also shown experimentally that variations in the substrate streaming potential with particle coverage for theta < 0.3 and zeta(p)/zeta(i) < 0 are characterized by a large slope, which enables the precise detection of particles attached to interfaces. However, measurements at high coverage and various pH values revealed that the apparent zeta potential of covered surfaces is 1/2(1/2) smaller than the bulk zeta potential of particles (in absolute terms). This is valid for arbitrary zeta potentials of substrates and particles, including the case of negative particles on negatively charged substrates that mimics rough surfaces. Therefore, it was concluded that the streaming potential method can serve as an efficient tool for determining bulk zeta potentials of colloids and bioparticles.