Marta Sadowska

Deposition of colloid particles on protein layers: fibrinogen on mica.

Colloid particle deposition was applied to characterize fibrinogen (Fb) monolayers on mica, which were produced by controlled adsorption under diffusion transport. By adjusting the time of adsorption and the bulk Fb concentration, monolayers of desired surface concentration were obtained. The surface concentration of Fb was determined directly by AFM enumeration of single molecules adsorbed over the substrate surface. It was proven that Fb adsorbed irreversibly on mica both at pH 3.5 and at pH 7.4 with the rate governed by bulk transport. The electrokinetic properties of Fb monolayers produced in this way were studied using the streaming potential method. The dependence of the apparent zeta potential of Fb monolayers was determined as a function of the coverage. It was shown that for pH 3.5 the initial negative zeta potential of the mica substrate was converted to positive for Fb coverage exceeding 0.16. On the other hand, for pH 7.4, the zeta potential of a Fb-covered mica remained negative for the entire coverage range. The charge distribution in Fb monolayers was additionally studied using the colloid deposition method, in which negatively and positively charged polystyrene latex particles (ca. 800 nm in diameter) were used. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential was observed. Results of these experiments were quantitatively interpreted in terms of the fluctuation theory assuming that adsorption sites consisted of two and three Fb molecules, for pH 3.5 and 7.4, respectively. These results suggested that for pH 7.4, the distribution of charge on Fb molecules was heterogeneous, characterized by the presence of positive patches, whereas the average zeta potential was negative, equal to -19 mV. The utility of the colloid deposition method for studying Fb monolayers was further demonstrated in deposition experiments involving positive latex particles. It was shown that for a rather broad range of fibrinogen coverage, both the positive and the negative latex particles can adsorb on surfaces covered by Fb, which behaved, therefore, as superadsorbing surfaces. It was also concluded that the colloid deposition method can be used to determine the Fb bulk concentration for the range inaccessible for other methods.

Mechanism of Nanoparticle Deposition on Polystyrene Latex Particles

The deposition of positive amidine latex particles (98 nm in diameter) on negative polystyrene latex particles (820 nm in diameter) was studied by SEM imaging, microelectrophoretic and concentration depletion methods involving AFM. The role of ionic strength varied between 10(-4) and 10(-2) M and was systematically studied. The number of deposited positive latex particles (surface coverage) was evaluated by a direct counting procedure exploiting the SEM images. This allowed one to calibrate the results obtained from measurements of the electrophoretic mobility of larger latex particles covered by a controlled amount of the positive latex. These dependencies were quantitatively interpreted in terms of the 3D electrokinetic model previously used for planar interfaces. This allowed us to determine the coverage of nanoparticles on latex carriers under in situ conditions. Additionally, the maximum coverage of the positive latex was determined via AFM where the kinetics of the residual amidine latex deposition on mica was measured. The maximum coverage monotonically increased with ionic strength, attaining 0.52 for 10(-2) M NaCl. This effect was interpreted in terms of reduced electrostatic repulsion among positive latex particles and theoretically accounted for by the random sequential adsorption model. The obtained results have significance for basic science, indicating that the results obtained for curved interfaces (polymeric carrier particles) by the microelectrophoretic method can be exploited to interpret the deposition of nanoparticles and proteins on planar interfaces and vice versa.

Fibrinogen monolayer characterization by colloid deposition.

Colloid particle deposition was applied to characterize bovine and human fibrinogen (Fb) monolayers on mica produced by controlled adsorption under diffusion transport at pH 3.5. The surface concentration of Fb was determined by AFM enumeration of single molecules adsorbed over the substrate surface. The electrokinetic properties of Fb monolayers for various ionic strength were studied using the in situ streaming potential measurements. It was shown that Fb adsorbs irreversibly on mica for a broad range of ionic strength of 4 × 10(-4) to 0.15 M, NaCl. The overcharging of initially negative mica surface occurred for fibrinogen surface concentrations higher than 1400 μm(-2). The orientation of fibrinogen molecules in the monolayers was evaluated by the colloid deposition method involving negatively charged polystyrene latex microspheres, 820 nm in diameter. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential was observed, which contradicts the mean-field DLVO predictions. Measurable deposition was observed even at low ionic strength where the minimum approach distance of latex particles to the interface exceeds 70 nm (for 6 × 10(-4) M NaCl). This confirms that, at this pH, fibrinogen molecules adsorb end-on on mica assuming extended conformations with the positive charge located mostly in the end part of the αA chains. This agrees with previous experimental and theoretical results discussed in the literature (Santore, M. M.; Wertz Ch. F. Protein spreading kinetics at liquid-solid interfaces via an adsorption probe method. Langmuir 2005, 21, 10172-10178 (experimental); Adamczyk, Z.; Barbasz, J.; Cieśla, M.; Mechanisms of fibrinogen adsorption at solid substrates. Langmuir, 2011, 25, 6868-6878 (theoretical)). This unusual latex deposition on Fb monolayers was quantitatively interpreted in terms of the model developed in ref 55 (Jin, X.; Wang, N. H. L.; Tarjus, G.; Talbot, J. Irreversible adsorption on nonuniform surfaces: the random site model. J. Phys. Chem. 1993, 97, 4256-4258). It was concluded that the colloid deposition method is an efficient tool for revealing protein adsorption mechanisms at solid/electrolyte interfaces.

Revealing fibrinogen monolayer conformations at different pHs: electrokinetic and colloid deposition studies.

Adsorption mechanism of human fibrinogen on mica at different pHs is studied using the streaming potential and colloid deposition measurements. The fibrinogen monolayers are produced by a controlled adsorption under diffusion transport at pH of 3.5 and 7.4. Initially, the electrokinetic properties of these monolayers and their stability for various ionic strength are determined. It is shown that at pH 3.5 fibrinogen adsorbs irreversibly on mica for ionic strength range of 4×10(-4) to 0.15 M. At pH 7.4, a partial desorption is observed for ionic strength below 10(-2) M. This is attributed to the desorption of the end-on oriented molecules whereas the side-on adsorbed molecules remain irreversibly bound at all ionic strengths. The orientation of molecules and monolayer structure is evaluated by the colloid deposition measurements involving negatively charged polystyrene latex microspheres, 820 nm in diameter. An anomalous deposition of negative latex particles on substrates exhibiting a negative zeta potential is observed. At pH 3.5 measurable deposition of latex is observed even at low ionic strength where the approach distance of latex particles exceeded 70 nm. At pH 7.4 this critical distance is 23 nm. This confirms that fibrinogen monolayers formed at both pHs are characterized by the presence of the side-on and end-on oriented molecules that prevail at higher coverage range. It is also shown that positive charge is located at the end parts of the αA chains of the adsorbed fibrinogen molecules. Therefore, it is concluded that the colloid deposition method is an efficient tool for revealing protein adsorption mechanisms at solid/electrolyte interfaces.

Formation mechanism of human serum albumin monolayers on positively charged polymer microparticles

Human serum albumin (HSA) adsorption on positively and negatively charged polystyrene microparticles was studied at various pHs and NaCl concentrations. Thorough electrophoretic mobility measurements were carried out that enabled to monitor in situ the progress of protein adsorption. The maximum coverage of irreversibly adsorbed HSA on microparticles was determined by different concentration depletion methods, one of them involving AFM imaging of residual molecules. An anomalous adsorption of HSA on the positive microparticles was observed at pH 3.5 where the maximum coverage attained 1.0mgm-2 for NaCl concentrations of 0.05M despite that the molecules were on average positively charged. For comparison, the maximum coverage of HSA on negatively charged microparticles was equal to 1.3mgm-2 at this pH and NaCl concentration. At pH 7.4 the maximum coverage on positive microparticles was equal to 2.1mgm-2 for 0.05M NaCl concentration. On the other hand, for negative microparticles, negligible adsorption of HSA was observed at pH 7.4 and 9.7. These experimental data were adequately interpreted in terms of the random sequential adsorption approach exploiting the bead model of the HSA molecule. Different orientations of adsorbed molecules, inert alia, the edge-on orientation prevailing for positively charged microparticles at pH 7.4, were confirmed. This was explained in terms of a heterogeneous charge distribution over the HSA molecule prevailing for a wide range of pHs.

Kinetics of Human Serum Albumin Adsorption at Silica Sensor: Unveiling Dynamic Hydration Function

Adsorption kinetics of human serum albumin (HSA) at a silica substrate was studied using the QCM-D and AFM methods. Measurements were performed at pH 3.5 for various bulk suspension concentrations and ionic strengths. The QCM experimental data were compared with the dry coverage of HSA derived from AFM and from the solution of the mass transfer equation. In this way, the dynamic hydration functions and water factors of HSA monolayers were quantitatively evaluated as a function of dry coverage for various ionic strengths. Using the hydration functions, the HSA adsorption runs derived from QCM-D measurements were converted to the dry coverage vs. the time relationships. In this way, the maximum coverage of irreversibly bound HSA molecules was determined. It was equal to 0.35 and 1.4 mg m-2 for NaCl concentration of 0.001 and 0.15 M, respectively. These results agree with previous experimental data derived by streaming potential measurements for mica and with theoretical modeling. Therefore, the side-on mechanism of HSA adsorption at silica sensor at pH 3.5 was confirmed. Also, a quantitative analysis of the desorption runs allowed one to calculate the binding energy of the reversibly bound HSA fraction. Beside significance to basic science, these results enable to develop a robust technique of preparing HSA monolayers at silica sensor of well-controlled coverage and molecule orientation.

Formation of hematite nanoparticle monolayers of controlled coverage and structure at polymeric microparticles

The deposition of hematite nanoparticles (22nm and 29nm in diameter) on negatively charged polystyrene microspheres (820nm in diameter) was studied by micro-electrophoretic measurements and AFM. The influence of ionic strength, varied between 10-4 and 10-2M, was determined. Initially, the electrophoretic mobility change of microspheres upon the addition of controlled amount of hematite nanoparticles were measured. These dependencies were quantitatively interpreted in terms of the general electrokinetic model. This allowed to determine the coverage of nanoparticles on microspheres under in situ conditions, which increased with ionic strength attaining 0.35 for the ionic strength of 10-2M and 29 in diameter hematite particles. This effect, attributed to the decreasing range of lateral electrostatic repulsion among deposited particles, was accounted for by the random sequential adsorption model. However, the coverages attained for lower ionic strength exceeded the theoretical predictions. This effect was interpreted in terms of an additional electrostatic screening due to polymeric chains present at the microparticle surface. The acid base properties of the hematite monolayers were also acquired by applying thorough micro-electrophoretic measurements. The obtained results confirmed a feasibility of preparing hematite nanoparticle monolayers on polymeric carrier microspheres having well-defined coverage and structure.

Fibrinogen Monolayers of Controlled Coverage and Conformations for Biosensing Applications

Protein adsorption at solid interfaces is involved in cell adhesion, inflammatory response, artificial organ and biomaterial failure, plaque formation and fouling of membranes. On the other hand, a controlled protein deposition on various surfaces is important for their efficient separation and purification by chromatography, filtration, for biocatalysis, biosensing, immunological assays, etc. Fibrinogen remains since many years one of the most extensively studied protein because of its fundamental role in the blood clotting cascade, platelet adhesion, leucocyte binding, thrombosis, angiogenesis, inflammatory response, tumor growth, fouling of artificial organs, and so forth. Reliable information about fibrinogen’s geometrical dimensions and conformations stem from electron [1] and atomic force microscopy [2--5] imaging of single molecules deposited on various substrates. It was established that the molecule has a co-linear, trinodular shape with a total length of 47.5 nm. The two equal end domains of rather irregular shape are approximated by spheres having the diameter of 6.5 nm; and the middle domain has a diameter of 5 nm. This indicates that the fibrinogen molecule is highly anisotropic and elongated, characterized by the length to the width ratio above ten. Numerous experimental studies were also devoted to fibrinogen adsorption on various substrates [2,5--7] using the in situTIRF andAFM [5] or ellipsometry [2,6]. However, using these techniques one cannot derive relevant information about orientation of fibrinogen molecules in monolayers, which is of a vital practical significance. Therefore, in this work, an efficient method of analyzing fibrinogenmonolayers on solid substrates under the wet, in situ conditions is used. The technique is based on unspecific, electrostatically driven, deposition of colloid particles onto polyelectrolyte or protein monolayers [8]. A major advantage of this method is that the coverage of colloid can be directly determined via optical microscope or AFM imaging. In this way a unique functional dependence between the protein and the colloid particle coverages can be experimentally derived.