Mechanisms of fibrinogen adsorption on latex particles determined by zeta potential and AFM measurements

The adsorption of fibrinogen on polystyrene latex particles was studied using the concentration depletion method combined with the AFM detection of residual protein after adsorption. Measurements were carried out for a pH range of 3.5-11 and an ionic strength range of 10(-3)-0.15 M NaCl. First, the bulk physicochemical properties of fibrinogen and the latex particle suspension were characterized for this range of pH and ionic strength. The zeta potential and the number of uncompensated (electrokinetic) charges on the protein were determined from microelectrophoretic measurements. It was revealed that fibrinogen molecules exhibited amphoteric characteristics, being on average positively charged for pH <5.8 (isolectric point) and negative otherwise. However, the latex particles did not show any isoelectric point, remaining strongly negative for this pH range. Afterward, systematic measurements of the electrophoretic mobility of fibrinogen-covered latex were carried out as a function of the amount of adsorbed protein, expressed as the surface concentration. A monotonic increase in the electrophoretic mobility (zeta potential) of the latex was observed in all cases, indicating a significant adsorption of fibrinogen on latex for pH below 11. It was also proven that fibrinogen adsorption was irreversible, with the maximum surface concentration varying between 2.5 and 5 × 10(3) μm(-2) (weight concentration of a bare molecule was 1.4 to 2.8 mg m(-2)). These measurements revealed two main adsorption mechanisms of fibrinogen: (i) the unoriented (random) mechanism prevailing for lower ionic strength, where adsorbing molecules significantly penetrate the fuzzy polymeric layer on the latex core and (ii) the side-on adsorption mechanism prevailing for pH > 5.8 and a higher ionic strength of 0.15 M. It was also shown that in the latter case, variations in the zeta potential with the protein coverage could be adequately described in terms of the electrokinetic model, previously formulated for planar substrate adsorption. On the basis of these experimental data, an efficient procedure of preparing fibrinogen-covered latex particles of controlled monolayer structure and coverage was envisaged.     

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.     

Biomimetic particles: optimization of phospholipid bilayer coverage on silica and colloid stabilization

The effect of ionic strength and pH on phosphatidylcholine (PC) adsorption from vesicles on silica nanoparticles was investigated over a range of NaCl concentrations (0.1-150 mM) at pH 6.3 and 7.4 from determination of adsorption isotherms, colloid stability, particle sizing, and zeta-potentials. At and above 10 mM ionic strength, pH 6.3, high-affinity adsorption isotherms with limiting adsorption indicative of one-bilayer deposition on each silica particle were obtained. At 10 mM ionic strength, adsorption isotherms indicated lower affinity between PC and silica at pH 7.4 than at pH 6.3, suggesting a role of hydrogen bonding between silanol on silica and phosphate on PC in promoting bilayer deposition at low pH. Under conditions where high affinity and bilayer deposition were achieved, silica sedimentation documented from photographs was absent, suggesting particle stabilization induced by bilayer coverage. However, at physiological (150 mM NaCl) or close to physiological ionic strength (140 mM NaCl), the large colloid stability similarly achieved at pH 6.3 or 7.4 suggested the major role of van der Waals attraction between the PC bilayer vesicle and silica particle in determining bilayer deposition. The effect of increasing ionic strength was increasing van der Waals attraction, which caused PC vesicle disruption with bilayer deposition and bilayer-induced silica stabilization.     

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.     

Interaction of dissolved proteins with spherical polyelectrolyte brushes

We consider the adsorption of bovine serum albumin (BSA) on spherical polyelectrolyte brushes (SPB). The SPB consist of a solid polystyrene core of 100nm diameter onto which linear polyelectrolyte chains (poly(acrylic acid), (PAA)) are grafted. The adsorption of BSA is studied at a pH of 6.1 at different concentrations of added salt and buffer (MES). We observe strong adsorption of BSA onto the SPB despite the effect that the particles as well as the dissolved BSA are charged negatively. The adsorption of BSA is strongest at low salt concentration and decreases drastically with increasing amounts of added salt. The adsorbed protein can be washed out again by raising the ionic strength. The various driving forces for the adsorption are discussed. It is demonstrated that the main driving force is located in the electrostatic interaction of the protein with the brush layer of the particles. All data show that the SPB present a new class of carrier particles whose interaction with proteins can be tuned in a well-defined manner.     

Characterization of fibrinogen adsorption onto glass microcapillary surfaces by ELISA

Adsorption of biomolecules onto microchannel surfaces remains a critical issue in microfluidic devices. This paper investigates the adsorption of fibrinogen on glass microcapillaries using an immunoassay method (ELISA) and X-ray photoelectron spectroscopy (XPS). Various adsorption conditions such as protein concentrations and incubation times, buffer pH, buffer ionic strengths and effects of flow are presented. ELISA is successfully demonstrated as a facile and robust technique to examine these phenomena. The highest adsorption level occurs near the isoelectric point of fibrinogen (pH 5.0) and low buffer ionic strengths (0-8 mM). Microchannel surface saturation was achieved at a fibrinogen solution concentration of approximately 50 microg ml(-1). Fibrinogen adsorption under flow was always higher than that seen in static systems. The importance of diffusion phenomena in microchannels on protein adsorption was demonstrated. ELISA experiments using fused silica and PEEK have also confirmed significant adsorption on these mass spectrometer transfer line materials.     

Elaboration of poly(ethyleneimine) coated poly( , -lactic acid) particles. Effect of ionic strength on the surface properties and DNA binding capabilities

Poly( , -lactic acid) (PLA)-based particles, obtained by the emulsification–diffusion process, were surface-modified by electrostatic adsorption of poly(ethylenimine) (PEI). The amount of immobilized PEI and the conformation of the polycation at the interface were dependent on the ionic strength of the media. In the absence of salt, or at low ionic strength, the adsorbed amounts of PEI, the surface charge and the critical concentration for coagulation (CCC) of the modified particles were lower than when the adsorption was achieved at elevated ionic strength. Moreover, at low salt concentration, isotherms were of Langmuir type, suggesting the formation of monolayers. The differences in PEI surface conformation had consequences on the DNA binding capacity of the particles, on the plasmid DNA conformation at the interface and on the DNA release in various media. When PEI was adsorbed in a 50 mM phosphate buffer, the amount of bound plasmid and the strength of binding were higher than when PEI was adsorbed in water. From these differences in physico-chemical properties, one can expect differences in transfection or immunization performances of the vectors.     

Adsorbed Layers of Ferritin at Solid and Fluid Interfaces Studied by Atomic Force Microscopy

The adsorption of the iron storage protein ferritin was studied by liquid tapping mode atomic force microscopy in order to obtain molecular resolution in the adsorbed layer within the aqueous environment in which the adsorption was carried out. The surface coverage and the structure of the adsorbed layer were investigated as functions of ionic strength and pH on two different charged surfaces, namely chemically modified glass slides and mixed surfactant films at the air-water interface, which were transferred to graphite substrates after adsorption. Surface coverage trends with both ionic strength and pH indicate the dominance of electrostatic effects, with the balance shifting between intermolecular repulsion and protein-surface attraction. The resulting behavior is more complex than that seen for larger colloidal particles, which appear to follow a modified random sequential adsorption model monotonically. The structure of the adsorbed layers at the solid surfaces is random, but some indication of long-range order is apparent at fluid interfaces, presumably due to the higher protein mobility at the fluid interface. Copyright 2000 Academic Press.     

In-situ investigation of the adsorption of globular model proteins on stimuli-responsive binary polyelectrolyte brushes

Binary brushes constituted from two incompatible polymers can be used in the form of ultrathin polymeric layers as a versatile tool for surface engineering to tune physicochemical surface characteristics such as wettability, surface charge, chemical composition, and morphology and furthermore to create responsive surface properties. Mixed brushes of oppositely charged weak polyelectrolytes represent a special case of responding surfaces that are sensitive to changes in the pH value of the aqueous environment and therefore represent interesting tools for biosurface engineering. The polyelectrolyte brushes used for this study were composed of two oppositely charged polyelelctrolytes poly(2-vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). The in-situ properties and surface characteristics such as as surface charge, surface tension, and extent of swelling of these brush layers are functions of the pH value of the surrounding aqueous solution. To test the behavior of the mixed polylelctrolyte brushes in contact with biosystems, protein adsorption experiments with globular model proteins were performed at different pH values and salt concentrations (confinement of counterions) of the buffer solutions. The influence of the pH value, buffer salt concentration, and isoelectric points (IEP) of the brush and protein on the adsorbed amount and the interfacial tension during protein adsorption as well as the protein adsorption mechanism postulated in reference to recently developed theories of protein adsorption on polyelectrolyte brushes is discussed. In the salted regime, protein adsorption was found to be similar to the often-described adsorption at hydrophobic surfaces. However, in the osmotic regime the balance of electrostatic repulsion and a strong entropic driving force, "counterion release", was found to be the main influence on protein adsorption.     

Effect of thermal treatment, ionic strength, and pH on the short-term and long-term coalescence stability of beta-lactoglobulin emulsions

We present experimental results about the effects of thermal treatment, ionic strength, and pH on the protein adsorption and coalescence stability of freshly prepared (2 h after emulsification) and 6-day-stored emulsions, stabilized by the globular protein beta-lactoglobulin (BLG). In all emulsions studied, the volume fraction of the dispersed soybean oil is 30% and the mean drop diameter is d(32) approximately 40 microm. The protein concentration, C(BLG), is varied between 0.02 and 0.1 wt %, the electrolyte concentration, C(EL), between 1.5 mM and 1 M, and pH between 4.0 and 7.0. The emulsion heating is performed at 85 degrees C, which is above the denaturing temperature of BLG. The results show that, at C(BLG) > or = 0.04 wt %, C(EL) > or = 150 mM, and pH > or = 6.2, the heating leads to higher protein adsorption and to irreversible attachment of the adsorbed molecules, which results in enhanced steric repulsion between the protein adsorption multilayers and to higher emulsion stability. At low electrolyte concentration, C(EL) < or = 10 mM, the emulsion stability is determined by electrostatic interactions and is not affected significantly by the emulsion heating. The latter result is explained by electrostatic repulsion between the adsorbed protein molecules, which keeps them separated from each other and thus precludes the formation of disulfide covalent bonds in the protein adsorption layer. The coalescence stability of heated and nonheated emulsions is practically the same and does not depend on C(EL), when pH is around the isoelectric point (IEP) of the protein molecules. This is explained with the adsorption of uncharged BLG molecules, in compact conformation, which stores the reactive sulfhydryl groups hidden inside the molecule interior, thus preventing the formation of covalent intermolecular bonds upon heating. We studied also the effect of storage time on the stability of heated and nonheated emulsions. The stability of nonheated emulsions (C(BLG) = 0.1 wt %, C(EL) > or = 150 mM, and pH = 6.2) significantly decreases after 1 day of storage (aging effect). In contrast, no aging effect is observed after emulsion heating. FTIR spectra of heated and nonheated, fresh and aged emulsions suggest that the aging effect is caused by slow conformational changes of the protein molecules in the adsorption layer, accompanied with partial loss of the ordered secondary structure of the protein and with the formation of lateral noncovalent bonds (H-bonds and hydrophobic interactions) between the adsorbed molecules. After thermal treatment of the BLG emulsions, the molecules preserve their original secondary structure upon storage, which eliminates the aging effect.     

Adsorption of alpha-chymotrypsin onto mica in laminar flow conditions. Adsorption kinetic constant as a function of tris buffer concentration at pH 8.6

We examined the adsorption kinetics of alpha-chymotrypsin (pH 8.6, 10(-2) to 0.5 M Tris buffer) on muscovite mica in conditions of laminar flow through a slit. The range of buffer concentrations is between two limits: (i) no adsorption in 1 M Tris and (ii) no desorption in 10(-3) M Tris. Studying the dependence of adsorption kinetics on the wall shear rate leads to the determination of the interfacial adsorption kinetic constant ka and the diffusion coefficient. The obtained value for the diffusion coefficient is close to the one expected from the molecular size of alpha-chymotrypsin. The interfacial adsorption kinetic constant of alpha-chymotrypsin decreases when ionic strength increases, while the initial desorption constant (over a part of all the adsorbed population) shows the contrary. Although alpha-chymotrypsin is almost at its isoelectric point, the effect of ionic strength on the adsorption kinetics suggests the importance of electrostatic interactions between the protein and mica. We observed an increase in the adsorption rate, at a surface coverage near 0.14 microg cm(-2), for adsorption in 10(-2) M Tris and the low wall shear rates (<300 s(-1)). This change in the adsorption rate suggests a structural transition, that we assume again to be due to electrostatic interactions, but between proteins. The large dipole moment of the protein may induce this transition, illustrated here by the ferroelectric/antiferroelectric pattern. The variation of the zeta potential with interfacial concentration seems to be in agreement with such a model.     

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.     

Atomic force microscopy study of the adsorption and electrostatic self-organization of poly(amidoamine) dendrimers on mica

The adsorption behavior of poly(amidoamine) dendrimers to mica surfaces was investigated as a function of ionic strength and pH. The conformation and lateral distribution of the adsorbed dendrimers of generations G8 and G10 were obtained ex situ by tapping mode atomic force microscopy (AFM). The deposition kinetics of the dendrimers was found to follow a diffusion-limited process. Fractional surface coverage and pair correlation functions of the adsorbed dendrimers were obtained from the AFM images. The data are interpreted in terms of the random sequential adsorption (RSA) model, where electrostatic repulsion due to overlapping double layers is considered. Although the general trends typical for an RSA-determined process are well-reproduced, quantitative agreement is lacking at low ionic strengths.     

表面等离子体子共振技术实时研究蛋白质在修饰表面的静电吸附行为

研究蛋白质在固相表面的静电吸附特性,进而控制蛋白质在修饰表面的静电吸附尤为重要,表面等离子体子共振可以检测金属表面吸附物质厚度和折射率的变化^[1]。这种技术已在研究生物分子相互作用^[2]和考察自组装单层的形成^[3]及蛋白质在固体表面吸附行为^[9-11]等方面得到广泛的应用。对蛋白质在固体表面吸附行为的研究多为考察不同的蛋白质在不同的修饰表面的吸附行为。然而,对蛋白质在修饰表面静电吸附的本质影响因素的研究却少有报道^[4]。本文使用表面等离子体子共振技术实时研究了蛋白质在甲羧基化葡聚糖修饰表面的静电吸附与溶液pH值及离子强度的依赖关系。     

Energetics of lysozyme adsorption on mesostructured cellular foam silica: effect of salt concentration

The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.     

Adsorption of microcystin-Lr onto iron oxide nanoparticles

In this study, the adsorption of microcystin-LR onto iron oxide (maghemite) nanoparticles from water was examined. Factors influencing the sorption behavior included microcystin and maghemite concentration, pH, ionic strength, and the presence of natural organic matter. Adsorption of microcystin-LR was strongly affected by pH. The adsorption increased with decreasing pH, with a maximum adsorption around pH 3. Adsorption of microcystin-LR on maghemite was primarily attributed to electrostatic interactions, although hydrophobic interactions may also play a role. The extent of microcystin-LR adsorption onto maghemite increased with increasing ionic strength at pH 6.4, since salt ions screened the electrostatic repulsion between adsorbed microcystin molecules. Adsorption of microcystin-LR was not significantly affected by the presence of Suwannee River Fulvic acid (SRFA) below 2.5 mg/L. However, adsorption decreased at higher SRFA concentrations (2.5–25 mg/L) due to competitive adsorption between SRFA and microcystin-LR for limited sorption sites.     

Microchip micellar electrokinetic chromatography based on one functionalized ionic liquid and its excellent performance on proteins separation

Herein, one water-soluble functionalized ionic liquid (IL), 1-butyl-3-methylimidazolium dodecanesulfonate (BAS), was designed, investigated and successfully applied to microchip micellar electrokinetic chromatography (MEKC) construction. It possessed the properties of both IL and surfactant. A fairly stable pH value approximately 7.4, which was fit to pH values of general biological buffers, was nicely placed at the optimum concentration of 20mM BAS solution. While applying BAS solution as running buffer in poly(dimethylsiloxane) (PDMS) microfluidic systems, significantly enhanced electroosmotic flow (8-fold) and resolutions between analytes were obtained than that using other supporting electrolytes or surfactants. Pure BAS solution could conveniently complete the task of MEKC establishment: not only the ionic strength of the running buffer was guaranteed, but also the analytes adsorption to PDMS surface was conquered, as was demonstrated in the sensitive determination of fluorescent dyes and well-separated protein mixtures.     

Flocculation and stabilization of colloidal silica by the adsorption of poly-diallyl-dimethyl-ammoniumchloride (PDADMAC) and of copolymers of DADMAC with N-methyl-N-vinyl- acetamide (NMVA)

 The stabilization and flocculation behavior of colloidal silica-particles with cationic polyelectrolytes (PE) is investigated. The zetapotentials, diffusion coefficients and flocculation rate constants of silica particles have been measured as a function of the adsorbed amount of cationic polyelectrolytes poly(diallyl-dimethyl-ammoniumchloride) (PDADMAC) of different molar masses and of statistic copolymers of DADMAC and N-methyl-N-vinyl-acetamide (NMVA) of various compositions at different salt concentrations and pH-values. Very fast flocculation due to van der Waals attraction occurs if the zetapotential is small. At low ionic strength this condition occurs just below the plateau of the adsorption isotherms where the surface charges are screened by adsorbed polycations. Additionally with high molecular polycations slow mosaic flocculation is observed at lower PE concentrations. At high ionic strength fast flocculation takes place at low macroion concentration due to the screening of the surface charges by adsorbed polycations and salt ions. At medium concentrations of polycations below plateau adorption slow bridging flocculation is observed. At plateau adsorption the suspensions become stabilized up to high ionic strength. At low salt concentration charge reversal at full coverage with polycations results in electrostatic repulsion. At high ionic strength the particles are stabilized sterically due to the osmotic repulsion of the long adsorbed PE tails. Therefore macroions of high molar mass are necessary to stabilize the suspension at high ionic strength. Received: 27 January 1998 Accepted: 23 March 1988     

Solute distribution and stability in emulsion-based delivery systems: an EPR study

Adsorption and desorption of human serum albumin (HSA) from aqueous solutions on mica were studied using AFM and in situ streaming potential measurements. A quantitative interpretation of these experiments was achieved in terms of the theoretical model postulating a 3D adsorption of HSA molecules as discrete particles. These measurements, performed for various ionic strength, allowed one to determine the coverage of HSA as a function of the zeta potential of mica. This allowed one to determine the amount of irreversibly bound HSA as a function of the ionic strength. It was found that the coverage of irreversibly adsorbed HSA increased from 0.52 mg m(-2) for I=1.3×10(-3) M to 1.6 mg m(-2) for I=0.15M (pH=3.5). The significant role of ionic strength was attributed to the lateral electrostatic repulsion among adsorbed HSA molecules, positively charged at this pH value. This was quantitatively interpreted in terms of the effective hard particle concept previously used for colloid particles. The experimental results confirmed that monolayers of irreversibly bound HSA of a well-controlled coverage can be produced by adjusting the ionic strength of the suspension.     

Adsorption behavior and activity of hexokinase

The study on the adsorption of hexokinase (HK) onto silicon wafers was carried out by means of in situ ellipsometry and atomic force microscopy in the liquid. The thickness values of the adsorbed HK layer determined by both techniques were in excellent agreement and evidenced HK monolayer formation. The adsorption of HK onto Si wafers was favored at low ionic strength, indicating that the adsorption is mainly driven by electrostatic forces, since salt screens not only the segment-segment repulsion but also the segment-surface attraction when the salt concentration increases. The enzymatic activity of free HK and of adsorbed HK was measured as a function of time. Free HK in solution lost activity upon storage. Contrarily, adsorbed HK kept its activity level even after 48 h storage at room temperature. This outstanding behavior was attributed to specific orientation of the HK active site to the solution.