Fibrinogen adsorption on three silica-based surfaces: conformation and kinetics

Using AFM (atomic force microscopy) to probe protein conformation and arrangement, and TIRF (total internal reflectance fluorescence) to monitor kinetics, fibrinogen adsorption on three different silica-based surfaces was studied: the native oxide on silicon, acid-etched microscope slides, and acid-etched polished glass. The three are chemically similar, but the microscope slide is rougher and induces AFM tip instabilities that appear as high spots on the bare surface. Fibrinogen's conformation and transport-limited adsorption kinetics are found to be quantitatively similar on all three surfaces. Further, the number of adsorbed proteins in progressive AFM micrographs quantitatively match the coverages measured by TIRF during early adsorption. Surfaces appear full, via AFM, when adsorbed amounts are about an order of magnitude below their true saturation levels (via TIRF) because, above about 0.26 mg/m(2), individual proteins cannot be discerned. The results demonstrate how the appearance of AFM micrographs can be misleading regarding surface saturation. On all three surfaces, fibrinogen is, at most, slightly aggregated, showing limited, if any, surface mobility. The complexities of the microscope slide's surface landscape minimally impact adsorption.     

Irreversible adsorption of triple-helical soluble collagen monomers from solution to glass and other surfaces

The adsorption from various solutions of triple-helical soluble collagen monomers to solid surfaces was studied by labeling the collagen with ¹²⁵1. Adsorption to glass, siliconized glass, and Teflon, from aqueous solutions at various pH and ionic strength, was determined at collagen concentrations from 2 to 25 μg/ml. Adsorption was shown to be irreversible and little dependent on pH and ionic strength but increasing enormously as the surface is made more hydrophobic. Surface denaturation of the collagen by heat results in a substantial loss of material. The kinetics of adsorption suggest that the adsorption process may be selective and that not all collagen molecules which reach the surface are immediately adsorbed. Checking these results with earlier measurements of adsorbed layer thickness, a model for collagen adsorption is proposed.     

Contact angle kinetics of human albumin solutions at solid surfaces

Contact angle kinetics of sessile drops of albumin solution on hydrophilic acetal and hydrophobic FC 721 surfaces were measured using axisymmetric drop shape analysis. Young's equation is used to calculate the solid/liquid interfacial tension from measured contact angles and surface tensions as a function of time. The change in solid/liquid interfacial tension is a result of protein adsorption. It indicates that at the hydrophilic acetal surface the albumin molecules, interact only weakly, whereas the interaction with the hydrophobic FC 721 surface is quite strong.     

Graft polymerization from a silica surface initiated by adsorbed peroxide macroinitiators. I. Adsorption and structure of the adsorbed layer of peroxide macroinitiators on a silica surface

The adsorption features of two peroxide macroinitiators (PMIs) with various functionalities from their semi-dilute solutions on the silica surface were thoroughly investigated in the present work. These investigations include the study of the adsorption kinetics of PMI in diverse solvents and a detailed examination of the adsorbed layer structure with the aid of ellipsometry, scanning force microscopy (SFM), and contact angle measurements. Rearrangements of PMI macromolecules at the solid surface are supposed to be the main reason for the appearance of extremes on the kinetic curves and, besides, have a more pronounceable effect on adsorption rate than their diffusion rate to the surface even at the initial stage of the process. Both island-like and densely packed structures of absorbed layers were revealed by combining contact angle measurements and SFM. Surprisingly, even in the case when saturation of the adsorbed layer is reached, PMI does not completely occupy the substrate surface which is at least particularly reachable for the wetting liquids. PMIs adsorbed at the solid surface are intended for the formation of tethered polymer "brushes" via the initiation of "grafting from" polymerization.     

Solid state 13C MAS NMR as a tool for the study of reactions between compounds adsorbed on porous materials

It is shown that magic angle spinning (MAS) solid state ¹³C NMR spectroscopy is a valuable tool for the study of reactions between compounds adsorbed on porous materials because it allows the direct characterization of surface species. The mobility of the adsorbed species yields high-resolution ¹³C spectra at moderate spinning speeds (4 kHz) from which the reactions can be traced. Catalysis of KMnO₄ oxidation of alcohols and proton transfer by the solid support is demonstrated. Received: 22 July 1996 / Revised: 20 August 1996 / Accepted: 23 August 1996     

Solid state 13C MAS NMR as a tool for the study of reactions between compounds adsorbed on porous materials

It is shown that magic angle spinning (MAS) solid state ¹³C NMR spectroscopy is a valuable tool for the study of reactions between compounds adsorbed on porous materials because it allows the direct characterization of surface species. The mobility of the adsorbed species yields high-resolution ¹³C spectra at moderate spinning speeds (4 kHz) from which the reactions can be traced. Catalysis of KMnO₄ oxidation of alcohols and proton transfer by the solid support is demonstrated. Received: 22 July 1996 / Revised: 20 August 1996 / Accepted: 23 August 1996     

On the Degree to which the Contact Angle is Affected by the Adsorption onto a Solid Surface of Vapor Molecules Originating from the Liquid Drop

ABSTRACT

From surface tensions of liquids and Lifshitz-van der Waals (LW) and Lewis acid-base (AB) surface tension components and the AB electron-acceptor γ⁺ and electron-donor γ^˙ parameters determined by contact angle (θ) measurements (using the Young-Dupré equation for polar systems), the interfacial work of salvation (W_st) between various contact angle liquids (L) and a moderately polar solid (S), such as polymethylmethacrylate (PMMA) could be determined. From these W_SL -values the maximum values of the equilibrium association constant, K_a, are obtained for the adsorption of molecules of liquids, L, onto a solid substratum, S. From the K_a-values and the vapor pressures of the various liquids, the maximum number of liquid molecules adsorbed from the gaseous phase onto the solid surface can be determined, at 20^°C and 76cm Hg ambient atmospheric pressure. This yields the maximum value for the fraction, ?, of the surface area of the solid that will be covered by molecules of the liquid, L, emanating from the liquid drop, via the gaseous state. From these ?-values, using Cassie's approach, the maximum amount, Δθ, can be determined by which the observed contact angle is lower than the ideal contact angle, as a consequence of the coverage of the solid substratum by adsorbed molecules originating from the contact angle liquid.

For most of the contact angle liquids used, the maximum deviation, Δθ, is well under 1^°; for water on PMMA it is about 1½^°.     

A small angle neutron scattering study of the interface between solids and oil-continuous emulsions and oil-based microemulsions

We have measured the small angle neutron scattering (SANS) from slurries of powder in contact with surfactant solutions and emulsions to determine the fluid/solid interfacial structure. The slurry solids consisted either of graphite or pyrites particles; and the fluids were hexadecane containing the robust commercial polyisobutylenesuccinamide (PIBSA) surfactant, or a high internal phase emulsion of aqueous ammonium nitrate in hexadecane stabilised by PIBSA. To resolve the interfacial structure for both systems, combinations of deuterated and protonated materials were used.At low concentration in hexadecane, PIBSA forms a complete monolayer on graphite with a footprint per molecule of 103 Å² and a layer thickness of 19 Å. At higher concentrations, the complete monolayer of footprint is 61 Å² and 30 Å thick indicating compression of the PIBSA chain coil structure. Geometric exclusion effects caused by the stacking of the graphite particles also results in an excess of oil for ca. 160 Å above the surfactant monolayer.For pyrites in contact with surfactant in hexadecane, the oxidised surface layer, while smooth at the oil interface, is diffuse and/or rough at the interface with the bulk sulphide below. There is again a complete monolayer of surfactant adsorbed at the oxide surface, in a relatively compressed state with a footprint of 70 Å², more tightly bound than on graphite. The excess of oil phase above the adsorbed surfactant monolayer is observed for samples with larger pyrites particle sizes but not for a sample with smaller particles. This suggests that the oil excess does arise from purely geometric solid particle packing, but that the local particle surface curvatures are significantly higher than the overall particle size would suggest.The scattering from the pyrites/emulsion interface was modelled by a 30 Å thick monolayer of surfactant coating an oxide surface with a molecular footprint of 123 Å². For the larger particle size samples, there is a 30 Å thick layer of oil above the pyrites particle surface before a bulk emulsion/pyrites mixture is reached.These results extend previous reflectometry experiments on the silicon/emulsion interface, indicating that for stable emulsions the structures are qualitatively similar for three dissimilar solid surfaces. They show that useful results on surfactant structure and emulsion layering at the solid/emulsion and other solid/fluid interfaces can be simply obtained by SANS on powder samples variously contrasted by deuteration. SANS can be applied to a much greater range of solid interfaces than reflectometry since large neutron-transparent single crystals are not required, although the variety of faces in a powdered material degrades the quality of the information.     

Structure of Protein Layers during Competitive Adsorption

The formation of protein layers during competitive adsorption was studied with ellipsometry. Single, binary, and ternary protein solutions of human serum albumin (HSA), IgG, and fibrinogen (Fgn) were investigated at concentrations corresponding to blood plasma diluted 1/100. As a model surface, hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer modified silica was used. By using multiambient media measurements of the bare substrate prior to protein adsorption the adsorbed amount as well as the thickness and refractive index of the adsorbed protein layer could be followedin situand in real time. Under conditions used in these experiments neither IgG nor fibrinogen could fully displace serum albumin from the interface. The buildup of the protein layer occurred via different mechanisms for the different protein systems. Fgn adsorbed in a rather flat orientation at low adsorbed amounts, while at higher surface coverage the protein reoriented to a more upright orientation in order to accommodate more molecules in the adsorbed layer. IgG adsorption proceeded mainly end-on with little reorientation or conformational change on adsorption. Finally, for HSA an adsorbed layer thickness greater than the molecular dimensions was observed at high concentrations (although not at low), indicating that aggregates or multilayers formed on HMDSO plasma polymer surfaces. For all protein mixtures the adsorbed layer structure and buildup indicated that Fgn was the protein dominating the adsorbed layer, although HSA partially blocked the adsorption of this protein. At high surface concentration, HSA/Fgn mixtures show an abrupt change in both adsorbed layer thickness and refractive index suggesting, e.g., an interfacial phase transition of the mixed protein layer. A similar but less pronounced behavior was observed for HSA/IgG. For IgG/Fgn and HSA/IgG/Fgn a buildup of the adsorbed layer similar to that displayed by Fgn alone was observed.     

Solid/liquid interfacial tension as a tool to study stability of lysozyme on adsorption to solid surfaces

This work proposes the use of solid/liquid interfacial tension to study the stability of adsorbed lysozyme films on a solid surface using the contact angle of a liquid at the three phase contact line, in the presence of a denaturant, urea.Results suggest a direct correlation between this method with a standard technique like the fluorescence emission spectra and is measured with the same observable error as in the spectral methods. Further the technique provides a simple and direct handle to evaluate the homogeneity and degree of polarity of protein films on solid surfaces.     

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

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

Solid phase immune reactions as monitored by sedimentation field-flow fractionation

Summary Sedimentation field-flow fractionation was shown to permit the precise evaluation of surface concentrations of human IgG, adsorbed to polystyrene latex spheres of different sizes. Unlike conventional techniques for measuring protein uptake by colloidal substrates, this method allowed a direct evaluation of mass adsorbed per unit area, without the need for potentially destructive labelling reactions. Thus, a four hour adsorption of IgG from a 3–10 fold excess of protein in solution yielded surface concentrations which were 1.4±0.1 mg/m² on a 272 nm latex and 1.9±0.1 mg/m² on a latex with a diameter of 142 nm. The lower value coincided with the estimated monolayer surface coverage. The IgG-PS 272 nm adsorption complex was shown to take up negligible amounts of HSA from a 10 mg/mL solution, while its specific uptake of a polyclonal rabbit anti-human IgG was 2.6 molecules per molecule of adsorbed antigen. The same ratio was found for the smaller particles. The surface concentration of adsorbed second antibody, often crucial in immunodiagnostic quantifications, was therefore found to be significantly enhanced by the increased substrate curvature presented by the smaller particles.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Adsorption of bovine serum albumin at solid/aqueous interfaces

Adsorption of soluble serum proteins on hydrophilic and hydrophobic solid surfaces is important for biomaterials and chromatographic separations of proteins. The adsorption of bovine serum albumin (BSA) from aqueous solutions was studied with in situ ATR-IR spectroscopy, and with ex situ ATR-IR, ellipsometry, and water wettablity measurements. The results were used to quantitatively determine the adsorbed film thickness and surface density of BSA on hydrophilic silicon oxide/silicon surfaces, and on these surfaces covered with a hydrophobic lipid monolayer of dipalmitoylphosphatidylcholine (DPPC). The water contact angles were 5° for silicon oxide, 47° ± 1° for the DDPC monolayer, and 53° ± 1° for the BSA monolayers. At 25 °C, and with 0.01–1 wt% BSA in water, the surface densities range from Γ = 2.6–5.0 mg/m², and the film thicknesses range from d = 2.0–3.8 nm, on the assumption that the film is as dense as bulk protein. These results, and certain changes in the IR amide I and II bands of the protein, indicate that the protein adsorbs as a side-on monolayer, with some flattening due to unfolding or denaturation. The estimated -helical content for protein in buffer solutions is 15% higher than for solutions in water. The adsorption density reaches a steady-state value within 10 min for the lowest concentration, but does not appear to reach a steady-state value after 3 h f‘or the higher concentrations. Adsorption of BSA on a silicon oxide surface covered with a monolayer of DPPC leads to an adsorbed protein film of about half the thickness and surface density than on silicon oxide, but the same contact angle, indicating more protein unfolding on the hydrophobic than on the hydrophilic surface.     

Elimination of Human Chorionic Gonadotropin in Sandwich Enzyme Immunoassay for Human Thyroid-Stimulating Hormone in Serum

Abstract

A method to eliminate human chorionic gonadotropin (hCG) in the sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. hTSH in serum containing hCG was reacted with dinitrophenyl monoclonal mouse anti-hTSH β-subunit IgG₁, and the complex formed between the dinitrophenyl IgG₁ and hTSH was trapped onto affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. hCG in the test serum was largely eliminated by washing the polystyrene balls. Subsequently, the complex on the polystyrene balls was reacted with affinity-purified rabbit anti-hCG Fab′-peroxidase conjugate followed by washing. The complex of the dinitrophenyl IgG₁, hTSH and the conjugate was eluted with dinitrophenyl-L-lysine from the polystyrene balls, to which hCG had been nonspecifically adsorbed, and was trapped onto clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. Peroxidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls in the absence and presence of hTSH was not significantly affected by the presence of up to 75,000 IU of hCG per liter of serum. As a result, serum hTSH could be sensitively measured with little interference by hCG.     

Monolayers at solid–solid interfaces probed with infrared spectroscopy

The sensitivities of infrared spectra of thin adsorbate layers measured in either transmission, internal reflection or external reflection can be greatly increased if a light incidence medium with a high refractive index such as an IR-transparent solid material is used. This increase in sensitivity is due to the strong enhancement of the perpendicular electric field in a thin layer of low refractive index sandwiched between two high refractive index materials. Based on model calculations of a hypothetical sample layer, the influence and optimization of experimental parameters such as incidence angle, sample layer thickness and optical contact between layers are investigated. Under optimized conditions, this enhancement can exceed a factor of 100 when compared to conventional surface IR techniques. In addition, the spectra of sandwiched sample layers are governed by a uniform surface selection rule, such that only the perpendicular vibrational components are enhanced, and they permit a straightforward, substrate-independent analysis of surface orientations. Experimental examples of monolayer spectra of long-chain hydrocarbon compounds adsorbed onto gold and silicon substrates and contacted with a germanium crystal used as the incidence medium demonstrate the simple experimental realization and unprecedented sensitivity of this sandwich technique, and they offer novel insights into the chemistry and structure of monolayers confined and compressed between two solid surfaces. Figure IR reflection spectrum of a monolayer of a fatty acid methyl ester sandwiched between silicon and germanium.     

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10^?7-10^?11 g ml^?1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10^?7-10^?11 g ml^?1.     

A comprehensive study of concepts and phenomena of the nonspecific adsorption of beta-lactoglobulin.

We investigate nonspecific protein adsorption processes by comparing experimentally measured adsorption kinetics of beta-lactoglobulin with mathematical models. The adsorption and desorption behavior of this protein on a hydrophilic glass surface in citrate buffer (pH 3.0), monitored for a large set of different bulk concentrations (0.5x10(-8) M-1.5x10(-6) M) using a supercritical angle fluorescence (SAF) biosensor, is reported. Increasing adsorption rates and overshootings in the beginning of the adsorption are observed as well as a transition to an almost irreversibly bound state of the protein in the long term. Furthermore, rinsing experiments prove that adsorbed proteins abruptly change their desorption behavior from irreversible to reversible when a critical surface coverage theta(crit) is reached. Based on all experimental observations, a mathematical model composed of three adsorbed states differing in their surface affinity is proposed. Terms to account for lateral interactions between surface-bound proteins are included, which yield an excellent fit of the measured kinetics. For the first time, several phenomena that have been discussed in theoretical studies are confirmed by comparing experimental data with a single model.     

Diffusion of cyclohexane in native and surface-modified mesoporous glasses

Diffusion of cyclohexane in mesoporous silica materials with different degrees of surface silanization has been probed by means of pulsed field gradient nuclear magnetic resonance. The self-diffusion coefficients have been measured at various pore fillings from about 10% of one monolayer coverage to complete pore saturation by the capillary-condensed phase. It is found that the surface modification, namely grafting of dimethyloctadecylmethoxysilane molecules to the silica surface, reduces diffusivities of guest molecules as compared to the native sample. The contribution of the Knudsen molecular diffusion to the measured diffusivity has been assessed using the model of fast molecular exchange between the adsorbed phase on the pore walls and the molecules in the gaseous phase in the pore interior. The diffusivity data were correlated with the degree of the surface modification, with the latter being probed by measuring ¹H and ¹³C spectra using magic angle spinning (MAS) solid state NMR, nitrogen adsorption and thermogravimetry.     

Adsorption of Acetone onto MgO: Experimental and Theoretical Evidence for the Presence of a Surface Enolate

A new band at 1640 cm ⁻¹ is revealed by diffuse reflectance FT‐IR spectroscopy of acetone adsorbed on a MgO surface (shown schematically). On the grounds of ab initio quantum‐mechanical calculations, this band is assigned to an adsorbed enolate species. This evidence proves the catalytic role of the metal oxide surface in the condensation reaction mechanism.     

Layer thickness of hydrophobin films leads to oscillation in wettability

In nanobiotechnology, the properties of surfaces are often key to sensor applications. If analytes possess a low tolerance or affinity regarding the sensory substrate (surface), then the setup of mediators may be indicated. Hydrophobins enable biocompatible surface functionalization without significant restrictions of the physicochemical substrate properties. Because of the imperfect formation of hydrophobin films, a high variation in surface properties is observed. In this study, we report on the relation between the film thickness of hydrophobin-coated solid surfaces and their wettability. We found that the wettability of protein-coated surfaces strictly depends on the amount of adsorbed protein, as reflected in an oscillation of the contact angles of hydrophobin-coated silicon wafers. Fusion proteins of Ccg2 and HFBI, representatives of class I and II hydrophobins, document the influence of fused peptide tags on the wettability. The orientation of the first crystal nuclei plays a decisive role in the formation of the growing hydrophobin layers. Here, a simple method of deducing the film thickness of hydrophobin assemblies on solid surfaces is presented. The determination of the static contact angle allows the prediction of which part of the protein is exposed to possible analytes.