Stefan Welin

A wettability gradient method for studies of macromolecular interactions at the liquid/solid interface

A method to make a surface energy gradient along silicon or glass plates, 20 mm long, has been developed. The plates are hydrophobic at one end and hydrophilic at the other with a gradient of increasing wettability in between. The wettability gradient has a sigmoidal character and can be visualized by the use of a capillary rise method. With the use of ellipsometry with sufficient lateral resolution, it is then possible to study quantitative aspects of macromolecular adsorption and interaction at liquid/solid interfaces and to relate continuously the different observed effects to solid surface wettability. In this study the gradient method was applied to the analysis of the surface energy dependence of the adsorption of human fibrinogen, γ-globulin, and lysozyme on the solid surfaces. Detergent-induced desorption of adsorbed fibrinogen and γ-globulin was studied by incubating the gradient plates in Tween 20 and sodium dodecyl sulfate (SDS). Only small amounts of protein were desorbed by incubation in Tween for contact angles higher than 80°. Desorption with Tween was instead maximal at about 70° for fibrinogen. The maximum of Tween-induced desorption was shifted to the hydrophilic side of the gradient for γ-globulin. Tween 20 had very little desorption effect at contact angles lower than 30°. SDS caused effective desorption of fibrinogen and γ-globulin on both the hydrophobic and the hydrophilic sides of the gradient. The desorption induced by addition of 4 M urea and acid buffer (pH 2.3) was also studied and was shown to be maximal at the hydrophilic side of the gradients although there was a considerable amount of protein also desorbed at hydrophobic parts of the gradient. There were other qualitative differences in the desorption pattern of γ-globulin and fibrinogen which may be partly explained by assuming different degrees of surface-induced conformational changes of the adsorbed protein molecules.

Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three‐dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM‐results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.

The interaction of proteins and cells with affinity ligands covalently coupled to silicon surfaces as monitored by ellipsometry

Two methods for the chemical binding of biomolecules to silicon surfaces are described. The first method utilizes an alkyl silane and a nucleophilic reagent to join the biomolecule to the silicon surface; the second method involves crosslinking with glutaraldehyde in order to couple the biomolecule and albumin molecules, which have first been physically adsorbed. The course of binding to the silicon surface has been followed with the aid of ellipsometry. This optical measuring technique estimates the thicknesses of, e.g., organic layers, by measuring the polarization properties of a light beam before and after reflection at surfaces. The method by which the binding of a biomolecule to its corresponding affinity ligand on silicon wafers can be followed with this technique is reported. The systems studied are concanavalin A-Saccharomyces cerevisiae cells, immunoglobulin G-Staphylococcus aureus cells, and an NAD-analog-lactate dehydrogenase. With ellipsometry it was possible to assess how the incubation time and the concentration of the cells and the biomolecules added influenced the results. It was found that an increasing time of incubation and higher concentration resulted in a more complete coverage of the silicon wafer surfaces.

Adsorption of fibrinogen as a measure of the distribution of methyl groups on silicon surfaces

Abstract It is shown that the amount of adsorbed fibrinogen can be used to indirectly determine the wettability of silicon with different amounts of methyl groups on its surface. This observation is used to evaluate surface wettability gradients along silicon surfaces created by a recently developed diffusion method.

Reflectometry in kinetic studies of immunological and enzymatic reactions on solid surfaces

Abstract An optical method is described, by means of which immunological and enzymatic reactions can be followed at a primary level on a solid surface, without labelling procedures. When plane-polarized light is reflected at a solid surface, there is a minimum in reflectance at a certain angle of incidence, the pseudo-Brewster angle. For example, a layer of protein adsorbed on a silicon surface increases the reflectance with increasing amount of adsorbed material. High sensitivity is obtained because of the large difference in refractive index between silicon and organic material; about 0.1 μg cm −2 adsorbed protein can be detected. In a model system of human IgG and anti-human IgG, the primary adsorption of IgG on a hydrophobic surface is first measured, and on this IgG-coated surface the binding kinetics of anti-IgG could be measured. The kinetics of proteolytic degradation of IgG-coated surfaces by trypsin was also investigated.

[35] Coupling of biomolecules to silicon surfaces for use in ellipsometry and other related techniques

Publisher Summary The formation of a biomolecular layer on a silicon surface can easily be followed by ellipsometry. This chapter presents several examples of the use of ellipsometry for the study of biomolecular interactions between receptor-cell and enzyme–coenzyme. By coupling one of the interacting components to the silicon surface the binding of the other can be monitored specifically and sometimes also quantitatively. The immobilization techniques employed all include formation of a hydrocarbon layer on top of the silicon oxide present on all silicon surfaces. By binding with established immobilization methods to this hydrocarbon layer, biomolecules, such as coenzymes, antibodies, or other active proteins can be attached to the silicon surface. The properties of the Si surface should not affect the biomolecules, leading, for instance, to denaturation, and that nonspecific adsorption which might impede any biospecific binding does not occur. The introduction of the hydrocarbon layer significantly limits nonspecific adsorption and thus protects the coupled biomolecules.

REFLECTANCE METHOD FOR IMMUNOASSAY ON SOLID SURFACES

In a solid phase immunoassay, one specifically wants to measure antibody adsorption from a liquid phase onto an antigen covered surface or vice versa. The objective is therefore to characterize and quantify adsorbed organic material on a solid substrate surface. Quantification is the most important task from a clinical point of view. We can either use the rate of adsorption or the total adsorbed amount of material as data. Most methods for quantification of binding reactions on solid surfaces, e. g. radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) (Engwall and Pesce, 1978), do not permit continuous (kinetic) measurement of binding reactions. In clinical studies one is therefore confined to the type of measurements in which the reaction under study either is terminated after a certain time or alternatively allowed to run to an equilibrium. In this communication we will refer to this type of measurements as steadystate measurements, although it is not always an equilibrium situation we study. We will here describe a simple optical technique by means of which it is possible to do either kinetic or steady-state measurements.