Inger Rönnberg

Bioanalysis with surface plasmon resonance

Abstract Research and development work has made surface plasmon resonance (SPR) into an accurate, sensitive and fast method with several bioanalytical applications. The first commercial instrument has also recently been introduced on the market. The present contribution reviews the use of SPR for biospecific interaction analysis and describes a recently developed SPR instrument. Special attention is paid to the use of a dextran layer on the surface of the thin metal film (gold) used for SPR. It is shown that with such a matrix on the surface, the evanescent electric field outside the metal surface is used more efficiently, which improves the analytical performance of the method. Furthermore, the matrix provides a convenient way of covalently binding biomolecules to the sensing surface, thus providing its necessary biospecificity. The chemistry of the coupling matrix is described as well as the optical and liquid-handling systems used. Finally, a few applications of the SPR instrument are demonstrated.

Adsorption of immunoglobulin G, protein A, and fibronectin in the submonolayer region evaluated by a combined study of ellipsometry and radiotracer techniques

The adsorption of the proteins: immunoglobulin G, protein A, and fibronectin was quantitated in a study applying both ellipsometry and radiotracer techniques. 14C- and 125I-labeled proteins at different concentrations were adsorbed to silica surfaces with three different solid surface tensions. The interaction of antibodies with a preadsorbed antigen film was also investigated. The surfaces were characterized by ellipsometry, contact angle measurements, and scanning electron microscopy. The proteins were characterized by gel electrophoresis. A linear relationship for the change in the ellipsometric parameter Δ and adsorbed amount was found for immunoglobulin G and protein A. A deviation from linearity was found for fibronectin adsorbed to two types of surfaces. The result was interpreted as similarities in the structure of the adsorbed IgG film on the different types of surfaces, whereas the structure of the adsorbed HFN film showed dependence on the solid surface tension. The results were discussed in view of the ellipsometric theories for adsorbed molecules in the submonolayer region. The amounts of adsorbed proteins obtained from the radiolabeling experiments agreed well with those calculated from ellipsometric data.

Chemical vapour deposition of silanes

Abstract Chemical surface modification of oxide-covered surfaces by silanization is a well-known technique in such fields as gas and liquid chromatography, electro-chemistry and immobilization of biomolecules. In the commonly used silanization technique the silane is reacted with the surfaces in a liquid phase. If strict anhydrous conditions do not prevail, however, this technique often results in polymerization, irreproducibility and instability of the silane films. We report here on a gas phase silanization of silicon surfaces at elevated temperatures. The method comprises a washing and surface activation step followed by silanization at about 0.5–1 Nm −2 and 80–190 °C depending on the type of silane. The silanized surfaces were characterized by ellipsometry, contact angle measurements and scanning electron microscopy, which revealed smooth, stable and reproducible silane films of monolayer character. A comparison of surfaces that were silanized in the gas phase with those that were silanized in the liquid phase was also made.

Dextran modified gold surfaces for surface plasmon resonance sensors: immunoreactivity of immobilized antibodies and antibody-surface interaction studies

Abstract Recently, the modification of gold surfaces with covalently bound carboxylated dextran was described, for use in biospecific interaction studies in an analytical system based on surface plasmon resonance (SPR). In this study, the antigen binding immunoreactivity of antibodies immobilized on the modified surfaces was investigated with the SPR-based system. Four different monoclonal antibodies specific against the antigens β 2 -microglobulin, luteinizing hormone, human transferrin, and immunoglobulin E, were covalently attached by amine coupling to the dextran layer and the antigen binding capacities were evaluated for different amounts of bound antibody. High immunoreactivities were obtained with molar activities ranging from 0.6 to 1.5 over a wide range of surface concentrations. Interaction studies between carboxymethylated dextran surfaces and antibodies showed that, in comparison with unmodified gold surfaces, the modified surface had low nonspecific adsorption and high capacity for antibody immobilization.

Immunoglobulin G and secretory fibronectin adsorption to silica: The influence of conformational changes on the surface

Abstract The adsorption isotherms for immunoglobulin G (IgG) and secretory fibronectin (HFN) on silica with two different surface free energies were compared by in situ ellipsometry. The isotherms were obtained by either direct- or successive-addition of the proteins. The influence of the initial concentration, the incubation time, and the ionic strength were investigated. The adsorption isotherms for IgG were also compared with isotherms obtained for IgG interacting with a surface immobilized film of Protein A. A significant difference between the direct- and successive-addition isotherms was found for both proteins on hydrophobic silica, whereas the isotherms essentially coincide for the proteins on hydrophilic silica. The same qualitative behavior was found for both proteins on both types of surfaces for the two different ionic strengths tested. The results were interpreted as time-dependent conformational changes in the adsorbed protein film, where the degree of changes was dependent on the solid surface free energy. These changes were most pronounced on hydrophobic silica.

Flow-injection ellipsometry — an in situ method for the study of biomolecular adsorption and interaction at solid surfaces

Abstract An ellipsometric technique for the study of biomolecular interactions at the solid—liquid interface is described. The technique consists of a flow-injection system and a micro flow cell with low dead volume together with a dynamic ellipsometric set-up. The kinetics of biomolecular adsorption and interactions, as well as the effect of flow rate and rinsing, may be studied in an easy and reproducible way. The system was exemplified by the reversible adsorption of lysozyme on silica surfaces and by the reversible interaction of Immunoglobulin G with an immobilized film of Protein A.

[34] Surface immobilization techniques in combination with ellipsometry

Publisher Summary The rapid development of bioanalytical methods and biosensors involving immobilized enzymes or antibodies focuses interest on the interaction of biomolecules at inorganic surfaces. It is also of importance to use efficient, gentle, and durable immobilization techniques and that the techniques are suited for large-scale production of sensor devices. The function of several surface-sensitive sensors based on electrochemical, optical, and gravimetric methods requires a proper immobilization of biomolecules to inorganic surfaces. Most such surfaces do not carry suitable chemically reactive groups. Such groups may, however, be introduced by chemical surface modification such as silanization. This process is commonly performed by deposition of the silane from a liquid phase. Several problems, such as nonuniformity of the deposited silane film thickness, codeposition of polymeric silane particles, and hydrolytic removal of the deposited film, occur using this method and are not acceptable for optimum performance of the surface-sensitive measuring devices. Vapor phase silanization can overcome some of these problems. The chemical vapor deposition technique is used for alkoxysilanes. The technique is exemplified by the covalent binding of protein A to silanized silica surfaces and the interaction of the immobilized protein A with immunoglobulins. The result is evaluated by in situ ellipsometry, an optical technique based on reflection of polarized light from a surface.

Butyl(3-trimethoxysilylpropyl)disulfide. A new effective reagent for silicon surface modification and protein immobilization; preparation procedure.

Abstract The synthesis of a new reagent for silicone and similar surface modification and protein immobilization - (n-butyl(3-trimethoxysilylpropyl)disulfide) - and its phenylanalog is presented.