Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.

Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.

We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.

Immobilization of immunoglobulins on silica surfaces. Stability.

The development of new immunosensors based on surface-concentration-measuring devices requires a stable and reproducible immobilization of antibodies on well-characterized solid surfaces. We here report on the immobilization of immunoglobulin G (IgG) on chemically modified silica surfaces. Such surfaces may be used in various surface-oriented analytical methods. Reactive groups were introduced to the silica surfaces by chemical-vapour deposition of silane. The surfaces were characterized by ellipsometry, contact-angle measurements and scanning electron microscopy. IgG covalently bound by the use of thiol-disulphide exchange reactions, thereby controlling the maximum number of covalent bonds to the surface, was compared with IgG adsorbed on various silica surfaces. This comparison showed that the covalently bound IgG has a superior stability when the pH was lowered or incubation with detergents, urea or ethylene glycol was carried out. The result was evaluated by ellipsometry, an optical technique that renders possible the quantification of amounts of immobilized IgG. The results outline the possibilities of obtaining a controlled covalent binding of biomolecules to solid surfaces with an optimal stability and biological activity of the immobilized molecules.

Immobilization of immunoglobulins on silica surfaces. Kinetics of immobilization and influence of ionic strength.

The kinetics of, and the influence of ionic strength on, the immobilization of rabbit immunoglobulin G (IgG) on different types of well-characterized silica surfaces were investigated. Adsorptive immobilization was compared with covalent attachment via thiol-disulphide exchange reactions. The amount of immobilized IgG on five different types of silica surfaces as a function of IgG concentration, at two different ionic strengths, was determined. The IgG-solid-surface interaction involved different types of interaction forces, depending on the surface chemistry of the solid surface. The solid-surface chemistry is an important parameter determining the immobilized amount of IgG. When conditions for covalent attachment of IgG to the surfaces were fulfilled, the IgG showed high affinity and the immobilized amount of IgG showed a fast saturation. Changes in ionic strength showed no significant influence on the kinetics of immobilization on these surfaces. The amount of covalently attached IgG was partially ionic-strength-dependent, indicating that adsorptive interactions were involved. The results are of fundamental interest for the development of new immunosensors based on surface-concentration-measuring devices.