Oriented immobilization of immunoglobulin G onto the cuvette surface of the resonant mirror biosensor through layer-by-layer assembly of multilayer films

A new method for oriented immobilization of immunoglobulin G (IgG) onto the cuvette surface of the resonant mirror biosensor through layer-by-layer (LBL) assembly of multilayer films composed of avidin/gold nanoparticles (GNp)/protein A/IgG was developed. First, avidin was added in the biotin cuvette, and then injected GNp, followed by the injection of protein A for oriented immobilization of IgG. The rinsing with PBS was applied at the end of each assembly deposition for dissociating the weak adsorption. Second, IgG was added in the protein A-coated cuvette, and regenerated by incubation with 0.1 M glycine-HCL buffer. Third, different concentrations of IgG were measured by repeating the second process. Film assembling and properties of the interaction between protein A and IgG were studied by resonant mirror biosensor and electrochemical measurements. Results confirmed that IgG was successfully oriented on the protein A-coated cuvette Surface by LBL assembly of multilayer films. The interaction response was dose-dependent which showed a linear range of 0.1 - 1.6 g L-1 IgG, with a detection limit of 8.7 mg L-1 estimated at a signal-to-noise ratio of 3. Moreover, the assay for oriented immobilization of IgG exhibited a good reproducibility and a favorable reusability. This method can provide a promising platform for fabricating immunoassay and immunosensor systems, protein reactors or protein-modified substrates, and affinity probes. (c) 2007 Elsevier B.V. All rights reserved.