Immobilization of antibodies on a photoactive self-assembled monolayer on gold

Abstract

This paper presents a strategy for immobilizing biomolecules on a photoactivable surface. A self-assembled monolayer is prepared by adsorbing an ω-functionalized dialkyl disulfide on gold. Functional groups of this monolayer are converted in two steps into a benzophenone derivative with an overall yield of 50 ± 10%. Several independent techniques (ellipsometry, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, radiolabel assay, and autoradiography) characterize the reaction and photoimmobilization of antibodies on this surface. The photoimmobilized antibodies cover the surface as a homogeneous and dense monolayer that could not be disrupted by vigorous washing with the detergent Tween 20. Immunoassays demonstrated specific recognition of the immobilized immunoglobulins as measured by their complexation with alkaline phosphatase-linked antibodies. The method of photoimmobilization used here leads to a homogeneous single layer of IgGs, in which the proteins maximize their contact with the surface. Residual adsorption of IgG on the nonirradiated surface of benzophenone remains one limitation of this approach. Progressively higher coverages of IgGs on the surface did not lead to strictly proportional changes of the biological activity of these surfaces, probably because of interactions between the IgGs in the film. This method of photoimmobilization is nonetheless useful as an experimental system to immobilize other proteins because it is simple, flexible, and efficient.