High-density immobilization of antibodies onto nanobead-coated cyclic olefin copolymer plastic surfaces for application as a sensitive immunoassay chip.

Our research efforts have been devoted to development of nanobead multilayer-based sensitive immunoassays on cyclic olefin copolymer (COC) plastic surfaces. To facilitate nanobead attachment and impart antibiofouling properties to a COC substrate, we used an amphiphilic copolymer comprising benzyl, polyethylene glycol, and reactive ester moieties to coat the hydrophobic COC surface in an aqueous environment. Subsequently, NH2-modified polystyrene nanobeads were reacted with the polymer-coated COC surface and further assembled into multilayers that increased the overall surface area available for attaching capture antibodies. After treatment of the nanobead multilayers with an amine-reactive homobifunctional crosslinker, a model capture antibody (anti-rabbit IgG) was covalently immobilized onto the activated surface of nanobeads. Finally, a sandwich immunoassay was carried out using rabbit IgG as a target analyte and rhodamine-labeled anti-rabbit IgG as a probe. Compared with a nanobead-free, polymer-coated COC surface, the nanobead multilayer-based immunoassay exhibited ~4-fold higher fluorescence intensity. In addition, our nanobead-based assay system exhibited a wide dynamic range of detection (0.1 to 1,000 ng/mL) and high specificity for rabbit IgG. Furthermore, much better detection sensitivity for rabbit IgG was attained in the nanobead multilayer-based immunoassay than with a conventional ELISA system (0.1 ng/mL versus 10 ng/mL), indicating the potential value of the proposed immunoassay system in plastic-based portable biochip applications.