ABSTRACT
The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer.
