Nanosphere-antibody conjugates with releasable fluorescent probes

Surface-functionalized, probe-containing, polymeric nanospheres with diameters between 10 nm and 40 nm (depending on the probe) were used to provide a fluorescent endpoint for nonextractive immunoassay. The probes inside the nanospheres were lanthanoid ions. Methyl methacrylate was used as the monomer to reduce random adsorption of proteins onto the nanosphere surfaces. The acid-group surface functionalization allowed the nanospheres to be conjugated to the amine groups on the antibodies (IgG). The surfaces were further functionalized with alcohol and ester groups to improve the suspension characteristics of the nanospheres. As many as four probe ions may be quantified simultaneously, although only three with a single ligand. Co-measurement of four probes required a combination of two ligands. The intensity of the fluorescence produced by these complexes allows detection with a sensitivity equivalent to enzyme-linked immunoassay.