Microarray Immunoassay for Phenoxybenzoic Acid Using Polymer Encapsulated Eu:Gd₂O₃Nanoparticles as Fluorescent Labels

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photobleaching and spectral overlaps we applied a new type of fluorophore, crystalline europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles, as labels in immunoassay microarrays. The Eu:Gd2O3 nanoparticles synthesized by spray pyrolysis offer narrow red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms). The amino functionalization of the particles was achieved by poly(L-lysine) (PL) encapsulation. The formation of a stable PL shell was confirmed by TEM analysis, colloidal stability studies, and quantification of the surface reactive amino groups. The PL-encapsulated particles were covalently conjugated to antibodies and successfully applied as reporters in a competitive fluorescence microimmunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to pyrethroid insecticides. Microarrays were fabricated by microcontact printing of BSA-PBA in line patterns (10 x 10 microm). Confocal fluorescence microscopy combined with internal standard (fluorescein) calibration was used for quantitative measurements. The microarray immunoassay demonstrated a limit of detection of 1.4 microg L(-1) PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and high-throughput screening.