Microarray Immunoassay for Phenoxybenzoic Acid Using Polymer Encapsulated Eu:Gd2O3 Nanoparticles as Fluorescent Labels

• Published in: Analytical chemistry (Washington) 2005-11, Vol.77 (21), p.6864-6873
• Main Authors: Nichkova, Mikaela, Dosev, Dosi, Gee, Shirley J, Hammock, Bruce D, Kennedy, Ian M
• Format: Article
• Language: English
• Subjects: Amino acids; Analytical chemistry; Antibodies; Benzoates - chemistry; Biological and medical sciences; Biosensors; Biotechnology; Chemistry; Dyes; Europium - chemistry; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Gadolinium - chemistry; General, instrumentation; Immunoassay - instrumentation; Immunoassay - methods; Methods. Procedures. Technologies; Microarray Analysis - methods; Miscellaneous; Nanoparticles; Nanoparticles - chemistry; Phenyl Ethers - chemistry; Polylysine - chemistry; Polymers; Polymers - chemistry; Spectrometric and optical methods; Various methods and equipments
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac050826p

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 × 10 μm). 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 μg 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.