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Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label

Fan, Ziyan, Keum, Young Soo, Li, Qing X., Shelver, Weilin L., Guo, Liang-Hong
Journal of environmental monitoring 2012 v.14 no.5 pp. 1345
DNA, antibodies, antigens, benzopyrene, cross reaction, detection limit, equipment performance, estradiol, ethers, fluorescent dyes, fluorescent labeling, glass, immunoassays, microarray technology, pollutants, proteins, river water, screening, water pollution
Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC₅₀ and calculated limit of detection (LOD) are 0.32 μg L⁻¹ and 0.022 μg L⁻¹ for E2, 37.2 μg L⁻¹ and 24.5 μg L⁻¹ for BaP, and 31.6 μg L⁻¹ and 2.8 μg L⁻¹ for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368–375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.