Submitted to: Journal of Environmental Monitoring
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/14/2012
Publication Date: 5/1/2012
Publication URL: handle.nal.usda.gov/10113/54134
Citation: Fan, Z., Keum, Y.S., Li, Q.X., Shelver, W.L., Guo, L.-H. 2012. Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label. Journal of Environmental Monitoring. 14:1345-1352. Interpretive Summary: Environmental organic contaminants have drawn a great deal of concern in recent years because many of them are persistent in the environment, bio-accumulative, and highly toxic. The three classes of organic contaminants studied in this work, polybrominated diphenyl ethers, polycyclic aromatic hydrocarbons, and environmental estrogens, are commonly present in the environment. A double-stranded DNA was first attached to the antibody. Fluorescent dyes were then allowed to bind to the DNA, forming a dye/DNA conjugate. Because the dye can bind to DNA at high ratios, the antibody is labeled with a large number of fluorescent dyes through the DNA. In this report, we describe our work on implementing DNA/dye conjugates to multiple labeled antibodies to increase the sensitivity of fluorescence immunoassays on protein microarrays, and to realize simultaneous detection of multiple environmental chemicals in one sample. Unlike fluorescent nanoparticles, the DNA/dye conjugation does not require any chemical synthesis. In addition, fluorescence detection of the conjugate can be performed on standard microarray scanners.
Technical Abstract: 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 antibody labels to increase the fluorescence signal and sensitivity. Epoxy modified glass slides were selected as the substrate for the production of 4x4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17B-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetra-bromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC50 and calculated limit of detection (LOD) are 0.32 ug/L and 0.022 ug/L for E2, 37.2 ug/L and 24.5 ug/L for BaP, 31.6 ug/L and 2.8 ug/L for BDE-47, respectively. The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, validating the assay. 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.