Cell-Free Bioassay for Measurement of Dioxins Based on Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled DNA Probe
- 1 September 2006
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 78 (20), 7138-7144
- https://doi.org/10.1021/ac060442e
Abstract
This study aims to develop a rapid and sensitive cell-free bioassay of dioxins. It is known that dioxin ligand can bind heterodimeric aryl hydrocarbon receptor (AhR) and triggers the formation of the complex of dioxin−AhR, AhR nuclear translocator (ARNT), and dioxin-responsive element (DRE) region of the DNA. The hypothesis of the proposed method is that if FITC were labeled at the DRE sequence, its fluorescence intensity would be enhanced when the complex forms because the interaction interface of the binding components (AhR, ARNT, and DRE) creates a rather hydrophobic condition that is in favor of FITC emission. Effects of modification site of FITC on the DNA probes on binding efficiency between the complex components and fluorescence emission enhancement were evaluated by surface plasmon resonance and fluorescence analysis, respectively. Results showed that the labeling site at the second base at the 5‘ end apart from the core region (5‘-TNGCGTG-3‘) of DRE did not obviously interfere with the binding between the DNA probe and dioxin−AhR/ARNT hybrid but presented significant fluorescence emission enhancement. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the typical toxin in this study. The method had a linear range of 1−100 pM, with detection limit of 0.1 pM (0.64 fg/assay) and coefficient of variation of 5.6% (n = 10, 50 pM TCDD in transformed cytosol). The whole detection cycle was ∼4 h. The method was also used to estimate the toxic equivalents (TEQ) of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD). Measurement of TEQs of the mixture of TCDD, PeCDD, and HxCDD were highly consistent with the predicted data. The average recovery using fly ash extract was ∼93%.Keywords
This publication has 27 references indexed in Scilit:
- A surface plasmon resonance immunosensor for detecting a dioxin precursor using a gold binding polypeptideTalanta, 2003
- Simple and rapid detection method using surface plasmon resonance for dioxins, polychlorinated biphenylx and atrazineAnalytica Chimica Acta, 2001
- Validation and use of the CALUX‐bioassay for the determination of dioxins and PCBs in bovine milkFood Additives & Contaminants, 1998
- Bioimmunoassay of Aryl Hydrocarbon (Ah) Receptor Transformation in Vitro by 2,3,7,8- Tetrachlorodibenzo-p-Dioxin(TCDD)Toxicology Mechanisms and Methods, 1996
- The Aryl Hydrocarbon Receptor ComplexAnnual Review of Pharmacology and Toxicology, 1995
- Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and proteinBiochemistry, 1992
- Polychlorinated Biphenyls (PCBs), Dibenzo-p-Dioxins (PCDDs), Dibenzofurans (PCDFs), and Related Compounds: Environmental and Mechanistic Considerations Which Support the Development of Toxic Equivalency Factors (TEFs)Critical Reviews in Toxicology, 1990
- Monoclonal antibodies for dioxin: Antibody characterization and assay developmentToxicology, 1987
- 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and Related Halogenated Aromatic Hydrocarbons: Examination of the Mechanism of ToxicityAnnual Review of Pharmacology and Toxicology, 1982
- Comparative induction of aryl hydrocarbon hydroxylase activity in vitro by analogues of dibenzo-p-dioxinFood and Cosmetics Toxicology, 1980