Cell-free bioassay for measurement of dioxins based on fluorescence enhancement of fluorescein isothiocyanate-labeled DNA probe.
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 approximately 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 approximately 93%.
You
Fan
F
Joint Research Group on Analytical Pathogen Microbiology Wuhan Institute of Virology, Wuhan 430071, China.
Zhou
Ya-Feng
YF
Zhang
Xian-En
XE
Huang
Zhi
Z
Bi
Li-Jun
LJ
Zhang
Zhi-Ping
ZP
Wen
Ji-Kai
JK
Chen
Yuan-Yuan
YY
Jiang
Gui-Bin
GB
Zheng
Ming-Hui
MH
D001681:Biological Assay / Q000379:methods*; D002474:Cell-Free System; D003031:Coal; D015342:DNA Probes / Q000032:analysis* / Q000737:chemistry; D004147:Dioxins / Q000032:analysis*; D005452:Fluoresceins / Q000032:analysis* / Q000737:chemistry; D005453:Fluorescence; D017879:Isothiocyanates / Q000032:analysis* / Q000737:chemistry; D012997:Solvents; D020349:Surface Plasmon Resonance
D003031:Coal; D015342:DNA Probes; D004147:Dioxins; D005452:Fluoresceins; D017879:Isothiocyanates; D012997:Solvents