A fluorescence biosensor with a dual-function DNA probe targeting the activated estrogen receptor for estrogenic activity evaluation

Rapid screening and evaluation of endocrine disruption chemicals including environmental estrogens (EEs) is crucial for environmental safety and public health. Conventional methods such as animal tests and cell assays are costly, time consuming, and hardly reproducible. In this work, a fluorescence biosensor mimicking the molecular interactions in the estrogen receptor (ER) signaling pathway was developed for the rapid evaluation of estrogenic activity of environmental chemicals. The key element of the biosensor is a dual-function DNA probe composed of an ER binding sequence and a dye-binding sequence. The ER binding sequence is part of the estrogen response element in the ER signaling pathway and used to bind the activated ER. The dye-binding sequence consists of six thymine bases which the OliGreen fluorescent dye binds to selectively and thus labels non-covalently. In the presence of an estrogenic chemical, ER is activated and then complexed with the DNA, leading to a reduction in the fluorescence anisotropy of OliGreen. Detection of estradiol produced a dose-response curve with an EC₅₀ of 3 nM and lower limit of 0.5 nM, whereas a known ER antagonist did not show any response. Five emerging contaminants including resorcinol bis(diphenyl) phosphate (RDP), triphenyl phosphate (TPHP), tris (2-chloroisopropyl) phosphate (TCIPP), perfluoro-nonenoxybenzene sulfonate (OBS) and perfluorooctanoic acid (PFOA) were evaluated by the biosensor, and the results were consistent with those of the cell assays. Detection of RDP, TPHP and OBS in spiked river water samples resulted in recovery rates of 103%, 93%, and 98%, respectively. The biosensor detection is significantly faster, more robust and easier to carry out than the cell assays, and may provide a new high throughput technique for the screening of estrogenic chemicals and environmental contaminants.