Effect-Targeted Mapping of Potential Estrogenic Agonists and Antagonists in Wastewater via a Conformation-Specific Reporter-Mediated Biosensor

Wastewater treatment plants (WWTPs) are regarded as the main sources of estrogens that reach the aquatic environment. Hence, continuous monitoring of potential estrogenic-active compounds by a biosensor is an appealing approach. However, existing biosensors cannot simultaneously distinguish and quantify estrogenic agonists and antagonists. To overcome the challenge, we developed an estrogen receptor-based biosensor that selectively screened estrogenic agonists and antagonists by introducing rationally designed agonist/antagonist conformation-specific reporters. The double functional conformation-specific reporters consist of a Cy5.5-labeled streptavidin moiety and a peptide moiety, serving as signal recognition and signal transduction elements. In addition, the conformation recognition mechanism was further validated at the molecular level through molecular docking. Based on the two-step "turn-off" strategy, the biosensor exhibited remarkable sensitivity, detecting 17β-estradiol-binding activity equivalent (E₂-BAE) at 7 ng/L and 4-hydroxytamoxifen-binding activity equivalent (4-OHT-BAE) at 91 ng/L. To validate its practicality, the biosensor was employed in a case study involving wastewater samples from two full-scale WWTPs across different treatment stages to map their estrogenic agonist and antagonist binding activities. Comparison with the yeast two-hybrid bioassay showed a strong liner relationship (r² = 0.991, p < 0.0001), indicating the excellent accuracy and reliability of this technology in real applications.