Effect-Based Photoelectrochemical Screening of Estrogenic Activity in Wastewater via Cell-Free ERα-ERE Assembly

Rapid and low-cost screening of estrogenic activity is urgently required for assessing endocrine disruption in complex aquatic environments. However, conventional instrumental methods, such as LC-MS/MS, cannot capture the integrated biological effects of chemical mixtures, whereas whole-cell reporter assays are often limited by matrix-induced cytotoxicity and membrane transport constraints. To address these limitations, we developed a cell-free photoelectrochemical (PEC) biosensor that targets the molecular initiating event of estrogenic toxicity, namely the specific assembly of human estrogen receptor α (hERα) on the estrogen response element (ERE). Mechanistic studies showed that the platform converts ligand-induced interfacial steric hindrance into a sensitive, low-background photocurrent signal, enabling differentiation and quantification of both agonistic and antagonistic effects. The biosensor showed subnanomolar sensitivity, with a half-maximal effective concentration (EC₅₀) of 0.065 nM for 17β-estradiol (E2) and a half-maximal inhibitory concentration (IC₅₀) of 96.5 nM for 4-hydroxytamoxifen (4-OHT). Screening of six representative chemicals produced estrogenic activity rankings that were consistent with binding free energies obtained from molecular docking. Application to real wastewater samples, including domestic, hospital, and aquaculture effluents, yielded E2-equivalent concentrations in good agreement with targeted UPLC-MS/MS measurements. By avoiding the limitations associated with cell-based assays, this platform provides an effect-based method for evaluating estrogenic activity in complex environmental samples and supports next-generation risk assessment of chemical mixtures.