Highly sensitive detection for endocrine disruptors using human cell biosensors expressing red fluorescence protein mScarlet3

Endocrine-disrupting chemicals (EDCs), such as 17β-estradiol (E2) and Bisphenol A (BPA), disrupt endocrine function and modulate critical physiological processes in the human body, even at low concentrations. This underscores the urgent need for efficient and rapid screening methods capable of detecting trace amounts of these compounds. Here, we generated a gene circuit to enhance EDC detection in whole-cell assays, utilizing classical nuclear Estrogen receptor (ER) response elements and the mScarlet3 red fluorescent protein reporter in human 293T and MCF7 cell lines. To further improve sensitivity, we integrated a galactose-regulated upstream promoter element (GAL4)- upstream activating sequence (UAS) system into these cell-based biosensors, initiating a cascaded amplification platform that achieved a limit of detection (LOD) of 10 pM for both E2 and BPA. Application of these biosensors to environmental samples successfully identified these EDCs at a LOD of 100 pM. The incorporation of cascaded amplifying circuits significantly enhances detection sensitivity and signal output, providing a method for evaluating the toxicity of environmental pollutants and supporting risk assessments.