A genetically encoded fluorescent whole-cell biosensor for real-time detecting estrogenic activities in water samples

Real-time monitoring of estrogenic activity in the aquatic environment is a challenging task. Current biosensors face difficulties due to their limited response speed and environmental tolerance, especially for detecting wastewater, the major source of estrogenic compounds in aquatic environments. To address these difficulties, this study developed a single fluorescent protein (FP) -based whole-cell bacterial biosensor named ER-Light, which was achieved by inserting the sensing domain of the estrogen receptor (ER) into the FP Citrine and expressing it in the periplasm of Escherichia coli. As designed, ER-Light enables the detection of net estrogenic activity in mixtures, represented by estradiol equivalent concentration (EEQ). ER-Light detects EEQ in 40 s with a detection limit of 4.55 x 10- 7 mu M and a maximum working range of 1.1 x 10-4 mu M, demonstrating sufficient response speed, sensitivity, and working range. In addition, the ER-Light can survive and tolerate wastewater effluent. Satisfactory recoveries (91.0 % to 102.1 %) eliminated concerns about the matrix effect of wastewater. EEQs (Not detected-2.9 x10- 5 mu M) measured by ER-Light from the effluent of 9 wastewater treatment plants validate its practicality in detecting wastewater. This is the first attempt to integrate ER into FP-based biosensors for environment monitoring. Our findings provide valuable design rules for real-time detection of bioactivity effects in the environment, contributing to the safeguarding of ecological and human health.