Triple functional small-molecule-protein conjugate mediated optical biosensor for quantification of estrogenic activities in water samples

Establishing biosensors to map a comprehensive picture of potential estrogen-active chemicals remains challenging and must be addressed. Herein, we describe an estrogen receptor (ER)-based evanescent wave fluorescent biosensor by using a triple functional small-molecule-protein conjugate as a signal probe for the determination of estrogenic activities in water samples. The signal probe, consisting of a Cy5.5-labelled streptavidin (STV) moiety and a 17 beta-estradiol (E-2) moiety, acts simultaneously as signal conversion, signal recognition and signal report elements. When xenoestrogens compete with the E-2 moiety of conjugate in binding to the ER, the unbound conjugates are released, and their STV moiety binds with desthiobiotin (DTB) modified on the optical fiber via the STV-DTB affinity interactions. Signal probe detection is accomplished by fluorescence emission induced by an evanescent field, which positively relates with the estrogenic activities in samples. Quantification of estrogenic activity expressed as E-2 equivalent concentration (EEQ) can be achieved with a detection limit of 1.05 mu g/L EEQ by using three times standard deviation of the mean blank values and a linear calibration range from 20.8 to 476.7 mu g/L EEQ. The optical fiber system is robust enough for hundreds of sensing cycles. The biosensor-based determination of estrogenic activities in wastewater samples obtained from a full-scale wastewater treatment plant is consistent with that measured by the two-hybrid recombinant yeast bioassay.