Negative selection assisted isolation of a highly selective DNA aptamer for the detection of 17α-ethinylestradiol

Abstract

Estrogenic compounds, including both natural estrogens such as estrone (E1), 17β estradiol (E2), and synthetic 17α ethinylestradiol (EE2), are widely recognized for their endocrine disrupting effects in the environment. EE2 is a main ingredient in oral contraceptives, and it poses environmental challenges due to its resistance to biodegradation and bioaccumulation in aquatic systems. In this work, EE2 was used as a target to select DNA aptamers, and extensive negative selections were employed using E2 as a counter target to obtain highly selective aptamers. After 18 rounds of selections, two families of aptamers were obtained. Among them, a sequence named EE2-1 has a predicted secondary structure containing a highly conserved loop region connecting two duplex stems. EE2-1 has a Kd of 200 nM based on thioflavin T (ThT) fluorescence spectroscopy and 138 nM based on isothermal titration calorimetry. Using this ThT fluorescence assay, a limit of detection of 40 nM was determined, and its binding to E2 was much weaker. This aptamer provides a promising molecular recognition element for development of biosensors and assays capable of detecting trace levels of EE2.