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Estradiol (E2) and related estrogens are emerging environmental contaminants that may adversely affect the health of humans, animals, and ecosystems. Many aptamers have been reported for the detection of E2, and our lab recently selected a series of high-affinity and short DNA aptamers that showed various binding orientations to E2, leading to different selectivity patterns. In this work, we report that using SYBR Green I (SGI) as a fluorescence probe, up to 200% fluorescence increase was achieved upon titration of E2 to these aptamers. Such enhancement was the highest among all reported small molecule binding aptamers using SGI for signal generation, although some metal-binding DNA can achieve even higher enhancement. By gradually shortening the stem region of an E2 binding aptamer, we concluded that the enhanced fluorescence was from the aptamer binding pocket upon target binding instead of from the duplexed stem region. Comparison was also made with a few other aptamers including those for caffeine, quinine, uric acid and cortisol, and none of them showed more than 20% fluorescence change. Using the SGI method, the detection limit was calculated to be 2.4 nM E2. We attributed the large fluorescence enhancement to the hydrophobic nature of E2 and the high-affinity binding of the aptamers. This study provides insights into the aptamers that can use SGI for their binding assays and biosensor development.