A highly sensitive biosensor for estradiol detection in environmental water samples using aptamer conformational switching and RCA-Cas12a amplification

Estradiol poses a potential threat to human health through bioaccumulation in aquatic food chains. The CRISPR-Cas system has revolutionized nucleic acid-based detection. However, adapting CRISPR-based diagnostics for small molecule detection remains a significant challenge. In this study, a biosensor for estradiol detection was developed using an aptamer-based strategy. This biosensor employs a high-affinity estradiol-binding aptamer (CN-Es2), which was previously identified by our group, to construct a “microbead-capture strand-blocker” functional probe. By integrating rolling circle amplification (RCA) with Cas12a-mediated nucleic acid cleavage, the system achieved robust signal amplification, enabling the highly sensitive and specific detection of estradiol. Moreover, this biosensor exhibited a dynamic detection range of 10 pM to 10 nM, with a detection limit of 19.2 pM (5.2 ng/L). Overall, this method provides a rapid and efficient strategy for monitoring trace levels of estradiol in complex matrices, addressing a critical requirement in the context of public food safety. • The “microbead-capture strand-blocker” functional probe based on aptamer ensures high specificity for E2 in complex samples. • A RCA-Cas12a-based biosensor enables ultrasensitive E2 detection. • The biosensor achieves a detection limit of 19.2 pM with a linear range of 50 pM–2 nM. • High recovery rates (89 %–110 %) confirm robustness in wastewater analysis. • This method provides a powerful tool for monitoring food safety.