Synergistic coupling of a spherical nucleic acid DNA walker and a redox cascade for self-calibrated ratiometric detection of endocrine disruptors.

Endocrine-disrupting chemicals (EDCs), such as 17β-estradiol (E2) and bisphenol A (BPA), are pervasive environmental contaminants that pose significant health risks even at trace concentrations. Their on-site monitoring remains challenging due to insufficient sensitivity and stability of conventional detection methods. To address this, we developed a universal ratiometric fluorescence biosensor by integrating a target-activated spherical nucleic acid (SNA) with an enzyme-nanozyme- mediated redox cascade for the ultrasensitive and self-calibrating detection of EDCs. The sensing mechanism is initiated by aptamer recognition, which triggers a Nb. BbvCI-powered DNA walking circuit on gold nanoparticles (AuNPs), resulting in the release of numerous alkaline phosphatase (ALP) molecules. These ALP then catalyze the conversion of ascorbic acid 2-phosphate (AAP) to ascorbic acid (AA), which subsequently reduces CoOOH nanoflakes (CoOOH NFs) in a g-C3N4/CoOOH nanohybrid. This redox cascade quenches the oxidase-mimicking activity of CoOOH NFs, suppresses oxidation-mediated emission at 556 nm, and simultaneously restores the intrinsic fluorescence of g-C3N4 nanosheets at 448 nm. This anti-correlated dual-signal output enables accurate and stable quantification against environmental interference. The biosensor achieved detection limits of 100 pM for E2 and 240 pM for BPA, outperforming conventional colorimetric methods by 2 orders of magnitude. Furthermore, the practical applicability was successfully validated using real water samples yielding high recovery rates and demonstrating excellent reliability. By synergizing SNA amplification with an enzyme-nanozyme-modulated ratiometric sensing strategy, this work provides a robust and generalizable platform for the on-site screening of trace-level EDCs, holding great promise for applications in environmental monitoring and public health diagnostics.