Aptamer-gated self-feedback CHA/DNAzyme molecular circuit for enzyme-free ultrasensitive fluorescent sensing of P-tau231

A highly sensitive fluorescent biosensor for P-tau231 detection is developed. • Aptamer-gated self-feedback CHA/DNAzyme circuit enables drastic amplification. • 0.45 pg mL −1 detection limit with high specificity against interferents is achieved. • The detection of trace P-tau231in diluted human serums is validated. Phosphorylated tau at threonine 231 (P-tau231) is a clinically informative blood-based biomarker for early Alzheimer's disease (AD), yet its extremely low abundance demands robust signal amplification for sensitive detection of P-tau231. Here, we develop an enzyme-free, target-activated fluorescence assay based on a continuous self-feedback DNA molecular circuit that integrates aptamer recognition, catalytic hairpin assembly, and a Mg 2+ -assisted DNAzyme. P-tau231 binding to an aptamer/ssDNA duplex releases a trigger strand that unlocks a DNAzyme-containing hairpin, initiating an autocatalytic amplification cascade. The activated DNAzyme repeatedly cleaves a fluorophore/quencher-labeled substrate hairpin, converting molecular recognition into a turn-on fluorescence output with substantial signal gain. Under optimized conditions, the proposed strategy enables highly sensitive P-tau231 assay (0.45 pg mL −1 detection limit), while maintaining high specificity against potential interferents. Feasibility in complex matrices is further supported by analysis in diluted human serum. This self-feedback CHA-DNAzyme circuit provides a modular framework for ultrasensitive fluorescence biosensing of low-abundance protein biomarkers relevant to early AD screening. Download: Download high-res image (73KB) Download: Download full-size image