Rational design of DNAzyme walker driven self-replication CHA amplification cascades for aptamer-based and highly sensitive fluorescent bioassay

A highly sensitive and aptamer-based fluorescent assay for flunixin is established. • DNAzyme walker/self-replication CHA circuit enables significant signal amplification. • Selective detection of fluxnixin in complex milk sample matrix by this assay is verified. Flunixin is a widely used veterinary non-steroidal anti-inflammatory drug, and its improper administration can lead to residue contamination in animal-derived foods, which poses a pressing need for rapid and sensitive residue monitoring. Herein, we develop an aptamer-based fluorescent assay that integrates a Mg 2+ -dependent DNAzyme walker with an autocatalytic catalytic hairpin assembly (CHA) circuit to achieve cascade signal amplification for flunixin determination. In the presence of flunixin, target binding to an aptamer/DNAzyme duplex activates the DNAzyme, which repeatedly walks on gold nanoparticle (AuNP) tracks and cleaves rA-containing substrate hairpins, continuously releasing single-stranded trigger sequences. These triggers initiate a three-hairpin CHA process to generate Y-shaped DNA products and simultaneously open FAM/Dabcyl-quenched reporter hairpins, restoring fluorescence. Notably, the Y-shaped products regenerate trigger-equivalent sequences, driving a self-replication CHA cycle that enables sustained reporter opening and markedly enhanced fluorescence output. Under optimized conditions, the proposed strategy achieves a detection limit of 0.41 nM with good selectivity against potential interferents. The assay was further validated by determining flunixin in milk samples, supporting its applicability for sensitive analysis of small-molecule veterinary molecule residues. Download: Download high-res image (44KB) Download: Download full-size image