A sensitive and visual colorimetric biosensor for liver cancer-associated miRNA using Exo-III assisted target...

The accurate detection of liver cancer-associated microRNAs (miRNAs) remains challenging due to their low abundance and high sequence homology. Herein, we report a colorimetric biosensor integrating Exo-III-assisted target recycling with a cyclization-functional DNAzyme walker for sensitive and highly specific miRNA detection. Notably, the assay incorporates two distinct miRNA recognition events: first, the target miRNA hybridizes with a stem-loop probe to trigger Exo-III-mediated cyclic cleavage; second, the released initiator strand activates the DNA walker on magnetic beads, where the miRNA must hybridize with a specific binding site on the tracker strand to reconstitute the split DNAzyme catalytic core. This dual-recognition mechanism dramatically enhances specificity, enabling the sensor to discriminate single- and double-nucleotide mismatches with high reliability. Coupled with Ag⁺-aptamer-mediated signal transduction, the platform produces a visible color change for naked-eye readout. With cascade amplification, the assay achieves a detection limit of 2.7 fM and a linear range of 5 fM to 100 pM. Successful spike-recovery tests in human serum (96.2-104.4%) suggest its potential for clinical application. This portable, visual colorimetric strategy offers a promising solution for early liver cancer screening.