Electrochemical ultrasensitive detection of miRNA-21 via enzyme-free ROMP amplification.

BACKGROUND: The early and accurate detection of miRNA-21 is of great significance for both lung cancer diagnosis and livestock health monitoring, given its conserved sequence across mammals. Herein, we report an enzyme-free electrochemical biosensor based on surface-initiated ring-opening metathesis polymerization (ROMP) for the ultrasensitive detection of miRNA-21. A thiolated peptide nucleic acid (PNA) probe was immobilized on a gold electrode to capture the target. The formed PNA-RNA hybrid duplex provided a high-density phosphate backbone that served as a coordination anchor for Zr(IV) ions, enabling directional immobilization of acrylic acid monomers via Zr-O-P/Zr-O-C bridges. Subsequent addition of a water-soluble ruthenium catalyst (AquaMet) initiated in situ ROMP of cyclopentenyl ferrocene (CFc) monomers, converting a single hybridization event into long polymer chains densely decorated with electroactive ferrocene units for substantial signal amplification.

RESULTS: Under optimized conditions, the biosensor exhibited a wide linear range from 0.1 fM to 0.1 nM, with an ultralow detection limit of 67 aM. It showed excellent selectivity against single-base mismatches, as well as good reproducibility and storage stability. Importantly, the sensor maintained satisfactory recovery rates in diluted human serum and demonstrated strong anti-interference capability in complex matrices.

SIGNIFICANCE: Owing to the sequence conservation of miRNA-21 across mammals and the mild, enzyme-free nature of the ROMP strategy, this platform holds great potential for both clinical lung cancer screening and on-site health monitoring in livestock and poultry.