Electrochemiluminescence\nBiosensor Based on a Duplex-Specific\nNuclease and Dual-Output Toehold-Mediated Strand Displacement Cascade\nAmplification Strategy for Sensitive Detection of MicroRNA-499

The\ntimely and accurate diagnosis of acute myocardial\ninfarction\n(AMI) is of great significance to reduce mortality and morbidity associated\nwith the condition. Herein, we developed an electrochemiluminescence\n(ECL) biosensor for the detection of the potential AMI biomarker microRNA-499\n(miRNA-499), which was based on duplex-specific nuclease-assisted\ntarget recycling and dual-output toehold-mediated strand displacement\n(TMSD). First, miRNA-499 was converted into a large amount of single-stranded\nDNA through the DSN-assisted target recycling, which was further incubated\nwith the DNA triple-stranded complex (S) to implement TMSD cycles.\nThus, the Ru(bpy)<sub>3</sub><sup>2+</sup>-labeled signal strands\nwere released and captured by the capture probe on the electrode surface,\nresulting in an intense ECL signal. Owing to the prominent cascade\nsignal amplification, the constructed biosensor exhibited a good linear\nresponse to miRNA-499 within the range of 100 aM–100 pM with\na detection limit of 69.99 aM. Furthermore, it demonstrated superior\nselectivity, stability, and reproducibility. In addition, the biosensor\nwas successfully applied to detect miRNA-499 in real human serum samples,\ndemonstrating its potential for nucleic acid detection in the early\ndiagnosis of diseases.