Single-base resolution detection of rotator cuff injury associated MicroRNA by a DSN-triggered Y-shaped dual-circular-probe DNA walker with ICP-MS readout

A DSN-triggered Y-shaped dual-circular-probe DNA walker is developed for RCI-associated miR-29a detection. • The Y-shaped steric hindrance structure effectively suppresses non-specific DSN cleavage, ensuring ultralow background. • The cascade amplification strategy achieves a detection limit as low as 0.26 fM for miR-29a. • The ICP-MS-based Rh readout provides label-free, photobleaching-free, and robust quantification. • The method exhibits excellent single-base mismatch discrimination and reliable performance in serum and cell samples. Rotator cuff injury (RCI) is a prevalent musculoskeletal disorder with limited early diagnostic options, partly due to the lack of sensitive and non-invasive biomarkers. Magnetic resonance imaging (MRI), the current mainstream imaging tool for RCI, suffers from high cost, limited accessibility, and an inability to reflect molecular-level pathological changes before structural damage becomes evident. MicroRNAs (miRNAs) have shown promise as diagnostic indicators, yet their reliable detection in clinical contexts requires methods with ultrahigh sensitivity and single-base resolution. Here, we report a duplex-specific nuclease (DSN)-triggered DNA walker with dual circular probes for highly specific and ultrasensitive analysis of RCI-associated miR-29a by inductively coupled plasma mass spectrometry (ICP-MS). The DNA walker is assembled on magnetic beads, where the walking track comprises two circular probes (c1 and c2) forming a Y-shaped steric hindrance structure that effectively suppresses non-specific DSN cleavage, ensuring an ultralow background. Upon target miR-29a hybridization, DSN specifically cleaves the DNA–RNA hybrid, initiating a cascade amplification involving self-priming, nicking/polymerase cycling, rolling circle amplification, and DNAzyme-mediated cleavage of a rhodium-labeled substrate. The released rhodium is quantified by ICP-MS, providing a photobleaching-free readout. The proposed method achieves a detection limit as low as 0.26 fM with outstanding single-base mismatch discrimination, a wide dynamic range from 1 fM to 100 pM, excellent recoveries in serum (93.2–103.2%), and high correlation with RT-PCR (R 2  = 0.9912) in real cell line samples. This DSN-triggered DNA walker offers a robust, sensitive, and specific platform for RCI-related miRNA detection, holding great promise for early diagnosis and prognosis monitoring of rotator cuff injuries. Download: Download high-res image (78KB) Download: Download full-size image