A self-feedback dual-walker nanosystem for sensitive miRNA detection and intracellular imaging

Abnormal expression of microRNA (miRNA) is closely associated with the occurrence and development of cancer and various other diseases. Therefore, obtaining quantitative information on miRNA expression, especially detecting miRNA expression levels in living cells, is of great significance for studying related physiological and pathological processes as well as diagnosing diseases. RNA-cleaving DNAzyme (RCD)-based DNA walkers have shown great potential in the detection of miRNA. However, their application is hindered by low intracellular delivery efficiency, limited catalytic efficiency, and the requirement for additional supply of cofactors.

In this study, we developed a novel self-feedback dual-walker (SFDW) nanosystem, loaded onto urchin-like nano-MnO₂ (UNM), for intracellular trace-level miR-21 imaging. The SFDW nanosystem incorporates a hairpin-structured capture probe and two mutually activating DNA walkers. During the detection process, the capture probe recognizes and binds to miR-21 in the matrix, opens the hairpin structure, and subsequently activates the DNA walkers. The cleavage product of each DNA walker in the nanosystem serves as the activator for the other, thereby establishing a biocircuit (self-feedback circuit) with cyclic amplification effect between the two. This mechanism significantly enhances the signal amplification capability. The UNM carrier not only promotes cellular uptake for the SFDW nanosystem, but supplies endogenous Mn²⁺ cofactors via intracellular glutathione-triggered degradation as well. The SFDW nanosystem achieved a low detection limit of 2.8 pM, and successfully enabled miRNA visualization in living cells.

The self-feedback circuit improved detection sensitivity and reduced the detection limit, while the introduction of UNM enhanced delivery efficiency and provided the necessary cofactors. This proposed strategy can address the limitations of RCD-based DNA walkers in intracellular miRNA detection and holds great potential for clinical diagnosis and early-stage disease screening.