Smart Hairpins@MnO2 Nanosystem Enables Target-Triggered Enzyme-Free Exponential Amplification for Ultrasensitive Imaging of Intracellular MicroRNAs in Living Cells

Sensitive and specific imaging of microRNA (miRNA) in living cells is of great value for disease diagnosis and monitoring. Hybridization chain reaction (HCR) and DNAzyme-based methods have been considered as powerful tools for miRNA detection, with low efficient intracellular delivery and limited amplification efficiency. Herein, we propose a Hairpins@MnO₂ nanosystem for intracellular enzyme-free exponential amplification for miRNA imaging. The enzyme-free exponential amplification is based on the synergistic cross-activation between HCR and DNAzymes. The MnO₂ nanosheets were employed as the carrier of three kinds of hairpin DNA probes and further provided appropriate Mn²⁺ as DNAzyme cofactors in the living cell. Upon entering cells and in the presence of highly expressed glutathione (GSH) in tumors, MnO₂ is reduced to release Mn²⁺ and the three kinds of hairpin DNA probes. In the presence of target miRNA, the released hairpin DNA H1 and H2 probes self-assemble via HCR into the wire-shaped active Mn²⁺-based DNAzymes which further catalyze the cleavage of H3 to generate numerous new triggers to reversely stimulate HCR amplifiers, thus offering tremendously amplified Förster resonance energy transfer readout. The method has a detection limit of 33 fM, which is 2.4 × 10⁴ times lower than that of the traditional HCR system. The developed method also has a high specificity; even miRNAs with a single base difference can be distinguished. Live cell imaging experiments confirmed that this Hairpins@MnO₂ nanosystem allows accurate differentiation of miRNA expression of cancer cells and normal cells. The method holds great potential in biological research of nucleic acids.