Localized Dual-Cycle Amplification Integrating Entropy-Driven Reaction and Catalytic Hairpin Assembly for miRNA Imaging in Living Cells and Tissues

Intracellular microRNA (miRNA) detection plays a crucial role in cancer diagnosis and treatment monitoring. However, current methods face challenges in sensitivity, specificity, and cellular application. Here, we present a novel localized dual-cycle amplification (LDA) system integrating localized entropy-driven DNA circuit reaction (LEDR) and localized catalytic hairpin assembly (LCHA) for sensitive miRNA imaging in living cells. This approach employs a dual-nanosphere platform functionalized with EDR and CHA units. The presence of target miR-155 initiates a cascade reaction of LEDR-LCHA process. The LDA system demonstrates several advantages: (1) enhanced sensitivity with a detection limit of 1.42 pM for miR-155; (2) enhanced reaction kinetics due to localized strategy, reaching signal plateau within 15 min under in vitro conditions; (3) efficient cellular uptake without transfection reagents and improved stability in biological fluids. The system successfully monitored intracellular miR-155 levels and discriminated between cancer and normal samples in both living cells and clinical tissues. This LDA system achieves a synergistic integration of sensitivity, rapid response, and practical applicability for intracellular miRNA detection, offering a robust platform for cancer diagnostics.