Enhanced through‐bond energy transfer‐based bioorthogonal probe enables catalytic‐amplified and sensitive detection of microRNA‐21 for clinical lung cancer diagnosis

The dysregulation of microRNA (miRNA) expression is closely linked to the pathogenesis of lung cancer, rendering the quantification of trace miRNAs clinically indispensable. However, achieving ultrasensitive detection of miRNAs in complex biological matrices, including living cells, tissues, and blood serum, remains significant challenges. To address this, we developed a fluorogenic detection platform for miR‐21 based on tetrazine‐mediated transfer (TMT) reactions. While TMT strategies have been previously explored, we report a refined probe design optimized through computational screening. By minimizing the energy gap (Δ E ) between the fluorophore's emissive state and the tetrazine's dark state, we achieved an efficient quenching mechanism with a low background (fluorescence quantum yield <0.01). By integrating hybridization‐mediated target recycling with the inverse electron demand Diels‐Alder reaction, the Inverse Diels‐Alder Cycloaddition Reaction (IDCR) probe achieves signal amplification. Although the theoretical catalytic turnover is significantly enhanced at low target concentrations, the practical detection limit is primarily governed by the high signal‐to‐background ratio afforded by our optimized probe. This mechanism yields an ultralow limit of detection (3.58 × 10 −18 M) with a broad linear range (100 aM to 100 nM). Building upon foundational bioorthogonal chemistry, the IDCR probe enables high‐contrast imaging in living cells and tissues and distinguishes lung cancer patients from healthy individuals.