Localized hybridization chain reaction on self-assembled DNA nanospheres overcomes the kinetics-stability-complexity trilemma in miRNA sensing.

Enzyme-free nucleic acid amplification circuits, such as the hybridization chain reaction (HCR), hold immense promise for molecular diagnostics but are fundamentally constrained by a persistent trilemma in biological applications: the trade-off between reaction kinetics, probe stability, and manufacturing complexity. Here, we overcome this challenge by introducing a Localized HCR Nanosphere (LHCR-NS), a self-assembling DNA nanodevice that leverages spatial confinement to simultaneously accelerate reaction speed, enhance biostability, and simplify fabrication. The LHCR-NS is constructed from a single palindromic DNA strand that spontaneously folds into a core-shell nanostructure, which then immobilizes hairpin probes. This localized architecture concentrates reactants, boosting the HCR kinetics by over an order of magnitude compared to conventional free-solution systems. The compact spherical structure provides steric shielding, rendering the nanoprobe exceptionally resistant to nuclease degradation even in raw serum. This robust platform achieved an attomolar limit of detection (LOD) for miR-21 with single-nucleotide specificity. Its superior stability and biocompatibility enabled real-time, high-contrast imaging of endogenous miRNA fluctuations within living cancer cells. Critically, the simplified one-pot synthesis and assay workflow allowed for the rapid and accurate quantification of miR-21 in clinical serum samples, perfectly discriminating cancer patients from healthy controls (AUC = 1.0). This work presents a new paradigm in DNA nanoprobe design, where architectural simplicity and physical principles, rather than chemical complexity, are harnessed to create powerful tools for both fundamental cell biology and clinical diagnostics.