Abstract
The pursuit of advanced mRNA detection methods has been driven by the need for sensitive, accurate approaches that are particularly suited for live-cell analysis. Herein, we proposed a cascaded and localized assembly (CLA) system, integrating branched catalytic hairpin assembly (bCHA) with a localized hybridization chain reaction (LHCR) for enhanced mRNA imaging. The CLA system employed a dual-nanosphere (NS) platform, NSABC and NS12, and the interaction between the target and NSABC initiated the bCHA process and activated a split trigger. The newly generated trigger served as the initiator for the LHCR on NS12, leading to amplified fluorescent signals. Notably, this work introduced the first integration of a splitting strategy in a bCHA-HCR cascaded system, reducing false-positive signals and enhancing specific detection. The dual-NS platform further minimized background noise and improved the reaction kinetics through spatial confinement. As a result, the system achieved a detection limit of 1.23 pM. With these advantages, the CLA system demonstrated successful application in both living cells and clinical tissues, underscoring its potential in biomolecular research and clinical diagnostics.
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