A tetrahedral DNA nanostructure-based DNAzyme fluorescent nanoprobe for intracellular Mn²⁺ imaging

Manganese (Mn) is an essential trace element in human body and acts as an enzyme cofactor, a neurotoxicant, and an activator of the cGAS-STING pathway. Monitoring intracellular Mn²⁺ is critical yet challenging due to its weak ligand-binding affinity and paramagnetic quenching effect. Here, we report a fluorescent nanoprobe (DS-TDN) that integrates a Mn²⁺-specific DNAzyme (Mn5V) into a tetrahedral DNA nanostructure (TDN) for turn-on imaging of intracellular Mn²⁺. In this design, a fluorophore (FAM) and a quencher (BHQ1) are placed on the DNAzyme and substrate strands, respectively, ensuring low background signal. The TDN scaffold protects the DNAzyme from nuclease degradation and enables transfection-free cellular entry via endocytosis. Upon encountering Mn²⁺, the DNAzyme catalytically cleaves the substrate strand, releasing the quencher and generating a significantly amplified fluorescence signal through multiple-turnover cleavage. The DS-TDN probe exhibits excellent analytical performance, with a detection limit as low as 0.26 nM and a linear range from 1 nM to 0.08 mM. It shows high selectivity against other metal ions and demonstrates reliable performance in complex biological matrices, with recovery rates ranging from 96.0% to 104.0%. Moreover, the probe displays low cytotoxicity and enables successful confocal imaging of Mn²⁺ in living RKO cells. This work provides a versatile and robust molecular tool for monitoring intracellular Mn²⁺, with promising applications in studying Mn²⁺-mediated signaling pathways, particularly in the context of cGAS-STING-driven immunotherapy.