Light-Inducible Bispecific DNAzymes Reveal Mitochondrial Fe²⁺/Fe³⁺ Dynamics During Ferroptosis

Mitochondrial iron redox homeostasis plays critical roles in cellular function and disease progression; however, spatiotemporal measurement of Fe²⁺ and Fe³⁺ dynamics in mitochondria remains challenging, largely due to limited analytical tools. Here we report a light-inducible bispecific DNAzyme sensor (LiBD) that enables simultaneous and spatiotemporally controlled imaging of mitochondrial labile Fe²⁺ and Fe³⁺ in living cells and in vivo. LiBD integrated two orthogonal iron-specific DNAzymes into a single DNA scaffold, incorporating photocleavable (PC) linkers, rendering DNAzymes inactive until light-triggered activation. Coupled with mitochondria-targeted nanocarriers, LiBD demonstrated high spatial and temporal resolution for monitoring Fe²⁺ and Fe³⁺ level changes in mitochondria. We revealed remarkable mitochondrial Fe²⁺ accumulation in tumor cells during drug-induced ferroptosis, whereas chemotherapy-resistant tumor cells exhibited substantially decreased Fe²⁺ in mitochondria, both accompanied by slight increases in Fe³⁺. Moreover, an NIR-activatable LiBD demonstrated in vivo visualization of mitochondrial Fe²⁺ and Fe³⁺ level changes during ferroptosis in tumor-bearing mice. Collectively, this work presents LiBD as a powerful platform for investigating mitochondrial iron biology with spatiotemporal precision and provides insights into iron-dependent mechanisms of ferroptosis and chemoresistance.