Tandem-Gated DNA Nanomachine Enables In Vivo Visualization of Mitochondrial Nickel Ions from Nickel Alloy Implants.

No approach has been reported for the in vivo detection of mitochondrial nickel ions (Ni2+), despite their crucial importance for elucidating the early timing, dynamic progression, and potential intervention windows of nickel-induced mitochondrial damage. Therefore, a tandem-gated DNA nanomachine (C-12S-Zyme-tFNA) that integrates spatially resolved sequential activation and enzymatic recycling amplification was developed for the accurate in vivo imaging of mitochondrial Ni2+. C-12S-Zyme-tFNA employs mitochondrial cytochrome c (Cyt c) and 12S rRNA as sequential endogenous triggers, with Cyt c recognition initiating the first conformational unlocking and 12S rRNA subsequently releasing the preinhibited Ni2+-dependent DNAzyme, thereby enabling spatially confined and sequentially activated imaging of mitochondrial Ni2+in vivo. The nanoprobe is activated via stepwise recognition of Cyt c, 12S rRNA, and Ni2+, which triggers a cascade of conformational rearrangements that progressively separate the fluorophore from its quencher, ultimately yielding a strong fluorescence readout. Experimental results demonstrate that C-12S-Zyme-tFNA affords a low detection limit (LOD = 0.05 μM) and demonstrates exceptional selectivity for Ni2+ over a panel of relevant metal ions. In particular, C-12S-Zyme-tFNA not only enables precise detection of mitochondrial Ni2+ at the cellular level but also permits in vivo visualization of Ni2+ in foot, subcutaneous, and intraosseous nickel alloy implants, with no appreciable organ toxicity detected.