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

Abstract 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.