A sequentially activated DNA biosensor for in vivo molecular imaging of nickel ion release from nickel alloy implants

Existing fluorescent probes for nickel ions (Ni²⁺) are inadequate for in vivo monitoring of Ni²⁺ release kinetics and deep-tissue tracing of nickel alloy implants intended for clinical use. Therefore, in this study, a sequentially activated DNA nanomachine (18S-Zyme-tFNA) was developed for in vivo imaging of cytoplasmic Ni²⁺.

The 18S-Zyme-tFNA utilizes a Ni²⁺-dependent DNAzyme as its core recognition module. Its catalytic domain is pre-hybridized with a lock strand complementary to 18S rRNA, thereby maintaining the DNAzyme in an inactive state prior to cytoplasmic entry. Upon cellular internalization, the abundant endogenous 18S rRNA competitively displaces the lock strand via sequence-specific hybridization, restoring the catalytic conformation of the DNAzyme. In the presence of Ni²⁺, the activated DNAzyme site-specifically cleaves the riboadenosine (rA) residue within the substrate strand, inducing spatial separation of the fluorophore and quencher and consequently eliciting a fluorescence signal. Critically, each enzyme molecule catalyzes multiple substrate cleavage events, thereby achieving robust signal amplification via an enzymatic turnover mechanism. Experimental results demonstrate that 18S-Zyme-tFNA achieved a low limit of detection (LOD = 0.09 μM) and demonstrated excellent selectivity for Ni²⁺ over other metal ions. In particular, 18S-Zyme-tFNA enabled in vivo visualization of Ni²⁺ in foot, subcutaneous, and intraosseous nickel alloy implants, with no significant organ toxicity observed.

This study develops a sequentially activated DNAzyme-tFNA biosensor that enables selective and amplified imaging of cytoplasmic Ni²⁺ in living systems. By integrating 18S rRNA-mediated intracellular activation with Ni²⁺-dependent catalytic signal generation, the platform improves the specificity and biological applicability of DNA-based metal-ion sensing. Importantly, it extends Ni²⁺ imaging from cells to mammalian nickel alloy implant models, providing a useful tool for evaluating implant-associated nickel release and biosafety.