<i>In Vivo</i> Molecular Imaging of Nickel Ions at Inflammatory Sites Using Programmable Allosteric DNA Nanomachine with Dual-Enzyme-Mediated Signal Amplification

Accurate and <i>in situ</i> visualization of nickel ions (Ni²⁺) at inflammatory sites is crucial for early diagnosis, progression monitoring, and targeted intervention of nickel-induced pathological conditions. However, no reports have been reported on the <i>in situ</i> detection of Ni²⁺ at inflammatory sites. Herein, a programmable allosteric DNA nanomachine (ES-AP-tFNA) with dual-enzyme-mediated signal amplification for <i>in vivo</i> molecular imaging of Ni²⁺ at inflammatory sites was developed. ES-AP-tFNA can be sequentially cleaved by apurinic/apyrimidinic endonuclease 1 (APE1), which is highly expressed in inflammatory cells, and by the Ni²⁺-dependent DNAzyme, thereby enabling the <i>in situ</i> detection of Ni²⁺ within inflammatory microenvironments. First, the locked DNAzyme (S5) is released following the cleavage of the AP site in ES-AP-tFNA by APE1. Subsequently, the DNAzyme hybridizes with S6 through base complementarity and, in the presence of Ni²⁺, specifically cleaves riboadenosine (rA), resulting in the separation of the fluorophore and quencher. Furthermore, the liberated DNAzyme can repeatedly cleave rA sites on multiple S6 strands, enabling fluorescence signal amplification through a catalytic recycling mechanism. Experimental results demonstrate that ES-AP-tFNA exhibits high sensitivity (LOD: 0.08 U/mL for APE1, 0.25 μM for Ni²⁺) and specificity. Furthermore, ES-AP-tFNA can rapidly and specifically visualize Ni²⁺ in inflammatory cells at the implantation sites without causing significant organ toxicity in mouse models implanted with a nickel alloy.