Engineering a programmable cascaded DNAzyme platform for sensitive detection of tobramycin via ligand-induced assembly

A novel cascade dual-DNAzyme amplification platform was engineered. • The aptasensor utilized a non-competitive, strand displacement-free transduction mechanism. • The aptasensor exhibits excellent analytical performance with the LOD of 21.0 nM. • Practical applicability was validated in real food samples Tobramycin (TOB), a widely employed broad-spectrum antibiotic in animal husbandry, leaves residual traces that exert inevitable adverse effects on human health, including kidney impairment, neuromuscular blockage, respiratory paralysis, and cross-allergic reactions. Herein, we constructed a novel ligand-induced assembled E6-split DNAzyme cascaded 8–17 DNAzyme aptasensor and applied it to the highly sensitive detection of TOB. In this sensing design, we incorporated the aptamer sequence with a short stem into the input strand. In the absence of the target TOB, the structurally flexible aptamer-programmed input strand fails to form a stable complex with the E6-split DNAzyme. Upon binding of TOB to the aptamer, the input strand undergoes a transition to a compact and rigid conformation, thereby forming a stable three-strand complex consisting of the input strand, E6-split DNAzyme 1, and E6-split DNAzyme 2; this complex assembles a catalytically active domain and restores the substrate cleavage activity. Upon activation, the DNAzyme cleaves a rationally designed hairpin DNA containing an RNA cleavage site to release an extra 8–17 DNAzyme sequence. The liberated DNAzyme subsequently drives the continuous cleavage of fluorophore-quencher labeled reporter probes, resulting in the progressive accumulation of fluorescence signals. Under the optimized reaction conditions, the detection range displays a well-defined linear relationship from 100 nM to 200 μM, with a limit of detection (LOD) of 21.0 nM. By circumventing equilibrium constraints and obviating protein enzymes or nanomaterial modifications, this cascade DNAzyme architecture offers a versatile, sequence-independent, and robust strategy for small-molecule antibiotic monitoring, with broad potential for adaptation to diverse non-nucleic acid targets in food safety. Download: Download high-res image (135KB) Download: Download full-size image