DNAzyme walker induced DNAzyme working cascade signal amplification strategy for sensitive detection of protein

Sensitive detection of disease-related proteins is crucial to better illustrate their roles in physiological and pathological processes and to further evaluate their functions in disease diagnosis and clinical research. Herein, we developed a DNAzyme walker induced DNAzyme working cascade signal amplification strategy for sensitive detection of disease-related protein thrombin. In this work, the DNAzyme walker was constructed by co-modifying walking strands and track strands onto gold nanoparticle surface. The walking strand is a single-stranded DNA containing DNAzyme sequence, which is pre-locked by thrombin aptamer sequence. The track strand is specially designed hairpin DNA, which embeds an RNA cleavage site and seals the same DNAzyme sequence. When the thrombin specifically bound to its aptamer sequence, the walking strand was unlocked and then the liberated DNAzyme catalyzed the cleavage of track strands to drive the DNA walker operation, exposing plenty of new DNAzyme buried in track strands. Subsequently, the exposed DNAzyme catalyzed the continuous cleavage of reporter probes labeled with fluorophore and quencher, accompanying by fluorescence signals accumulation. For thrombin detection, a wide linear ranging from 0.02 nM to 15 nM and a detection limit of 4.5 pM were achieved. Benefiting from the cascade amplification effect of DNA walker operation and DNAzyme cleavage reaction, the detection sensitivity was significantly improved. The proposed strategy will provide an alternative method for sensitive analyzing of proteins and hold great application potential in disease diagnosis and clinical research.