A dual-localized DNA walker (dlDW) was constructed by utilizing multiple split DNAzymes with probes, and their substrates are separately localized on streptavidin and AuNPs, serving as walking pedals and tracks, respectively. Upon recognition of the target, the split DNAzymes are self-assembled and activate catalytic activity, enabling rapid walking and signal output. By simply altering the probes, quantitative analysis of protein and nucleic acid disease markers can be achieved in both buffer and serum. This versatile platform shows great potential to be used in clinical disease diagnosis.

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