Self-Replicating\nCatalytic Hybridization Assembly\nof Bipedal DNAzyme Walkers for Enhanced Electrochemiluminescence Bioanalysis

Dynamic DNA nanodevices, particularly\nDNA walkers, have\nproven\nto be versatile tools for target recognition, signal conversion, and\namplification in biosensing. However, their ability to detect low-abundance\nanalytes in complex biological samples is often compromised by limited\namplification depth and severe signal leakage. To address these challenges,\nwe developed a simple yet highly efficient strategy to engineer a\nself-replicating bipedal DNAzyme (SEDY) walker for sensitive and selective\nelectrochemiluminescence (ECL) bioanalysis. Unlike conventional DNA\nwalkers that are typically constructed by catalytic DNA assembly in\na single direction, the SEDY walker integrates a self-replicating\nfeedback mechanism that greatly enhances both the selectivity and\nsensitivity of bioanalysis. First, the SEDY walker is assembled through\na target-triggered, enzyme-free, self-replicating catalytic approach,\nminimizing the risk of undesired side reactions and signal leakage\nby simplifying reactant complexity. Furthermore, the SEDY walker features\nnewly exposed trigger sequences that facilitate its autonomous replication,\nleading to a robust and exponential amplification of its products.\nOur experiments demonstrate that the SEDY walker can sensitively and\nselectively detect acetamiprid by navigating specific probes within\ncross-shaped DNA orbits. The ECL biosensor offers a linear detection\nrange from 1 × 10<sup>–15</sup> M to 1 × 10<sup>–9</sup> M, with a limit of detection as low as 5.8 × 10<sup>–16</sup> M. We anticipate that the SEDY walker will be a powerful tool for\ndetecting various analytes in biological applications.