A rolling circle amplification-assisted DNA walker triggered by multiple DNAzyme cores for highly sensitive electrochemical biosensing.

DNA walkers from monopodial to multipedal types have usually one cleavage site to power the walking system along with the track. Herein, a multipedal DNA walker (m-DNA walker) with multiple DNAzyme cores was constructed with the assistance of rolling circle amplification (RCA) for highly sensitive electrochemical biosensing. Firstly, a three-component DNA complex as a swing strand was prepared by integrating a padlock, an RCA primer and a block DNA as a recognition element in the DNA walker system. After ferrocene-labeled track DNA (trDNA) and capture DNA were fixed on a gold electrode, the three-component DNA complex was imported onto the electrode as a swing arm to form a m-DNA walker. In the presence of target DNA and a RCA kit, the block was displaced from the complex and RCA was initiated to form multiple DNAzyme strands. Upon hybridization with trDNA, the m-DNA walker was motivated by the cleavage of multiple DNAzyme cores in the presence of manganese ions to free signal molecules. Under the optimal circumstances, the electrochemical m-DNA walker showed a linear range from 1.0 fM to 1.0 nM with a detection limit of 0.28 fM. Moreover, the m-DNA walker demonstrated a rapid cleavage rate and a low ratio of the swing strand to the track, which is more excellent than a single foot walker and a bipedal DNA walker. The practicality of the proposed strategy was also confirmed by detecting target DNA in 10% human serum, showing promising applications in clinical diagnosis.