Development of a regenerable dual-trigger tripedal DNA walker electrochemical biosensor for sensitive detection of...

Since microRNAs (miRNAs) are valuable biomarkers for disease diagnosis and prognosis, the pursuit of enhanced detection sensitivity through signal amplification strategies has emerged as a prominent focus in low-abundance miRNA detection research. DNA walkers, as dynamic DNA nanodevice, have gained significant attention for their applications as signal amplification strategies. To overcome the limitations of unipedal DNA walkers with a restricted signal amplification efficiency, there is a great need for multi-pedal DNA walkers that offer improved walking and signal amplification capabilities. Here, we employed a combination of catalytic hairpin assembly (CHA) and APE1 enzymatic cleavage reactions to construct a tripedal DNA walker, driving its movement to establish a cascade signal amplification system for the electrochemical detection of miRNA-155. The biosensor utilizes tumor cell-endogenous microRNA-155 and APE1 as dual-trigger for DNA walker formation and walking movement, leading to highly efficient and controllable signal amplification. The biosensor exhibited high sensitivity, with a low detection limit of 10 pM for microRNA-155, and successfully differentiated and selectively detected microRNA-155 from other interfering RNAs. Successful detection in 20 % serum samples indicates its potential clinical application. In addition, we harnessed strand displacement reactions to create a gentle yet efficient electrode regeneration strategy, to addresses the time-consuming challenges during electrode modification processes. We have successfully demonstrated the stability of current signals even after multiple cycles of electrode regeneration. This study showcased the high-efficiency amplification potential of multi-pedal DNA walkers and the effectiveness and versatility of strand displacement in biosensing applications. It opens a promising path for developing regenerable electrochemical biosensors. This regenerable strategy for electrochemical biosensors is both label-free and cost-effective, and holds promise for detecting various disease-related RNA targets beyond its current application.