Development of a regenerable dual-trigger tripedal DNA walker electrochemical biosensor for sensitive detection of microRNA-155

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.