An integrated fluorescence biosensor for microRNA detection based on exponential amplification reaction-triggered three-dimensional bipedal DNA walkers

Sensitive and specific miRNA detection is essential for the early cancer diagnosis. In this work, we design a fluorescent microRNA biosensor based on exponential amplification reaction (EXPAR) and nicking endonuclease-powered three-dimensional (3-D) bipedal DNA walkers (BDW). Target microRNA initiates EXPAR with the help of polymerase and nicking endonuclease to generate the large number of BDW in solution. The newly generated BDW can be continuously assembled onto polystyrene microsphere track co-modified with fluorescence-labeled DNA strand. Thus, in the presence of nicking endonuclease, the walking machine is activated to produce enhanced fluorescent signal in the supernatant. Besides, we prove that BDW holds the faster walking speed than single-legged DNA walker (SDW) based on comparative study. Under optimal conditions, the proposed amplification method owns a wide linear range from 10 fM to 5 nM with a detection limit down to 5.2 fM. The reaction time of the assay takes about 70 min. The combination of enzyme-assisted EXPAR in solution and enzyme-powered BDW on particle significantly increases the signal amplification efficiency and improves the detection sensitivity. Therefore, our method has enormous potential for the application of BDW-related biosensors.