Integrated nicking enzyme-powered numerous-legged DNA walker prepared by rolling circle amplification for fluorescence detection of microRNA.

MicroRNAs (miRNAs) have been accepted as promising non-invasive biomarkers for cancer early diagnosis. Developing amplified sensing strategies for detecting ultralow concentration of miRNAs in clinical samples still requires much effort. Herein, an integrated fluorescence biosensor using nicking enzyme-powered numerous-feet DNA walking machine was developed for ultrasensitive detection of miRNA. A long numerous-feet walker produced by target-triggered rolling circle amplification autonomously moves along the defined DNA tracks on gold nanorods (AuNRs) with the help of nicking enzyme, leading to the recovery of fluorescence. This results in an amplified fluorescence signal, typically measured at 518 nm emission wavelength. Benefiting from the long walker that dramatically improves movement range, the homogenous and one-step strategy realizes ultrahigh sensitivity with a limit of detection of 0.8 fM. Furthermore, this walking machine has been successfully used to quantification of miRNA in clinical serum samples. The consistency of the gained results between of the developed strategy and reverse transcription quantitative polymerase chain reaction (RT-qPCR) shows that the sensing method has great promise for tumor diagnostics based on nucleic acid. Schematic representation of the fluorescent biosensing strategy, numerous-legged DNA walker prepared by rolling circle amplification on gold nanorods (AuNRs) for microRNA analysis, which can be applied in real samples with good results.