Efficient and Exponential Rolling Circle Amplification Molecular Network Leads to Ultrasensitive and Label-Free Detection of MicroRNA

MicroRNAs (miRNAs) are useful biomarkers for the diagnosis of a variety of cancers. However, it is a major challenge to detect miRNAs, considering their high sequence similarity, low concentration, and small size nature. With the establishment of an efficient rolling circle amplification (RCA) molecular network by target-driven polymerization/nicking reactions, we present here an exponential amplification strategy for detecting miRNA in a label-free way with ultrahigh sensitivity. The target miRNA sequences can bind two ssDNA probes to form a junction structure to initiate a dual polymerization/nicking cyclic reaction for the production of many primers, which further trigger multiple RCA reactions in a drastically amplified sequence replication and extension mode for the yield of substantial dsDNAs with various sizes. The SYBR Green I then binds these dsDNAs to induce significantly magnified fluorescence emission for detecting the target miRNA sequences with a detection limit down to 0.86 fM in the linear range between 1 fM and 10 pM. Because of the involvement of the presynthesized circular DNA template, the RCA efficiency is further improved, and such a method can also be used for detecting miRNA in diluted human serum samples, demonstrating its great potential and universality for detecting different nucleic acid sequences for biochemical research and clinical diagnosis applications.