A sensing system constructed by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification for highly sensitive miRNA detection

Abstract

The detection of disease-related biomarkers, including microRNA (miRNA), is of crucial importance in reducing the morbidity and mortality of cancer. Thus, there is a great desire to develop an efficient and simple sensing method to fulfill the detection of miRNAs. In this study, a novel amplification assay strategy is demonstrated for the highly sensitive detection of miRNA-21 by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification (SMB-NRCA). A circular padlock probe (CPP) contains a target recognition sequence, two binding sites for nicking endonuclease and three hybridization sites for SMBs. miRNA-21 can hybridize with the CPP and act as polymerization primer that initiates the rolling circle amplification (RCA) reaction and two different nicking-mediated RCA processes, releasing a large amount of SMBs and leading to a significantly amplified fluorescence signal originating from the restoration of pre-quenched fluorescence via their structural switching. Via the signal amplification based on the combination of RCA, nicking and SDA, this assay system can quantitatively detect miRNA-21 in a linear change of three orders of magnitude with a detection limit of 1 pM. The assay specificity is very high so that there is no interference from coexisting miRNAs. Moreover, the sensing system possesses ideal anti-interference ability in complicated milieux such as human serum. The novel sensing strategy shows tremendous prospects for application in tumor diagnosis and clinical therapy guidance.