A one-pot strategy for the sensitive detection of miRNA by catalyst-oligomer-mediated enzymatic amplification-based fluorescence biosensor

MiRNAs have become an ideal class of biomarker candidates for cancer classification, diagnosis, and prognosis. However, traditional approaches for the detection of miRNAs are usually laborious and time-consuming with a low sensitivity. We developed an ultrasensitive one-pot miRNA detection strategy that uses catalyst-oligomer-mediated enzymatic amplification (CMEA) sensor to circumvent the aforementioned limitations in miRNA detection. In this assay, we employed three oligonucleotides, DNA1, protector-oligomer, and catalyst-oligomer. One target miRNA can generation thousands of DNA1/catalyst-oligomer duplex. Since the DNA1/catalyst-oligomer duplex contains an endonuclease sites for Nt.CviPII, DNA1 can be cleaved into two pieces by this NEase, which leads to the denaturation of the DNA1/catalyst-oligomer duplex and thus results in a significant fluorescence signal intensity increase. Notably, this method can sensitively measure Let-7a with a detection limit of 10 fM, which has improved by as much as 7 orders of magnitude higher than that of the photoinduced electron transfer (PET) method without any amplification. Importantly, this method can be used to measure Let-7a in A549 cell extracts and might be further applied for the detection of various miRNA.