MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are associated with various diseases. Here we develop a rapid, highly sensitive, and specific miRNA assay based on the two-stage exponential amplification reaction (EXPAR) and a single-quantum-dot (QD)-based nanosensor. The two-stage EXPAR involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide. Importantly, different miRNAs can be converted to the same reporter oligonucleotides, which can hybridize with the same set of capture and reporter probes to form sandwich hybrids. These sandwich hybrids can be assembled on the surface of 605 nm emission QDs (605QDs) to form the 605QD/reporter oligonucleotide/Cy5 complexes, where the 605QD functions as both a fluorescence resonance energy transfer donor and a target concentrator. Upon excitation with a wavelength of 488 nm, distinct Cy5 signals can be observed in the presence of target miRNA. This assay is highly sensitive and specific with a detection limit of 0.1 aM and can even discriminate single-nucleotide differences between miRNA family members. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same set of capture and reporter probes. This highly sensitive and specific assay has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.
© 2011 American Chemical Society