Nucleic Acid Amplification-Free Bioluminescent Detection of MicroRNAs with High Sensitivity and Accuracy Based on Controlled Target Degradation

Accurate and sensitive detection of microRNAs is crucial to clinical diagnosis and therapy. Most of microRNA assays require target conversion in combination with nucleic acid amplification to improve the detection sensitivity, which may compromise the assay accuracy and specificity. Herein we report a sensitive bioluminescent method for microRNA assay on the basis of controlled target degradation without target conversion and nucleic acid amplification. In this assay, the target microRNA can be specifically degraded by exonuclease III after hybridization to its complementary probe, releasing adenosine monophosphate (AMP) from microRNA itself. The AMP then triggers an efficient bioluminescence generation system to produce a strong bioluminescence signal. This assay is highly sensitive with zero-background signal and a detection limit of 7.6 fM even without target amplification, and it can discriminate the single-nucleotide difference among microRNA family members with extremely high discrimination ratio. With the assistance of magnetic separation to eliminate the interference of endogenous ATP, ADP, and AMP in sample matrix, this assay can be further applied to absolute quantification of microRNAs in cancer cells and tissues from lung cancer patients, holding great potential in biomedical research and clinical diagnosis.