Single-molecule catalytic hairpin assembly for rapid and direct quantification of circulating miRNA biomarkers

The emergence of circulating miRNAs as potential biomarkers for cancer necessitates reliable approaches to detect miRNAs with high sensitivity and specificity. We disclose a highly sensitive method for rapid and direct quantification of circulating miRNA in serum by combining dynamic DNA circuit and single-molecule fluorescence detection. The product of DNA circuits based amplification is detected by total internal reflection fluorescence microscopy (TIRFM). The single-molecule counting allows the quantification of miRNA targets. Owing to the high sensitivity for fluorophore labeled nucleic acids of TIRFM, the products generated by 15 min amplification are sufficient for quantification. Meanwhile, the fast detection also addresses the problem of leakage because non-target triggered DNA circuits is relatively slow. There miRNA biomarkers miR-141, miR-21, miR-16 were detected with remarkable sensitivity as detection limits of 0.017, 0.012, 0.006 fM, respectively. This approach was applied for the direct quantification of the circulating miRNAs in human serum. The results of 29 health samples, 18 prostate cancer samples, 23 breast cancer samples imply that miR-141 and miR-21 are up-regulated in the prostate cancer samples and the breast cancer samples, respectively, and as reference miR-16 shows no difference in health and patient samples.