A digital nonenzymatic nucleic acid amplification assay for ultrasensitive detection of cell-free microRNA in human serum

Few point-of-care (POC) molecular methods exist that are as sensitive as polymerase chain reaction (PCR) while maintaining the simplicity, portability, and robustness for detecting specific nucleic acids in complex sample media. Here, we developed an isothermal nonenzymatic amplification cascade, named sequential nonenzymatic amplification (SENA), and its digital assay version (dSENA), for the ultrasensitive detection of cell-free microRNAs (miRNAs) in diluted human serum with a >95% recovery rate. SENA consists of two layers of DNA circuit-based amplifiers, in which the hybridization chain reaction (HCR) and catalyzed hairpin assembly (CHA) were concatenated to amplify the signals by more than 4000-fold. The sensitivity was further improved in dSENA, where a limit of detection (LOD) down to 5 fM was achieved under the optimized conditions. SENA and dSENA together demonstrated a broad detection dynamic range over 6 logs of analyte concentrations (10 fM – 10 nM), and high specificity for discriminating target miRNAs from point mutations and other interference sequences. dSENA was demonstrated to quantify expression levels of miR-21 and miR-92 in colorectal cancer patient serum with accuracy comparable to RT-PCR. Given its simplicity, compactness, and PCR-like performance, SENA holds great potential in POC miRNA or ssDNA analysis.