A Digital Nonenzymatic Nucleic Acid Amplification Assay for Ultrasensitive Detection of Cell-Free microRNA in Human Serum

Abstract

Detection and quantification of specific nucleic acids is one of the most important point-of-care (POC) diagnostic methods. However, few POC molecular methods exist that are as sensitive as polymerase chain reaction (PCR) while maintaining the simplicity, portability, and robustness for 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 human serum. 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 to differentiate target microRNAs from their single-base mutations and other interference sequences. Both SENA and dSENA assays worked robustly in spiked serum samples that were diluted as few as 10x and also in the dehydrated state by completely drying the assay components on the digital chip (>95% recovery rate for fM-pM targets). Finally, the expression levels of miR-21 and miR-92 in colorectal cancer (CRC) patient serum samples were quantified with the dSENA setup, and the detection accuracy is comparable to the standard RT-PCR. Given its simplicity, compactness, and PCR-like sensitivity and specificity, SENA and its digital format hold great potentials in POC miRNA or ssDNA analysis.