Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers

There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance clinical diagnostics beyond the single biomarker model. Here we perform nanopore sequencing of DNA-barcoded molecular probes engineered to recognize a panel of analytes. This allows for highly multiplexed and simultaneous quantitative detection of at least 40 targets, such as microRNAs, proteins and neurotransmitters, on the basis of the translocation dynamics of each probe as it passes through a nanopore. Our workflow is built around a commercially available MinION sequencing device, offering a one-hour turnaround time from sample preparation to results. We also demonstrate that the strategy can directly detect cardiovascular disease-associated microRNA from human serum without extraction or amplification. Due to the modularity of barcoded probes, the number and type of targets detected can be significantly expanded. This study establishes a method for multiplexed detection of up to 40 different biomarkers in a single assay by combining nanopore sequencing with analyte-selective barcoded probes, including cardiac disease-associated microRNA directly from blood serum.