Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific analysis of a DNA sample. Solid-state nanopore sensors are especially promising for this type of single-molecule sensing because of the ability to analyze patterns in the ionic current signatures of DNA molecules. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify 1 two different DNA targets in a background mixture of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.

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Currently, most blood tests in a clinical setting only investigate a handful of markers. A low-cost, rapid, and highly multiplexed platform for the quantitative detection of blood biomarkers has the potential to advance clinical diagnostics beyond the single biomarker paradigm. In this study, we perform nanopore sequencing of barcoded molecular probes that have been engineered to recognise a panel of biological targets (miRNAs, proteins, and small molecules such as neurotransmitters), allowing for highly multiplexed simultaneous detection. Our workflow is rapid, from sample preparation to results in 1 hour. We also demonstrate that the strategy can be used to detect biomarkers directly from human serum without extraction or amplification. The established method is easily adaptable, as the number and type of targets detected can be greatly expanded depending on the application required.