ddRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform.

Digital nucleic acid amplification tests (digital NAATs) have emerged as a popular tool for nucleic acid detection due to their high sensitivity and specificity. Most current digital NAAT platforms, however, are limited to a "one-color-one-target" approach wherein each target is encoded with a specific fluorescently-labeled probe for single-plex fluorometric detection. This approach is difficult to multiplex due to spectral overlap between any additional fluorophores, and multiplexability of digital NAATs has therefore been limited. As a means to scale multiplexability, we have developed a multiplexed digital NAAT platform, termed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), via a padlock probe-based nucleic acid detection assay which encodes each nucleic acid target with a unique combination of 2 fluorophores. We detect this encoded two-color fluorescence signature of each target by performing digital amplification in microfluidic droplets. To demonstrate the utility of our platform, we have synthesized 6 distinct padlock probes, each rendering a unique two-color fluorescence signature to a nucleic acid target representing a clinically important sexually transmitted infection (STI). We proceed to demonstrate broad-based, two-plex, four-plex, and six-plex detection of the STI targets with single-molecule resolution. Our design offers a cost-effective approach to scale up multiplexability by simply tuning the number of molecular beacon binding sites on the padlock probe without redesigning amplification primers or fluorescent molecular beacons. With further development, our platform has the potential to enable highly multiplexed detection of nucleic acid targets, with potentially unrestricted multiplexability, and serve as a diagnostic tool for many more diseases in the future.