SBIR Phase I: Low-cost Detection and Enrichment of Nucleic Acids by Interfacing with Commercially Available Cell Sorters

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project will be to enable affordable detection and enrichment of rare DNA or RNA molecules from a large background population. Currently, such detection is performed on expensive, dedicated instruments that have low sensitivity and are not able to enrich. The proposed strategy is to encapsulate the fluorescently-identified target molecules in thin oil shells so that they mimic cells, and then process them on sensitive and high-throughput cell-sorting instruments that are readily available. By offloading the detection and enrichment to such machines, all that is required by researchers is a simple tool that encapsulates the molecules in thin oil shells. Applications afforded by high sensitivity and enrichment are numerous, from rare pathogen or cell detection to target capture of uncommon mutant sequences with the goal of downstream sequencing. Between the reduced cost and expansion of capabilities, this approach has the potential to become a widely adopted method. This SBIR Phase I project proposes to enable high-throughput digital PCR (dPCR) at a dramatic cost reduction by interfacing with existing flow cytometers (FC). The main research objectives center on characterizing and optimizing the dPCR droplets for use on a wide variety of FC instruments originally intended for cell sorting. First, the biological, chemical, mechanical, and optical properties of the dPCR droplets will be optimized to guarantee compatibility with different FC systems while maximizing earlier PCR efficiency and specificity. Next, serial dilutions of target sequences will be run to test and improve detection thresholds. Those same samples also will be benchmarked against currently commercially available dPCR systems. In tandem, the droplet-on-FC system will be used in a research collaboration to quantitate and characterize HIV latency in an infected cell population. Initial results with FC-processed droplets are promising, as FC systems are already remarkably flexible due to the range of cell types they must be capable of accepting.