Project Summary/Abstract The 21st century has seen a rise in sexually transmitted infections (STIs) to epidemic proportions with recent estimates of over 1 million curable STIs acquired daily. To combat the spread of STIs, diagnostics that are fast, sensitive, and affordable are needed to accurately identify pathogens and link patients to the appropriate treatment. Antimicrobial resistance (AMR), particularly in Neisseria gonorrhoeae (NG), threatens to aggravate the spread of STIs with untreatable infections, so tests that also characterize the AMR profile of infections are needed to identify the proper treatment and promote antimicrobial stewardship. Currently available diagnostics are either too slow to guide prescribed treatment, too expensive for widespread adoption, or too limited to provide a complete diagnosis. In this Phase I SBIR proposal, we will develop a 12-plex assay for detection of the 3 most prevalent STI pathogens- NG, Chlamydia trachomatis (CT), Trichomonas vaginalis (TV)- along with genotypic characterization of NG AMR targets for 4 types of antimicrobials. This assay will be integrated into low-cost magnetofluidic cartridges for fully automated sample-to-answer operation in a portable instrument for use at the point-of-care. Magnetofluidic cartridges leverage magnetic transfer of functionalized beads through preloaded reagents to capture, purify, and transport analytes without the need for complex fluidic circuitry and controls that increase cost of diagnostic platforms currently on the market. Our simple extruded thermoplastic well design enables easily scalable manufacture and focused heating using a miniaturized heat block for rapid PCR with turnaround times under 25 minutes. High degrees of multiplexing will be achieved in part using our recent developments in two-dimensional/two-dye (2D)-melt assays which identify amplified PCR targets using probes encoded with both fluorophore dye color and hybridization melt temperature. Another key innovation for multiplexing within our cartridges augments our previously demonstrated magnetofluidic capabilities to enable distribution of captured DNA between multiple reaction wells via a sequential multi-elution technique. The proposed work in this Phase I project will lay the foundation for a clinically relevant test with 2D- melt assay development (Aim 1), expansion of our Prompt magnetofluidic platform to run the 2D-melt assays (Aim 2), and validation of our assays on reference genomes and clinical isolates (Aim 3). Subsequent Phase II funding will allow design refinement for manufacturing cartridges with automated assembly, prolonged shelf-life, and human factors engineering.