Summary: Treatment decisions for respiratory infections, diarrheal diseases, sepsis, and urinary tract infections (UTIs) are tied to the identification and differentiation of the many possible infection-causing pathogen(s). Nucleic acids (NAs) are effective biomarkers for pathogen identification, but detecting nucleic acid sequences typically requires some variation of the polymerase chain reaction (PCR), necessitating complex instrumentation and trained staff that are only found in centralized laboratories. The long-term goal of the proposed project is to develop a point- of-care (POC)-compatible microfluidic device for DNA amplification and detection of 9 different UTI-causing pathogens. The proposed project will focus on developing the amplification and detection components, which in future efforts will be integrated with sample preparation. In the herein proposed system, the user will add extracted DNA to a disposable cartridge, external instruments will actuate fluid handling, thermal control, and imaging, and the results will be available 1 hr later. This method will use isothermal nucleic acid amplification, which is more suitable for POC settings than PCR because it requires no thermocycling, resulting in less expensive and more robust systems. However, isothermal nucleic acid amplification is usually not suitable for higher order multiplexing (> 2 or 3 NA sequences). To achieve high-order multiplexing, the proposed method will combine the advantages of spatial multiplexing, where the sample is divided into and amplified within distinct compartments, and color-based multiplexing, where color of a unique oligonucleotide detection probe is used to identify an amplified sequence. However, we will circumvent the limitations of these two approaches, such as loss of sensitivity in spatial multiplexing due to sample dilution, and limited filter space to differentiate excitation and emission of multiple fluorophores in color multiplexing. We will use clonal isothermal nucleic acid amplification inside a water-in-oil emulsion with fluorescently encoded microbeads, followed by detection in a microchannel. In Aim 1, we will establish the required processes to generate the droplet-bead emulsions and isothermally amplify NAs within each droplet, resulting in amplicons bound to the microbeads, followed by breaking open the emulsion, and isolating the beads for imaging. In Aim 2, we will design and fabricate a microfluidic device appropriate for use at the point-of-care to execute the processes developed in Aim 1. In Aim 3, we will test the device with extracted DNA from UTI pathogens to validate the device's accuracy in identifying the correct pathogen. In future work, we will create a small compact instrument (< 1 ft3) that autonomously actuates the fluid handling, thermal control, and imaging components in an integrated user friendly format with the microfluidic device developed here. This device will also be coupled with upstream sample preparation and we will test the entire sample-to-answer process with actual clinical UTI samples.