SUMMARY The current rapid antigen detection tests for group A strep lack sensitivity and result in upwards of 10 million unnecessary antibiotic treatments provided to children each year. Current diagnostic methods utilized when a patient presents at a clinic with a sore throat include a throat swab and rapid strep test. A positive rapid test results in antibiotic prescription, but due to the low sensitivity of existing rapid strep immunoassays, physicians often prescribe antibiotics as a precaution while waiting for culture results after a negative rapid test. It is estimated that up to 70% of these doses are unnecessary, contributing to the rise in antimicrobial resistance. While ELISAs and molecular (PCR-based) assays with improved sensitivity have been reported to reduce unnecessary antibiotic treatment, these tests rely on laboratory instrumentation and significant infrastructure, making them unsuitable for point-of-care settings and rural clinics, and impossible to implement as at-home testing protocols for telemedicine purposes. Thus, there is an urgent unmet need for an improved, ultrasensitive rapid strep test that could reduce the unnecessary use of antibiotics, stemming the increase in antimicrobial resistance and preserving the gut microbiome of children. Furthermore, if an improved rapid strep test could be optimized for robustness and ease of use, as well as sensitivity, it could be performed at home enabling telemedicine protocols and reducing the need for a potentially infected individual to expose frontline healthcare workers. The outcome of this project will be a proof-of- principle demonstration of an ultrasensitive rapid strep test with onboard reagents that requires a simple swab insertion step to initiate to completion with limits of detection 10-100x lower than current dipstick methods. Improved sensitivity will be achieved through use of the Capillary-Driven Immunoassay (CaDI) technology in which we are able to integrate all steps of highly sensitive, laboratory-performed ELISAs (washing, labeling, and amplification) on a simple microfluidic device without requiring instrumentation or highly trained personnel. This first-in-its-class microfluidic point-of-care (POC) device will be realized through the following aims. First, proof of concept for a Strep-CaDI rapid strep test will be demonstrated for the ultrasensitive detection of Streptococcus pyogenes antigen in spiked buffer through antibody screening and assay parameter optimization. Secondly, the bacterial extraction method and corresponding CaDI parameters and device design will be optimized for use with clinical samples spiked with whole bacteria. The final, optimized design will be compared to current market solutions to compare assay sensitivity (LOD), time to result, and complexity (steps to result). Lastly, to increase the likelihood of approval for at-home use and to provide a clear market advantage over current solutions, we will prototype (through modeling, 3D ...