Project Summary In this proposal, we aim to create and validate a novel, inexpensive, convenient-to-use, point-of- care, ultrasensitive yet highly specific Urine Tuberculosis (TB) Antigen Rapid Detection Test capable of detecting pg to sub-pg/ml of Mycobacterium tuberculosis-specific antigens. This breakthrough is achieved by an enrichment step using large volume of urine sample (≥2 ml) and functionalized superparamagnetic nanoparticles (MNPs) in combination with a lateral flow rapid test for visual result readout by naked eye. Very high affinity antibodies developed in our laboratory and specific to three M. tuberculosis-specific markers known to be present in urine, will be utilized to functionalize MNPs, allowing the MNPs to capture M. tuberculosis antigens in urine efficiently even at very low concentrations (pg to sub-pg/ml). The functionalized MNPs with captured cargoes will be magnetically concentrated quickly and loaded onto a lateral flow rapid test to assay for the presence of M. tuberculosis-specific markers. The MNPs have intense color, allowing visual result readout by the lateral flow rapid test. The materials used in the ultrasensitive assay (the MNPs & the antibodies), and the design of the assay will be optimized. The rapid assays will also be designed for different M. tuberculosis-antigen detection schemes – singly or in combinations. The prototypes will then be validated with clinical urine samples collected from active TB patients.