PROJECT SUMMARY The ability to measure extremely low levels of biomolecules accurately and rapidly is essential for diagnosing and monitoring many diseases. While sufficient for certain biomarkers, the sensitivities of most existing diagnostic systems are inadequate for measuring many protein biomarkers that exist in easily accessible biofluids at concentrations below the picomolar range. In this application, we propose to engineer and refine a new ultrasensitive single molecule protein analysis platform that will be able to routinely measure attomolar protein concentrations, which we call Molecular On-bead Signal Amplification for Individual Counting (MOSAIC). MOSAIC transforms single molecule measurements into a simplified assay format via on-bead signal localization, which has the potential to be integrated into a point of care (POC) device. In MOSAIC, a non- diffusible signal is generated on each bead carrying a target molecule, creating an on-bead signal that remains attached for prolonged periods of time, thereby enabling alternative detection schemes to be employed that do not require bead confinement into microwells or droplets to localize signals. A key challenge to be addressed in the proposed work will be to ensure that this MOSAIC platform can consistently outperform current ultrasensitive protein detection technologies in sensitivity by one to two orders of magnitude across many protein analytes, which in turn lays the foundation for future work in translating this enhanced analytical sensitivity to improved clinical sensitivity and specificity for diagnostic applications. In Aims 1 and 2, we will optimize signal generation and readout methods for MOSAIC and expand its multiplexing capabilities. In Aim 3, we will evaluate the diagnostic utility of MOSAIC in a proof-of-principle clinical application to detect very low abundance Mycobacterium tuberculosis antigens in urine as a potential triage test for tuberculosis. The resulting biosensing technology will provide an ultrasensitive diagnostic platform that will open up protein analysis to previously inaccessible biomarkers and also be readily and affordably utilized across both research and clinical laboratories using common laboratory instruments.

PROJECT NARRATIVE The ability to accurately and rapidly measure extremely low levels of biomolecules is essential for diagnosing and monitoring many diseases, as well as for detecting pathogens and toxins in food and in the environment. While sufficient for certain biomarkers, the sensitivities of most existing diagnostic systems are inadequate for measuring many protein biomarkers that exist in easily accessible biofluids at concentrations much lower than the picomolar range. In this application, we propose to engineer and refine a new ultrasensitive single molecule protein analysis platform, Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), that will be able to routinely measure attomolar protein concentrations and perform high-multiplex measurements, using common laboratory infrastructure.