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 much lower than their detection limits, which remain in the picomolar range. In this application, we propose to develop, design, 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 loading into microwells or well sealing 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 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 in 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 plan to integrate MOSAIC into a disposable cartridge and a portable instrument with automated sample processing, liquid handling, incubation, imaging, and sample analysis. The resulting sample-to-answer biosensing technology will provide an ultrasensitive diagnostic platform that can be readily and affordably utilized in the central clinical laboratory, in emergency departments, and in resource-limited settings, enabling rapid, accurate disease diagnosis and early treatments.