PROJECT SUMMARY Over 6 million Americans currently live with Alzheimer's disease (AD), and this number is projected to increase to 16 million by 2050. The consensus is arising in the field that in order to treat AD effectively, early detection at the MCI stage is a must. Thus, there is a critical need for a novel minimally invasive and cost-effective diagnostic assay capable of such early detection. Cell-secreted extracellular vesicles (EVs) recently gained significant attention in the fields of liquid biopsy and AD research. Within the scope of biomarker detection, EVs offer numerous benefits for clinical analysis, including non-invasive collection, a suitable sample source for longitudinal disease monitoring, ability to cross the blood-brain barrier, higher stability and sample volumes, faster processing times and lower cost. Multiple reports linked EVs to neurodegeneration and have been able to couple exosome load in CSF to AD progression. However, the analysis of plasma exosomes and other EV has not really been possible for early AD detection. The procedures for plasma biomarker analysis are very long and cumbersome due to extremely low target levels and high background of free plasma proteins. In order to enable better biomarker discovery and AD diagnosis, a more reliable and efficient approach is needed, capable of enriching potential AD-associated proteins with higher purity. In the NIH SBIR Phase I portion of this study, we have developed a novel method for fast and reproducible capture and isolation of EVs with >95% recovery yield and >99.9% purity from plasma samples. We then implemented this EV capture approach to discover novel potential AD markers in plasma EVs with, which showed a clear and consistent differentiation from non-AD samples. In the Phase II portion of this project, we propose to validate our discovered protein biomarkers in a much larger cohort and translate the new marker panel into a clinically relevant ELISA format for the intended clinical use of early screening and AD/ADRD progression monitoring. The following aims will be completed in the Phase II of the proposal: Aim #1: Validate the discovered plasma EV biomarkers for Alzheimer's disease and refine the panel. Aim #2: Carry out analytical evaluation and validate the EXrich instrument for automated EV enrichment. Aim #3: Adapt the developed plasma EV biomarker panel into a clinically relevant ELISA format. By the completion of this project, a minimally invasive plasma-based early Alzheimer's disease detection assay will be validated that can overcome the limitations of current approaches, and thus could have an enormous public health impact and market potential.