Extracellular vesicles (EVs) present new opportunities for molecular diagnostics from non-invasive liquid biopsies. These cell-derived membrane-bound vesicles are abundantly present in biological fluids. EVs carry cell-specific cargos (e.g., lipids, proteins, and genetic materials), which can be harnessed to probe the molecular status of their cellular origins. EV analyses, however, pose unique technical challenges due to EVs' nanometer-sizes and presence in a vast biological background. EV analyses, however, pose unique technical challenges due to EVs' nanometer-sizes and presence in a vast biological background. While new technologies for EV analysis have been developed, fundamental limitations still remain, including i) low sensitivity limited to bulk analyses; ii) necessities of EV lysis for detecting markers inside of EVs; iii) lack of multiplexed analysis on protein and RNA markers; and iv) a separate EV isolation process required prior to the assay. The overall goal of this application is to overcome these technical challenges and develop a new platform that enables multiplexed analyses of EV protein and RNA markers in individual EVs. We previously developed a nanoplasmonic EV sensing platform based on transmission surface plasmon resonance through periodic nanohole gratings. We showed that the nanoplasmonic sensors could rapidly and sensitively detect disease-specific EVs directly from clinical samples. In this project, we will further advance the technology for robust multiplexed EV analysis and implement on-chip EV isolation to achieve simple assay procedures and good reproducibility. We will validate the system using well-established preclinical and clinical samples to demonstrate the feasibility and potential of the new technology for clinical applications. Successful completion of the project will produce a highly sensitive sensing platform for multiplexed EV analysis. The development of such a technology could offer additional insight into understanding subtypes, heterogeneity, and production dynamics of EVs during disease development and progression. The gained insights will pave the way for expanding EV studies to various diseases, further broadening the scope of EV applications in clinical settings.