One exciting new strategy to probe disease biomarkers focuses on extracellular vesicles (EVs). These membrane-delimited nanoparticles are shed by all cells, harbor the molecular content of their cellular origins, and circulate within patient biofluids. These features make EVs an attractive source of biomarkers. However, a major technical hurdle is stalling their clinical implementation: it is challenging to isolate and robustly characterize specific EV subpopulations. Our laboratory has been focused on assessing EVs with methods that operate at the molecular level. To that end, quantitative single-molecule localization microscopy (qSMLM) is a powerful method to characterize EV size, molecular content, and heterogeneity. Here, we present a novel qSMLM-based assay for characterizing multiple EV subpopulations on a single microscope coverslip. First, we optimized the analytical assay, including the fluorescent labeling of EVs and chemical functionalization of microscope coverslips. Second, we imaged either the total EV population (nonspecific pulldown of EVs) or specific EV subpopulations (affinity pulldown of EVs on antibody coated spots). Third, we analyzed these images with new algorithm tools developed in our laboratory. Using EVs isolated from either cultured cell lines or patient biofluids, we quantified tetraspanin-enriched (CD9, CD63, CD81) and growth factor receptor-enriched (e.g., EGFR, HER2) EV subpopulations. Our ultimate goal is to develop this assay into a platform for performing personalized and molecular level diagnoses.

Multiplexed single molecule assay for extracellular vesicle characterization

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.