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Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level., Riazanski et al describe a platform to capture extracellular vesicles (EVs) using a nanoporous silicon nitride membrane, investigate the expression of NHE1 protein on the surface of EVs and monitor the transport of Na+ and H+ at the single EV level. The authors report a mechanistic function of the proteins found in EVs and specifically identify NHE1 on a single EV, where it maintains pH neutrality within single vesicles.