(375f) Electrokinetic Quantification of Extracellular Vesicles

Diseased cells transfer their surface molecules to extracelluar vesicles (EVs--- about 20 nm to 200 nm in size) that circulate in the blood. The colocalized proteins on these vesicles often reflect their parent cells' proteomic signature for specific pathological pathways. There is hence considerable interest in profiling the proteins on intact EVs from a blood sample. There are, however, several technical obstacles to this non-invasive EV diagnostics. Their relative large dimension increases the transport time of any surface assay by orders of magnitude, relative to dispersed protein affinity assays. Yet, they are below the diffraction limit to allow fluorescence or other optical characterization. In the last 3 years, our lab has developed an electrokinetic sensing technology for EVs that have led to new screening tests for cancer, neurodegenerative and infectious diseases. We first render EV capture transport limited by using high-density antibody probes on the sensor to achieve multivalent docking with the multiple EV proteins. We then reduce the transport time by using a small (~100 micron) sensor instead of a surface assay. A transient diffusion front rapidly depletes EVs within the sensor depletion volume within 30 minutes until a quasi-steady concentration gradient is established to produce a slow steady-state diffusive flux. This separation of time scale allows us to capture the EVs in the nL depletion volume robustly and rapidly. The sensor is an anion-exchange membrane that can deplete the ions by a field-induced external concentration polarization phenomenon. We hence deionize the sensor electrokinetic depletion volume, which is identical to the diffusion depletion volume, to eliminate Debye screening of the EV charge. This depletion action amplifies the voltage drop due to the charged EV (~1 V) that depends logarithmically on the total EV charge. In particular, an electro-convective instability , due to a vortex pair in the depletion region, allows easy measurement of this EV induced voltage signal. The result is an EV sensor with fM sensitivity and 4 log dynamic range. By docking different nanoparticle charge reporters on the EVs, we obtain additional voltage signals that allow us to profile the EV proteins. Based on the classical Guoy-Chapman theory, we are able to develop a universal calibration curve for the EV and protein concentrations that collapse data for different proteins on various EVs: HDL, exosomes, supermeres etc, thus enabling EV diagnostics for the first time by discovering new pathological colocalized proteins on them. The universality results from the coincidence of diffusion, electrokinetic and hydrodynamic length scales (depletion volumes) of a small point-like sensor that results from the 1/r Laplace monopole.

[Sensale et al, JPhysChem B 125: 1906(2021); Ellis et al, Small 2201330 (2022); Kumar et al, Nature Comm 14:557 (2023)]