Modeling and optimization of parallelized immunomagnetic nanopore sorting for surface marker specific isolation of extracellular vesicles from complex media

The isolation of specific subpopulations of extracellular vesicles (EVs) based on their expression of surface markers poses a significant challenge due to their nanoscale size (< 800 nm), their heterogeneous surface marker expression, and the vast number of background EVs present in clinical specimens (10¹⁰-10¹² EVs/mL in blood). Highly parallelized nanomagnetic sorting using track etched magnetic nanopore (TENPO) chips has achieved precise immunospecific sorting with high throughput and resilience to clogging. However, there has not yet been a systematic study of the design parameters that control the trade-offs in throughput, target EV recovery, and ability to discard background EVs in this approach.

Researchers at the University of Pennsylvania combine finite-element simulation and experimental characterization of TENPO chips to elucidate design rules to isolate EV subpopulations from blood. They demonstrate the utility of this approach by reducing device background > 10× relative to prior published designs without sacrificing recovery of the target EVs by selecting pore diameter, number of membranes placed in series, and flow rate. The researchers compare TENPO-isolated EVs to those of gold-standard methods of EV isolation and demonstrate its utility for wide application and modularity by targeting subpopulations of EVs from multiple models of disease including lung cancer, pancreatic cancer, and liver cancer.

Characterization of TENPO isolation of EV subpopulations

(A) Schematic of track-etched magnetic nanopore EV isolation. EVs are first labeled with biotinylated capture antibodies followed by anti-biotin magnetic nanoparticles (50 nm). EV-MNP complexes are magnetically captured as they flow vertically through parallelized magnetic nanopores. (B) Illustrations of tradeoffs in TENPO isolation. Adjusting the design parameters—pore diameter d, flow rate ɸ, and number of membranes n—results in trade-offs that can be used to tailor TENPO to isolate particular EV subpopulations from clinical specimens. (C) Photograph of an assembled TENPO chip (left) and SEM micrographs of the TENPO magnetic nanopores (center and right) with an EV immobilized on-chip (right). (D) A schematic of the workflow of this study.