Electroformed Inverse‐Opal Nanostructures for Surface‐Marker‐Specific Isolation of Extracellular Vesicles Directly from Complex Media

Abstract

be reflective of their cells of origin. For a wide range of clinical questions across diseases including cancer, infectious diseases, and traumatic brain injury, EVs have demonstrated unique clinical potential as a blood-based source of biomarkers that can be used as an adjunct or alternative to standard-of-care tissue biopsy. [1][2][3][4][5][6][7] Unlike liquid biopsy analytes such as circulating tumor cells (CTCs), EVs are abundant in circulation (1-10 CTCs mL −1 in cancer patients [8] vs up to 10 12 EVs mL −1 in serum [9] ); however, precise numbers for the concentration of most disease-associated EVs in patient blood have not been established. Moreover, single EVs contain multiple proteins and nucleic acid cargoes from their cells of origin, [10] yielding a more comprehensive view of the complex, heterogeneous, and often dynamically changing disease states, especially when compared to single-analyte readouts such as circulating serum-based antigens (e.g., carcinoembryonic antigen, CEA). [11] Also, EVs are a growing area of biological inquiry, in particular for their role in intercellular interactions, including interactions with immune cells [12] and metastatic sites in cancer. [13] Despite their widely appreciated potential as biomarkers, EVs have yet to be translated to widespread clinical use beyond proofof-concept studies. One fundamental hurdle to the translation of EV biomarkers to address clinical questions is the lack of an adequately specific, robust, and reproducible isolation technology for relatively sparse disease-associated EVs from the large background of EVs present in the blood. [2,6,10] To solve this problem, we were inspired by the field of CTC isolation, where rare cells (as rare as 1 in 10 9 hematologic cells in patient blood) tagged with antibodyfunctionalized magnetic nanoparticles (MNPs) have been precisely isolated from or quantified in whole blood by matching the feature size of the sorting or detection element on microfluidic chips to the given target analyte (e.g., 9-19 µm for a CTC). [14][15][16] The nanoscale size (30-200 nm for exosomes, [17] 50 nm − 1 µm for microvesicles [5] ) of EVs, however, has made it challenging to develop technology analogous to what has been successful in CTCs due to the difficulty and expense of fabricating nanoscale devices and their susceptibility to clogging. As a result, many Extracellular vesicles (EVs) -nanoscale membranous particles that carry multiple proteins and nucleic acid cargoes from their mother cells of origin into circulation -have enormous potential as biomarkers. However, devices appropriately scaled to the nanoscale to match the size of EVs (30-200 nm) have orders of magnitude too low throughput to process clinical samples (10 12 EVs mL −1 in serum). To address this challenge, we develop a novel approach that incorporates billions of nanomagnetic sorters that act in parallel to precisely isolate sparse EVs based on immunomagnetic labeling directly from clinical samples at flow rates billions of times greater than that of a single na...

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

“…4 C) (SI Fig. 18 ) 5 , 37 .

Figure 4 In vitro benchmarking of TENPO to gold standard technologies and in different biological systems.

…”

“…The results from the magnetic field and laminar flow simulation were then combined using the Particle Tracking for Fluid Flow module to perform particle tracking simulations. Similar to our previous work 37 , the relative permeabilities of the material layers on TENPO in COMSOL were set to approximate the saturation magnetization values of the 200 nm layer of NiFe in the device (~ 7900 Gauss) 46 at an input field of 341,000 A/m 37 . A 200 nm diameter sphere was chosen to model EV-MNP complexes because its volume was equivalent to a 150 nm diameter EV + 15 MNPs with a 50 nm diameter.…”

Parallelized immunomagnetic nanopore sorting: modeling, scaling, and optimization of surface marker specific isolation of extracellular vesicles from complex media

“…The results from the magnetic eld and laminar ow simulation were then combined using the Particle Tracking for Fluid Flow module to perform particle tracking simulations. Similar to our previous work [31], the relative permeabilities of the material layers on TENPO in COMSOL were set to approximate the saturation magnetization values of the 200 nm layer of NiFe in the device (~ 7900 Gauss) [41] at an input eld of 341000 A/m [31]. A 200 nm diameter sphere was chosen to model EV-MNP complexes because its volume was equivalent to a 150 nm diameter EV + 15 MNPs with a 50 nm diameter.…”

“…Cell culture media was prepared per a protocol detailed in our previous work, which is reproduced here in full via [31]. The Panc1 (pancreatic cancer), SNU449 (liver cancer), and the lung cancer cell lines H322, H358, H1975, H460, H1299, and H1264 were used; all cell lines listed were purchased from ATCC.…”

“…EV Characterization Methods: On-Chip EV SEM Scanning electron microscope experiments were carried out at the Cell and Developmental Biology Microscopy Core (Perelman School of Medicine, University of Pennsylvania) via a protocol previously reported by our group [31] which is reproduced here. Following addition of sample and completion of sample owing and washing steps, EV-MNP complexes were immobilized on-chip.…”