A solution to the biophysical fractionation of extracellular vesicles: Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER)

Abstract

High-precision isolation of small extracellular vesicles (sEVs) from biofluids is essential toward developing next-generation liquid biopsies and regenerative therapies. However, current methods of sEV separation require specialized equipment and time-consuming protocols and have difficulties producing highly pure subpopulations of sEVs. Here, we present Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER), which allows single-step, rapid (<10 min), high-purity (>96% small exosomes, >80% exomeres) fractionation of sEV subpopulations from biofluids without the need for any sample preprocessing. Particles are iteratively deflected in a size-selective manner via an excitation resonance. This previously unidentified phenomenon generates patterns of virtual, tunable, pillar-like acoustic field in a fluid using surface acoustic waves. Highly precise sEV fractionation without the need for sample preprocessing or complex nanofabrication methods has been demonstrated using ANSWER, showing potential as a powerful tool that will enable more in-depth studies into the complexity, heterogeneity, and functionality of sEV subpopulations.

Do different exosome biogenesis pathways and selective cargo enrichment contribute to exosomal heterogeneity?

“…The current Human plasma Small EV subpopulations: Exomeres (30-50 nm) Exo-S (60-80 nm) Exo-L (90-150 nm) J. Zhang et al (2022) methods like density gradient are difficult to be scaled for the clinical and therapeutic options. Exosome secretion in blood and the ability to maintain the stability of its constituents like different RNA species, protein complexes, and mitochondrial DNA make exosomes an excellent non-invasive biomarker to identify disease specific signatures.…”

“…Acoustic Nanoscale Separation via Wave‐pillar Excitation Resonance (ANSWER) is a newly developed technique that allows single‐step, rapid fractionation of sEV subpopulations from biofluids without the need of sample pre‐processing. This method involves the formation of surface acoustic waves depending on the biophysical nature of small vesicles and has precisely separated 96% small exosomes and >80% exomere populations in less than 10 min (Tayebi et al., 2021; J. Zhang et al., 2022). Electric‐hydraulic principle based cascaded microfluidic circuits to integrate cell‐removal circuit and an EV‐isolation circuit have been designed for pulsatile filtration of tumor derived EVs.…”

Recent advances of liquid biopsy: Interdisciplinary strategies toward clinical decision‐making

“…(H) Acoustophoresis based isolation. Reproduced with permission 141 . Copyright 2022, American Association for the Advancement of Science.…”

Direct isolation of small extracellular vesicles from human blood using viscoelastic microfluidics

“…Passive separation methods do not require the application of external forces but rather isolate sEVs through the use of size-dependent hydrodynamic forces (29)(30)(31)(32)(33) or complex channel structures [e.g., nanoporous membranes (34,35) and nanopillar arrays (36,37)]. Conversely, active separation methods require the application of external force fields, most notably acoustic (38)(39)(40), electric (41,42), and magnetic (43,44) fields to manipulate sEVs.…”