Acoustic Separation and Isolation of Viruses, Small Extracellular Vesicles and Other Nanoscale Bioparticles

The isolation of small extracellular vesicles (sEVs), viruses and other nanoscale lipid particles from biofluids offers actionable possibilities for advancing disease diagnosis, drug delivery, regenerative medicine, personalized medicine and immunotherapy. Several methods are available to isolate sEVs from biofluids and acoustic techniques provide distinct advantages. Challenges constraining its wider application encompass the absence of adequate procedures for fabrication, implementation and performance validation. These issues impede the development of protocols applicable to nanoscale bioparticles experiencing acoustic isolation effects. Here we present a detailed protocol for acoustic separation of nanoscale bioparticles from biofluids, including plasma and saliva, achieving both high purity and throughput suitable for routine application. This protocol offers a comprehensive, step-by-step guide for the design and fabrication of the acoustic separation device, the establishment of the experimental setup and the isolation of bioparticles. To ensure reliability, rigor and reproducibility, we delineate essential procedures, including acoustic field optimization, channel fabrication and biofluid preparation, subsequently validating the protocol and its performance across different operators. Our protocol further encompasses procedures for data collection and analysis, which are essential for characterizing viruses and sEVs, as well as for evaluating their quality and integrity. This protocol enables researchers to perform high-quality isolation of nanoscale bioparticles, providing access to reliable acoustic separation techniques. Standardizing this technique will pave the way for discoveries in virology and intercellular communication research, with applications in medicine, biology, and materials science. This protocol covers the acoustics-based separation of biological particles such as viruses and single extracellular vesicles from biofluids, including plasma and saliva, with high purity.