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Acoustic separation and isolation of viruses, small extracellular vesicles and other nanoscale bioparticles

Nature Protocols (2026)Cite this article

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Abstract

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.

Key points

  • This protocol provides a guide for designing and fabricating the acoustic separation device, including channel fabrication, the experimental setup and acoustic field optimization, and the biofluid preparation and isolation of nano-sized biological particles.

  • Alternative approaches for biological nanoparticle separation include differential ultracentrifugation; however, this has a low yield and is time consuming and labor intensive. Another alternative is size exclusion chromatography, which has drawbacks such as sample dilution and limited resolution below 70 nm.

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Fig. 1: Summary of the fabrication and sample processing for the acoustic nanoparticle-separation device, corresponding to the protocol steps.
Fig. 2: Fabrication of the microfluidic channel with Bessel interdigital transducers and the setup for the acoustic separation device.
Fig. 3: Sample preparation steps for tissue, plasma, saliva or cell culture medium.
Fig. 4: Evaluation of the rigor and reproducibility of the acoustic EV separation device.

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Data availability

The authors declare that all data supporting the findings of this study are available within the article. The sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE235349. Further information is available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We acknowledge support from the Shared Materials Instrumentation Facility (SMIF) at Duke University. We used ChatGPT (OpenAI) to help improve the clarity and readability of part of the manuscript after the initial draft was completed. We acknowledge support from the National Institutes of Health (R01HD103727, R01AG084098, R01GM141055, R01GM143439, R01GM145960, R01GM144417, U18TR003778 and UH3TR002978), and the National Science Foundation (CMMI-2104295). This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (no. S10 OD026929).

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Authors and Affiliations

  1. Thomas Lord Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA

    Jianping Xia, Brandon Lu, Shujie Yang, Ying Chen & Tony Jun Huang

  2. Harvard Medical School, Harvard University; Renal Division and Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    Shujie Yang & Luke P. Lee

  3. Ascent Bio-Nano Technologies, Morrisville, NC, USA

    Arindam Ghosh & Chaoran Chang

  4. Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Diego, San Diego, CA, USA

    Abbas Hakim & Louise C. Laurent

  5. Alfred E. Mann Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, USA

    John D. H. Mai

  6. Center for Oral/Head and Neck Oncology Research, School of Dentistry, University of California, Los Angeles, CA, USA

    David T. W. Wong

  7. Scintillon Institute, San Diego, CA, USA

    John P. Nolan

  8. Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Ming Dao & Subra Suresh

  9. Department of Pediatrics, Stanford University, Palo Alto, CA, USA

    Yoel Sadovsky

  10. Department of Bioengineering, University of California, Berkeley, CA, USA

    Luke P. Lee

  11. Department of Electrical Engineering and Computer Science, University of California, Berkeley, CA, USA

    Luke P. Lee

  12. School of Engineering, Brown University, Providence, RI, USA

    Subra Suresh

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  1. Jianping Xia
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Contributions

J.X., L.P.L. and T.J.H. designed the research. J.X., B.L., A.G., C.C., J.P.N. and L.C.L. performed the research. J.X., B.L., A.G., C.C., S.Y., J.P.N. and L.C.L. analyzed data. J.X., B.L. and L.P.L. drew the figures. All authors wrote and edited the manuscript.

Corresponding authors

Correspondence to David T. W. Wong, John P. Nolan, Louise C. Laurent, Ming Dao, Yoel Sadovsky, Luke P. Lee, Subra Suresh or Tony Jun Huang.

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Competing interests

T.J.H. has cofounded a start-up company, Ascent Bio-Nano Technologies Inc., to commercialize technologies involving acoustofluidics and acoustic tweezers. J.P.N. is founder of Cellarcus Biosciences, which provides products and services for EV research. The remaining authors declare no competing interests.

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Nature Protocols thanks Stephanie Descroix and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Xia, J. et al. ACS Nano 18, 22596–22607 (2024): https://doi.org/10.1021/acsnano.4c09692

Wu, M. et al. Proc. Natl Acad. Sci. USA 114, 10584–10589 (2017): https://doi.org/10.1073/pnas.1709210114

Wang, Z. et al. Microsyst. Nanoeng. 7, 20 (2021): https://doi.org/10.1038/s41378-021-00244-3

Shi, J. et al. Lab Chip 9, 3354–3359 (2009): https://doi.org/10.1039/B915113C

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Figs. 1–8 and Table 1.

Reporting Summary

Source data

Source Data Fig. 1 and Fig. 4

NTA results in Fig. 1d is provided. Vesicles flow cytometry results in Fig. 4f and 4g are provided. RNA sequencing results in Fig. 4h–j are provided.

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Xia, J., Lu, B., Yang, S. et al. Acoustic separation and isolation of viruses, small extracellular vesicles and other nanoscale bioparticles. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01286-x

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