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. 2021 Apr 13;11:8034. doi: 10.1038/s41598-021-86910-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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The Immunomagnetic sequential ultrafiltration (iSUF) platform. (A) Schematic representation of iSUF stages 1, 2, 3, including tangential flow filtration (TFF) purification, centrifugal unit enrichment, and tEV immunoaffinity isolation, respectively. In stage 1, biofluids were processed using a 500 kDa TFF filter, EVs were retained in the retentate and enriched in 7 mL while free proteins and nucleic acids permeated through the TFF filter. Then the PBS valve was opened to start TFF diafiltration until removing up to 99% of free proteins. Finally, EVs were recovered in 2 mL of PBS after flushing the TFF system with air. In stage 2, EVs were centrifuged using a 10 kDa centrifugal unit at 3000×g and enriched in 100 μL. In stage 3, tEVs were captured using antibodies immobilized to streptavidin-coated magnetic beads (i.e., EGFR) and subsequently pulled out with a magnet. (B) iSUF stage 1 purification performance for different biofluids. Cell supernatant (CCM), urine, and serum took 80, 100, and 120 min to obtain up to 99% efficiency of free protein and nucleic acid removal.