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Volume 26
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10.3390/ijms262311324
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Profiling of CD63 and EpCAM Membrane Proteins of Extracellular Vesicles on Tannic Acid-Coated Magnetic Beads Using Conventional Flow Cytometry

by
Ekaterina Moiseeva
1,
Igor Sergeev
1,
Vasiliy Chernyshev
1,2,
Olga Zaborova
3,
Daria Kohzevnikova
1,
Alexander Yakovlev
4,5,
Olesya Kuznetsova
6,
Alexey Tryakin
6,
Aleksei Komlev
7,
Dmitry Gorin
1 and
Alexey Yashchenok
1,*
1
Skolkovo Institute of Science and Technology, Skolkovo Innovation Center, 121205 Moscow, Russia
2
National Medical Research Center for Obstetrics, Gynecology and Perinatology Named After Academician V.I. Kulakov, 117997 Moscow, Russia
3
Faculty of Chemistry, Moscow State University, 119991 Moscow, Russia
4
Moscow Research and Clinical Center for Neuropsychiatry, 115419 Moscow, Russia
5
Department of Functional Biochemistry of Nervous System, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117865 Moscow, Russia
6
N.N. Blokhin National Medical Research Center of Oncology, 115522 Moscow, Russia
7
Faculty of Physics, Moscow State University, 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(23), 11324; https://doi.org/10.3390/ijms262311324 (registering DOI)
Submission received: 2 October 2025 / Revised: 14 November 2025 / Accepted: 19 November 2025 / Published: 23 November 2025
(This article belongs to the Section Molecular Biophysics)
Versions Notes

Abstract

Extracellular vesicles (EVs) are considered to be a promising tool in disease diagnosis. However, the clinical translation of EV-based liquid biopsy faces significant challenges due to the lack of inexpensive, rapid, and high-throughput methods of EV analysis. Bead-based platforms, combined with conventional flow cytometry, allow for the simultaneous capture and immunolabeling of EVs. In this study, we present a new approach based on the label-free isolation of EVs by tannic acid-coated superparamagnetic beads (TASPMB) combined with immunofluorescence detection of EV membrane proteins using flow cytometry. First, we tested the molecular profiling capabilities of the approach using EVs derived from human breast and colorectal cancer cell lines and from plasma of colorectal cancer patients to recognize the tetraspanin protein CD63 and the epithelial cell adhesion molecule (EpCAM). Subsequently, the developed approach was validated to identify proteins on EVs enriched with TASPMB from the conditioned media of SKBR3 and HT29 cell cultures without preliminary purification by a size-exclusion chromatography (SEC) column. The developed approach demonstrates a high capacity for isolating EVs and subsequently profiling of their membrane proteins, with a total assay time of approximately 2 h. The approach presented here can be a promising tool for rapid detection of EV membrane proteins using conventional instruments, such as flow cytometry.
Keywords: exosomes; small extracellular vesicles; superparamagnetic beads; cancer diagnostics; liquid biopsy exosomes; small extracellular vesicles; superparamagnetic beads; cancer diagnostics; liquid biopsy

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MDPI and ACS Style

Moiseeva, E.; Sergeev, I.; Chernyshev, V.; Zaborova, O.; Kohzevnikova, D.; Yakovlev, A.; Kuznetsova, O.; Tryakin, A.; Komlev, A.; Gorin, D.; et al. Profiling of CD63 and EpCAM Membrane Proteins of Extracellular Vesicles on Tannic Acid-Coated Magnetic Beads Using Conventional Flow Cytometry. Int. J. Mol. Sci. 2025, 26, 11324. https://doi.org/10.3390/ijms262311324

AMA Style

Moiseeva E, Sergeev I, Chernyshev V, Zaborova O, Kohzevnikova D, Yakovlev A, Kuznetsova O, Tryakin A, Komlev A, Gorin D, et al. Profiling of CD63 and EpCAM Membrane Proteins of Extracellular Vesicles on Tannic Acid-Coated Magnetic Beads Using Conventional Flow Cytometry. International Journal of Molecular Sciences. 2025; 26(23):11324. https://doi.org/10.3390/ijms262311324

Chicago/Turabian Style

Moiseeva, Ekaterina, Igor Sergeev, Vasiliy Chernyshev, Olga Zaborova, Daria Kohzevnikova, Alexander Yakovlev, Olesya Kuznetsova, Alexey Tryakin, Aleksei Komlev, Dmitry Gorin, and et al. 2025. "Profiling of CD63 and EpCAM Membrane Proteins of Extracellular Vesicles on Tannic Acid-Coated Magnetic Beads Using Conventional Flow Cytometry" International Journal of Molecular Sciences 26, no. 23: 11324. https://doi.org/10.3390/ijms262311324

APA Style

Moiseeva, E., Sergeev, I., Chernyshev, V., Zaborova, O., Kohzevnikova, D., Yakovlev, A., Kuznetsova, O., Tryakin, A., Komlev, A., Gorin, D., & Yashchenok, A. (2025). Profiling of CD63 and EpCAM Membrane Proteins of Extracellular Vesicles on Tannic Acid-Coated Magnetic Beads Using Conventional Flow Cytometry. International Journal of Molecular Sciences, 26(23), 11324. https://doi.org/10.3390/ijms262311324

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