Standardized Microplate‐Based Assays for Quantifying Antioxidant Activity in Extracellular Vesicles and Nanovesicles Across Biological Kingdoms
11 affiliations
- 1Laboratory of Epigenetics of Lipid Metabolism Instituto Madrileño de Estudios Avanzados (IMDEA)‐Nutrición, CEI UAM+CSIC Madrid Spain
- 2Bioactivity and Nutritional Immunology Group (BIOINUT), Faculty of Health Science Universidad Internacional de Valencia (VIU) Valencia Spain
- 3Department of Nutrition and Metabolism, Institute of Food Science, Technology and Nutrition Spanish National Research Council, CSIC‐ICTAN Madrid Spain
- 4Department of Pharmaceutical Sciences Università Degli Studi di Milano Milan Italy
- 5Laboratory of Functional Foods Instituto Madrileño de Estudios Avanzados (IMDEA)‐Nutrición, CEI UAM+CSIC Madrid Spain
- 6Department of Molecular Medicine University of Padova Padova Italy
- 7Facultad de Ciencias de la Salud Universidad Tecnológica Atlántico Mediterráneo ‐ UTAMED Málaga Spain
- 8Laboratory of Food & Health, Research Group on Quality, Safety, and Bioactivity of Plant Foods, CEBAS‐CSIC Campus Universitario de Espinardo Murcia Spain
- 9Nutraceuticals and Neuroendocrine and Neurodegenerative Diseases Group (NUENE), Institute of Biomedicine. Faculty of Environmental Sciences and Biochemistry University of Castilla‐La Mancha Toledo Spain
- 10Plant Molecular Genetics Department, National Centre for Biotechnology (CNB) Consejo Superior de Investigaciones Cientificas (CSIC) Madrid Spain
- 11Consorcio CIBER de la Fisiopatología de la Obesidad y Nutrición (CIBERObn) Instituto de Salud Carlos III (ISCIII) Madrid Spain
Abstract
The antioxidant activity of extracellular vesicles (EVs) and nanovesicles are increasingly linked to their physiological and therapeutic functions, yet its accurate quantification remains limited by low EV yield, co‐isolated contaminants and the lack of standardized methodologies. Here, we establish optimized microplate‐based protocols for ABTS and ORAC assays specifically adapted to highly purified EVs and nanovesicles obtained by ultracentrifugation followed by size‐exclusion chromatography (UC + SEC). By minimizing sample input, implementing protein‐based normalization (TEAC/g EV protein), and introducing assay‐specific controls—including a self‐blank correction for ORAC—we achieve sensitive and reproducible measurements across plant‐, human plasma‐ and bacterial‐derived EVs and nanovesicles. Our results demonstrate that conventional isolation methods substantially overestimate antioxidant activity due to non‐vesicular contaminants, underscoring the need for rigorous purification. Using a detergent‐based disruption strategy compatible with ABTS, we further distinguish surface‐associated from cargo‐derived antioxidant activity, revealing a two‐tier functional organization of EVs. Notably, we report for the first time measurable antioxidant activity in bacterial extracellular vesicles (BEVs). Application of these methods to a human dietary intervention shows that EV antioxidant capacity is dynamically modulated in vivo. Collectively, this work provides a standardized and broadly applicable framework for the reliable assessment of EV and nanovesicles antioxidant properties across biological kingdoms.
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BibTeX
@article{nieto2026standardized,
title = {{Standardized Microplate‐Based Assays for Quantifying Antioxidant Activity in Extracellular Vesicles and Nanovesicles Across Biological Kingdoms}},
author = {Juan Antonio Nieto and María‐Carmen López de las Hazas and Victoria Martín‐Santiago and Laura Rubio‐Silvan and Carmen Mazarío‐Gárgoles and Ludovica Attanasio and Benedetta Mercuriali and João Tomé‐Carneiro and Giulia Bernabè and Andrea del Saz‐Lara and Carmen Crespo and María Ángeles Ávila‐Gálvez and Emma Burgos‐Ramos and Juan Carlos Espín and Andrea Chini and Alberto Dávalos},
journal = {Journal of extracellular vesicles},
year = {2026},
month = nov,
doi = {10.1002/jev2.70392},
url = {https://europepmc.org/article/PMC/PMC13730445},
note = {CC BY},
}
RIS
TY - JOUR
TI - Standardized Microplate‐Based Assays for Quantifying Antioxidant Activity in Extracellular Vesicles and Nanovesicles Across Biological Kingdoms
AU - Juan Antonio Nieto
AU - María‐Carmen López de las Hazas
AU - Victoria Martín‐Santiago
AU - Laura Rubio‐Silvan
AU - Carmen Mazarío‐Gárgoles
AU - Ludovica Attanasio
AU - Benedetta Mercuriali
AU - João Tomé‐Carneiro
AU - Giulia Bernabè
AU - Andrea del Saz‐Lara
AU - Carmen Crespo
AU - María Ángeles Ávila‐Gálvez
AU - Emma Burgos‐Ramos
AU - Juan Carlos Espín
AU - Andrea Chini
AU - Alberto Dávalos
PY - 2026
DA - 2026/11/01
JO - Journal of extracellular vesicles
DO - 10.1002/jev2.70392
UR - https://europepmc.org/article/PMC/PMC13730445
AB - The antioxidant activity of extracellular vesicles (EVs) and nanovesicles are increasingly linked to their physiological and therapeutic functions, yet its accurate quantification remains limited by low EV yield, co‐isolated contaminants and the lack of standardized methodologies. Here, we establish optimized microplate‐based protocols for ABTS and ORAC assays specifically adapted to highly purified EVs and nanovesicles obtained by ultracentrifugation followed by size‐exclusion chromatography (UC + SEC). By minimizing sample input, implementing protein‐based normalization (TEAC/g EV protein), and introducing assay‐specific controls—including a self‐blank correction for ORAC—we achieve sensitive and reproducible measurements across plant‐, human plasma‐ and bacterial‐derived EVs and nanovesicles. Our results demonstrate that conventional isolation methods substantially overestimate antioxidant activity due to non‐vesicular contaminants, underscoring the need for rigorous purification. Using a detergent‐based disruption strategy compatible with ABTS, we further distinguish surface‐associated from cargo‐derived antioxidant activity, revealing a two‐tier functional organization of EVs. Notably, we report for the first time measurable antioxidant activity in bacterial extracellular vesicles (BEVs). Application of these methods to a human dietary intervention shows that EV antioxidant capacity is dynamically modulated in vivo. Collectively, this work provides a standardized and broadly applicable framework for the reliable assessment of EV and nanovesicles antioxidant properties across biological kingdoms.
SN - 2001-3078
ER -