{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T16:28:12Z","timestamp":1788280092802,"version":"build-2803163510"},"reference-count":62,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"HUN-REN Research Centre for Natural Sciences"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Extracellular vesicles (EVs) hold strong potential as non-invasive biomarkers for early detection of diseases. However, their clinically relevant quantitative flow cytometric (FCM) analysis remains limited by the lack of suitable calibration particles with low single-particle fluorescence values. Although fluorescent labeling of silica particles is well established, traditional approaches typically yield heterogeneous fluorophore distributions in the low MESF (Molecules of equivalent soluble fluorophore) range, which is required for single-EV fluorescence calibration. To this end, we introduce a labeling strategy that overcomes this limitation by using fluorescently tagged silica-binding peptides (R5-FITC) to produce submicron silica particles with homogeneous low-level fluorophore coverage. Silica particles of three different sizes were synthesized and labeled in a fast and straightforward manner. After comprehensive characterization, FCM investigations revealed that the resulting dim fluorescent silica particles (as low as 278 MESF with narrow distribution) are suitable for calibrating the fluorescence intensity of EVs isolated from red blood cells. Our findings show that fluorescent particles resembling EVs in both size and fluorescence can improve calibration accuracy and foster standardization in FCM-based EV characterization.<\/jats:p>","DOI":"10.1038\/s41598-026-60804-3","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T15:00:36Z","timestamp":1788274836000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Low-MESF fluorescent reference particles for flow cytometry analysis of extracellular vesicles"],"prefix":"10.1038","volume":"16","author":[{"given":"Marcell","family":"P\u00e1lmai","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viet Thuc","family":"Dang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anik\u00f3","family":"Ga\u00e1l","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kinga","family":"Ily\u00e9s","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Istv\u00e1n","family":"M\u00e1ndity","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Judith","family":"Mih\u00e1ly","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andy I.","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Preston T.","family":"Snee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zolt\u00e1n","family":"Varga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"60804_CR1","doi-asserted-by":"publisher","first-page":"1907","DOI":"10.1016\/j.jprot.2010.06.006","volume":"73","author":"S Mathivanan","year":"2010","unstructured":"Mathivanan, S., Ji, H., Simpson, R. J. & Exosomes Extracellular organelles important in intercellular communication. J. Proteom. 73, 1907\u20131920 (2010).","journal-title":"J. Proteom."},{"key":"60804_CR2","doi-asserted-by":"publisher","first-page":"172","DOI":"10.1016\/j.tcb.2016.11.003","volume":"27","author":"SLN Maas","year":"2017","unstructured":"Maas, S. L. N., Breakefield, X. O. & Weaver, A. M. Extracellular vesicles: Unique intercellular delivery vehicles. Trends Cell Biol. 27, 172\u2013188 (2017).","journal-title":"Trends Cell Biol."},{"key":"60804_CR3","doi-asserted-by":"publisher","first-page":"2667","DOI":"10.1007\/s00018-011-0689-3","volume":"68","author":"B Gy\u00f6rgy","year":"2011","unstructured":"Gy\u00f6rgy, B. et al. Membrane vesicles, current state-of-the-art: Emerging role of extracellular vesicles. Cell. Mol. Life Sci. 68, 2667\u20132688 (2011).","journal-title":"Cell. Mol. Life Sci."},{"key":"60804_CR4","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1124\/pr.112.005983","volume":"64","author":"E van der Pol","year":"2012","unstructured":"van der Pol, E. et al. Functions, and clinical relevance of extracellular vesicles. Pharmacol. Rev. 64, 676\u2013705 (2012).","journal-title":"Pharmacol. Rev."},{"key":"60804_CR5","doi-asserted-by":"publisher","first-page":"236","DOI":"10.1038\/s41577-022-00763-8","volume":"23","author":"EI Buzas","year":"2023","unstructured":"Buzas, E. I. The roles of extracellular vesicles in the immune system. Nat. Rev. Immunol. 23, 236\u2013250 (2023).","journal-title":"Nat. Rev. Immunol."},{"key":"60804_CR6","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1007\/s00281-011-0250-3","volume":"33","author":"TH Lee","year":"2011","unstructured":"Lee, T. H. et al. Microvesicles as mediators of intercellular communication in cancer\u2014the emerging science of cellular \u2018debris\u2019. Semin. Immunopathol. 33, 455\u2013467 (2011).","journal-title":"Semin. Immunopathol."},{"key":"60804_CR7","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1016\/j.semcdb.2015.02.010","volume":"40","author":"VR Minciacchi","year":"2015","unstructured":"Minciacchi, V. R., Freeman, M. R. & Di Vizio, D. Extracellular vesicles in cancer: Exosomes, microvesicles and the emerging role of large oncosomes. Semin. Cell Dev. Biol. 40, 41\u201351 (2015).","journal-title":"Seminars Cell Developmental Biology"},{"key":"60804_CR8","doi-asserted-by":"publisher","first-page":"2267","DOI":"10.1002\/glia.23445","volume":"66","author":"SN Blandford","year":"2018","unstructured":"Blandford, S. N., Galloway, D. A. & Moore, C. S. The roles of extracellular vesicle microRNAs in the central nervous system. Glia 66, 2267\u20132278 (2018).","journal-title":"Glia"},{"key":"60804_CR9","doi-asserted-by":"publisher","first-page":"4800","DOI":"10.3390\/ijms16034800","volume":"16","author":"P Joshi","year":"2015","unstructured":"Joshi, P., Benussi, L., Furlan, R., Ghidoni, R. & Verderio, C. Extracellular vesicles in Alzheimer\u2019s disease: Friends or foes? Focus on A\u03b2-vesicle interaction. Int. J. Mol. Sci. 16, 4800\u20134813 (2015).","journal-title":"Int. J. Mol. Sci."},{"key":"60804_CR10","doi-asserted-by":"publisher","first-page":"292","DOI":"10.1007\/s11481-017-9768-z","volume":"13","author":"AM DeLeo","year":"2018","unstructured":"DeLeo, A. M. & Ikezu, T. Extracellular vesicle biology in Alzheimer\u2019s disease and related tauopathy. J. Neuroimmune Pharmacol. 13, 292\u2013308 (2018).","journal-title":"J. Neuroimmune Pharmacol."},{"key":"60804_CR11","doi-asserted-by":"crossref","unstructured":"De Toro, J., Herschlik, L., Waldner, C. & Mongini, C. Emerging roles of exosomes in normal and pathological conditions: New insights for diagnosis and therapeutic applications. Front. Immunol. 6 (2015).","DOI":"10.3389\/fimmu.2015.00203"},{"key":"60804_CR12","doi-asserted-by":"publisher","first-page":"397","DOI":"10.1016\/j.nantod.2015.04.004","volume":"10","author":"G Fuhrmann","year":"2015","unstructured":"Fuhrmann, G., Herrmann, I. K. & Stevens, M. M. Cell-derived vesicles for drug therapy and diagnostics: Opportunities and challenges. Nano Today 10, 397\u2013409 (2015).","journal-title":"Nano Today"},{"key":"60804_CR13","doi-asserted-by":"publisher","first-page":"1649","DOI":"10.1161\/CIRCRESAHA.117.310752","volume":"120","author":"F Jansen","year":"2017","unstructured":"Jansen, F., Nickenig, G. & Werner, N. Extracellular vesicles in cardiovascular disease. Circul. Res. 120, 1649\u20131657 (2017).","journal-title":"Circul. Res."},{"key":"60804_CR14","doi-asserted-by":"publisher","first-page":"147","DOI":"10.1186\/s12943-018-0897-7","volume":"17","author":"Z Sun","year":"2018","unstructured":"Sun, Z. et al. Effect of exosomal miRNA on cancer biology and clinical applications. Mol. Cancer 17, 147 (2018).","journal-title":"Mol. Cancer"},{"key":"60804_CR15","doi-asserted-by":"publisher","first-page":"e1835","DOI":"10.1002\/wnan.1835","volume":"15","author":"KP De Sousa","year":"2023","unstructured":"De Sousa, K. P. et al. Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy. WIREs Nanomed. Nanobiotechnol. 15, e1835 (2023).","journal-title":"WIREs Nanomed. Nanobiotechnol."},{"key":"60804_CR16","doi-asserted-by":"crossref","unstructured":"Willms, E., Caba\u00f1as, C., M\u00e4ger, I., Wood, M. J. A. & Vader, P. Extracellular vesicle heterogeneity: Subpopulations, isolation techniques, and diverse functions in cancer progression. Front. Immunol. 9 (2018).","DOI":"10.3389\/fimmu.2018.00738"},{"key":"60804_CR17","doi-asserted-by":"publisher","first-page":"1816641","DOI":"10.1080\/20013078.2020.1816641","volume":"9","author":"JA Welsh","year":"2020","unstructured":"Welsh, J. A. et al. Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration. J. Extracell. Vesicles 9, 1816641 (2020).","journal-title":"J. Extracell. Vesicles"},{"key":"60804_CR18","doi-asserted-by":"publisher","first-page":"32945","DOI":"10.3402\/jev.v5.32945","volume":"5","author":"C Gardiner","year":"2016","unstructured":"Gardiner, C. et al. Techniques used for the isolation and characterization of extracellular vesicles: Results of a worldwide survey. J. Extracell. Vesicles. 5, 32945 (2016).","journal-title":"J. Extracell. Vesicles"},{"key":"60804_CR19","doi-asserted-by":"publisher","first-page":"1917","DOI":"10.1021\/acs.chemrev.7b00534","volume":"118","author":"H Shao","year":"2018","unstructured":"Shao, H. et al. New technologies for analysis of extracellular vesicles. Chem. Rev. 118, 1917\u20131950 (2018).","journal-title":"Chem. Rev."},{"key":"60804_CR20","doi-asserted-by":"publisher","first-page":"2400024","DOI":"10.1002\/ppsc.202400024","volume":"41","author":"EJ Morris","year":"2024","unstructured":"Morris, E. J. et al. The Physical characterization of extracellular vesicles for function elucidation and biomedical applications: A review. Particle Particle Syst. Charact. 41, 2400024 (2024).","journal-title":"Particle Particle Syst. Characterization"},{"key":"60804_CR21","doi-asserted-by":"publisher","first-page":"2032","DOI":"10.1016\/j.jtha.2023.04.042","volume":"21","author":"BA Bettin","year":"2023","unstructured":"Bettin, B. A., Varga, Z., Nieuwland, R. & van der Pol, E. Standardization of extracellular vesicle concentration measurements by flow cytometry: The past, present, and future. J. Thromb. Haemost. 21, 2032\u20132044 (2023).","journal-title":"J. Thromb. Haemost."},{"key":"60804_CR22","doi-asserted-by":"publisher","first-page":"294","DOI":"10.1002\/cyto.990100308","volume":"10","author":"RF Vogt Jr","year":"1989","unstructured":"Vogt, R. F. Jr., Cross, G. D., Henderson, L. O. & Phillips, D. L. Model system evaluating fluorescein-labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers. Cytometry 10, 294\u2013302 (1989).","journal-title":"Cytometry"},{"key":"60804_CR23","doi-asserted-by":"publisher","first-page":"5066","DOI":"10.1021\/bi00234a033","volume":"30","author":"SP Fay","year":"1991","unstructured":"Fay, S. P., Posner, R. G., Swann, W. N. & Sklar, L. A. Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry. Biochemistry 30, 5066\u20135075 (1991).","journal-title":"Biochemistry"},{"key":"60804_CR24","doi-asserted-by":"publisher","first-page":"83","DOI":"10.6028\/jres.107.009","volume":"107","author":"A Schwartz","year":"2002","unstructured":"Schwartz, A. et al. Quantitating fluorescence intensity from fluorophore: The definition of MESF assignment. J. Res. Natl. Inst. Stand. Technol. 107, 83\u201391 (2002).","journal-title":"J. Res. Natl. Inst. Stand. Technol."},{"key":"60804_CR25","doi-asserted-by":"publisher","first-page":"102720","DOI":"10.1016\/j.nano.2023.102720","volume":"56","author":"E Lozano-Andr\u00e9s","year":"2024","unstructured":"Lozano-Andr\u00e9s, E. et al. Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry. Nanomed. Nanotechnol. Biol. Med. 56, 102720 (2024).","journal-title":"Nanomed. Nanotechnol. Biol. Med."},{"key":"60804_CR26","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1016\/j.nima.2005.01.312","volume":"545","author":"YJ Park","year":"2005","unstructured":"Park, Y. J. et al. The preparation of uranium-adsorbed silica particles as a reference material for the fission track analysis. Nucl. Instrum. Methods Phys. Res., Sect. A. 545, 493\u2013502 (2005).","journal-title":"Nucl. Instrum. Methods Phys. Res., Sect. A"},{"key":"60804_CR27","doi-asserted-by":"publisher","first-page":"112","DOI":"10.1002\/ppsc.200900094","volume":"27","author":"O Couteau","year":"2010","unstructured":"Couteau, O. et al. A colloidal silica reference material for nanoparticle sizing by means of dynamic light scattering and centrifugal liquid sedimentation. Particle Particle Syst. Charact. 27, 112\u2013124 (2010).","journal-title":"Particle Particle Syst. Characterization"},{"key":"60804_CR28","doi-asserted-by":"publisher","first-page":"3895","DOI":"10.1007\/s00216-013-7554-1","volume":"406","author":"R Grombe","year":"2014","unstructured":"Grombe, R. et al. Production of reference materials for the detection and size determination of silica nanoparticles in tomato soup. Anal. Bioanal. Chem. 406, 3895\u20133907 (2014).","journal-title":"Anal. Bioanal. Chem."},{"key":"60804_CR29","doi-asserted-by":"publisher","first-page":"12966","DOI":"10.1021\/acs.analchem.4c00021","volume":"96","author":"D Bartczak","year":"2024","unstructured":"Bartczak, D. et al. Determination of the nanoscale silica mass fraction by AF4\/ICP-MS with isotope dilution analysis using 29Si-enriched silica nanoparticles. Anal. Chem. 96, 12966\u201312972 (2024).","journal-title":"Anal. Chem."},{"key":"60804_CR30","doi-asserted-by":"publisher","first-page":"34","DOI":"10.1016\/j.jcis.2012.09.025","volume":"390","author":"M P\u00e1lmai","year":"2013","unstructured":"P\u00e1lmai, M. et al. Preparation, purification, and characterization of aminopropyl-functionalized silica sol. J. Colloid Interface Sci. 390, 34\u201340 (2013).","journal-title":"J. Colloid Interface Sci."},{"key":"60804_CR31","doi-asserted-by":"publisher","first-page":"2747","DOI":"10.5012\/bkcs.2013.34.9.2747","volume":"34","author":"W He","year":"2013","unstructured":"He, W. et al. Surface modification of colloidal silica nanoparticles: Controlling the size and grafting process. Bull. Korean Chem. Soc. 34, 2747\u20132752 (2013).","journal-title":"Bull. Korean Chem. Soc."},{"key":"60804_CR32","doi-asserted-by":"publisher","first-page":"1426","DOI":"10.3390\/molecules16021426","volume":"16","author":"UOS Seker","year":"2011","unstructured":"Seker, U. O. S. & Demir, H. V. Material binding peptides for nanotechnology. Molecules 16, 1426\u20131451 (2011).","journal-title":"Molecules"},{"key":"60804_CR33","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1016\/j.tibtech.2015.02.005","volume":"33","author":"A Care","year":"2015","unstructured":"Care, A., Bergquist, P. L. & Sunna, A. Solid-binding peptides: Smart tools for nanobiotechnology. Trends Biotechnol. 33, 259\u2013268 (2015).","journal-title":"Trends Biotechnol."},{"key":"60804_CR34","doi-asserted-by":"publisher","first-page":"4738","DOI":"10.1021\/acsbiomaterials.2c00651","volume":"8","author":"Y Ruan","year":"2022","unstructured":"Ruan, Y. et al. Applications of material-binding peptides: A review. ACS Biomater. Sci. Eng. 8, 4738\u20134750 (2022).","journal-title":"ACS Biomaterials Science Engineering"},{"key":"60804_CR35","doi-asserted-by":"publisher","first-page":"1304","DOI":"10.3390\/ijms26031304","volume":"26","author":"GT Lim","year":"2025","unstructured":"Lim, G. T. & Jo, B. H. Exploiting silica-binding and silica-forming proteins as versatile tools for one-step enzyme immobilization on siliceous materials. Int. J. Mol. Sci. 26, 1304 (2025).","journal-title":"Int. J. Mol. Sci."},{"key":"60804_CR36","doi-asserted-by":"publisher","first-page":"6356","DOI":"10.1021\/nn301866q","volume":"6","author":"V Puddu","year":"2012","unstructured":"Puddu, V. & Perry, C. C. Peptide adsorption on silica nanoparticles: Evidence of hydrophobic interactions. ACS Nano 6, 6356\u20136363 (2012).","journal-title":"ACS Nano"},{"key":"60804_CR37","doi-asserted-by":"publisher","first-page":"227","DOI":"10.1021\/la403242f","volume":"30","author":"V Puddu","year":"2014","unstructured":"Puddu, V. & Perry, C. C. Interactions at the silica\u2013peptide interface: The influence of particle size and surface functionality. Langmuir 30, 227\u2013233 (2014).","journal-title":"Langmuir"},{"key":"60804_CR38","doi-asserted-by":"publisher","first-page":"1609","DOI":"10.1021\/acs.jpclett.1c03306","volume":"13","author":"C Liu","year":"2022","unstructured":"Liu, C., Steer, D. L., Song, H. & He, L. Superior binding of proteins on a silica surface: Physical insight into the synergetic contribution of polyhistidine and a silica-binding peptide. J. Phys. Chem. Lett. 13, 1609\u20131616 (2022).","journal-title":"J. Phys. Chem. Lett."},{"key":"60804_CR39","doi-asserted-by":"publisher","first-page":"1129","DOI":"10.1126\/science.286.5442.1129","volume":"286","author":"N Kr\u00f6ger","year":"1999","unstructured":"Kr\u00f6ger, N., Deutzmann, R. & Sumper, M. Polycationic peptides from diatom biosilica that direct silica nanosphere formation. Science 286, 1129\u20131132 (1999).","journal-title":"Science"},{"key":"60804_CR40","doi-asserted-by":"publisher","unstructured":"Knecht, M. R. & Wright, D. W. Functional analysis of the biomimetic silica precipitating activity of the R5 peptide from Cylindrotheca fusiformis. Chem. Commun. 3038\u20133039. https:\/\/doi.org\/10.1039\/B309074D (2003).","DOI":"10.1039\/B309074D"},{"key":"60804_CR41","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez, F., Glawe, D. D., Naik, R. R., Hallinan, K. P. & Stone, M. O. Study of the chemical and physical influences upon in vitro peptide-mediated silica formation. Biomacromolecules 5, 261\u2013265 (2004).","DOI":"10.1021\/bm034232c"},{"key":"60804_CR42","doi-asserted-by":"publisher","first-page":"2607","DOI":"10.1039\/C4TB01679C","volume":"3","author":"L Senior","year":"2015","unstructured":"Senior, L. et al. Structure and function of the silicifying peptide R5. J. Mater. Chem. B. 3, 2607\u20132614 (2015).","journal-title":"J. Mater. Chem. B"},{"key":"60804_CR43","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1016\/0021-9797(68)90272-5","volume":"26","author":"W St\u00f6ber","year":"1968","unstructured":"St\u00f6ber, W., Fink, A. & Bohn, E. Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 26, 62\u201369 (1968).","journal-title":"J. Colloid Interface Sci."},{"key":"60804_CR44","doi-asserted-by":"publisher","first-page":"385701","DOI":"10.1088\/1361-6528\/ad568b","volume":"35","author":"R Sch\u00fcrmann","year":"2024","unstructured":"Sch\u00fcrmann, R. et al. Comparing novel small-angle x-ray scattering approaches for absolute size and number concentration measurements of spherical SiO2 particles to established methods. Nanotechnology 35, 385701 (2024).","journal-title":"Nanotechnology"},{"key":"60804_CR45","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1007\/s11051-010-0085-1","volume":"13","author":"CO Metin","year":"2011","unstructured":"Metin, C. O., Lake, L. W., Miranda, C. R. & Nguyen Q. P. Stability of aqueous silica nanoparticle dispersions. J. Nanopart. Res. 13, 839\u2013850 (2011).","journal-title":"J. Nanopart. Res."},{"key":"60804_CR46","doi-asserted-by":"publisher","first-page":"111053","DOI":"10.1016\/j.colsurfb.2020.111053","volume":"192","author":"Z Varga","year":"2020","unstructured":"Varga, Z. et al. Size measurement of extracellular vesicles and synthetic liposomes: The impact of the hydration shell and the protein corona. Colloids Surf. B. 192, 111053 (2020).","journal-title":"Colloids Surf., B"},{"key":"60804_CR47","doi-asserted-by":"publisher","first-page":"308","DOI":"10.1038\/nnano.2011.24","volume":"6","author":"JL Fraikin","year":"2011","unstructured":"Fraikin, J. L., Teesalu, T., McKenney, C. M. & Ruoslahti, E. Cleland, A. N. A high-throughput label-free nanoparticle analyser. Nat. Nanotechnol. 6, 308\u2013313 (2011).","journal-title":"Nat. Nanotechnol."},{"key":"60804_CR48","doi-asserted-by":"publisher","first-page":"459","DOI":"10.1016\/j.bbamem.2016.12.005","volume":"1859","author":"J Mih\u00e1ly","year":"2017","unstructured":"Mih\u00e1ly, J. et al. Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and CH stretching vibrations. Biochimica et Biophys. Acta (BBA) - Biomembranes. 1859, 459\u2013466 (2017).","journal-title":"Biochimica et Biophys. Acta (BBA) - Biomembranes"},{"key":"60804_CR49","doi-asserted-by":"publisher","first-page":"1568780","DOI":"10.1080\/20013078.2019.1568780","volume":"8","author":"A Gualerzi","year":"2019","unstructured":"Gualerzi, A. et al. Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality. J. Extracell. Vesicles. 8, 1568780 (2019).","journal-title":"J. Extracell. Vesicles"},{"key":"60804_CR50","doi-asserted-by":"publisher","first-page":"19868","DOI":"10.1038\/s41598-019-56375-1","volume":"9","author":"D Kitka","year":"2019","unstructured":"Kitka, D., Mih\u00e1ly, J., Fraikin, J. L., Beke-Somfai, T. & Varga, Z. Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection. Sci. Rep. 9, 19868 (2019).","journal-title":"Sci. Rep."},{"key":"60804_CR51","doi-asserted-by":"publisher","first-page":"977","DOI":"10.1038\/s41598-022-04915-7","volume":"12","author":"T Bebesi","year":"2022","unstructured":"Bebesi, T. et al. Storage conditions determine the characteristics of red blood cell derived extracellular vesicles. Sci. Rep. 12, 977 (2022).","journal-title":"Sci. Rep."},{"key":"60804_CR52","doi-asserted-by":"publisher","first-page":"114366","DOI":"10.1016\/j.colsurfb.2024.114366","volume":"246","author":"T Bebesi","year":"2025","unstructured":"Bebesi, T. et al. Surface-enhanced infrared spectroscopic study of extracellular vesicles using plasmonic gold nanoparticles. Colloids Surf. B. 246, 114366 (2025).","journal-title":"Colloids Surf., B"},{"key":"60804_CR53","doi-asserted-by":"crossref","unstructured":"Dehghani, M. & Gaborski, T. R. Chapter Two - Fluorescent labeling of extracellular vesicles. in Methods in Enzymology (eds Spada, S. & Galluzzi, L.) vol. 645 15\u201342 (Academic, 2020).","DOI":"10.1016\/bs.mie.2020.09.002"},{"key":"60804_CR54","doi-asserted-by":"publisher","first-page":"569","DOI":"10.1002\/cyto.a.23782","volume":"97","author":"JA Welsh","year":"2020","unstructured":"Welsh, J. A. et al. FCMPASS Software aids extracellular vesicle light scatter standardization. Cytometry Part. A. 97, 569\u2013581 (2020).","journal-title":"Cytometry Part. A"},{"key":"60804_CR55","doi-asserted-by":"publisher","first-page":"1574","DOI":"10.1021\/j150570a026","volume":"62","author":"T Nash","year":"1958","unstructured":"Nash, T. Interactions of un-ionized fluorescein. I. Color discharge by solvents and colloids. J. Phys. Chem. 62, 1574\u20131578 (1958).","journal-title":"J. Phys. Chem."},{"key":"60804_CR56","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/cyto.b.10066","volume":"57B","author":"A Schwartz","year":"2004","unstructured":"Schwartz, A. et al. Formalization of the MESF unit of fluorescence intensity. Cytometry Part. B: Clin. Cytometry 57B, 1\u20136 (2004).","journal-title":"Cytometry Part. B: Clin. Cytometry"},{"key":"60804_CR57","doi-asserted-by":"publisher","first-page":"1646","DOI":"10.1111\/jth.14193","volume":"16","author":"Z Varga","year":"2018","unstructured":"Varga, Z. et al. Hollow organosilica beads as reference particles for optical detection of extracellular vesicles. J. Thromb. Haemost. 16, 1646\u20131655 (2018).","journal-title":"J. Thromb. Haemost"},{"key":"60804_CR58","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1186\/s11671-024-03956-3","volume":"19","author":"J Deumer","year":"2024","unstructured":"Deumer, J. et al. Traceable characterization of hollow organosilica beads as potential reference materials for extracellular vesicle measurements with optical techniques. Disc. Nano 19, 14 (2024).","journal-title":"Discover Nano"},{"key":"60804_CR59","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1002\/anie.199101133","volume":"30","author":"E Bayer","year":"1991","unstructured":"Bayer, E. Towards the chemical synthesis of proteins. Angewandte Chemie Int. Ed. Engl. 30, 113\u2013129 (1991).","journal-title":"Angewandte Chemie Int. Ed. Engl."},{"key":"60804_CR60","doi-asserted-by":"publisher","first-page":"4397","DOI":"10.1021\/ja00063a082","volume":"115","author":"LA Carpino","year":"1993","unstructured":"Carpino, L. A. 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397\u20134398 (1993).","journal-title":"J. Am. Chem. Soc."},{"key":"60804_CR61","doi-asserted-by":"publisher","first-page":"e12404","DOI":"10.1002\/jev2.12404","volume":"13","author":"JA Welsh","year":"2024","unstructured":"Welsh, J. A. et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J. Extracell. Vesicles 13, e12404 (2024).","journal-title":"J. Extracell. Vesicles"},{"key":"60804_CR62","unstructured":"https:\/\/tsapps.nist.gov\/srmext\/certificates\/1932.pdf"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-60804-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-60804-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-60804-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T16:04:52Z","timestamp":1788278692000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-026-60804-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,1]]},"references-count":62,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["60804"],"URL":"https:\/\/doi.org\/10.1038\/s41598-026-60804-3","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,1]]},"assertion":[{"value":"7 January 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"30 June 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"1 September 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"27406"}}