AFM-Based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles.
Anal Chem
; 92(15): 10274-10282, 2020 08 04.
Article
en En
| MEDLINE
| ID: mdl-32631050
ABSTRACT
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Microscopía de Fuerza Atómica
/
Nanotecnología
/
Ensayos Analíticos de Alto Rendimiento
/
Vesículas Extracelulares
Tipo de estudio:
Diagnostic_studies
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Screening_studies
Límite:
Animals
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Humans
Idioma:
En
Revista:
Anal Chem
Año:
2020
Tipo del documento:
Article
País de afiliación:
Italia