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Updated: Mar 11, 2026

Imaging of Extracellular Vesicles by Atomic Force Microscopy
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The Comparison between Different Extracellular Vesicle Isolation Methods by AFM-IR Nanospectroscopy.

Jéssica Verônica da Silva1, Otávio Berenguel2, Raquel Silva Neres-Santos1

  • 1Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC, Av. dos Estados 5001, Santo André, SP 09210-580, Brazil.

Analytical Chemistry
|March 10, 2026
PubMed
Summary

Extracellular vesicle (EV) isolation methods impact their molecular signatures. Atomic force microscopy-infrared nanospectroscopy revealed differences in biomolecule preservation and structural integrity based on isolation techniques.

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Area of Science:

  • Biophysics
  • Nanotechnology
  • Biochemistry

Background:

  • Extracellular vesicles (EVs) are crucial biomarkers due to their cell-specific signatures.
  • Characterizing EVs at the nanoscale is challenging due to their small size and limitations of conventional spectroscopy.
  • Atomic force microscopy-infrared nanospectroscopy (AFM-IR) offers a potential solution for nanoscale EV analysis.

Purpose of the Study:

  • To investigate the molecular signatures of EVs isolated using different methods.
  • To correlate EV topography and biochemical properties with isolation techniques.
  • To evaluate the impact of isolation methods on EV integrity and biomolecule preservation.

Main Methods:

  • EVs were isolated from mouse serum and plasma using Total Exosome Isolation Reagent (TEIR), ultracentrifugation (UC), and size-exclusion chromatography (SEC).
  • Fourier-transform infrared (FTIR) spectroscopy and AFM-IR were used for analysis.
  • Multivariate statistical analysis (PCA, PLS-DA) was employed to interpret spectral data.

Main Results:

  • Isolation methods significantly altered phosphate groups and glycosidic linkages in EVs.
  • SEC depleted biomolecules above 1150 cm-1 (e.g., α-helix, fatty acids) but preserved protein conformations.
  • TEIR affected nucleic acids, phospholipids, and carbohydrates, while UC preserved most biomolecules but caused membrane damage.

Conclusions:

  • The choice of EV isolation method critically influences the resulting molecular composition and integrity.
  • AFM-IR is a valuable tool for detailed biophysical and biochemical characterization of EVs.
  • Understanding these method-dependent variations is essential for reliable EV biomarker discovery and application.