From AFM to TERS: Unveiling the Nanoscale Morphological and Chemical Composition of Bovine Milk-Derived Extracellular Vesicles

Tip-enhanced Raman Spectroscopy (TERS) is an advanced technique that combines Raman spectroscopy and Atomic Force Microscopy (AFM) to perform local chemical analysis of sample surfaces at nanoscale resolution. This work demonstrates the innovative application of TERS for analyzing bovine milk derived extracellular vesicles (mEVs). These nanostructures have significant potential in drug delivery and therapeutic applications. Traditional Raman spectroscopy has been used to analyze mEVs, providing a detailed spectrum that identifies the “signature” of mEVs based on their characteristic molecular vibrations, revealing their chemical composition. However, TERS advances this analysis a step further by allowing the study of individual mEVs and specific locations on their surfaces with nanometer lateral resolution. AFM and Transmission Electron Microscopy (TEM) provide critical data on the size, distribution and morphology of the mEVs, confirming their spherical shape and structural integrity. In particular, AFM analysis provided detailed topographical images, while TEM confirmed the average size of 86 ± 34 nm. The characteristic peaks obtained in the Raman spectra indicated the presence of various molecular components such as proteins, lipids and nucleic acids[1]. These analyses provided a comprehensive chemical profile of the mEVs, which served as a reference for the subsequent TERS analysis. TERS characterisation was then carried out to obtain Raman spectra of isolated mEVs and specific locations on their surfaces. The TERS technique uses a silver-coated AFM tip to amplify the Raman signal through plasmonic effects, enabling the acquisition of spectra from nano-sized volumes[2]. This study focused on the 600-1800 cm-1 and 2600-3200 cm-1 spectral ranges typical of biological structures. TERS spectra showed distinct peaks correlating with chemical bonds identified in standard Raman spectra, with additional peaks and improved resolution, demonstrating the superior sensitivity and spatial resolution of the technique. The potential of TERS for nanoscale chemical mapping was further demonstrated by probing different locations on the surface of a single mEV. In particular, the analysis focused on the CH stretching region (2750-3050 cm-1) due to the high intensity of the peaks in this region. By collecting spectra from multiple points on the mEV surface, it was possible to detect variations in the intensity of the CH2 and CH3 stretching peaks, indicating compositional heterogeneity at the nanoscale[3]. This ability to perform detailed chemical mapping of individual mEVs highlights the value of TERS for advanced studies at the molecular level, such as investigating the mechanisms of mEV loading and their functional properties in drug delivery. In conclusion, this work highlights the power of TERS to provide a comprehensive nanoscale characterisation of mEVs, integrating morphological and chemical information. The ability to perform chemical analysis at the level of individual vesicles and specific surface locations paves the way for more detailed studies of the composition and functionality of extracellular vesicles, enhancing their potential applications in biotechnology and medicine. [1] G. Pezzotti, J. Raman Spectrosc. 2021, 52, 2348. [2] Z. Zhang, & al., Anal. Chem. 2016, 88, 9328. [3] L. Buccini & al., Nanoscale. 2024, 16, 8132.