Chemical spectroscopy of individual human milk extracellular vesicles

Abstract

Extracellular vesicles (EVs) are nanosized particles, which are associated with various physiological and pathological functions. They play a key role in intercell communication and are used as transport vehicles for various cell components. In human milk, EVs are believed to be important for the nutrition of infants and for the development of their acquired immunity. State of the art analysis methods are not able to provide label free chemical information at the single-vesicle level, hence new analysis techniques are required to study the chemical difference within EV (sub-)population. We introduce a protocol to profile structure and composition of individual EVs with the help of photothermal scanning probe infrared spectroscopy (AFM-IR). First, EVs are immobilized onto a silicone surface using microcontact printed Anti-CD9 antibodies.AFM-IR then provides both EV size and mid-infrared spectra for individual vesicles. The received spectra compare favorably to bulk reference spectra. In addition, AFM-IR images provide spectral information for multiple EVs within a single measurement. Stacking images taken at different wavenumbers yields hyperspectral chemical images of vesicles at nanoscale spatial resolution.