Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy

Beekman, Pepijn; Enciso-Martinez, Agustin; Rho, Hoon Suk; Pujari, Sidharam Pundlik; Lenferink, Aufried; Zuilhof, Han; Terstappen, Leon W.M.M.; Otto, Cees; Gac, Séverine Le


Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 μm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.