## 3 Results and Discussion
### Interaction between Extracellular Vesicles and Microcantilevers  
Microantilevers are nanomechanical based sensors designed to measure surface stress changes induced by biomolecular recognition and adsorption occurring on their surface (static mode) and/or contemporarily measure the resonance frequency shifts due to the related mass change (dynamic mode) [13]. These peculiar characteristic prompted successful application of MC sensors, and other tensiometric biosensors to label-free study of molecular collective surface nanomechanics [33; 34; 35; 36], opening new insights in surfaces confined molecular transformations and machines [37; 38; 39; 40; 32], up to single cell investigation [41; 42; 43]. Experiments exploiting static deflection of MC beams are reported in literature to study the chemi- or physisorption of synthetic lipid bilayer on a cantilever surface, on either gold (Au) or silicon dioxide (SiO\({}_{2}\)) surfaces [17; 18]. Moreover, measurements of the resonance frequency shift induced upon phospholipid vesicle adsorption on oscillating cantilevers immersed in a liquid have been described [30]. In the case of static deflection, the bending of the cantilever is caused by the difference in the surface stress between the upper and lower surface. Usually, to reach this asymmetry,  
Figure 2: Characterization of extracellular vesicles (EVs) samples. **(A)** Aggregation index (AI) of EV preparation. AI is inversely proportional to the preparation purity grade; **(B)** western blot of EV samples and parent cells (H) with the EV markers Alix, CD63, HSP70, annexin V, and GM130 as negative control; **(C)** Atomic Force Microscopy (AFM) image of EVs (scale bar = 200 nm) and **(D)** EV size distribution. A total of 108 objects with a size between 30 nm and 160 nm were analyzed; **(E,F)** Colorimetric nanoplasmonic (CONAN) analysis of molar concentration of EVs. Refer to the main text for the details. the cantilevers are Au coated on one side and show a Si surface on the opposite side. The two surfaces allow selective use of either thiol chemistry to functionalize/passivate the Au side or silane chemistry for the Si side [44]. Typical experiments exploit the well-known fact that liposomes readily adsorb and fuse on glass or silicon dioxide, giving rise to extended, homogeneous lipid double layers [45; 46]. The common functionalization choice is then to protect the Au side of cantilevers and drive the vesicles fusion primarily on the SiO\({}_{2}\) surface [18]. Stability of liposomes on Au surfaces is instead not well understood: electrostatic (image-charge) attraction should provide strong adhesion leading to vesicles rupture and bilayer formation, yet adsorbed lipids exhibit negligible lateral diffusion. It is demonstrated that vesicles partially fuse, to some extent, on gold, but the resulting membrane lacks long-range lateral mobility over the time scale of several hours [47; 48]. In the first set of experiments, we addressed the interaction of the EVs with the Si surface. We employed the array of eight replicate MCs. Four of them used as internal reference and therefore with both faces passivated with PEG to prevent vesicles fusion [49] and protein adsorption [50]. The other four MCs instead had the Au surface passivated with 6-mercapto-1-hexanol, to drive and limit EVs to interact with the Si surface (Figure 3A). The MCs mean differential deflection signal between the four active MCs absolute deflection and the four reference MCs absolute deflection is reported in Figure 3B together with the errors (as standard deviation of the mean) at selected points. The first part of the signal is characterized by typical background noise deflection of few nm. When the EV solution is injected into the measurement chamber, the equilibrium interaction between the EVs and the MCs drives significant and chaotically oscillating deflections up to hundreds of nm.