## 3 Results and Discussion
### Interaction between Extracellular Vesicles and Microcantilevers
This behavior characterizes the whole 30 min the MCs are incubated with the EV solution (stopped flow), and is in agreement to what previously observed with liposomes [17]. When the flow is restarted and the solution is replaced by the pure buffer, excess and weakly adsorbed EVs are washed away and the MCs stabilize with an upward differential deflection of \(\Delta\)z = (38 \(\pm\) 5) nm, which probes the formation of a stable EV layer. By substituting \(\Delta\)z into Equation (1) we learn that the layer formation involves a surface work that appears as a surface stress change of \(\Delta\sigma\) = (8 \(\pm\) 1) mJ/m\({}^{2}\). This result is about half the value reported in the literature for the surface stress measured for the formation of supported lipid bilayer (SLB) on SiO\({}_{2}\) surface from POPC liposomes [17; 18]. This indicates that the formed layer is not continuous and/or that the formation of the EV layer is less energetic. Very likely a combination of both. Figure 3: Static microcantilever (MC) experiments. (**A**) Scheme of MCs and their deflection due to the interaction between the EVs and their silicon bottom face; (**B**) MCs mean differential deflection driven by the interaction of the EVs with the MCs. The error bars represent the standard deviation of the mean. To explore the hypotheses formulated after the results of the first set of experiments, we exploited the dynamic mode of MCs actuation, measuring the mass contribution of the EV layer during the EV interaction with the MC surface. For this set of experiments, we used bare MCs without any passivation, in order to maximize the adsorbed EVs and obtain significant results with respect to the limit of detection (viz. the largest frequency shift achievable). Figure 4B shows the resonant frequency signal of a representative MC. After injection, the EV solution was allowed to flow in the measurement chamber for about 2 min, corresponding to the first peak in the frequency signal. Then the flow was stopped for about 30 min allowing equilibrium interaction of the EVs with the MCs. During this period the frequency signal decreases due to the increased adsorbed mass. After this period the flow was restarted, corresponding to the second peak in the signal, until the buffer has completely removed the excess and weakly adsorbed EVs and the signal reaches its final value. Three replicates of this experiment were performed with three different MC arrays. The value of added mass was determined by Equation (2) yielding a mean value of m = (110 \(\pm\) 40) ng/cm\({}^{2}\). The interpretation of this measured added mass can be assisted by calculating a theoretical value of added mass. Let us consider a MC surface occupied by a continuous double layer of phospholipids, with a single phospholipid head of 0.7 nm\({}^{2}\), a mass of 1.26 \(\times\) 10\({}^{-24}\) kg and a packing factor onto the surface between 0.5 and 1. The theoretical estimation of the value can range between 180 ng/cm\({}^{2}\) and 360 ng/cm\({}^{2}\). This calculation underestimates the real value, due to the following assumptions: (a) it does not take into account the contribution of the proteins that EVs harbor on the surface, and (b) it assumes similar contribution in weight of the two faces of the MC, neglecting the possibility to have intact vesicles on the Au surface that would pack in the same surface area, with a \(>\) 5 times higher mass. Taking into account these considerations, we expected an experimental value higher than the theoretical one, instead the result was slightly lower. This can be ascribed to the formation of non-continuous layers of EVs on the surface, maybe both on the Au and Si faces, supporting the results indicated by the first set of experiments. Figure 4: Simultaneous MCs experiments in static and dynamic mode. (**A**) Scheme of the interaction between MCs and EVs; (**B**) representative MC frequency signal due to the interaction of EVs and MC surfaces; (**C**) absolute deflection signal of the MC of panel B.