*3.2. Spatial Distribution of the Antibody at the Surface of Electrospun Nanofibers*

After the optimization of the anti-TNF-α antibody immobilization, its spatial distribution at the surface of electrospun NFMs was evaluated by fluorescence microscopy (Figure 2). The fluorescence images display a random mesh-like arrangement comparable to the typical morphology of the electrospun NFM (Figure 2A), showing that the antibody immobilization was successfully performed and uniformly distributed along the surface of the nanofibers (Figure 2B). To ensure that the secondary antibody was only bound to the immobilized primary antibody, a control experiment was defined, in which all biofunctionalization steps were performed, except for incubation with the primary antibody. The absence of a fluorescent signal proves that Alexa Fluor 488® secondary antibody was not immobilized at the surface of activated and functionalized NFMs (data not shown), confirming the specific bond between the primary and secondary antibodies.

**Figure 2.** SEM micrograph of a biofunctionalized NFM (**A**). Fluorescence micrograph of a NFM with the TNF-α antibody immobilized at the maximum concentration (6 μg/mL) (**B**).
