*3.3. Quantification of Available Amine Groups*

Antibody immobilization was achieved by the amine groups inserted at the nanofibers' surface, which provides binding sites. Consequently, the availability of this functional group influences directly the primary antibody immobilization. To confirm the success of aminolysis functionalization, as well as to determine if the binding points were enough for primary antibody immobilization, quantification of available amine groups was performed. For this, SH groups were inserted at the surface of the aminolysis-treated NFMs and determined by the Ellman's reagent method [49]. Table 1 presents SH groups quantification at the surface of bare, UV-O irradiated, aminolysis-treated, and fully biofunctionalized (with primary antibody immobilized) NFMs. The concentration of free SH groups at the surface of electrospun NFMs is at its maximum value for the aminolysis-treated NFM, whereas the bare NFM presents the minimum value, as expected. These values are in agreement with the ones described in the literature [49] and confirm that the insertion of amine groups was successful. Moreover, it was also proved that the amine groups inserted at the surface of activated NFM were enough for primary antibody immobilization, since free amine groups were still present. Indeed, only around 48% of the inserted amine groups were used in the immobilization of the TNF-α antibody at 6 μg/mL.


**Table 1.** Quantification of the free SH groups at the surface of untreated, activated, aminolys-treated, and biofunctionalized NFMs.
