*2.3. Antibody Immobilization*

The TNF-α antibody was immobilized at the surface of activated and functionalized electrospun NFMs. To determine the substrate's maximum immobilization capacity, a wide range of primary antibody concentrations were considered (from 0 to 12 μg/mL). The TNF-α antibody was firstly activated by a 15-minute incubation with a solution of EDC/NHS (50 mM EDC and 200 mM NHS), dissolved in a 0.1 M 2-(*N*-morpholino)-ethanesulfonic acid (MES) buffer with 0.9% (*w*/*w*) NaCl, followed by pH adjustment to 4.7 [48].

The functionalized NFMs were incubated with 200 μL of the activated TNF-α antibody overnight at 4 ◦C. Biofunctionalized NFMs were washed three times with PBS. A 3% bovine serum albumin (BSA) solution was used as a blocking step. To determine the degree of TNF-α antibody immobilization, biofunctionalized NFMs were incubated with an Alexa Fluor® 488 solution diluted in PBS (1:200 ratio). As a negative control, NFMs with no antibody immobilized (plotted as 0 μg/mL) were used. The fluorescence of the supernatant was determined in triplicate, using an excitation of 495/20 nm and an emission of 519/20 nm, and used to quantify the amount of antibody effectively immobilized.
