is possible to conclude that both polymers chitosan and pcl were able to immobilize the desired antibodies in this case anti vegf after determining the maximum antibody concentration immobilized at the surface of the activated and functionalized electrospun nanofibers a standard curve can be determined for each antibody with the remaining solution of each secondary antibody it is possible to determine the amount of unbound secondary antibody leading indirectly to the concentration of primary antibody that is immobilized a linear regression standard curve fitting those data points allows obtaining a r 2 above 0 98 for every antibody as represented in fig5 these standard curves are further used for the quantification of the immobilized antibodies in next assays fig6 shows the spatial distribution of the tgf u03b21 bfgf and vegf antibodies at the surface of activated and functionalized electrospun nanofibers primary antibodies are immobilized at the previous optimized concentrations in fig6 a 12 u03bcg ml of anti tgf u03b21 in fig6 b 8 u03bcg ml of anti bfgf and in fig6 c 4 u03bcg ml of anti vegf in the case of the tgf u03b21 and vegf antibodies the secondary antibody alexa fluor u00ae 594 is used whereas the secondary antibody alexa fluor u00ae 488 is used for the bfgf antibody the negative controls represented in fig6 d e and f are subjected to all the steps except the incubation with the primary antibodies in an embodiment all the immobilized antibodies seem to be uniformly distributed through the nanofibers surface resembling the random mesh like arrangement of the electrospun nfm structure the tgf u03b21 antibody seems to have a more intense and densely distributed fluorescence than the other immobilized antibodies.