Fabrication of an electrochemical biosensor for detection of dopamine, employing tyrosinase immobilized polymeric nanofibers

Nanofibers of polyacrylonitrile (PAN)/polyurethane (PU)/poly(m-anthranilic acid) (P3ANA) blend were fabricated by the electrospinning process. UV-Visible spectroscopy was performed to confirm the incorporation of each individual polymer into the nanofibers. This is followed by a Fourier Transform Infrared Spectroscopy – Attenuated Total Reflectance (FTIR-ATR) analysis, which displayed an increasing intensity in the bands specific to poly(m-anthranilic acid), in correlation with its increasing amounts in the blend. Nanofibers with the highest amount of P3ANA were then used for enzyme immobilization, considering it provides more carboxyl groups for enzymes to bind onto. Next, tyrosinase immobilization on these nanofibers was carried out by 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS) activation procedure. Covalent binding of tyrosinase onto nanofiber mats was confirmed by FTIR-ATR analysis, and a subsequent bicinchoninic acid (BCA) assay revealed the amount of enzyme immobilized. Morphology and composition of nanofibers before and after enzyme immobilization were characterized by Scanning Electron Microscopy (SEM)/Energy Dispersive X-ray Spectroscopy (EDX). The surface morphology of the tyrosinase immobilized PAN/PU/P3ANA nanofibers became smoother and the diameters of the fibers increased. Elemental analyses and EDX-mapping of nitrogen and copper atoms showed that the enzyme was evenly distributed on the surface of the nanofibers.Then, Electrochemical Impedance Spectroscopy (EIS) analysis was performed to observe the effects of immobilized enzyme on electrochemical properties of nanofibers and the data were fitted with an equivalent electrical circuit model. The changes in structure, composition and morphology of nanofibers after tyrosinase immobilization affected their electrochemical behavior. The solution resistance and charge transfer resistance of nanofibers decreased after enzyme immobilization.