Electrochemical monitoring of the layer-by-layer assembly process carried out for fabrication of immunosensor, including the cleaned bare gold (blue), chemical modification with linkers MUA/MPA (red), activation of linkers with EDC/NHS (green), immobilization of cis P-tau mAb (Purple), and deposition of cis P-tau (orange). (A) Electrochemical impedance spectroscopy (EIS). The imaginary component of EIS (Z″) was plotted against the real component of EIS (Z′) in a Nyquist plot (resistance of solution, (Rsol = 33 Ω). (B) Cyclic voltammetry (CV) voltammogram. The electrochemical measurements were conducted in PBS (pH 7.5) containing 10 mM K4Fe(CN)6/K3Fe(CN)6 (1:1 ratio) by using a three-electrode set up including the modified Au surface as working electrode, a Pt wire as an auxiliary electrode, and Ag/AgCl as the reference electrode.