Oriented immobilization of antibody through carbodiimide reaction and controlling electric field

Abstract

A novel and simple approach for oriented immobilization of antibody (Ab) was proposed through carbodiimide reaction and controlling electric field (CEF). The electrode was modified by self-assembled monolayer of cysteine (CYS SAM) with amino group stretching outside, and Fc region of Ab was activated via carbodiimide reaction. By adjusting pH of solution and applying a proper electric field, Abs were manipulated owing to their charged nature and immobilized on the electrode via the activated Fc region. The stepwise modified electrodes were characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results proved that Abs could be successfully immobilized on the surface of electrode. Under optimal experimental parameters, the immunosensor could detect antigen (Ag) in a range from 0.04 to 400 ng/mL. Furthermore, the immunosensors fabricated with and without CEF were comparatively studied, and the results showed that the former had better detection properties than the later. Finally, the effect of CEF on Ab was studied by polarization modulation infrared reflection–absorption spectroscopy (PM-IRRAS) and scanning electron microscope (SEM); the results indicated that Abs were arranged in better order under electric field, this may be the reason for the better properties of the immunosenor. In a word, Ab immobilization through carbodiimide reaction and CEF was very simple, fast, and easy to perform, and it would find wide application in the immune-based assay systems.