Controlled immobilization of IgG1 on carboxymethyl and amino-dextran SPR chips under external vertical electric fields

Abstract

Aiming at producing surface plasmon resonance (SPR) sensor chips of improved performance, a novel approach for controlled immobilization of antibody (Ab) molecules has been developed through the implementation of surface functionalization and external vertical electric field (EVEF) assisted immobilization techniques. Carboxymethyl dextran (CMD) chips were reacted with ethylenediamine to create surface amino groups and subsequently reacted with the activated carboxyl group of the fragment crystallisable (Fc) region of Ab. An external vertical electric field was applied to control the movement of antibody molecules toward the chip and their orientation on the surface. The EVEF imparted a current density of 4.17 μA cm−2 through the solution. Immobilization was achieved via a carbodiimide reaction as the antibody molecules reached the functionalized surface. Physical characterization was performed through ATR-FTIR, AFM and surface plasmon resonance (SPR) analyses. Results show that the orientation of antibody molecule on the amino dextran (AmDex) surface is better than on the CMD surface in terms of antibody accessibility for interaction with toxin molecule. Moreover, applying EVEF during the antibody immobilization stage leads to an increased density of the antibody layer and enhanced accessibility of antibody binding domain due to improved orientation of antibody in the direction of the electric field. Discrete-Time-Fourier-Transformation analysis of the chip surface line profiles obtained by AFM analysis clearly show an increased order of Ab molecules after implementation of EVEF. The new findings obtained in this study are expected to be useful for controlling Ab molecule orientation on the immobilizing surface.