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.

Single-step label-free nanowell immunoassay accurately quantifies serum stress hormones within minutes

“…Recently, AC electric field has found use in capturing, alignment, and enhanced detection of biomolecules (34,39). The electric field entrapment has previously been combined with the antigen-antibody reaction to facilitate selective detection, and it has been used to immobilize antibody probe (40)(41)(42). Here, in fig.…”

Hapten-Grafted Programmed Probe as a Corecognition Element for a Competitive Immunosensor to Detect Acetamiprid Residue in Agricultural Products

“…As a competitor for acetamiprid, HGPP can adsorb effectively on the surface of antibody by specific recognition interaction. Moreover, the fabrication of the proposed acetamiprid biosensor is illustrated in Scheme B, and such an immunosensor is developed based on the capture antibody-immobilized gold electrode via carbodiimide method . The self-assembled monolayer of dithiol-functionalized DHLA enhanced gold surface-binding .…”

“…Moreover, the fabrication of the proposed acetamiprid biosensor is illustrated in Scheme 1B, and such an immunosensor is developed based on the capture antibody-immobilized gold electrode via carbodiimide method. 40 The self-assembled monolayer of dithiol-functionalized DHLA enhanced gold surface-binding. 41 Finally, MCH was assembled on electrode surface to hinder extra active sites and reduce nonspecific adsorption.…”

Orientation and characterization of immobilized antibodies for improved immunoassays (Review)

“…Carrigan et al 51 used EDC/NHS in two ways, (1) for the cross-linking of polyethylenimine and carboxymethyl cellulose, and (2) for activation of this substrate for protein binding targeting amine and carboxyl functionality. Sun et al 52 took a novel approach to EDC/NHS coupling by introducing NHS reactive groups to random sites on an antibody then using an electric field to preferentially orient the antibody, via its intrinsic dipole, before reaction to the free amines on cysteine immobilized to a gold electrode.…”