Investigation of spacer length effect on immobilizedEscherichia coli pili-antibody molecular recognition by AFM

Abstract

The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Poly(ethylene glycol) (PEG) and Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to silicon wafer surfaces for the purpose of improving the orientation of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a variety of linear polymers were used to covalently attach the antibodies to silicon surfaces. Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical composition at each reaction step. The effect of spacer length in improving the specificity of immobilized antibody was investigated by attaching E. coli on the end of an AFM tip. The distribution of unbinding force and rupture distance from the force-distance curves obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the incidence of interactions by up to 90%. J600 proved to be the most effective spacer overcoming the steric hindrance seen with direct immobilization of antibody. In addition, the force spectroscopy reveals the elementary force quantum of E. coli-antibody to be 0.3 nN.

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

“…Considering merely the steric environment, the reaction of the surface-immobilized NHS groups most likely takes place at the flexible N-terminal residues 35 . However, as shown for other ligands, the length and the flexibility of the linker might enable interaction with otherwise sterically hindered binding sites, thus compensating for unfavorable orientation 36,37 . In unpublished work we compared the one-step with a two-step immobilization strategy.…”

Trends and Perspectives in Immunosensors

“…The spacer between the Ab and the immobilisation surface also plays a role in the final Abantigen interaction [4]. Direct conjugation of Abs to surfaces often hinders antigen binding (steric hindrances) and limits the mobility of the bound Abs.…”

“…Direct conjugation of Abs to surfaces often hinders antigen binding (steric hindrances) and limits the mobility of the bound Abs. Immobilising Abs via flexible spacer chains can lead to target antigens being captured twice as efficiently as directly-linked Abs [4]. This technology is adapted by Biacore (GE Healthcare) where they use carboxymethylated dextran as a linker, which is attached to a gold sensor surface.…”

Functionalization of AlN surface and effect of spacer density on Escherichia coli pili-antibody molecular recognition

“…While these studies have illustrated the contribution of PEG spacers in improving the function of immobilized biomolecules compared to direct immobilization, only a few have systematically investigated the influence of PEG spacer length and density on solid substrates. The effect of spacer length has been investigated in our previous research [20], while the effect of surface density of spacer molecules on the efficiency of immobilized proteins, which may be one of the principal factors in determining the bioactivity, has not been fully elucidated. Covalent grafting of an organosilane self-assembled monolayer (SAM) has been used extensively as the first step of surface functionalization [21].…”