Controlling Antibody Orientation on Charged Self-Assembled Monolayers

Abstract

The orientation of antibodies on surfaces is critical to the performance of biosensors. Due to the charge distribution within an antibody molecule, it is hoped that the orientation of adsorbed antibodies can be controlled by adjusting microenvironments (e.g., surface and solution properties). In this work, we investigated the orientations of two types of monoclonal anti-human chorionic gonadotropin (anti-hCG) adsorbed on NH2 (positively charged) and COOH (negatively charged) terminated self-assembled monolayers (SAMs) by surface plasmon resonance (SPR) biosensors. Adsorbed antibody molecules on surfaces were also characterized by atomic force microscopy. Results show that the orientation of the IgG1 type antibody is better on the NH2 surface than on the COOH surface, as indicated by a higher hCG to adsorbed anti-hCG ratio on the NH2 surface from SPR experiments. However, for the IgG2a type antibody, the antigen-to-antibody ratio, and thus the orientation of the adsorbed IgG2a, is similar on the NH2- and COOH-terminated SAM surfaces. Surface charge affects the orientation of IgG2a less than IgG1 due to the smaller dipole moment of IgG2a compared to IgG1. This work provides not only a fundamental understanding of how microenvironments affect protein behavior on surfaces but also a useful guide to designing surfaces for applications, such as biosensors and biomaterials.

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

“…Differences in adsorption and or CE due to pH value variation are clear indicators of electrostatic forces and physical adsorption (Chen et al 2003). For this experiment (Fig.…”

“…The lowest pH (pH 5) resulted in the lowest CE. A possible explanation might be the positive charge of the applied antibodies at pH 5 and the assumption that antibodies bind stronger to negatively charged surfaces at lower ionic strength with head-on orientation rather than with end-on orientation, thus resulting in lower reactive particles (Chen et al 2003). At pH 6, a small percentage of polyclonal antibodies exhibits isoelectric behaviour, so that hydrophobic forces exceed the electrostatic ones.…”

“…Firstly, buffers with medium and low salt concentration (mM to 0.1 M) that directly influence electrostatic forces and therefore physical adsorption (Chen et al 2003). In general, it could be stated for liquid-solid interfaces that lower ionic strength results in higher electrostatic attraction or repulsion.…”

Molecular Engineering of Surfaces for Sensing and Detection

“…Recently, Chen et al [24] showed that the orientation of adsorbed anti-hCG can be controlled by appropriately tuning the microenvironments of the adsorption process (i.e., surface and solution properties). The IEP of anti-hCG (pH 6.8) lies between those of its F(ab') 2 (pH 8.5) and F c (pH 6.1) fragments.…”

A new strategy for label-free detection of lymphoma cancer cells

“…The use of Id specific peptide as molecular probe could highly improve affinity and selectivity of the capture surface. In addition, it simplifies the biochip functionalization procedure with respect to that employed for antibodies in which controlling protein orientation is still very challenging [19]. In this setting, we constructed an Id-peptide-based biosensor by using as substrate both planar and porous silicon.…”