Abstract
Three calix[4]arene (Cal-4) derivatives which separately contain ethylester (1), carboxylic acid (2), and crownether (3) at the lower rim with a common reactive thiol at the upper rim were synthesized and constructed to self-assembled monolayers (SAMs) on Au films. After spectroscopic characterization of the monolayers, surface coverage and orientation of antibody immobilized on the Cal-4 derivative SAMs were studied by surface plasmon resonance (SPR) technique. Experimental results revealed that the antibody could be immobilized on the Cal-4 derivatives spontaneously. The orientation of absorbed antibody on the Cal-4 derivative SAMs is related to the SAM’s dipole moment. The possible orientations of the antibody immobilized on the Cal-4 derivative 1 SAM are lying-on or side-on, while on the Cal-4 derivative 2 and Cal-4 derivative 3 head-on and end-on respectively. These experimental results demonstrate the surface dipole moment of Cal-4 derivative appears to be an important factor to antibody orientation. Cal-4 derivatives are useful in developing site direct protein chips.
Highlights
Immunosensors are among the most important diagnostic tools, which are widely used in medical diagnostics, forensic medicine, environmental analysis, and so on
The formation of each Cal-4 derivative self-assembled monolayers (SAMs) was characterized by Fourier Transform Infrared
In the case of the indirect immobilization assay using the protein G terminated antibody immobilization method, 10 μg/mL of protein G was first immobilized on the Cal-4 derivative SAMs and incubated for 30 min, followed by the steps used in the direct method
Summary
Immunosensors are among the most important diagnostic tools, which are widely used in medical diagnostics, forensic medicine, environmental analysis, and so on. In order to avoid random antibody immobilization and improve antigen binding site availability, site-directed immobilization methods are being constantly developed via, e.g., proteins binding the Fc region of immunoglobulins, using the antibody fragments and oxidized oligosaccharide moieties. Han et al [9,10], found that Cal-4 crown ether may have a unique surface chemistry allowing the formation of a self-assembled monolayer (SAM) on gold surface and tight binding of the captured proteins to the crown moiety of the linker molecules. We confirmed the controlled antibody orientation on the Cal-4 crown SAM by surface plasmon resonance (SPR) study [11]. Immobilization characteristics of Cal-4 derivatives and surface coverage of adsorbed protein on the molecular linker system were precisely measured by SPR. The crown ether Cal-4 derivative was very effective in promoting immunoactivity of immobilized antibody. The dipole moment of Cal-4 derivatives could be an important factor for antibody immobilization, which involved dipole-dipole interactions between the antibody and Cal-4 derivatives SAMs
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