Immobilization of Antibodies on a Photoactive Self-Assembled Monolayer on Gold

Abstract

This paper presents a strategy for immobilizing biomolecules on a photoactivable surface. A self-assembled monolayer is prepared by adsorbing an ω-functionalized dialkyl disulfide on gold. Functional groups of this monolayer are converted in two steps into a benzophenone derivative with an overall yield of 50 ± 10%. Several independent techniques (ellipsometry, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, radiolabel assay, and autoradiography) characterize the reaction and photoimmobilization of antibodies on this surface. The photoimmobilized antibodies cover the surface as a homogeneous and dense monolayer that could not be disrupted by vigorous washing with the detergent Tween 20. Immunoassays demonstrated specific recognition of the immobilized immunoglobulins as measured by their complexation with alkaline phosphatase-linked antibodies. The method of photoimmobilization used here leads to a homogeneous single layer of IgGs, in which the proteins maximize their contact with the surface. Residual adsorption of IgG on the nonirradiated surface of benzophenone remains one limitation of this approach. Progressively higher coverages of IgGs on the surface did not lead to strictly proportional changes of the biological activity of these surfaces, probably because of interactions between the IgGs in the film. This method of photoimmobilization is nonetheless useful as an experimental system to immobilize other proteins because it is simple, flexible, and efficient.

Study of Factors Affecting the Performance of Voltammetric Copper Sensors Based on Gly‐Gly‐His Modified Glassy Carbon and Gold Electrodes

“…The area ratio between the peaks at 399.0, 400.3 and 401.4 eV was approximately 1 : 3.4 : 1, which is in reasonable agreement with the ratio of 1 : 3 : 1 predicted for Gly-Gly-His peptides coupled to the 4-carboxyphenyl monolayer via the formation of amide bonds. The highest binding energy peak in the N1s spectrum at 402.5 eV was assigned to the nitrogen of unreacted NHS esters remaining on the surface, since such a positive shift is consistent with the presence of the electronegative oxygen attached to the succinimidyl nitrogen [37,38]. It should be noted that some unidentified nitrogen species already present in very low levels on the bare GC electrode may contribute to the N1s scan shown in Figure 2c.…”

Molecular Recognition of Avidin on Biotin-Functionalized Gold Surfaces Detected by FT-IRRAS and Use of Metal Carbonyl Probes

“…In the case of Co-avidin (Fig. 1F), only the mode e band was detected, whereas the mode a 1 of the cyclopentadienyl Mn(CO) 3 probe was overlapped by one of the three bands of the alkyne Co 2 (CO) 6 probes of Co-avidin. Hence, avidin molecules immobilized onto the biotin layer retained a good affinity for the biotinylated ligand Mn-biotin and, more importantly, this molecular association was successfully transduced by FT-IRRAS.…”

“…Planar gold surfaces appear as attractive solid supports owing to the ability to build well-defined self-assembled monolayers (SAMs) of functionalized thiolates (2) that are subsequently used to couple various kinds of biomolecules, including proteins (3)(4)(5)(6) and oligonucleotides (7), without impairing their biological activity. Such surfaces also enable both optical (8) and electrical (9) transduction schemes.…”

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

“…It has been shown repeatedly that optimization of physical interactions between immobilized macromolecules, e.g., antigens, antibodies, peptides, and their corresponding target ligands affects sensitivity [31,32]. There is currently a substantial variety of procedures for oriented immobilization: ionic interaction, specific covalent binding, apoenzyme reconstituted on the surface that binds to a prostetic group, receptor/ligand interactions, specific affinity motifs engineered into the surface of proteins, etc.…”