title: Reversible immobilization of peptides: surface modification and in situ detection by attenuated total reflection FTIR spectroscopy.
abstract: A generic method is described for the reversible immobilization of polyhistidine-bearing polypeptides and proteins on attenuated total reflecting (ATR) sensor surfaces for the detection of biomolecular interactions by FTIR spectroscopy. Nitrilotriacetic acid (NTA) groups are covalently attached to self-assembled monolayers of either thioalkanes on gold films or mercaptosilanes on silicon dioxide films deposited on germanium internal reflection elements. Complex formation between Ni2+ ions and NTA groups activates the ATR sensor surface for the selective binding of polyhistidine sequences. This approach not only allows a stable and reversible immobilization of histidine-tagged peptides (His-peptides) but also simultaneously allows the direct in situ quantification of surface-adsorbed molecules from their specific FTIR spectral bands. The surface concentrations of both NTA and His-peptide on silanized surfaces were determined to be 1.1 and 0.4 molecules nm-2, respectively, which means that the surface is densely covered. A comparison of experimental FTIR spectra with simulated spectra reveals a surface-enhancement effect of one order of magnitude for the gold surfaces. With the presented sensor surfaces, new ways are opened up to investigate, in situ and with high sensitivity and reproducibility, protein-ligand, protein-protein, protein-DNA interactions, and DNA hybridization by ATR-FTIR spectroscopy.
authors.affiliation: Institute of Biomolecular Science, Swiss Federal Institute of Technology Lausanne, 1015 Ecublens, Switzerland.
authors.forename: Per
authors.initials: P
authors.lastname: Rigler
authors.forename: Wolf-Peter
authors.initials: WP
authors.lastname: Ulrich
authors.forename: Patrik
authors.initials: P
authors.lastname: Hoffmann
authors.forename: Michael
authors.initials: M
authors.lastname: Mayer
authors.forename: Horst
authors.initials: H
authors.lastname: Vogel
mesh_terms: D000327:Adsorption; D006639:Histidine; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D012821:Silanes / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013499:Surface Properties
chemical_list: D010455:Peptides; D011506:Proteins; D012821:Silanes; D006639:Histidine