Probing the Orientation of β-Lactoglobulin on Gold Surfaces Modified by Alkyl Thiol Self-Assembled Monolayers

Abstract

The adsorption of a globular protein on chemically well controlled surfaces was investigated in order to correlate its orientation to the surface properties. To this end, three different alkyl thiols, differing by their end group (−COOH, −CH3, and −NH2), were used to build up self-assembled monolayers (SAMs) on gold substrates. β-Lactoglobulin (βLG) was then adsorbed on these SAMs by immersion in a phosphate buffer solution. The surface modification with alkyl thiols and the subsequent adsorption of proteins were characterized ex situ by polarization modulated infrared reflection–absorption spectroscopy (PM-IRRAS) and X-ray photoelectron spectroscopy (XPS). The adsorption behavior of proteins was also monitored in situ using quartz crystal microbalance with dissipation measurements (QCM-D). Direct evidence regarding the protein orientation in the adsorbed state was obtained by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA), performed on the ToF-SIMS results, enables to separate the samples and shows that the proteins display different distributions of amino acids at the surface depending on the conditioning thiol layer. Our results revealed that the adsorption mode of the protein is influenced by the thiol end groups, and specific orientations of the protein on the surface are proposed for the different substrates.

Adsorption of Milk Proteins (β-Casein and β-Lactoglobulin) and BSA onto Hydrophobic Surfaces

“…Overall, the arrangements proposed in Figure 6 in the case of BSA and -lactoglobulin are consistent with previously-reported experimental results (see [ 13 , 34 , 35 ] for BSA and [ 13 , 36 , 37 ] for -lactoglobulin). In the case of -casein films, we can found both examples of monolayer or multilayer formation in the literature [ 34 , 36 , 38 ].…”

Gallium‐Loaded Dissolvable Microfilm Constructs that Provide Sustained Release of Ga3+ for Management of Biofilms

“…First, treatment of as-assembled PEMs with acid converted the COO − groups (that were electrostatically linked to NH 3 + of PAH in as-assembled PEMs) to COOH, as evidenced by the diminished asymmetric and symmetric COO − bands (at 1575 cm −1 and 1404 cm −1,[35] respectively) and enhanced COOH bands at 1715 cm −1[36] (in comparison to the spectrum of the as-assembled PEMs). The formation of COOH groups in acid-treated PEMs is also accompanied by generation of unbound NH 3 + groups at 1563 cm −1,[37] consistent with prior reports. [33,38] Second, for Ga 3+ -loaded PEMs, we observed an interaction between Ga 3+ and unbound carboxylic acid/carboxylate groups as seen by the shifts of COOH and symmetric COO − bands to higher wavenumbers (~15 cm −1 ) as compared to the bands of acid-treated PEMs.…”

Application of Polarization Modulation Infrared Reflection Absorption Spectrosocpy Under Electrochemical Control for Structural Studies of Biomimetic Assemblies

“…Indeed, IRS is a superb technique allowing studies of the secondary structure of proteins and its changes [14,15,163,164]. PM IRRAS is sensitive to the structure of species adsorbed at a solid surface and it has been used to study the adsorption of proteins at stainless steel|air [162] and gold|air interfaces [165,166]. The analysis of the amide I modes of PM IRRA spectra of albumin adsorbed on the stainless steel surface show that the increase in the surface concentration of the adsorbed protein leads to a gradual loss of the helical structure and unfolding of the adsorbed protein [162].…”