Probing ligand-protein recognition with sum-frequency generation spectroscopy: the avidin-biocytin case.

Infrared/visible sum-frequency generation (SFG) spectroscopy is used to study the recognition of a protein (avidin) by a derived vitamin (biocytin) adsorbed on a calcium fluoride substrate. The specificity of the process is tested by replacing avidin with bovine serum albumin or presaturated avidin. The SFG spectroscopy shows drastic modifications in the CH and NH spectral ranges only upon exposure of the biocytin film to avidin. The comparison of the SFG data with Fourier transform infrared reflection absorption spectra (FT-IRRAS) in the same spectral ranges illustrates the advantages of nonlinear spectroscopy for studying and detecting recognition between biomolecules.     

Binding of Biotin to Gold Surfaces Functionalized by Self-Assembled Monolayers of Cystamine and Cysteamine: Combined FT-IRRAS and XPS Characterization

As part of our project of developing a new IR-based immunosensor, we investigated the functionalization of gold substrates with thin organic films containing biotin ligands. A two-step procedure was developed consisting of the chemisorption of short amine-terminated organosulfur compounds, followed by their reaction at the solid liquid interface with an activated ester derivative of biotin. Covalent binding of biotin to these attachment layers was assessed by Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS). The interaction of activated biotin with alcohol- and carboxylic acid-terminated monolayers was also investigated, and, as expected, no binding occurred. Moreover, mixed layers of short alcohol- and amine-terminated thiolates were successfully constructed at the gold surfaces and were shown to be the most efficient for the covalent binding of biotin thanks to the blocking effect of the thioalcohol, which prevented direct adsorption of biotin to the gold surface. Copyright 2001 Academic Press.     

Characterization of bovine serum albumin adsorption on chromium and AISI 304 stainless steel, consequences for the Pseudomonas fragi K1 adhesion

Adsorption of BSA on the surface of chromium and 304 stainless steel, has been characterized by Contact Angle Measurements, X-ray Photoelectron Spectroscopy (XPS) and Infrared Reflection Absorption Spectroscopy (IRRAS). Bacterial adhesion has been tested and compared on these two materials before and after pre-conditioning the surface with BSA. Chromium and stainless steel surfaces, when covered by a natural oxide layer, exhibit different energetic characteristics as shown by their γ_s^- and γ_s^LW respective values. These data vary upon immersion in BSA solutions, tending towards common values for duration of immersions. After immersion in BSA solutions, the evolution of the N 1s XPS signal, specific for the BSA, suggests that the surface is nearly saturated in a few minutes. Longer times of immersion only lead to a re-ordering of the adsorbed layer. Immersion tests in dilute BSA solutions (0.01 g/l) enabled us to make clear a higher reactivity of chromium towards the protein compared to stainless steel. These differences are cancelled at higher BSA concentrations (1 g/l). IRRAS spectra of BSA adsorbed on the two substrates demonstrated the appearance of amide I and amide II bands with small shifts and intensity variations supporting orientation changes of the protein when the concentration or immersion time varies. A model for the building up of the BSA layer is proposed, which accounts for these data. Chromium and stainless steel surfaces, also have different behaviours towards adhesion of Pseudomonas fragi K1, whereas surfaces that are pre-conditioned by BSA behave in a similar way. The overall number of adherent bacteria is decreased on stainless steel, whereas it is hardly affected on chromium. On both surfaces, the fraction of viable cells is increased.     

An Experimental and Theoretical Approach to Investigate the Effect of Chain Length on Aminothiol Adsorption and Assembly on Gold

Despite the numerous studies on the self‐assembled monolayers (SAMs) of alkylthiols on gold, the mechanisms involved, especially the nature and influence of the thiol–gold interface are still under debate. In this work the adsorption of aminothiols on Au(111) surfaces has been studied by using surface IR and X‐ray photoelectron spectroscopy (XPS) as well as by density functional theory (DFT) modeling. Two aminothiols were used, cysteamine (CEA) and mercaptoundecylamine (MUAM), which contain two and eleven carbon atoms, respectively. By combining experimental and theoretical methods, it was possible to draw a molecular picture of the thiol–gold interface. The long‐chain aminothiol produced better ordered SAMs, but, interestingly, the XPS data showed different sulfur binding environments depending on the alkyl chain length; an additional peak at low binding energy was observed upon CEA adsorption, which indicates the presence of sulfur in a different environment. DFT modeling showed that the positions of the sulfur atoms in the SAMs on gold with similar unit cells [(2√3×2√3)R30°] depended on the length of the alkyl chain. Short‐chain alkylthiol SAMs were adsorbed more strongly than long‐chain thiol SAMs and were shown to induce surface reconstruction by extracting atoms from the surface, possibly forming adatom/vacancy combinations that lead to the additional XPS peak. In the case of short alkylthiols, the thiol–gold interface governs the layer, CEA adsorbs strongly, and the mechanism is closer to single‐molecule adsorption than self‐assembly, whereas for long chains, interactions between alkyl chains drive the system to self‐assembly, leading to a higher level of SAM organization and restricting the influence of the sulfur–gold interface.     

A convenient biomimetic synthesis of optically active putative neurotoxic metabolites of MDMA ("ecstasy") from R-(-)- and S-(+)-N-methyl-α-methyldopamine precursors

(±)-3,4-Methylenedioxymethamphetamine (MDMA, also known as "ecstasy") is a psychoactive drug with selective neurotoxic potential toward brain serotonin (5-HT) neurons. One hypothesis holds that MDMA neurotoxicity may at least partially be a consequence of its metabolism. In most species (including primates), O-demethylenated MDMA metabolites such as N-methyl-α-methyldopamine (HHMA) have been postulated to serve as precursors for toxic thioether conjugates. As yet, chirality of MDMA was not considered in previously reported in vivo studies because HHMA was used as the racemate. Since the stereochemistry of this chiral drug needs to be considered, the total synthesis of enantiomerically pure precursors, R-(-)-HHMA and S-(+)-HHMA, was envisioned with the ultimate goal to prepare substantial amounts of optically active thioether conjugates. Recently, we reported the first total synthesis of the R-enantiomer. In this paper, a novel synthesis of the S-enantiomer is described, in 45% overall yield (six steps) and 99% ee, using commercially available l-Boc-alanine (99% ee) as the chiral source. Having at our disposal suitable amounts of R-(-)-HHMA and S-(+)-HHMA precursors, a straightforward one-pot electrochemical procedure has been further developed for the synthesis of several catechol-thioether conjugates in acceptable yields (40-53%) and high degree of purity (99%), with complete diastereoselectivity. The availability of these newly synthesized optically active catechol-thioether conjugates is crucial for ongoing future in vivo studies about their role in MDMA neurotoxicity.     

Covalent immobilization of lysozyme on stainless steel. Interface spectroscopic characterization and measurement of enzymatic activity

A new strategy aiming at the protection of metallic surfaces against the growth of biofilms is presented here. This work reports the grafting of primary amines by aminosilanization of oxidized stainless steel followed by chemical coupling of the glycosidase lysozyme from hen egg white using glutaraldehyde as homobifunctional cross-linking agent. Controlled characterization of a stainless steel surface by X-ray photoelectron spectroscopy and Fourier transform infrared reflection-absorption spectroscopy at each step enabled the mode of binding, coverage, and orientation of the grafted molecules to be addressed. As a result, the stainless steel samples covered with a covalently immobilized layer of lysozyme showed some lytic activity on a suspension of bacteria Micrococcus lysodeikticus.     

Adsorption on stainless steel surfaces of biosurfactants produced by gram-negative and gram-positive bacteria: Consequence on the bioadhesive behavior of Listeria monocytogenes

The ability of adsorbed biosurfactants (Pf and Lb) obtained from gram-negative bacterium (Pseudomonas fluorescens) or gram-positive bacterium (Lactobacillus helveticus) to inhibit adhesion of four listerial strains to stainless steel was investigated. These metallic surfaces were characterized using the following complementary analytical techniques: contact-angle measurements (CAM), atomic force microscopy (AFM), polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS) and X-ray photoelectron spectroscopy (XPS). Contact-angles with polar liquids (water and formamide) indicated that the stainless steel surface covered with adsorbed biosurfactant was more hydrophilic and electron-donating than bare stainless steel. The surface characterization by XPS and PM-IRRAS revealed that conditioning the stainless steel changes the substrate in two ways, by modifying the surface alloy composition and by leaving an thin adsorbed organic layer. AFM observations enabled to say that the layer covered entirely the surface and was probably thicker (with patches) in the case of Pf-conditioned surfaces compared to the Lb-conditioned ones, which seemed to be less homogeneous. Though the added layer was thin, significant chemical changes were observed that can account for drastic modifications in the surface adhesive properties. As a matter of fact, adhesion tests showed that both used biosurfactants were effective by decreasing strongly the level of contamination of stainless steel surfaces by the four strains of Listeria monocytogenes. The more important decrease concerned the CIP104794 and CIP103573 strains (>99.7%) on surface conditioned by L. helveticus biosurfactant. A less reduced phenomenon (75.2%) for the CIP103574 strain on stainless steel with absorbed biosurfactant from P. fluorescens was observed. Whatever the strain of L. monocytogenes and the biosurfactant used, this antiadhesive biologic coating reduced both total adhering flora and viable and cultivable adherent bacteria on stainless steel surfaces. This study confirms that biosurfactants constitute an effective strategy to prevent microbial colonization of metallic surfaces by pathogenic bacteria like the food-borne pathogen L. monocytogenes.     

Interaction of ZnII Porphyrin with TiO2 Nanoparticles: From Mechanism to Synthesis of Hybrid Nanomaterials

The mechanism of interaction of Zn porphyrin (ZnPP) with TiO₂ surfaces is investigated with a view to optimizing the synthesis of hybrid nanomaterials. The strategy consists of studying the adsorption of ZnPP on TiO₂ flat surfaces by taking advantage of complementary surface characterization techniques. Combining a detailed X‐ray photoelectron spectroscopic analysis with AFM imaging allows ZnPP–surface and ZnPP intermolecular interactions to be discriminated. Probing the adsorption of ZnPP on TiO₂ nanoparticles (NPs) reveals the dominant role of ZnPP‐mediated interactions, which are associated with the formation of ZnPP multilayers and/or with the state of aggregation of NPs. These preliminary investigations provide a guideline to synthesizing a novel ZnPP–TiO₂ hybrid nanomaterial in a one‐step protocol. In this material, ZnPP molecules are presumably involved in the TiO₂ lattice rather than on the NP surface. Furthermore, ZnPP molecules preserve their electronic properties within the TiO₂ NPs, and this makes the ZnPP–TiO₂ hybrid nanomaterial an excellent candidate for nanomedicine and related applications, such as localization of nanoparticles in cells and tissues or in photodynamic therapy.