Michele Sferrazza

Protein Adsorption on Poly(N-isopropylacrylamide) Brushes: dependence on grafting density and chain collapse

The protein resistance of poly(N-isopropylacrylamide) brushes grafted from silicon wafers was investigated as a function of the chain molecular weight, grafting density, and temperature. Above the lower critical solution temperature (LCST) of 32 °C, the collapse of the water-swollen chains, determined by ellipsometry, depends on the grafting density and molecular weight. Ellipsometry, radio assay, and fluorescence imaging demonstrated that, below the lower critical solution temperature, the brushes repel protein as effectively as oligoethylene oxide-terminated monolayers. Above 32 °C, very low levels of protein adsorb on densely grafted brushes, and the amounts of adsorbed protein increase with decreasing brush-grafting-densities. Brushes that do not exhibit a collapse transition also bind protein, even though the chains remain extended above the LCST. These findings suggest possible mechanisms underlying protein interactions with end-grafted poly(N-isopropyl acrylamide) brushes.

Adsorption and displacement of a globular protein on hydrophilic and hydrophobic surfaces

Abstract The adsorption of the globular protein β -lactoglobulin on hydrophilic and hydrophobic surfaces has been investigated. Spectroscopic ellipsometry and Fourier transform infrared spectroscopy in the attenuated total reflection mode were used in our study. β -lactoglobulin adsorbed in surface concentrations less than or equal to those corresponding to a closely packed monolayer of molecules. The amount of β -lactoglobulin removed from hydrophilic surfaces upon elution with buffer solution, as a fraction of the final amount reached upon adsorption, was observed to decrease at longer adsorption times. This provides evidence that the conformation of the adsorbed protein changes relatively slowly once adsorbed, leading to more irreversibly adsorbed states with stronger binding to the surface. The fraction of protein irreversibly adsorbed to the surface was generally higher on hydrophobic surfaces than hydrophilic surfaces, confirming the general principle that globular proteins exhibit stronger binding to hydrophobic surfaces than hydrophilic ones. The displacement of pre-adsorbed layers of the protein from hydrophobic and hydrophilic surfaces by the non-ionic surfactant octaethylene glycol monododecyl ether (C 12 E 8 ) was also studied. We observed that the non-ionic surfactant caused partial displacement of adsorbed protein from hydrophobic surfaces. The displacement kinetics also reveal that the protein layers are more strongly bound to hydrophobic surfaces at longer than at shorter adsorption times, showing that slow protein binding changes and ageing effects are also important at hydrophobic surfaces.

Applications of environmental scanning electron microscopy to colloidal aggregation and film formation

Abstract Environmental scanning electron microscopy (ESEM) is a rather new form of electron microscopy, which permits the observation of hydrated samples in their native state, and also does not require that insulators are coated with a conducting layer. These two factors make it ideal for studying colloidal dispersions as they aggregate and/or film form. This paper describes the application of ESEM to three situations involving aggregating latices. Firstly the nature of fractal structures grown from aggregating acrylic latices is discussed, with a comparison given of the behaviour with and without added salt as the screening between particles is altered. Secondly the behaviour of vinyl latices is considered. The impact of the addition of starch, both modified and unmodified, upon the particle size distribution and ability to film form is examined. Finally, the structures which form when the hard inorganic component silica is added to acrylic latices are explored. Together these three examples illustrate some of the many strengths of the ESEM in the field of colloidal dispersions and aggregates.

Crystallisation kinetics in thin films of dihexyl-terthiophene: the appearance of polymorphic phases

The presence of surface-induced crystal structures is well known within organic thin films. However, the physical parameters responsible for their formation are still under debate. In the present work, we present the formation of polymorphic crystal structures of the molecule dihexyl-terthiophene in thin films. The films are prepared by different methods using solution-based methods like spin-coating, dip-coating and drop-casting, but also by physical vapour deposition. The thin films are characterised by various X-ray diffraction methods to investigate the crystallographic properties and by microscopy techniques (atomic force microscopy and optical microscopy) to determine the thin film morphologies. Three different polymorphic crystal structures are identified and their appearance is related to the film preparation parameters. The crystallisation speed is varied by the evaporation rate of the solvent and is identified as a key parameter for the respective polymorphs present in the films. Slow crystallisation speed induces preferential growth in the stable bulk structure, while fast crystallisation leads to the occurrence of a metastable thin-film phase. Furthermore, by combining X-ray reflectivity investigations with photoelectron spectroscopy experiments, the presence of a monolayer thick wetting layer below the crystalline film could be evidenced. This work gives an example of thin film growth where the kinetics during the crystallisation rather than the film thickness is identified as the critical parameter for the presence of a thin-film phase within organic thin films.

The role of cholesterol in the activity of pneumolysin, a bacterial protein toxin

The mechanism via which pneumolysin (PLY), a toxin and major virulence factor of the bacterium Streptococcus pneumoniae, binds to its putative receptor, cholesterol, is still poorly understood. We present results from a series of biophysical studies that shed light on the interaction of PLY with cholesterol in solution and in lipid bilayers. PLY lyses cells whose walls contain cholesterol. Using standard hemolytic assays we have demonstrated that the hemolytic activity of PLY is inhibited by cholesterol, partially by ergosterol but not by lanosterol and that the functional stoichiometry of the cholesterol-PLY complex is 1:1. Tryptophan (Trp) fluorescence data recorded during PLY-cholesterol titration studies confirm this ratio, reveal a significant blue shift in the Trp fluorescence peak with increasing cholesterol concentrations indicative of increasing nonpolarity in the Trp environment, consistent with cholesterol binding by the tryptophans, and provide a measure of the affinity of cholesterol binding: K(d) = 400 +/- 100 nM. Finally, we have performed specular neutron reflectivity studies to observe the effect of PLY upon lipid bilayer structure.

Synthesis of Polystyrene Thin Films by Means of an Atmospheric-Pressure Plasma Torch and a Dielectric Barrier Discharge

In this paper, the deposition and characterization of plasma-polymerized polystyrene (pp-PS) using PECVD under atmospheric pressure on a variety of substrates was investigated. An atmospheric RF plasma torch and an HF dielectric-barrier-discharge (DBD) system were used to deposit thin pp-PS coatings on PTFE, HDPE, stainless steel, glass, and silicon wafer. The styrene vapor was carried by Ar or He. The pp-PS films were characterized by Fourier transform infrared spectroscopy (FTIR) (infrared reflection absorption spectroscopy), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), static secondary ion mass spectroscopy (SSIMS), and optical microscopy, and the plasma phase was studied by optical-emission spectroscopy. The major features that characterize PS are present in the FTIR, SSIMS, and XPS spectra of our films, although some differences are observed between pp-PS and their conventionally polymerized counterparts: oxygenation, branching, degree of cross-linking, and unsaturation. According to the WCA and XPS results, the films deposited by the RF plasma torch (placed in a Plexiglass chamber) are more oxygenated than those deposited by DBD, which is operated under a much more controlled atmosphere. A comparison of the chemical structure of the deposited coatings (branching, cross-linking) as a function of the nature of the carrier gas was established by FTIR: pp-PS synthesized in the presence of Ar (for both processes) exhibit more branching and a higher degree of cross-linking than pp-PS synthesized with He as the main plasma gas. The optical microscopy points out a diversity of structures that depend on the nature of the substrate and the plasma parameters.

On the pearl size of hydrophobic polyelectrolytes

Hydrophobic polyelectrolytes have been predicted to adopt a unique pearl-necklace conformation in aqueous solvents. We present in this letter an attempt to characterise quantitatively this conformation with a focus on Dp, the pearl size. For this purpose polystyrenesulfonate (PSS) of various effective charge fractions feff and chain lengths N has been adsorbed onto oppositely charged surfaces immersed in water in condition where the bulk structure is expected to persist in the adsorbed state. In situ ellipsometry has provided an apparent thickness happ of the PSS layer. In the presence of added salts, we have found happ ~ aN0feff−2/3 (a is the monomer size) in agreement with the scaling predictions for Dp in the pearl-necklace model if one interprets happ as a measure of the pearl size. At the lowest charge fractions we have found happ ~ aN1/3 for the shorter chains, in agreement with a necklace/globule transition.

Neutron Reflectometry Elucidates Density Profiles of Deuterated Proteins Adsorbed onto Surfaces Displaying Poly(ethylene glycol) Brushes: Evidence for Primary Adsorption

The concentration profile of deuterated myoglobin (Mb) adsorbed onto polystyrene substrates displaying poly(ethylene glycol) (PEG) brushes is characterized by neutron reflectometry (NR). The method allows to directly distinguish among primary adsorption at the grafting surface, ternary adsorption within the brush, and secondary adsorption at the brush outer edge. It complements depth-insensitive standard techniques, such as ellipsometry, radioactive labeling, and quartz crystal microbalance. The study explores the effect of the PEG polymerization degree, N, and the grafting density, σ, on Mb adsorption. In the studied systems there is no indication of secondary or ternary adsorption, but there is evidence of primary adsorption involving a dense inner layer at the polystyrene surface. For sparsely grafted brushes the primary adsorption involves an additional dilute outer protein layer on top of the inner layer. The amount of protein adsorbed in the inner layer is independent of N but varies with σ, while for the outer layer it is correlated to the amount of grafted PEG and is thus sensitive to both N and σ. The use of deuterated proteins enhances the sensitivity of NR and enables monitoring exchange between deuterated and hydrogenated species.

Linking transitions between the highly deformed states and the yrast states of normal deformation in 133Nd

Abstract Transitions linking the highly deformed band to low deformation states have been unambiguously established in 133 Nd in the very first experiment performed with the GASP array. the highly deformed band becomes yrast at 29 2 + , where it mixes with the positive parity band built upon the [404] 7 2 Nilsson state. The experimentally deduced interaction amounts to 13 keV. The measurement of the spins in the highly deformed intruder band sheds some light upon the reported backbend anomaly in this mass region.

Interface induced crystal structures of dioctyl-terthiophene thin films.

Temperature dependent structural and morphological investigations on semiconducting dioctyl-terthiophene (DOTT) thin films prepared on silica surfaces reveals the coexistence of surface induce order and distinct crystalline/liquid crystalline bulk polymorphs. X-ray diffraction and scanning force microscopy measurements indicate that at room temperature two polymorphs are present: the surface induced phase grows directly on the silica interface and the bulk phase on top. At elevated temperatures the long-range order gradually decreases, and the crystal G (340 K), smectic F (348 K), and smectic C (360 K) phases are observed. Indexation of diffraction peaks reveals that an up-right standing conformation of DOTT molecules is present within all phases. A temperature stable interfacial layer close to the silica-DOTT interface acts as template for the formation of the different phases. Rapid cooling of the DOTT sample from the smectic C phase to room temperature results in freezing into a metastable crystalline state with an intermediated unit cell between the room temperature crystalline phase and the smectic C phase. The understanding of such interfacial induced phases in thin semiconducting liquid crystal films allows tuning of crystallographic and therefore physical properties within organic thin films.