X-ray microscopy studies of protein adsorption on a phase-segregated polystyrene/polymethyl methacrylate surface. 1. Concentration and exposure-time dependence for albumin adsorption

X-ray photoemission electron microscopy using synchrotron radiation illumination has been used to measure the spatial distributions of albumin on a phase-segregated polystyrene/poly(methyl methacrylate) (PS/PMMA) polymer thin film following adsorption from unbuffered, deionized aqueous solutions under a range of solution concentrations and exposure times. Chemical mapping of the albumin, PS, and PMMA shows that the distribution of albumin on different adsorption sites (PS, PMMA, and the interface between the PS and PMMA domains) changes depending on the concentration of the albumin solution and the exposure time. The preferred sites of absorption at low concentration and short exposure are the PS/PMMA interfaces. Albumin shows a stronger preference for the PS domains than the PMMA domains. The exposure-time dependence suggests that a dynamic equilibrium between albumin in solution and adsorbed on PS domains is established in a shorter time than is required for equilibrating albumin between the solution and the PMMA domains. The explanation of these preferences in terms of possible adsorption mechanisms is discussed.     

Effect of solvent power on the adsorption of polystyrene onto a metal surface

The adsorption of linear polystyrenes from cyclohexane solutions onto a chrome plate of 35 (the theta temperature), 40, and 45°C was studied by ellipsometry. The adsorbance decreases with increasing temperature, while the extension of the adsorbed polymer layer increases. The adsorbance is almost independent of the molecular weight at these temperatures. The slope of a double logarithmic plot of extension versus molecular weight is 0.5 at the theta temperature, whereas at the higher temperatures the slope is a little larger. Both the adsorbance and the extension of the adsorbed layer change reversibly over a cycle of temperature change, indicating that a reversible conformational change has occurred. The expansion factor α_t of the adsorbed layer is compared with the theoretical predictions of Hoeve and of Jones and Richmond. The expansion factor α_t according to Hoeve's theory, was smaller than the measured value, whereas the expansion calculated by the Jones–Richmond theory is much larger than the measured value. It is concluded that the tail portions of adsorbed chains predominantly govern the extension of the adsorbed layer.     

Effect of pH and applied potential on the adsorption of DNA on highly oriented pyrolytic graphite electrodes. Atomic force microscopy surface characterisation

Single-stranded and double-stranded DNA electrochemical biosensors prepared by adsorption during 3 min on HOPG, with or without an applied potential, at pH 5.3 and 7.0, were characterised by MAC mode AFM. During adsorption DNA condenses on the substrate forming complex network films with pores exposing the HOPG surface. The thin films formed in pH 5.3 acetate buffer always presented a better coverage of the HOPG surface with DNA molecules than films formed in pH 7.0 phosphate buffer. The application of a positive potential of 300 mV during adsorption enhanced the robustness and stability of the DNA films with the formation of bigger network holes and a more condensed and compact self-assembled DNA lattice. The knowledge of the morphology of adsorbed DNA on electrode surfaces explains non-specific adsorption on the electrode surface and can be used to improve and develop DNA-electrochemical biosensors.     

X-ray diffraction and scanning electron microscopy studies of calcium carbonate electrodeposited on a steel surface

Calcium carbonate was deposited on a stainless steel surface with the use of an electrical potential of 10 V. The crystals formed on the surface were examined with X-ray diffraction and with scanning electron microscopy, which revealed that calcite, vaterite and amorphous calcium carbonate was formed. Two different surface active polymers were added to the solution and their effect on the crystal structure was investigated. It was found that the more hydrophilic of the two polymers promoted calcite growth and suppressed vaterite growth. The more hydrophobic polymer completely inhibited vaterite growth. Both polymers decreased the amount of crystals formed on the steel surface, the more hydrophobic polymer being the most effective. The crystal inhibition efficiency was enhanced close to the cloud point of the polymers. The results were compared with the effect of poly(acrylic acid), a commonly used antiscalant. It was found that poly(acrylic acid) was about as efficient as the more hydrophobic polymer in decreasing the amount of calcium carbonate. At higher concentrations of poly(acrylic acid), almost all of the calcium carbonate precipitated in the amorphous form.     

Honeycomb-patterned films of polystyrene/poly(ethylene glycol): preparation,surface aggregation and protein adsorption

Highly ordered honeycomb-patterned polystyrene (PS)/poly(ethylene glycol) (PEG) films were prepared by a water-assisted method using an improved setup, which facilitated the formation of films with higher regularity, better reproducibility, and larger area of honeycomb structures. Surface aggregation of hydrophilic PEG and adsorption of bovine serum albumin (BSA) on the honeycomb-patterned films were investigated. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to observe the surface morphologies of the films before and after being rinsed with water. As confirmed by the FESEM images and the AFM phase images, PEG was enriched in the pores and could be gradually removed by water. The adsorption of fluorescence-labeled BSA on the films was studied in visual form using laser scanning confocal microscopy. Results clearly demonstrated that the protein-resistant PEG was selectively enriched in the pores. This water-assisted method may be a latent tool to prepare honeycomb-patterned biofunctional surfaces. Supported by the National Natural Science Foundation of China (Grant No. 50803053), the National Natural Science Foundation of China for Distinguished Young Scholars (Grant No. 50625309), the National Postdoctoral Science Foundation of China (Grant Nos. 20070421172 & 20081466) and the National Undergraduate Innovative Test Program     

Vibrational spectroscopic studies on fibrinogen adsorption at polystyrene/protein solution interfaces: hydrophobic side chain and secondary structure changes

Structural changes of fibrinogen after adsorption to polystyrene (PS) were examined at the PS/protein solution interface in situ using sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Different behaviors of hydrophobic side chains and secondary structures of adsorbed fibrinogen molecules have been observed. Our results indicate that upon adsorption, the hydrophobic PS surface induces fast structural changes of fibrinogen molecules by aligning some hydrophobic side chains in fibrinogen so that they face to the surface. Such structural changes of fibrinogen hydrophobic side chains are local changes and do not immediately induce significant changes of the protein secondary structures. Our research also shows that the interactions between adsorbed fibrinogen and the PS surface can induce significant changes of protein secondary structures or global conformations which occur on a much longer time scale.     

Effect of pH on protein adsorption capacity of strong cation exchangers with grafted layer

The effect of pH on the static adsorption capacity of immunoglobulin G, human serum albumin, and equine myoglobin was investigated for a set of five strong cation exchangers with the grafted tentacle layer having a different ligand density. A sharp maximum of adsorption capacity with pH was observed for adsorbents with a high ligand density. The results were elucidated using the protein structure and calculations of pK(a) of ionizable groups of surface basic residues. Inverse size-exclusion experiments were carried out to understand the relation between the adsorption capacity and pore accessibility of the investigated proteins.     

pH dependence of tailing in reversed-phase chromatography of a cationic dye: measurement of the strong adsorption site surface density

A question that has interested Dr. J.J. Kirkland is addressed: what is the nature of the silanols that cause tailing to persist at low pH in reversed-phase chromatography? Chromatograms for a cationic dye, 1,1'-didodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI), were studied at varying pH using an Agilent SB-C8 column and 80% ACN/water for six DiI concentrations ranging from 0.9 to 316 microM. The chromatograms showed increased retention and tailing from pH 1 to 5, as expected. Simulations of the chromatograms agreed well with experiment for a bi-Langmuir isotherm with weak (C8) and strong (silica) adsorption sites. The simulation parameters revealed that the number of strong adsorption sites decreases by 40% from pH 1 to 5, which indicates that the silanols causing tailing are in the SiOH, not the SiO-, form. This seems paradoxical because tailing increases with increasing pH. The simulation parameters reveal that this increased tailing from pH 1 to 5 owes to doubling of the partition coefficient for DiI to the strong adsorption site, which more than compensates for the decreasing number of sites. We attribute this increased partition coefficient to increased long-range coulombic interactions with the increasingly abundant SiO- groups at higher pH, which boosts DiI's partition coefficient for both the C8 and SiOH sites. The picture thus emerges that for DiI, higher pH causes increased tailing because the SiO- groups exacerbate tailing that actually originates from adsorption to SiOH groups.     

Computational studies on polymer adhesion at the surface of gamma-Al2O3. I. The adsorption of adhesive component molecules from the gas phase

We calculate the minimum energy paths and reaction energies of the adsorption of the epoxide adhesive components diglycidylesterbisphenol A (DGEBA), diethyltriamine (DETA), and the adhesion promoter 3-aminopropylmethoxysilane (AMEO) at two different sites on a model of the native Al2O3 surface, using the nudged elastic band algorithm in conjunction with self-consistent charge-density functional based tight binding. Our results show that the chosen combination of methods is well suited to obtain an overview of the reaction mechanisms and kinetics of the adsorption of organic molecules on inorganic surfaces. The obtained MEP-s show that there is preference for the adsorption of the adhesion promoter, AMEO, over the resin, DGEBA, while the adsorption of the curing agent, DETA, is unfavorable. Our approach also gives an insight into the ranges of the mechanical and electronic influences of the adsorption process on the interface, which neither full ab initio methods nor force field approaches can provide. These results will help to develop a quantum mechanics-molecular mechanics multiscale embedding scheme for more detailed studies of organic/inorganic hybrid interface reactions.     

Effect of surface defects on the adsorption rate of O2 on polycrystalline rhodium

By using a method of secondary ion mass-spectrometry, the initial sticking coefficient of oxygen (S_o) has been measured when O₂ is adsorbed on two types of the polycrystalline rhodium surface. These surfaces are: 1. defective, i. e. subjected to ion bombardment and 2. smooth, i.e. annealed after bombardment. Values obtained for the defective and smooth surfaces are S_o 0.35 at sample temperatures T=295–1000 K and S_o 4×10^–3 at T=680–870 K, respectively.

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- (S_o) - : — , -. S_o0,35 295–1000 K, 4×10^–3 T=680–870 K.

     

FTIR studies of CO adsorption on Rh-Ge/Al2O3 catalysts prepared by surface redox reactions

A series of bimetallic Al2O3-supported Rh-Ge catalysts was prepared by surface redox reactions under controlled hydrogen atmosphere. The surface properties of these catalysts were probed via in-situ FTIR spectroscopic studies of adsorbed CO and were compared to those of monometallic Rh catalysts that had undergone similar treatments. The results indicate that Ge addition results in the formation and stabilization of smaller rhodium ensembles at the expense of larger Rh0 surfaces. A charge-transfer mechanism from Ge to Rh is also inferred by the IR results for the high Ge loading samples. Air exposure of the catalysts leads to an irreversible segregation of the two metals and formation of large Rh crystallites.     

Equilibrium, FTIR, scanning electron microscopy and small wide angle X-ray scattering studies of chromates adsorption on modified bentonite

The study presents a discussion about the adsorption mechanism of chromate anions on bentonite modified by hexadecyltrimethylammonium bromide (HDTMA-Br). The formation of alkylammonium chromates: HDTMAHCrO₄, (HDTMA)₂Cr₂O₇ and to the lesser extent (HDTMA)₂CrO₄ at the water–bentonite interface is examined based on the Scanning Electron Microscopy and surface tension measurements. The histograms of HDTMA/Cr(VI) molar ratio on the bentonite surface, found from Scanning Electron Microscopy (SEM) measurements, show that for the majority of points of bentonite surface the value of this ratio is in 1–2 range. FTIR spectra of modified bentonite samples show the change from gauche to trans conformation in the surfactant arrangement in the clay interlayer accompanying its concentration increase.

In turn Small Wide Angle X-Ray Scattering (SWAXS) patterns evidently suggest incorporation of chromate anions into the interlamellar space of bentonite structure.     

Role of association on protein adsorption isotherms. Beta-lactoglobulin A adsorbed on a weakly hydrophobic surface

This paper explores the role of association on the adsorption isotherms of beta-lactoglobulin A on a weakly hydrophobic stationary phase at 4 degrees C and mobile phases of 0.85 M and 1 M ammonium sulfate, pH 4.5. The isotherms, obtained by frontal analysis, show an S-shape and the corresponding Scatchard plots indicate positive cooperativity. The slopes and intercepts of the Scatchard plots at low solute concentration are analyzed in terms of two species--a promoter and a higher order stronger adsorbing species. An explicit equation of the isotherm is developed based on this model, and this expression is shown to reproduce the isotherm shape using the appropriate derived parameters. It is further shown from this equation that a Langmuir-shaped adsorption isotherm can be obtained if the higher order associate or aggregate binds weaker to the support than the promoter. These results indicate that protein-protein interactions and the formation of associates can play a significant role on the shape of the isotherm and ultimately on the behavior of the species in preparative scale chromatography.     

Fracture surface morphology and phase relationships of polystyrene/poly(methyl methacrylate) systems. I. Low-molecular-weight polystyrene in poly(methyl methacrylate)

Samples of low-molecular-weight polystyrene (PS) in poly(methyl methacrylate) (PMMA) were prepared by first dissolving PS in methyl methacrylate monomer and then polymerizing the monomer. Forty-three specimens of varying number-average molecular weight (2100–49,000) and composition (5–40 wt %) of PS were prepared, and the surface morphology and phase relationships studied by scanning electron microscopy. Four distinct types of phase relationships were observed: (i) a single phase consisting of PS dissolved in PMMA; (ii) PS dispersed in PMMA; (iii) PMMA dispersed in PS; and (iv) regions of PS dispersed in PMMA coexisting with regions of PMMA dispersed in PS. Values of the size and population density of the dispersed particles are reported. Finally, the size and distribution of the dispersed particles and the various types of phase relationships are discussed in terms of the ternary polystyrene/poly(methyl methacrylate)/methyl methacrylate phase diagram.     

Chemical imaging of protein adsorption and crystallization on a wettability gradient surface

The use of self-assembled monolayers is an established method to study the effect of surface properties on proteins and other biological materials. The generation of a monolayer with a gradient of chemical properties allows for the study of multiple surface properties simultaneously in a high throughput manner. Typically, in order to detect the presence of proteins or biological material on a surface, the use of additional dyes or tags is required. Here we present a novel method of studying the effect of gradient surface properties on protein adsorption and crystallization in situ through the use of ATR-FTIR spectroscopic imaging, which removes the need for additional labeling. We describe the successful application of this technique to the measurement of the growth of a gradient monolayer of octyltrichlorosilane across the surface of a silicon ATR element. ATR-FTIR imaging was also used to study the adsorption of lysozyme, as a model protein, onto the modified surface. The sensitivity of measurements obtained with a focal plane array (FPA) detector were improved though the use of pixel averaging which allowed small absorption bands to be detected with minimal effect on the spatial resolution along the gradient. Study of the effect of surface hydrophobicity on both adsorption of lysozyme to the element and lysozyme crystallization revealed that more lysozyme adsorbed to the hydrophobic side of the ATR element and more lysozyme crystals formed in the same region. These findings strongly suggest a correlation exists between surface protein adsorption and protein crystallization. This method could be applied to the study of other proteins and whole cells.     

Effect of salt concentrations on the displacement adsorption enthalpies of denatured protein folding at a moderately hydrophobic surface

Lead(II) complexes of reduced glutathione (GSH) of general composition [Pb(L)(X)]·H₂O (where L=GSH; X=Cl, NO₃, CH₃COO, NCS) have been synthesized and characterized by elemental analyses, infrared spectra and electronic spectra. Thermogravimetric (TG) and differential thermal analytical (DTA) studies have been carried out for these complexes. Infrared spectra indicate deprotonation and coordination of cysteinyl sulphur with metal ion. It indicates the presence of water molecule in the complexes that has been supported by TG/DTA. The thermal behaviour of complexes shows that water molecule is removed in first step-followed removal of anions and then decomposition of the ligand molecule in the subsequent steps. Thermal decomposition of all the complexes proceeds via first order kinetics. The thermodynamic activation parameters, such as E*, A, ΔH*, ΔS* and ΔG* have been calculated. The geometry of the metal complexes has been studied with the help of molecular modeling for energy minimization calculation.     

Thermodynamic analysis of polymerization-induced phase separation of a polystyrene in epoxy/monoamine–diamine systems. Effect of monoamine–diamine proportion on the phase diagram

Polymerization-induced phase separation of a polystyrene in various epoxy-amine systems where the amino groups were provided by a monoamine and a diamine mixed in different proportions was thermodynamically studied. A model based on the Flory–Huggins theory extended by Koningsveld and Staverman approach where the interaction parameter was dependent on temperature, composition and conversion, and polydispersity of the components was considered, was used. A general equation for the evolution during polymerization of the epoxy-amine species distributions according to the monoamine–diamine ratio was derived from the Stockmayer distribution. The interaction parameters continuously decreased with conversion. Phase diagrams of the blends were obtained and the critical composition was between 5 and 6 vol.% PS in all blends.     

Effect of temperature on self-assembly of bovine beta-casein above and below isoelectric pH. Structural analysis by cryogenic-transmission electron microscopy and small-angle X-ray scattering

beta-Casein is one of the main proteins in milk, recently classified as an intrinsically unstructured protein. At neutral pH, it is composed of a highly polar N-terminus domain and a hydrophobic C-terminus tail. This amphiphilic block-copolymer-like structure leads to self-organization of the protein monomers into defined micelles. Recently, it has been shown that at room temperature, beta-casein also self-organizes into micelles in an acidic environment, but the effect of temperature on the micelles' formation and properties at the low pH regime were not explored. In the present study, we used two complementary techniques, cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS), to characterize at high-resolution the micelles' shape, dimensions, and aggregation numbers and to determine how these properties are affected by temperature between 1 and 40 degrees C. Two different regimes were studied: highly acidic pH where the protein is cationic, and neutral pH, where it is anionic. We found that flat disk-like micelles with low aggregation numbers formed at low temperature in the two pH regimes. Close to neutral pH increase in temperature involves a transition in the micelles' shape and dimensions from flat disks to bulky, almost spheroidal micelles, coupled with a sharp increase in the micelles' aggregation number. In contrast, no effects on the micelles' morphology or aggregation number were detected in the acidic environment within the entire temperature range studied. The self-organization into disk micelles and the lack of effect of temperature in the acidic environment are linked to the unstructured character of the protein and to the charge distribution map. The latter indicates that below the isoelectric pH (pI), beta-casein loses the distinct separation of hydrophobic and hydrophilic domains, thereby suggesting that it may no longer be considered as a classical head-tail block-copolymer amphiphile as in neutral pH.