X-ray microscopy studies of protein adsorption on a phase segregated polystyrene/polymethylmethacrylate surface. 2. Effect of pH on site preference

X-ray photoemission electron microscopy (XPEEM) using synchrotron radiation illumination has been used to study the adsorption of human serum albumin (HSA) onto a phase segregated polystyrene/polymethylmethacrylate (PS/PMMA) blend surface from solutions of five different pH values. The absolute coverage of albumin on each of three chemically distinct components of the surface, PS domains, PMMA domains, and the interface between the domains, was determined from a quantitative analysis of C 1s image sequences. At all pH values, the preferred adsorption site is the interface. At neutral pH (7.0), albumin showed a slight preference for PS regions relative to PMMA. At strongly acidic pH (2.0) and strongly basic pH (10.0), similar amounts of albumin adsorb on the PS and PMMA regions. However, at pH 4.0, the amount of albumin adsorbed on PMMA domains is approximately 1.6 times greater than that on PS domains, while at pH 8.6 the amount of albumin adsorbed on PMMA is one-half that adsorbed on PS domains. The pH dependence of the site preference is rationalized in terms of the known changes of albumin conformation with pH [Peters, T., Jr. All About Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: New York, 1995]. We infer from our results that the site preference of albumin adsorption on PS/PMMA blends is related mainly to changes in hydrophobic interactions, which are driven by pH-dependent electrostatic effects, that is, changes to the protein surface structure as the charge on the protein changes. The results provide insight into changes in the secondary structure of albumin in acid and basic media.     

Elucidation of protein adsorption behavior on polymeric surfaces: toward high-density, high-payload protein templates

The elucidation of protein adsorption behavior on polymeric surfaces is very important, since their use as arrays and carriers of biomolecules is ever growing for a wide variety of bioapplications. We evaluate protein adsorption characteristics on chemically homogeneous and heterogeneous polymeric surfaces by employing polystyrene-block-polymethylmethacrylate (PS-b-PMMA) diblock copolymer, PS homopolymer, PMMA homopolymer, and PS/PMMA blend as protein templates. We also investigate distance-dependent protein adsorption behavior on the interfacial region between PS and PMMA. We observe selective protein adsorption exclusively onto PS areas for the chemically heterogeneous PS-b-PMMA and PS/PMMA blend templates. On blend films, protein adsorption is highly favored on the PS regions located near the PS:PMMA interface over that on the PS areas situated away from the interface. Protein density on PS domains is inversely proportional to the separation distance between two neighboring PS:PMMA interfaces. We also observe a higher protein density on the PS-b-PMMA than on the PS or PMMA homopolymer templates. This effect is due to the fact that chemically heterogeneous PS-b-PMMA presents periodically spaced PS:PMMA interfaces on the nanometer scale, whereas no such interfaces are present on homopolymer films. The density of protein molecules on the heterogeneous PS-b-PMMA surface is approximately 3-4-fold higher than on the homogeneous PS surface for the identical experimental conditions. These results demonstrate that self-assembling, chemically heterogeneous, nanoscale domains in PS-b-PMMA diblock copolymers can be used as excellent, high-payload, high-density protein templates. The unique advantages of the diblock copolymer may prove the spontaneously constructed protein nanotemplates to be highly suitable as functional substrates in many proteomics applications.     

Nano-organized collagen layers obtained by adsorption on phase-separated polymer thin films

The organization of adsorbed type I collagen layers was examined on a series of polystyrene (PS)/poly(methyl methacrylate) (PMMA) heterogeneous surfaces obtained by phase separation in thin films. These thin films were prepared by spin coating from solutions in either dioxane or toluene of PS and PMMA in different proportions. Their morphology was unraveled combining the information coming from X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurements. Substrates with PMMA inclusions in a PS matrix and, conversely, substrates with PS inclusions in a PMMA matrix were prepared, the inclusions being either under the form of pits or islands, with diameters in the submicrometer range. The organization of collagen layers obtained by adsorption on these surfaces was then investigated. On pure PMMA, the layer was quite smooth with assemblies of a few collagen molecules, while bigger assemblies were found on pure PS. On the heterogeneous surfaces, it appeared clearly that the diameter and length of collagen assemblies was modulated by the size and surface coverage of the PS domains. If the PS domains, either surrounding or surrounded by the PMMA phase, were above 600 nm wide, a heterogeneous distribution of collagen was found, in agreement with observations made on pure polymers. Otherwise, fibrils could be formed, that were longer compared to those observed on pure polymers. Additionally, the surface nitrogen content determined by XPS, which is linked to the protein adsorbed amount, increased roughly linearly with the PS surface fraction, whatever the size of PS domains, suggesting that adsorbed collagen amount on heterogeneous PS/PMMA surfaces is a combination of that observed on the pure polymers. This work thus shows that PS/PMMA surface heterogeneities can govern collagen organization. This opens the way to a better control of collagen supramolecular organization at interfaces, which could in turn allow cell-material interactions to be tailored.     

Polystyrene-coated micro-sized particles by "in situ" surface initiated polymerization in accord with Langmuir model adsorption

This study describes the mechanism of adsorption of polystyrene chains PS "in situ" growth from micro-sized commercial silicate particles, i.e. Feldspar. The main aim is to derive adsorption isotherms from thermal gravimetric analysis (TGA) and size exclusion chromatography (SEC) data obtained during the direct polymerization of PS initiated by a pre-adsorbed radical initiator onto the inorganic surface. The adsorption isotherm plot indicates that the PS adsorption is in accord with the Langmuir Model. The amount of PS monolayer coverage increases with polymerization time, and it is highly dependent on the monomer diffusion to the surface during the hybrid inorganic/organic synthesis. Such behavior depends on the concentration gradient between monomer concentration in solution and that adsorbed by polymerization onto the micro-sized particles surface of Feldspar.     

Surface structures and properties of polystyrene/poly(methyl methacrylate) blends and copolymers

Sum frequency generation (SFG) vibrational spectroscopy has been applied to study the molecular surface structures of polystyrene (PS)/poly(methyl methacrylate) (PMMA) blends and the copolymer between PS and PMMA (PS-co-PMMA) in air, supplemented by atomic force microscopy (AFM) and contact angle goniometer. Both the blend and the copolymer have equal weight amounts of the two components. SFG results show that both components, PS and PMMA, can segregate to the surface of the blend and the copolymer before annealing, although PMMA has a slightly higher surface tension. Upon annealing both SFG results and contact angle measurements indicate that the PS segregates to the surface of the PS/PMMA blend more but no change occurs on the PS-co-PMMA surface. AFM images show that the copolymer surface is flat but the 1:1 PS/PMMA blend has a rougher surface with island like domains present. The annealing effect on the blend surface morphology has also been investigated. We collected amide SFG signals from interfacial fibrinogen molecules at the copolymer or blend/protein solution interfaces as a function of time. Different time-dependent SFG signal changes have been observed, showing that different surfaces of the blend and the copolymer mediate fibrinogen adsorption behavior differently.     

Isotherm analysis of phenol adsorption on polymeric adsorbents from nonaqueous solution

Macroporous poly(methyl methacrylate-co-divinylbenzene) (PMMA), interpenetrating polymer adsorbent based on poly(styrene-co-divinylbenzene) (PS) and poly(methyl methacrylate-co-divinylbenzene) (PMMA/PS), and macroporous cross-linked poly(N-p-vinylbenzyl acetylamide) (PVBA) were prepared for the adsorption of phenol from cyclohexane. The sorption isotherms of phenol on the three polymeric adsorbents were measured and fitted to Langmuir and Freundlich isotherms. It is shown that the Langmuir isotherm, which is based on a homogeneous surface model, is unsuitable to describe the sorption of phenol on the adsorbents from nonaqueous solution and the Freundlich equation fits the tested three adsorption systems well. The isosteric enthalpy was quantitatively correlated with the fractional loading for the sorption of phenol onto the three polymeric adsorbents. The surface energetic heterogeneity patterns of the adsorbents were described with functions of isosteric enthalpy. The results showed that the tested three polymeric adsorbents exhibited different surface energetic heterogeneity patterns. The initial isosteric enthalpy of phenol sorption on polymeric adsorbent has to do with the surface chemical composition and is free from the pore structure of the polymeric adsorbent matrix. Forming hydrogen bonds between phenol molecules and adsorbent is the main driving force of phenol sorption onto PVBA and PMMA adsorbent from nonaqueous solution. When phenol is adsorbed on PMMA/PS, pi-pi interaction resulting from the stacking of the benzene rings of the adsorbed phenol molecules and the pendant benzene ring of adsorbent is involved.     

PMMA adsorption over previously adsorbed PS studied by polarized FTIR-ATR

Polarized infrared spectroscopy in attenuated total reflection was used to investigate the adsorption of PMMA (polymethylmethacrylate) onto PS (polystyrene) that was previously adsorbed onto oxidized silicon from dilute solution in carbon tetrachloride at 30°C. The carbonyl group of PMMA forms hydrogen bonds with surface silanol groups, giving segment-surface interaction energy of 4 kT as against 1 kT for PS (k is the Boltzmann constant, T the absolute temperature). The formation of hydrogen-bonding by PMMA was unaffected, in rate or amount, by preadsorbed PS, but a lesser total amount of PMMA adsorbed onto PS, resulting in a higher average bound fraction. The histogram of the mass adsorbed, attributable to subpopulations of chains with different bound fractions, was inferred by subtracting infrared spectra acquired at successive times. Whereas this histogram was broad and bimodal for adsorption onto bare surface, it was narrower and unimodal for adsorption onto preadsorbed PS. © 1995 John Wiley & Sons, Inc.     

Scaling aspects of block co-polymer adsorption on curved surfaces from nonselective solvents

In this paper, we have developed a geometric-based scaling model that describes the adsorption of diblock copolymer chains from good solvents and theta-solvents onto reactive surfaces of varying curvatures. To evaluate the impact of particle size on the adsorption process, we probed the adsorption of poly(styrene-b-methymethacrylate) (PS-PMMA) diblock copolymers from solvents with different degrees of selectivity on aluminum oxide (Al(2)O(3)) surfaces belonging to particles of different sizes. When the adsorbed PMMA layer is dense enough (in the case of a theta-solvent for the PMMA block), our results show good correlation between the theory and experimental results, pointing to the formation of a PMMA adsorption layer and a brushlike PS layer. Conversely, when adsorption occurs from a nonpreferential solvent, particularly on particles with high curvature, the PMMA adsorption layer at the surface becomes less dense and the grafted PS moiety exhibits a transitional morphology consisting of several layers of increasingly sparsely spaced blobs.     

PDMS微芯片吸附牛血清白蛋白的研究

材料表面的物理和化学性质对蛋白质的吸附具有很大的影响[1].对蛋白质吸附的研究是研制生物传感器、生物芯片和生物材料的基础.     

The influence of nanoparticle fillers on the morphology of a spin-cast thin film polymer blend

Polymer/nanoparticle composite films are receiving growing attention thanks to their potential for application in ultra-thin electronic and optical devices. Polymer blend demixing has been shown to be a suitable technique for the structuring of polymer thin films and the patterning of nanoparticles (NP) within them. In this work we show that the morphology of thin polymer films made by spin-casting a polymer blend solution containing NP fillers on a surface depends strongly on the concentration of NP fillers. More specifically, polystyrene/polymethylmethacrylate (PS/PMMA) films formed from a toluene solution, and which demix following a nucleation and growth mechanism, were studied. It was found that both the height and the surface density of PMMA domains increased as the concentration of CoPt:Cu NPs in the film was increased. We find that similar effects are induced in a NP-free PS/PMMA demixed film upon increasing the molecular weight of the PS molecules. This suggests that under certain conditions the NPs and the polymer molecules in the blend do not behave as separate species but form aggregates.     

Shifts in polystyrene particle surface charge upon adsorption of the Pluronic F108 surfactant

Electrical field-flow fractionation (ElFFF) and sedimentation field-flow fractionation (SdFFF) were used in combination to study the adsorption of the triblock polymeric surfactant, Pluronic F108 [(EO)129-(PO)56-(EO)129] to 200 nm polystyrene (PS) latex spheres. The SdFFF technique allowed an accurate determination of the mass of surfactant adsorbed on each particle from a solution of given concentration. To complement this isotherm study, we show that ElFFF can be used to measure fractional coverages of the formed electrically neutral surfactant layers on the charged PS particles. Through a combination of the two techniques it is possible to gain information about the structure of the adsorbate layer. Thus, when Pluronic F108 is taken up by the PS surface from solutions of low concentration, all three blocks appear to adhere to the surface as long as there is free space available. As the solution concentration increases and the fractional coverage reaches approximately 20%, the surface turns crowded enough to let the strongly adsorbing PPO blocks competitively displace the weakly adherent PEO blocks, which gradually rise to extend into the aqueous phase until the surface is fully saturated.     

A novel and simple method for finding the heterogeneity of adsorbents on the basis of adsorption kinetic data

The heterogeneity of adsorbent was characterized by a novel and simple method that uses adsorption kinetic data from solution. On the basis of different simulated adsorption kinetic data for heterogeneous adsorbents with two different adsorption sites, it was found that there is a deviation from linearity in pseudo-second-order adsorption kinetic models. The patterns of obtained diagrams (t/qt vs t) shows five main classes of adsorption. On the basis of this novel and simple method it is possible to show that the surface of adsorbents is homogeneous or heterogeneous, and in the case of heterogeneous surfaces it is possible to find the differences between adsorption sites. For finding kinetic constants of adsorption on heterogeneous surfaces with two different sites, a new equation was presented and called the two-site pseudo-second-order (TS-PSO) model. Finally, some experimental kinetic data of adsorption were analyzed by the new method.     

Effect of surface charge on adsorption of bovine serum albumin 2. Interaction of protein molecules with an anionic monolayer,as studied by ellipsometry,radiotracer and surface tension measurements

The adsorption of bovine serum albumin (BSA) onto an anionic monolayer of sodium docosylsulfate (SDocS) spread at the air/water interface was studied by ellipsometry. The adsorption behavior of BSA was estimated from the observed changes in phase differences and in the ratio of reflection coefficients. The dynamic process of BSA adsorption was measured after the injection of BSA solution into the aqueous substrate of SDocS monolayer. The gentle stirring of the substrate solution for 10 min was found to be enough to make the solution homogeneous without damaging the monolayer. The adsorption characteristics of BSA onto a negatively charged surface was compared with that onto a positively charged surface previously reported.The amount of adsorption depended on time and showed a maximum with an initial rapid rise, followed by gradual decrease toward the ultimate equilibrium value. The amount and time of the maximum adsorption depended on the concentration of BSA added to the aqueous substrate.Separate radiotracer measurement, using³⁵S-labeled SDocS monolayer, which is insoluble by itself, revealed that SDocS is solubilized into the bulk solution when BSA is added to the aqueous substrate.     

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.     

Formation of "snowmanlike" polystyrene/poly(methyl methacrylate)/toluene droplets dispersed in an aqueous solution of a nonionic surfactant at thermodynamic equilibrium

"Snowmanlike" polystyrene (PS)/poly(methyl methacrylate) (PMMA) composite particles were prepared by evaporation of toluene from PS/PMMA/toluene droplets dispersed in an aqueous solution of polyoxyethylene nonylphenyl ether surfactant (Emulgen 911). Partitioning experiments revealed that the Emulgen 911 concentration was higher in the droplets than in the aqueous solution during toluene evaporation. As a consequence, the interfacial tensions between the polymer phases (PS and PMMA) and the aqueous phase (gammaP-T/W) were extraordinarily low (approximately 10(-1) mN/m). The interfacial tension between the PS and PMMA phases containing toluene (gammaPS-T/PMMA-T) measured by the spinning drop method was not affected by the presence of Emulgen 911. Based on minimization of the total interfacial free energy at a polymer weight fraction in the toluene droplet of 0.17, the formation of spherical droplets is expected, in agreement with experiment. The subsequent morphology change of the PS/PMMA/toluene droplets from spherical to snowmanlike during toluene evaporation under thermodynamic equilibrium is attributed to (i) the low values of gammaP-T/W, which explains the increase in the interfacial area between the droplets and the aqueous phase, and (ii) the increase in gammaPS-T/PMMA-T with increasing polymer weight fraction.     

Breath figures in polymer and polymer blend films spin-coated in dry and humid ambience

We investigate effects of two spin-coating parameters, relative humidity (5% < or = RH < or = 80%) in ambient atmosphere and water content (3 wt % < or = f(H2O) < or = 20 wt %) in solution (rich in tetrahydrofuran), on the structure of breath figures (BF) formed in spin-cast films of polar poly(methyl methacrylate) (PMMA) and PMMA mixed with nonpolar polystyrene (PS). Film morphologies, examined with atomic and lateral force microscopy, are analyzed with integral geometry analysis to yield morphological BF measures. In PMMA, water added to solution has much stronger impact than that from moisture on formed BFs, which could be ordered (with conformational entropy S approximately 0.9-1.0). In PMMA/PS, BFs decorate exclusively polar PMMA domains, resulting in morphologies with two length scales (sub-micrometer BFs and domains >10 microm). This suggests a novel strategy for herarchic structure formation in multicomponent polymer films. In PS/PMMA, BFs are better developed than in pure PMMA spin-coated in identical conditions. These observations show that the air boundary layer facing the spin-cast polymer film (region) is more important than the ambient atmosphere.     

Surface and interface morphology of polystyrene/poly(methyl methacrylate) thin‐film blends and bilayers

The surface and interface morphologies of polystyrene (PS)/poly(methyl methacrylate) (PMMA) thin‐film blends and bilayers were investigated by means of atomic force microscopy (AFM) and X‐ray photoelectron spectroscopy. Spin‐coating a drop of a PS solution directly onto a PMMA bottom layer from a common solvent for both polymers yielded lateral domains that exhibited a well‐defined topographical structure. Two common solvents were used in this study. The structure of the films changed progressively as the concentration of the PS solution was varied. The formation of the blend morphology could be explained by the difference in the solubility of the two polymers in the solvent and the dewetting of PS‐rich domains from the PMMA‐rich phase. Films of the PS/PMMA blend and bilayer were annealed at temperatures above their glass‐transition temperatures for up to 70 h. All samples investigated with AFM were covered with PS droplets of various size distributions. Moreover, we investigated the evolution of the annealed PS/PMMA thin‐film blend and bilayer and gave a proper explanation for the formation of a relatively complicated interface inside a larger PS droplet. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 9–21, 2006     

Revisiting the morphology development of solvent-swollen composite polymer particles at thermodynamic equilibrium

Morphology of polystyrene (PS)/poly(methyl methacrylate) (PMMA)/toluene droplets, in which phase separation proceeds, dispersed in SDS aqueous solution was examined. It changed from ex-centered PS-core/PMMA-shell to hemisphere with increasing SDS concentration. At low polymer weight fraction (wp), PS and PMMA phases contained non-negligible amount of PMMA and PS, respectively. The small amount of PS and PMMA in PMMA and PS phases, respectively, affected significantly the interfacial tension between polymer/toluene and aqueous solutions. Interfacial tension between PS and PMMA phases at low wp was measured by the spinning drop method, showing a quite low value ( approximately 10-2 mN/m). Predicted morphology obtained from calculation of minimum total interfacial free energy of the droplets using the interfacial tensions agreed well with the experimental observation.     

Influence of buffer composition on protein adsorption on agarose (Sepharose 6B)

Summary The adsorption of albumin on a commercial, high quality agarose (Sepharose^® 6B) was studied in different buffers of low pH (4.00) and low molarity (9–11 mM). Eluents containing 0.025% albumin were introduced onto small gel columns until saturation and the adsorption capacity of the gel in different buffers calculated. Adsorption was high in solutions of monobasic acids. Although cation-exchange interactions between the protein and the gel matrix were found to be influenced essentially by the sodium ion concentration or ionic strength of the buffer, adsorption in cirate buffers is weak compared to that in formate, acetate or propionate buffers of about the same ionic strength (I=0.01).     

Integral geometry analysis of fluorescence micrographs for quantitative relative comparison of protein adsorption onto polymer surfaces

Most methods developed to study protein binding to distinct surfaces can only determine the average amount of adsorbed protein or merely provide (qualitative) information on its spatial distribution. Both these features can be characterized rigorously by integral geometry analysis of fluorescence micrographs. This approach is introduced here to compare the relative protein adsorption onto various polymer surfaces: polystyrene (PS), poly(methyl methacrylate) (PMMA), poly( n-butyl methacrylate) (PnBMA), poly( tert-butyl methacrylate) (PtBMA), and PS(PETA) and cross-linked poly(ethylene oxide) (PEO*(PETA)), admixed with pentaerythritol triacrylate (PETA). The polymeric surfaces were incubated for 15 min in phosphate-buffered saline (pH 7.4) containing 125 mug/mL fluorescently labeled lectins, either lentil lectin (LcH) or concanavalin A (ConA). Fluorescence images were recorded at identical conditions (physiological buffer, same exposure time, magnification, gain). For each image, taken a few times for each polymer, the distribution and average value of the normalized intensity were determined. The results show that the binding of LcH to PS(PETA), PtBMA, PS, PnBMA, PMMA, and PEO*(PETA) can be expressed by the ratio of the following values (mean +/- 95% confidence interval): 0.356 +/- 0.022, 0.298 +/- 0.030, 0.241 +/- 0.014, 0.083 +/- 0.008, 0.039 +/- 0.008, and 0.010 +/- 0.006, respectively. In turn, the relative adsorption of ConA is described by the values 0.252 +/- 0.016, 0.217 +/- 0.014, 0.222 +/- 0.016, 0.046 +/- 0.006, 0.116 +/- 0.008, and 0.006 +/- 0.002, respectively. Low dispersions of fluorescence intensity around average values indicate homogeneous distribution of adsorbed proteins. The introduced approach enables a fast and easy way not only to quantify the relative amount of bound proteins but also to characterize quantitatively the organization of their surface distribution, as demonstrated for patchlike protein adsorption onto the polymer blend surface.