Nanoscale organization of adsorbed collagen: influence of substrate hydrophobicity and adsorption time

The adsorption of collagen on polystyrene (PS) and polystyrene oxidized by oxygen plasma discharge (PSox) was studied as a function of time using radiolabeling, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Radiolabeling and XPS indicated that the initial step of adsorption was faster on PS than on PSox. AFM imaging under water revealed very different supramolecular organization of the adsorbed films depending on time and on the nature of the substrate: PS showed patterns of collagen aggregates at all adsorption times (from 1 min to 24 h); PSox was covered with a smooth layer except at long adsorption times (24 h), for which a mesh of collagen structures was observed. After fast drying, the collagen layer remained continuous and showed a morphology which recalled that observed under water. The mechanical stability of the adsorbed films was assessed under water by scraping with the AFM probe at different loading forces: no perturbations were created on PSox; in contrast, the layer adsorbed on PS was sensitive to scraping, the minimum force required to alter the collagen layer morphology increasing with time. These differences in the film properties were correlated with force measurements upon retraction: multiple adhesion forces were observed with collagen adsorbed on PS samples, whereas such an effect was never observed on PSox. The results show that the amount adsorbed and the organization of the adsorbed film respond differently to the adsorption time and that this is influenced by surface hydrophobicity. The quick initial adsorption on PS, compared to PSox, is thought to leave dangling collagen segments that are responsible for the observed morphology, for adhesion forces, and for lower mechanical resistance of the adsorbed layer.     

Influence of collagen denaturation on the nanoscale organization of adsorbed layers

Adsorption (at 37 degrees C) of type I collagen, in native and heat-denatured (30 min at 40 and 90 degrees C) forms, on polystyrene was studied using quartz crystal microbalance with energy dissipation monitoring (QCM-D), atomic force microscopy (AFM) in tapping mode and X-ray photoelectron spectroscopy (XPS). The significance of the parameters deduced from QCM-D data was examined by comparing different approaches. The adsorbed layer of native collagen has a complex organization consisting of a thin mat of molecules near the surface, in which fibrils develop depending on concentration and time, and of a thicker overlayer containing protruding molecules or bundles which modify noticeably the local viscosity. As a result of drastic denaturation, the ability of collagen to assemble into fibrils in the adsorbed phase is lost and the protrusion of molecules into the aqueous phase is much less pronounced. The adsorbed layer of denatured collagen appears essentially as a monolayer of flattened coils. At low concentration, this is easily displaced upon drying, leading to particular dewetting figures; at high concentration, aggregates add to the first layer. Moderate denaturation leads to an adsorbed phase which shows properties intermediate between those observed with native and extensively denatured collagen, regarding the ability to form fibrillar structures and the adlayer thickness and viscosity.     

Wettability changes in the formation of polymeric multilayers on cellulose fibres and their influence on wet adhesion

Elucidating the assembly mechanism of the collagen at interfaces is important. In this work, the structures of type I collagen molecules adsorbed on bare mica and on LB films of propanediyl-bis(dimethyloctadecylammonium bromide) transferred onto mica at zero surface pressure was characterized by AFM. On mica, the granular morphologies randomly distributed as elongated structures were observed, which were resulted from the interlacement of the adsorbed collagen molecules. On the LB films, the topographical evolution of the adsorbed collagen layers upon the increasing adsorption time was investigated. After 30 s, the collagen assembled into network-like structure composed of the interwoven fibrils, called as the first adlayer, which was attributed to its adsorption on the LB film by means of a limited number of contact points followed by the lateral association. One minute later, the second adlayer was observed on the top of the first adlayer. Up to 5 min, collagen layers, formed by inter-twisted fibrils, were observed. Under the same conditions after 1 min adsorption on LB film, the AFM image of the layer obtained in the diluted hydrochloric acid solution is analogous to the result of the sample dried in air, indicating that it is the LB film that leads to the formation of the network structure of collagen and the formation of the network structures of collagen layers is tentatively ascribed to the self-assembly of type I collagen molecules on LB film, not to the dewetting of the collagen solution during drying.     

Nanostructured collagen layers obtained by adsorption and drying

The supramolecular organization of collagen adsorbed from a 7 microg/ml solution on polystyrene was investigated as a function of the adsorption duration (from 1 min to 24 h) and of the drying conditions (fast drying under a nitrogen flow, slow drying in a water-saturated atmosphere). The morphology of the created surfaces was examined by atomic force microscopy (AFM), while complementary information regarding the adsorbed amount and the organization of the adsorbed layers was obtained using radioassays, X-ray photoelectron spectroscopy (XPS), and wetting measurements. The collagen adsorbed amount increased up to an adsorption duration of 5 h and then leveled off at a value of 0.9 microg/cm2. For samples obtained by fast drying, modeling of the N/C ratios obtained by XPS in terms of thickness and surface coverage, in combination with the adsorbed amount, water contact angle measurements and AFM images, indicated that the adsorbed layer formed a felt starting from 30 min of adsorption, the density and/or the thickness of which increased with the adsorption time. Upon slow drying, the collagen layers formed after adsorption times up to about 2 h underwent a strong reorganization. The obtained nanopatterns were attributed to dewetting, the liquid film being ruptured and adsorbed collagen being displaced by the water meniscus. At higher adsorption times, the organization of the collagen layer was similar to that obtained after fast drying, because the onset of dewetting and/or collagen displacement were prevented by the high density of the collagen felt.     

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.     

Interaction forces between particles stabilized by a hydrophobically modified inulin surfactant

The adsorption isotherm of a hydrophobically modified inulin (INUTEC SP1) on polystyrene (PS) and poly(methyl methacrylate) (PMMA) particles was determined. The results show a high affinity isotherm for both particles as expected for a polymeric surfactant adsorption. The interactions forces between two layers of the hydrophobically modified inulin surfactant adsorbed onto a glass sphere and plate was determined using a modified atomic force microscope (AFM) apparatus. In the absence of any polymer, the interaction was attractive although the energy of interaction was lower than predicted by the van der Waals forces. The results between two layers of the adsorbed polymer confirms the adsorption isotherms results and provides an explanation to the high stability of the particles covered by INUTEC SP1 at high electrolyte concentration. Stability of dispersions against strong flocculation could be attributed to the conformation of the polymeric surfactant at the solid/liquid interface (multipoint attachment with several loops) which remains efficient at Na(2)SO(4) concentration reaching 1.5 mol dm(-3). The thickness of the adsorbed polymer layer in water determined both by AFM and rheology measurements, was found to be about 9 nm.     

Detection of interfacial phenomena with osteoblast-like cell adhesion on hydroxyapatite and oxidized polystyrene by the quartz crystal microbalance with dissipation

The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (μ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial μ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.     

In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy

The adsorption of a 14-amino acid amphiphilic peptide, LK14, which is composed of leucine (L, nonpolar) and lysine (K, charged), on hydrophobic polystyrene (PS) and hydrophilic silica (SiO2) was investigated in situ by quartz crystal microbalance (QCM), atomic force microscopy (AFM), and sum frequency generation (SFG) vibrational spectroscopy. The LK14 peptide, adsorbed from a pH 7.4 phosphate-buffered saline (PBS) solution, displayed very different coverage, surface roughness and friction, topography, and surface-induced orientation when adsorbed onto PS versus SiO2 surfaces. Real-time QCM adsorption data revealed that the peptide adsorbed onto hydrophobic PS through a fast (t < 2 min) process, while a much slower (t > 30 min) multistep adsorption and rearrangement occurred on the hydrophilic SiO2. AFM measurements showed different surface morphologies and friction coefficients for LK14 adsorbed on the two surfaces. Surface-specific SFG spectra indicate very different ordering of the adsorbed peptide on hydrophobic PS as compared to hydrophilic SiO2. At the LK14 solution/PS interface, CH resonances corresponding to the hydrophobic leucine side chains are evident. Conversely, only NH modes are observed at the peptide solution/SiO2 interface, indicating a different average molecular orientation on this hydrophilic surface. The surface-dependent difference in the molecular-scale peptide interaction at the solution/hydrophobic solid versus solution/hydrophilic solid interfaces (measured by SFG) is manifested as significantly different macromolecular-level adsorption properties on the two surfaces (determined via AFM and QCM experiments).     

Adsorption of trypsin on hydrophilic and hydrophobic surfaces

The adsorption of trypsin onto polystyrene and silica surfaces was investigated by reflectometry, spectroscopic methods, and atomic force microscopy (AFM). The affinity of trypsin for the hydrophobic polystyrene surface was higher than that for the hydrophilic silica surface, but steady-state adsorbed amounts were about the same at both surfaces. The conformational characteristics of trypsin immobilized on silica and polystyrene nanospheres were analyzed in situ by circular dichroism and fluorescence spectroscopy. Upon adsorption the trypsin molecules underwent structural changes at the secondary and tertiary level, although the nature of the structural alterations was different for silica and polystyrene surfaces. AFM imaging of trypsin adsorbed on silica showed clustering of enzyme molecules. Rinsing the silica surface resulted in 20% desorption of the originally adsorbed enzyme molecules. Adsorption of trypsin on the surface of polystyrene was almost irreversible with respect to dilution. After adsorption on silica the enzymatic activity of trypsin was 10 times lower, and adsorbed on polystyrene the activity was completely suppressed. The trypsin molecules that were desorbed from the sorbent surfaces by dilution with buffer regained full enzymatic activity.     

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.     

Kinetics of conformational changes of fibronectin adsorbed onto model surfaces

Fibronectin (FN), a large glycoprotein found in body fluids and in the extracellular matrix, plays a key role in numerous cellular behaviours. We investigate FN adsorption onto hydrophilic bare silica and hydrophobic polystyrene (PS) surfaces using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) in aqueous medium. Adsorption kinetics using different bulk concentrations of FN were followed for 2h and the surface density of adsorbed FN and its time-dependent conformational changes were determined. When adsorption occurs onto the hydrophilic surface, FN molecules keep their native conformation independent of the adsorption conditions, but the amount of adsorbed FN increases with time and the bulk concentration. Although the protein surface density is the same on the hydrophobic PS surface, this has a strong impact on the average conformation of the adsorbed FN layer. Indeed, interfacial hydration changes induced by adsorption onto the hydrophobic surface lead to a decrease in unhydrated beta-sheet content and cause an increase in hydrated beta-strand and hydrated random domain content of adsorbed FN. This conformational change is mainly dependent on the bulk concentration. Indeed, at low bulk concentrations, the secondary structures of adsorbed FN molecules undergo strong unfolding, allowing an extended and hydrated conformation of the protein. At high bulk concentrations, the molecular packing reduces the unfolding of the stereoregular structures of the FN molecules, preventing stronger spreading of the protein.     

Factors and mechanisms determining the formation of fibrillar collagen structures in adsorbed phases

The adsorption of collagen (type I from calf skin) was studied, comparing different collagen sources and using substrates which differ according to surface hydrophobicity (polystyrene, either native, with OH substitution of each repeat unit, with COOH substitution of a small fraction of repeat units, or surface modified by oxygen plasma discharge). The atomic force microscopy observation of the adsorbed layers showed that aggregation in the solution acts in competition with the formation of fibrils in the adsorbed phase; more aggregated solutions behave like less concentrated solutions regarding adsorption. The fibrils formed in the adsorbed phase are much smaller than the fibrils formed in the suspension, and, in contrast with the latter, do not show regular band pattern. It is confirmed that fibrils formation occurs more readily on more hydrophobic surfaces, which is tentatively attributed to a greater mobility of individual molecules adsorbed on more hydrophobic substrates. This interpretation is supported by previously published radiochemical measurements. However, the comparison of strongly different adsorption procedures (progressive on the one hand; quick and massive on the other hand) did not provide any additional clue.     

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.     

Proton spin-lattice relaxation study of polymer solutions

Proton spin-lattice relaxation times of solvent molecules were measured on ternary mixtures of a polymer and two solvents by the adiabatic rapid-passage method. The selective adsorption of a good solvent was verified by this experimental technique for the systems benzene—cyclohexane—polystyrene(PS), benzene—carbon tetrachloride—poly(methyl methacrylate)(PMMA), and chloroform—carbon tetrachloride—PMMA. The number of molecules of adsorbed benzene per monomeric unit of PS was estimated to be about four, which is almost identical with that determined previously by thermodynamic measurements. The number of molecules of benzene and chloroform adsorbed on PMMA were also determined to be about five and four, respectively. It was found that the interaction between chloroform and PMMA has the greatest effect on the molecular motion of the solvent, whereas the benzene—PS interaction is weak.     

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.     

Adsorption kinetics of ultrathin polymer films in the melt probed by dielectric spectroscopy and second-harmonic generation

We studied the adsorption kinetics of supported ultrathin films of dye-labeled polystyrene (l-PS) by combining dielectric spectroscopy (DS) and the interface-specific nonlinear optical second harmonic generation (SHG) technique. While DS is sensitive to the fraction of mobile dye moieties (chromophores), the SHG signal probes their anisotropic orientation. Time-resolved measurements were performed above the glass transition temperature on two different sample geometries. In one configuration, the l-PS layer is placed in contact with the aluminum surface, while in the other one, the deposition is done on a strongly adsorbed layer of neat PS. From the time dependence of the dielectric strength and SHG signal of the l-PS layer in contact with the metal, we detected two different kinetics regimes. We interpret these regimes in terms of the interplay between adsorption and orientation of the adsorbing labeling moieties. At early times, dye moieties get adsorbed adopting an orientation parallel to the surface. When adsorption proceeds to completeness, the kinetics slows down and the dye moieties progressively orient normal to the surface. Conversely, when the layer of l-PS layer is deposited on the strongly adsorbed layer of neat PS, both the dielectric strength and the SHG signal do not show any variation with time. This means that no adsorption takes place.     

A new insight into the adsorption of bovine serum albumin onto porous polyethylene membrane by zeta potential measurements, FTIR analyses, and AFM observations

The mode of adsorption of bovine serum albumin (BSA) on porous polyethylene (PE) membrane was studied as a function of time and concentration, which may contribute to the surface coverage. An improved physical model for adsorption is initiated based on the results of the adsorptional and desorptional measurements, FTIR analysis, and AFM observations as well as streaming potential measurements. The results obtained indicate that the adsorptional mode depend on both time and concentration. It is shown that a critical concentration (about 1000 ppm here) exists in the adsorptional process. Below this concentration, the adsorption seems to be conducted in a normal side-on way but time elapse gives rise to greater conformational change than concentration increase; above this concentration, the aggregation of protein molecular plays a decisive role and the adsorption is in an aggregation way, which is similar to end-on, but a relative large gap between the adsorbed molecules exists due to aggregation. This conclusion is general and can be expected to apply in other globular protein-hydrophobic porous surface systems.     

Estimation of the adsorption state of nonionic emulsifier molecules onto styrene–methacrylic acid copolymer particles by in situ <Superscript>1</Superscript>H NMR measurements

The adsorption behavior of poly(oxyethylene) nonyl phenyl ether nonionic emulsifier molecules onto polystyrene (PS) and styrene-methacrylic acid copolymer [P(S-MAA)] particles dispersed in D₂O was evaluated by in situ ¹H NMR measurements at room temperature. The resonance due to the protons of the emulsifier molecules was only observed. Normalized NMR integrals of the resonance due to the protons of hydrophobic groups (nonyl and phenyl groups) and the hydrophilic group, poly(oxyethylene) chain, at a certain concentration of the emulsifier decreased with an increase in the total surface area of the PS particles dispersed in the system. The decrease for the hydrophobic groups was much larger than that for the hydrophilic groups. In the dispersion system of P(S-MAA) particles, ionized carboxyl groups at the particle surface decreased the amount of the emulsifier adsorbed.     

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.     

Molecular packing of lysozyme,fibrinogen, and bovine serum albumin on hydrophilic and hydrophobic surfaces studied by infrared-visible sum frequency generation and fluorescence microscopy

Infrared-visible sum frequency generation (SFG) vibrational spectroscopy, in combination with fluorescence microscopy, was employed to investigate the surface structure of lysozyme, fibrinogen, and bovine serum albumin (BSA) adsorbed on hydrophilic silica and hydrophobic polystyrene as a function of protein concentration. Fluorescence microscopy shows that the relative amounts of protein adsorbed on hydrophilic and hydrophobic surfaces increase in proportion with the concentration of protein solutions. For a given bulk protein concentration, a larger amount of protein is adsorbed on hydrophobic polystyrene surfaces compared to hydrophilic silica surfaces. While lysozyme molecules adsorbed on silica surfaces yield relatively similar SFG spectra, regardless of the surface concentration, SFG spectra of fibrinogen and BSA adsorbed on silica surfaces exhibit concentration-dependent signal intensities and peak shapes. Quantitative SFG data analysis reveals that methyl groups in lysozyme adsorbed on hydrophilic surfaces show a concentration-independent orientation. However, methyl groups in BSA and fibrinogen become less tilted with respect to the surface normal with increasing protein concentration at the surface. On hydrophobic polystyrene surfaces, all proteins yield similar SFG spectra, which are different from those on hydrophilic surfaces. Although more protein molecules are present on hydrophobic surfaces, lower SFG signal intensity is observed, indicating that methyl groups in adsorbed proteins are more randomly oriented as compared to those on hydrophilic surfaces. SFG data also shows that the orientation and ordering of phenyl rings in the polystyrene surface is affected by protein adsorption, depending on the amount and type of proteins.