Study of cell behaviour on a cellulose anti-adhesive substratum

Carboxymethylcellulose (CMC) is used as an anti-adhesive in several biomaterial applications. We have shown that an original bi-layered hydroxypropylmethylcellulose (HPMC)-CMC-coated substratum (CEL) is suitable for studying the molecular mechanisms underlying cell–substratum and cell–cell interactions. The present work was carried out to assess the anti-adhesive properties of a CEL substratum by analysing the behaviour of three types of adherent cells (Swiss 3T3 fibroblasts, MC-3T3-E1 pre-osteoblasts and B16F10 highly metastatic melanoma cells). Tissue culture polystyrene (tPS) was used as an adhesive control and poly(2-hydroxyethyl methacrylate) PolyHEMA as an anti-adhesive control. Cell morphology, growth, cell cycle analysis and apoptosis were compared. The proliferation of cells on CEL was significantly decreased, cells were arrested in the G1 phase and underwent apoptosis (except for melanoma cells) 48 h post-seeding. We conclude that CEL is an effective anti-adhesive cellulose biomaterial that gives reproducible and demonstratable results, especially for apoptosis. Its advantage as a coating for culture devices is its long shelf-live. It may therefore also be a very good candidate for other biomedical applications requiring adhesion-resistant substrata.     

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 amyloid beta-peptide at polymer surfaces: a neutron reflectivity study.

The adsorption of amyloid beta-peptide at hydrophilic and hydrophobic modified silicon-liquid interfaces was characterized by neutron reflectometry. Distinct polymeric films were used to obtain noncharged (Formvar), negatively (sodium poly(styrene sulfonate)) and positively charged (poly(allylamine hydrochloride)) hydrophilic as well as hydrophobic surfaces (polystyrene and a polysiloxane-dodecanoic acid complex). Amyloid beta-peptide was found to adsorb at positively charged hydrophilic and hydrophobic surfaces, whereas no adsorbed layer was detected on hydrophilic noncharged and negatively charged films. The peptide adsorbed at the positively charged film as patches, which were dispersed on the surface, whereas a uniform layer was observed at hydrophobic surfaces. The thickness of the adsorbed peptide layer was estimated to be approximately 20 A. The peptide formed a tightly packed layer, which did not contain water. These studies provide information about the affinity of the amyloid beta-peptide to different substrates in aqueous solution and suggest that the amyloid fibril formation may be driven by interactions with surfaces.     

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.     

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.     

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.     

Probing fibronectin-surface interactions: a multitechnique approach

The development of adhesive as well as antiadhesive surfaces is essential in various biomaterial applications. In this study, we have used a multidisciplinary approach that combines biological and physicochemical methods to progress in our understanding of cell-surface interactions. Four model surfaces have been used to investigate fibronectin (Fn) adsorption and the subsequent morphology and adhesion of preosteoblasts. Such experimental conditions lead us to distinguish between anti- and proadhesive substrata. Our results indicate that Fn is not able to induce cell adhesion on antiadhesive materials. On adhesive substrata, Fn did not increase the number of adherent cells but favored their spreading. This work also examined Fn-surface interactions using ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified tips. The results provided clear evidence of the advantages and limitations of each technique. All of the techniques confirmed the important adsorption of Fn on proadhesive surfaces for cells. By contrast, antiadhesive substrata for cells avoided Fn adsorption. Furthermore, ELISA experiments enabled us to verify the accessibility of cell binding sites to adsorbed Fn molecules.     

Ordering surfaces on the nanoscale: implications for protein adsorption

Monolayer-protected metal nanoparticles (MPMNs) are a newly discovered class of nanoparticles with an ordered, striped domain structure that can be readily manipulated by altering the ratio of the hydrophobic to hydrophilic ligands. This property makes them uniquely suited to systematic studies of the role of nanostructuring on biomolecule adsorption, a phenomenon of paramount importance in biomaterials design. In this work, we examine the interaction of the simple, globular protein cytochrome C (Cyt C) with MPMN surfaces using experimental protein assays and computational molecular dynamics simulations. Experimental assays revealed that adsorption of Cyt C generally increased with increasing surface polar ligand content, indicative of the dominance of hydrophilic interactions in Cyt C-MPMN binding. Protein-surface adsorption enthalpies calculated from computational simulations employing rigid-backbone coarse-grained Cyt C and MPMN models indicate a monotonic increase in adsorption enthalpy with respect to MPMN surface polarity. These results are in qualitative agreement with experimental results and suggest that Cyt C does not undergo significant structural disruption upon adsorption to MPMN surfaces. Coarse-grained and atomistic simulations furthermore elucidated the important role of lysine in facilitating Cyt C adsorption to MPMN surfaces. The amphipathic character of the lysine side chain enables it to form close contacts with both polar and nonpolar surface ligands simultaneously, rendering it especially important for interactions with surfaces composed of adjacent nanoscale chemical domains. The importance of these structural characteristics of lysine suggests that proteins may be engineered to specifically interact with nanomaterials by targeted incorporation of unnatural amino acids possessing dual affinity to differing chemical motifs.     

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography

The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol‐bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous‐acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show “U‐profile” characteristic of a dual hydrophilic interaction–reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton–donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.     

The adsorption of human serum albumin (HSA) on CO2 laser modified magnesia partially stabilised zirconia (MgO-PSZ)

Magnesia partially stabilised zirconia (MgO-PSZ), a bioinert ceramic, exhibits high mechanical strength, excellent corrosion resistance and good biocompatibility, but it does not naturally form a direct bond with bone resulting in a lack of osteointegration. The surface properties and structure of a biomaterial play an essential role in protein adsorption. As such, changes in the surface properties and structure of biomaterials may in turn alter their bioactivity. So, the fundamental reactions at the interface of biomaterials and tissue should influence their integration and bone-bonding properties. To this end, CO2 laser radiation was used to modify the surface roughness, crystal size, phase and surface energy of the MgO-PSZ. The basic mechanisms active in improving the surface energy were analysed and found to be the phase change and augmented surface area. The adsorption of human serum albumin (HSA), which is a non-cell adhesive protein, was compared on the untreated and CO2 laser modified MgO-PSZ. It was observed that the thickness of the adsorbed HSA decreased as the polar surface energy of the MgO-PSZ increased, indicating that HSA adsorbed more effectively on the hydrophobic MgO-PSZ surface than the hydrophilic surface. The current study provided important information regarding protein-biomaterial interactions and possible mechanisms behind the cell interaction and in vivo behaviour.     

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.     

Fibronectin conformation switch induced by coadsorption with human serum albumin

The dynamic adsorption of human serum albumin (HSA) and plasma fibronectin (Fn) onto hydrophobic poly(hydroxymethylsiloxane) (PHMS) and the structures of adsorbed protein layers from single and binary protein solutions were studied. Spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation monitoring (QCM-D) together with atomic force microscopy (AFM) were used to measure the effective mass, thickness, viscoelastic properties, and morphology of the adsorbed protein films. Adsorbed HSA formed a rigid, tightly bound monolayer of deformed protein, and Fn adsorption yielded a thick, very viscoelastic layer that was firmly bound to the substrate. The mixed protein layers obtained from the coadsorption of binary equimolecular HSA-Fn solutions were found to be almost exclusively dominated by Fn molecules. Further sequential adsorption experiments showed little evidence of HSA adsorbed onto the predeposited Fn layer (denoted as Fn ? HSA), and Fn was not adsorbed onto predeposited HSA (HSA ? Fn). The conformational arrangement of the adsorbed Fn was analyzed in terms of the relative availability of two Fn domains. In particular, (4)F(1)·(5)F(1) binding domains in the Hep I fragment, close to the amino terminal of Fn, were targeted using a polyclonal antifibronectin antibody (anti-Fn), and the RGD sequence in the 10th segment, in the central region of the molecule, was tested by cell culture experiments. The results suggested that coadsorption with HSA induced the Fn switch from an open conformation, with the amino terminal subunit oriented toward the solution, to a close conformation, with the Fn central region oriented toward the solution.     

Influence of nanobubbles on the adsorption of nanoparticles

A quartz crystal microbalance was used to study the influence of nanobubbles on the adsorption of polystyrene nanoparticles onto surfaces coated with gold, or coated with dodecanethiol or mercaptoundecanoic acid self-assembled monolayers (SAMs). Adsorption of the nanoparticles onto the surface causes the resonant frequency of the quartz crystal to decrease. We found that particles were adsorbed onto the gold-coated quartz crystal in air-rich water, but not in degassed water. This finding supports the long-standing hypothesis that nanobubbles play a key role in the long-range attractive force between hydrophobic surfaces in aqueous solutions. When the experiments were conducted using quartz crystals coated with a hydrophobic dodecanethiol SAM, the nanoparticles were adsorbed onto the surface even in degassed water due to the short-range hydrophobic interactions between the nanoparticles and the dodecanethiol molecules. In contrast, the nanoparticles were adsorbed to a lesser degree onto the hydrophilic mercaptoundecanoic acid-coated crystals due to electrostatic repulsive forces.     

Preparation of micron-sized composite polymer particles containing hydrophilic 2-hydroxyethyl methacrylate and their biomedical applications

Micron-sized polystyrene or PS particles were first prepared by dispersion polymerization. Then a series of polystyrene/poly(styrene-2-hydroxyethyl methacrylate) or PS/P(S-HEMA) composite polymer particles was prepared by seeded copolymerization using different amounts of 2-hydroxyethyl methacrylate (HEMA) at the constant core/shell ratio of 1/0.5. The produced PS seed and composite polymer particles were characterized by transmission electron microscopy. Adsorption behaviors of some biologically active macromolecules were studied under similar conditions. In each case the magnitude of adsorption on composite polymer particles decreased with the increase in HEMA content in the recipe, which means that the hydrophobic interaction between the surface of the particles and biomolecules decreased. The specific activities of trypsin aqueous solution and adsorbed trypsin on PS seed and composite polymer particles prepared with different HEMA contents were also measured and compared. The activity of adsorbed trypsin on composite polymer particles improved significantly with the incorporation of hydrophilic HEMA.     

The influence of electrostatic forces on protein adsorption

In this paper we investigate the importance of electrostatic double layer forces on the adsorption of human serum albumin by UV-ozone modified polystyrene. Electrostatic forces were measured between oxidized polystyrene surfaces and gold-coated atomic force microscope (AFM) probes in phosphate buffered saline (PBS) solutions. The variation in surface potential with surface oxygen concentration was measured. The observed force characteristics were found to agree with the theory of electrical double layer interaction under the assumption of constant potential. Chemically patterned polystyrene surfaces with adjacent 5 microm x 5 microm polar and non-polar domains have been studied by AFM before and after human serum albumin adsorption. A topographically flat surface is observed before protein adsorption indicating that the patterning process does not physically modify the surface. Friction force imaging clearly reveals the oxidation pattern with the polar domains being characterised by a higher relative friction compared to the non-polar, untreated domains. Far-field force imaging was performed on the patterned surface using the interleave AFM mode to produce two-dimensional plots of the distribution of electrostatic double-layer forces formed when the patterned polystyrene surfaces is immersed in PBS. Imaging of protein layers adsorbed onto the chemically patterned surfaces indicates that the electrostatic double-layer force was a significant driving force in the interaction of protein with the surface.     

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.     

Adsorption of different amphiphilic molecules onto polystyrene latices

In order to know the influence of the surface characteristics and the chain properties on the adsorption of amphiphilic molecules onto polystyrene latex, a set of experiments to study the adsorption of ionic surfactants, nonionic surfactants and an amphiphilic synthetic peptide on different latex dispersions was performed. The adsorbed amount versus the equilibrium surfactant concentration was determined. The main adsorption mechanism was the hydrophobic attraction between the nonpolar tail of the molecule and the hydrophobic regions of the latex surface. This attraction overcame the electrostatic repulsion between chains and latex surface with identical charge sign. However, the electrostatic interactions chain-surface and chain-chain also played a role. General patterns for the adsorption of ionic chains on charged latex surfaces could be established. Regarding the shape, the isotherms presented different plateaus corresponding to electrostatic effects and conformational changes. The surfactant size also affects the adsorption results: the higher the hydrophilic moiety in the surfactant molecule the lower the adsorbed amount.     

Effects of Serum on the Kinetics of CHO Attachment to Ultraviolet-Ozone Modified Polystyrene Surfaces

Attachment kinetics of Chinese hamster ovarian (CHO) cells were investigated on ultraviolet-ozone oxidized polystyrene (UVO-PS) dishes in the presence and absence of serum. The surface chemistry of UVO-PS has been extensively characterized. Although cells attached rapidly to the oxidized dishes with serum present it was found that serum actually inhibits the rate of attachment. Spreading of attached cells was favored by the presence of serum. It is suggested that the increased quantity of hydrophilic carboxyl groups on longer exposed UVO-PS leads to a change in the protein layer adsorbed from serum and also a higher affinity of the surface for extracellular proteins secreted by the attached cells. The UVO-PS surfaces present a new way of producing tissue culture grade polystyrene (TCPS) in a highly controllable method, which would ensure greater consistency in TCPS surfaces. Copyright 2001 Academic Press.     

Determining the conformation of an adsorbed Br-PEG-peptide by long period X-ray standing wave fluorescence

Long-period X-ray standing wave fluorescence (XSW) and X-ray reflectivity techniques are employed to probe the conformation of a Br-poly(ethylene glycol) (PEG)-peptide adsorbate at the hydrated interface of a polystyrene substrate. The Br atom on this Br-PEG-peptide construct serves as a marker atom allowing determination by XSW of its position and distribution with respect to the adsorption surface with angstrom resolution. Adsorption occurs on native or ion-beam-modified polystyrene films that are spin-coated onto a Si substrate and display either nonpolar or polar surfaces, respectively. A compact, oriented monolayer of Br-PEG-peptide can be formed with the peptide end adsorbed onto the polar surface and the PEG end terminating with the Br tag extending into the aqueous phase. The 108-141 A distance of the Br atom from the polystyrene surface in this oriented monolayer is similar to the estimated approximately 150 A length of the extended Br-PEG-peptide. This Br-polystyrene distance depends on adsorption time and surface properties prior to adsorption. Incomplete multilayers form on the polar surface after sufficient adsorption time elapses. By contrast, adsorption onto the nonpolar surface is submonolayer, patchy, and highly disordered with an isotropic Br distribution. Overall, this combination of X-ray surface scattering techniques with a novel sample preparation strategy has several advantages as a real space probe of adsorbed or covalently bound biomolecules at the liquid-solid interface.     

Adsorption of an endoglucanase from the hyperthermophilic Pyrococcus furiosus on hydrophobic (polystyrene) and hydrophilic (silica) surfaces increases protein heat stability

The interaction of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus with two types of surfaces, that is, hydrophobic polystyrene and hydrophilic silica, was investigated, and the adsorption isotherms were determined. The adsorbed hyperthermostable enzyme did not undergo loss of biological activity. A model was proposed for the mechanism of interaction of the enzyme with the surface based on the shape of the adsorption isotherm, the morphological characteristics of the enzyme, and the thermodynamic parameters of the system. The enzyme was irreversibly immobilized at the solid/liquid interface even at high temperatures, and most interestingly, it acquired further heat stabilization upon adsorption. The denaturation temperature increased from 108 degrees C in solution to 116 degrees C upon adsorption on hydrophilic silica particles. Adsorption on the hydrophobic polystyrene surface even shifted the denaturation temperature to 135 degrees C, the most extreme experimentally determined protein denaturation temperature ever reported. Maintenance of the biological function particularly at high temperatures is important for the development of solid substrate immobilized enzymes for applications in biocatalysis and biotechnology. This also presents an additional stabilization mechanism employed by nature where the extracellular hyperthermostable enzyme remains folded and active at the extreme temperatures of its natural environment by adsorption on the surface of rocks and other materials appearing in the surroundings of the microorganism.