Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity

Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self-assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance. Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy. The extent of lipase adsorption was nearly identical on either surface (approximately 240 ng cm⁻²), but its specific activity was sixfold higher on the methyl-terminated SAM, showing no activity loss upon immobilization. A uniform, 5.5 nm high, highly packed monolayer of CALB formed on the methyl-terminated SAM, while the adsorbed protein was disordered on the hydroxyl-terminated SAM. Hydrophobic surfaces thus may specifically orient the lipase in a highly active state.     

Inhibition of Escherichia coli biofilm formation by self-assembled monolayers of functional alkanethiols on gold

Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation. Escherichia coli was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl, L-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to E. coli biofilm formation; e.g., it repressed biofilm formation by E. coli DH5alpha by 99.5% +/- 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs, E. coli formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility.     

Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.     

Zein adsorption to hydrophilic and hydrophobic surfaces investigated by surface plasmon resonance

Zein, the prolamine of corn, has been investigated for its potential as an industrial biopolymer. In previous research, zein was plasticized with oleic acid and formed into sheets/films. Physical properties of films were affected by film structure and controlled in turn by zein-oleic acid interactions. The nature of such interactions is not well understood. Thus, protein-fatty acid interactions were investigated in this work by the use of surface plasmon resonance (SPR). Zein adsorption from 75% aqueous 2-propanol solutions, 0.05% to 0.5% w/v, onto hydrophilic and hydrophobic self-assembled monolayers (SAMs) formed by 11-mercaptoundecanoic acid and 1-octanethiol, respectively, was monitored by high time resolution SPR. Initial adsorption rate and ultimate surface coverage increased with bulk protein concentration for both surfaces. The initial slope of plotted adsorption isotherms was higher on 11-mercaptoundecanoic acid than on 1-octanethiol, indicating higher zein affinity for hydrophilic SAMs. Also, maximum adsorption values were higher for zein on hydrophilic than on hydrophobic SAMs. Flushing off loosely bound zein in the SPR cell allowed estimation of apparent monolayer values. Differences in monolayer values for hydrophobic and hydrophilic surfaces were explained in terms of zein adsorption footprint.     

Using Microcontact Printing to Pattern the Attachment of Mammalian Cells to Self-Assembled Monolayers of Alkanethiolates on Transparent Films of Gold and Silver

This paper describes a convenient methodology for patterning substrates for cell culture that allows the positions and dimensions of attached cells to be controlled. The method uses self-assembled monolayers (SAMs) of terminally substituted alkanethiolates (R(CH₂)_11–15S−) adsorbed on optically transparent films of gold or silver to control the properties of the substrates. SAMs terminated in methyl groups adsorb protein and SAMs terminated in oligo(ethylene glycol) groups resist entirely the adsorption of protein. This methodology uses microcontact printing (μCP)—an experimentally simple, nonphotolithographic process—to pattern the formation of SAMs at the micrometer scale; μCP uses an elastomeric stamp having at its surface a pattern in relief to transfer an alkanethiol to a surface of gold or silver in the same pattern. Patterned SAMs having hydrophobic, methyl-terminated lines 10, 30, 60, and 90 μm in width and separated by protein-resistant regions 120 μm in width were prepared and coated with fibronectin; the protein adsorbed only to the methyl-terminated regions. Bovine capillary endothelial cells attached only to the fibronectin-coated, methyl-terminated regions of the patterned SAMs. The cells remained attached to the SAMs and confined to the pattern of underlying SAMs for at least 5–7 days. Because the substrates are optically transparent, cells could be visualized by inverted microscopy and by fluorescence microscopy after fixing and staining with fluorescein-labeled phalloidin.     

Structural organization of DMPC lipid layers on chemically micropatterned self-assembled monolayers as biomimetic systems

The growth structure of DMPC lipid layers on hydrophobic and hydrophilic alkylsilane-based self-assembled monolayers adsorbed on silicon has been investigated by means of X-ray reflectometry and atomic force microscopy. Hydrophilic modification of hydrophobically terminated ODS-SAMs has been achieved by dose-controlled irradiation with DUV light. While island formation of small DMPC bilayer islands is observed on hydrophobic SAM surfaces, closed layers of DMPC monolayers are formed on hydrophilic SAM surfaces. Furthermore, DMPC adsorption on chemically micropatterned substrates with alternating hydrophobic/hydrophilic surface properties has been studied by imaging ellipsometry and photoemission microscopy. Indication for at least partial bridging of hydrophobic areas by an adsorbed DMPC monolayer has been found.     

Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.

X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A.     

Self-assembled monolayers of rigid thiols

The preparation, structure, properties and applications of self-assembled monolayers (SAMs) of rigid 4-mercapto-biphenyls are briefly reviewed. The rigid character of the biphenyl moiety results in a molecular dipole moment that affects both the adsorption kinetics on gold surfaces, as well as the equilibrium structure of mixed SAMs. Due to repulsive intermolecular interaction, the Langmuir isotherm model does not fit the adsorption kinetics of these biphenyl thiols, and a new Ising model was developed to fit the kinetics data. The equilibrium structures of SAMs and mixed SAMs depend on the polarity of the solution from which they were assembled. Infrared spectroscopy suggests that biphenyl moieties in SAMs on gold have small tilt angles with respect to the surfaces normal. Wetting studies shows that surfaces of these SAMs are stable for months, thus providing stable model surfaces that can be engineered at the molecular level. Such molecular engineering is important for nucleation and growth studies. The morphology of glycine crystals grown on SAM surfaces depends on the structure of the nucleating glycine layer, which, in turn, depends on the H-bonding of these molecules with the SAM surface. Finally, the adhesion of PDMS cross-linked networks to SAM surfaces depends on the concentration of interfacial H-bonding. This non-linear relationship suggests that the polymeric nature of the elastomer results in a collective H-bonding effect.     

Cellular-automata models of solid-liquid interfaces

A series of cellular-automata (CA) models have been created, simulating relationships between water (or aqueous solutions) and solid surfaces of differing hydropathic (i.e., hydrophilic or hydrophobic) nature. Both equilibrium- and dynamic-flow models were examined, employing simple breaking and joining rules to simulate the hydropathic interactions. The CA simulations show that water accumulates near hydrophilic surfaces and avoids hydrophobic surfaces, forming concave-up and concave-down meniscuses, resp., under equilibrium conditions. In the dynamic-flow simulations, the flow rate of water was found to increase past a wall surface as the surface became less hydrophilic, reaching a maximum rate when the solid surface was of intermediate hydropathic state, and then declining with further increase in the hydrophobicity of the surface. Solution simulations show that non-polar solutes tend to achieve higher concentrations near hydrophobic-wall surfaces, whereas other hydrophobic/hydrophilic combinations of solutes and surfaces do not show such accumulations. Physical interpretations of the results are presented, as are some possible biological consequences.     

Characterization and redox properties of cytochrome c552 from Thermus thermophilus adsorbed on different self-assembled thiol monolayers, used to model the chemical environment of the redox partner

The structure of cytochrome c552 (Cyt-c552) from Thermus thermophilus shows many differences to other c-type cytochromes. The rich lysine domain close to the heme does not exist in this cytochrome, allowing us to postulate that the interaction with its redox partner must be different to the cytochrome c/cytochrome c oxidase interaction. We report a study of Cyt-c552 adsorbed on self-assembled monolayers (SAMs) of functionalized alkanethiols used to mimic the chemical properties of its redox partner (ba3-oxydase). Hydrophilic (-COOH), polar (-OH), hydrophobic (-CH3), and mixed (-OH/-CH3) SAMs grafted on roughened silver electrodes were characterized by X-ray photoelectron spectroscopy. Surface enhanced resonance Raman spectroscopy (SERRS) was employed to determine the structure and the redox properties (E degrees and number of transferred electron) of the heme of Cyt-c552 adsorbed on roughened silver electrodes coated by the different SAMs. The surface that most closely models the environment of the ba3-oxidase is a mixed SAM formed by 50% polar [Ag-(CH2)5-CH2OH] and 50% hydrophobic [Ag-(CH2)5-CH3] alkanethiols. Only the native form B1(6cLS) of Cyt-c552 is detected by SERRS when the protein is adsorbed on such a surface that promotes a protein orientation favorable for the electron transfer (number of transferred electron = 1). We shall discuss the differences and similarities of the electron-transfer mechanism of Cyt-c552 compared to cyt-c.     

Strong repulsive forces between protein and oligo (ethylene glycol) self-assembled monolayers: a molecular simulation study

Restrained molecular dynamics simulations were performed to study the interaction forces of a protein with the self-assembled monolayers (SAMs) of S(CH2)4(EG)4OH, S(CH2)11OH, and S(CH2)11CH3 in the presence of water molecules. The force-distance curves were calculated by fixing the center of mass of the protein at several separation distances from the SAM surface. Simulation results show that the relative strength of repulsive force acting on the protein is in the decreasing order of OEG-SAMs > OH-SAMs > CH3-SAMs. The force contributions from SAMs and water molecules, the structural and dynamic behavior of hydration water, and the flexibility and conformation state of SAMs were also examined to study how water structure at the interface and SAM flexibility affect the forces exerted on the protein. Results show that a tightly bound water layer adjacent to the OEG-SAMs is mainly responsible for the large repulsive hydration force.     

Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.     

Binding preferences, surface attachment, diffusivity, and orientation of a family 1 carbohydrate-binding module on cellulose

Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.     

Adsorption of zein on surfaces with controlled wettability and thermal stability of adsorbed zein films

Adsorption characteristics of zein protein on hydrophobic and hydrophilic surfaces have been investigated to understand the orientation changes associated with the protein structure on a surface. The protein is adsorbed by a self-assembly procedure on a monolayer-modified gold surface. It is observed that zein shows higher affinity toward hydrophilic than hydrophobic surfaces on the basis of the initial adsorption rate followed by quartz crystal microbalance studies. Reflection absorption infrared (RAIR) spectroscopic studies reveal the orientation changes associated with the adsorbed zein films. Upon adsorption, the protein is found to be denatured and the transformation of alpha-helix to beta-sheet form is inferred. This transformation is pronounced when the protein is adsorbed on hydrophobic surfaces as compared to hydrophilic surfaces. Electrochemical techniques (cyclic voltammetry and impedance techniques) are very useful in assessing the permeability of zein film. It is observed that the zein moieties adsorbed on hydrophilic surfaces are highly impermeable in nature and act as a barrier for small molecules. The topographical features of the deposits before and after adsorption are analyzed by atomic force microscopy. The protein adsorbed on hydrophilic surface shows rod- and disclike features that are likely to be the base units for the growth of cylindrical structures of zein. The thermal stability of the adsorbed zein film has been followed by variable-temperature RAIR measurements.     

Use of Self-Assembled Monolayers of Different Wettabilities To Study Surface Selection and Primary Adhesion Processes of Green Algal (Enteromorpha) Zoospores

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH₃) or hydroxyl (OH) groups or mixtures of CH₃- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and “searching” above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to “sense” that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.     

Zwitterionic polymers exhibiting high resistance to nonspecific protein adsorption from human serum and plasma

This study examined six different polymer and self-assembled monolayer (SAM) surface modifications for their interactions with human serum and plasma. It was demonstrated that zwitterionic polymer surfaces are viable alternatives to more traditional surfaces based on poly(ethylene glycol) (PEG) as nonfouling surfaces. All polymer surfaces were formed using atom transfer radical polymerization (ATRP) and they showed an increased resistance to nonspecific protein adsorption compared to SAMs. This improvement is due to an increase in the surface packing density of nonfouling groups on the surface, as well as a steric repulsion from the flexible polymer brush surfaces. The zwitterionic polymer surface based on carboxybetaine methacrylate (CBMA) also incorporates functional groups for protein immobilization in the nonfouling background, making it a strong candidate for many applications such as in diagnostics and drug delivery.     

Analysis of accessible surface of residues in proteins

We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent-accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein-accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and beta-structures (outside beta-sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from beta-sheets are the most inaccessible to solvent (10% accessible). Hence, beta-sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.     

Thermoresponsive protein adsorption of poly(N-isopropylacrylamide)-modified streptavidin on polydimethylsiloxane microchannel surfaces

The control of protein adsorption on microchannel surfaces is important for biosensors. In this study, we demonstrated protein adsorption method that is controlled through temperature change, i.e., thermoresponsive protein adsorption, on polydimethylsiloxane (PDMS) microchannel surfaces using a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). To provide general protein adsorption control method, we adopted biotin-streptavidin chemistry and synthesized streptavidin covalently modified with PNIPAAm (PNIPAAm-StAv). Modification of streptavidin, a hydrophilic protein, with PNIPAAm induced successful thermoresponsive adsorption on a PDMS microchannel surfaces: PNIPAAm-StAv adsorbed at 37 degrees C and desorbed at 10 degrees C on the surfaces. We also demonstrated the thermoresponsive adsorption of biotinylated immunoglobulin G (IgG-b) using PNIPAAm-StAv. Conjugation of IgG-b with PNIPAAm-StAv induced successful thermoresponsive IgG-b adsorption on PDMS. Modification of PDMS surfaces with PNIPAAm reduced physical adsorption of the partially hydrophobic IgG-b on the surface and contributed to the high-contrast thermoresponsive adsorption of IgG-b: less than 1% of the IgG-b adsorbed at 37 degrees C was detected after the PNIPAAm-PDMS surface was washed at 10 degrees C. The controllable adsorption of this system is expected to be applied to the regeneration of biosensor chips and to on-chip protein manipulation.     

材料表面化学刺激对纤连蛋白构象及细胞的影响

生物材料的表面化学性质通过改变吸附蛋白的构象,影响细胞粘附铺展,产生不同的细胞行为.采用金-硫自组装单分子层技术(alkanethiol self-assembled monolayers,SAMs)构建了不同化学基团(-NH2、-COOH、-OH和-CH3)修饰的基底材料表面.运用X射线光能质谱(XPS)和接触角仪表...     

Molecular orientation of tropoelastin is determined by surface hydrophobicity

Tropoelastin is the precursor of the extracellular protein elastin and is utilized in tissue engineering and implant technology by adapting the interface presented by surface-bound tropoelastin. The preferred orientation of the surface bound protein is relevant to biointerface interactions, as the C-terminus of tropoelastin is known to be a binding target for cells. Using recombinant human tropoelastin we monitored the binding of tropoelastin on hydrophilic silica and on silica made hydrophobic by depositing a self-assembled monolayer of octadecyl trichlorosilane. The layered organization of deposited tropoelastin was probed using neutron and X-ray reflectometry under aqueous and dried conditions. In a wet environment, tropoelastin retained a solution-like structure when adsorbed on silica but adopted a brush-like structure when on hydrophobized silica. The orientation of the surface-bound tropoelastin was investigated using cell binding assays and it was found that the C-terminus of tropoelastin faced the bulk solvent when bound to the hydrophobic surface, but a mixture of orientations was adopted when tropoelastin was bound to the hydrophilic surface. Drying the tropoelastin-coated surfaces irreversibly altered these protein structures for both hydrophilic and hydrophobic surfaces.