Whole cells are attractive biocatalysts, particularly if the reaction requires cofactors or involves multiple transformations. Immobilization of the catalyst is often a prerequisite for continuous processes. The highly cationic chemically modified plasma protein bovine serum albumin (cBSA-147) has been applied for the electrostatically mediated immobilization of the planktonic bacterium E. coli BL21 star (DE3), and the resulting biofilms were superior to those formed on poly-L-lysine coated surfaces. The biocatalyst was immobilized in a capillary column (inside diameter of 530 μm and L=30 m) and evaluated in the enantioselective reduction of ethyl acetoacetate to R-(-)ethyl hydroxybutyrate. In continuous operation in the microreactor format, the productivity of the cells was about 30% higher than that determined in a bench-scale fermentation system. This increase is attributed to the improved mass transfer over short geometrical dimensions. The similarity in the results indicates that studies on a biofilm-coated microreactor can be used for the accelerated collection of data for process optimization.
Phosphoinositides are involved in a large number of processes in cells and it is very demanding to study individual protein-lipid interactions in vivo due to their rapid turnover and involvement in simultaneous events. Supported lipid bilayers (SLBs) containing controlled amounts of phosphoinositides provide a defined model system where important specific recognition events involving phosphoinositides can be systematically investigated using surface sensitive analytical techniques. The authors have demonstrated the formation and characterized the assembly kinetics of SLBs incorporating phosphatidylinositol 4,5-biphosphate (PIP(2); 1, 5, and 10 wt %) and phosphoinositol-3,4,5-triphosphate (1 wt %) using the quartz crystal microbalance with dissipation monitoring and fluorescence recovery after photobleaching. An increased fraction of phosphoinositides led to a higher barrier to liposome fusion, but full fluidity for the phosphatidylcholine lipids in the formed SLB. Significantly, the majority of phosphoinositides were shown to be immobile. X-ray photoelectron spectroscopy was used for the first time to verify that the PIP(2) fraction of lipids in the SLB scales linearly with the amount mixed in from stock solutions.
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a rapidly developing technique for the characterization of a wide range of materials. Recently, advances in instrumentation and sample preparation approaches have provided the ability to perform 3D molecular imaging experiments. Polyatomic ion beams, such as C60, and gas cluster ion beams, often Arn (n = 500–4000), substantially reduce the subsurface damage accumulation associated with continued bombardment of organic samples with atomic beams. In this review, the capabilities of the technique are discussed and examples of the 3D
imaging approach for the analysis of model membrane systems, plant single cell, and tissue samples are presented. Ongoing challenges for 3D ToF-SIMS imaging are also discussed along with recent developments that might offer improved 3D
imaging prospects in the near future.
Despite its medical applications, the mechanisms responsible for the osseointegration of bioactive glass (45S5) have yet to be fully understood. Evidence suggests that the strongest predictor for osseointegration of bioactive glasses, and ceramics, with bone tissue as the formation of an apatitic calcium phosphate layer atop the implanted material, with osteoblasts being the main mediator for new bone formation. Most have tried to understand the formation of this apatitic calcium phosphate layer, and other bioresponses between the host and bioactive glass 45S5 using Simulated Body Fluid; a solution containing ion concentrations similar to that found in human plasma without the presence of proteins. However, it is likely that cell attachment is probably largely mediated via the adsorbed protein layer. Plasma protein adsorption at the tissue bioactive glass interface has been largely overlooked. Herein, we compare crystalline and amorphous bioactive glass 45S5, in both melt-derived as well as sol-gel forms. Thus, allowing for a detailed understanding of both the role of crystallinity and powder morphology on surface ions, and plasma protein adsorption. It was found that sol-gel 45S5 powders, regardless of crystallinity, adsorbed 3-5 times as much protein as the crystalline melt-derived counterpart, as well as a greater variety of plasma proteins. The devitrification of melt-cast 45S5 resulted in only small differences in the amount and variety of the adsorbed proteome. Surface properties, and not material crystallinity, play a role in directing protein adsorption phenomena for bioactive glasses given the differences found between crystalline melt-cast 45S5 and sol-gel derived 45S5.
In order to develop biomedical materials with specific functionalities, thermoresponsive conjugates [poly(N-isopropylacrylamide)-C60 (PIPAAm-C60) ]of fullerene (C60) and PIPAAm with two different polymer chain lengths (4 and 20 kDa) were synthesized by atom transfer radical polymerization. The effects of the polymer chain length on the temperature-responsive phase transition behavior of the synthetic PIPAAm-C60 conjugates were probed by means of various physicochemical techniques. The coexistence of unimers and molecular assemblies of PIPAAm-C60 was observed by gel permeation chromatography and dynamic light scattering studies in two PIPAAm-C60 aqueous solutions below their lower critical solution temperatures (LCSTs). Additionally, below their LCSTs, differences in PIPAAm chain length gave rise to changes in the composition of the unimers and molecular assemblies. In response to temperature, the absorbance of the PIPAAm-C60 aqueous solution changed according to a two-step behavior profile. Increasing temperature during the primary stage, where a change in the absorbance of the PIPAAm-C60 aqueous solution took place, did not change the transition temperature, regardless of the solution concentration of PIPAAm-C60. This absorbance change was associated with the phase transition of the molecular assemblies of PIPAAm-C60. However, at the second stage, the transition temperature shifted to a higher value with the decrease in the concentration of PIPAAm-C60, in the same manner as free PIPAAm chains. The second change was associated with the phase transition of the unimeric PIPAAm-C60. Differences in PIPAAm chain length gave rise to the change in the phase transition behavior of PIPAAm-C60 aqueous solution. Therefore, the chain length of PIPAAm was found to be a predominant factor involved in the solution characteristics of PIPAAm-C60. Consequently, the PIPAAm-C60 is expected to be an intelligent biomaterial possessing heat-induced accumulation and bioactivities.
Textured surfaces obtained by UV laser ablation of poly(ethylene terephthalate) films were used to study the effect of shape and spacing of surface features on cellular response. Two distinct patterns, cones and ripples with spacing from 2 to 25 μm, were produced. Surface features with different shapes and spacings were produced by varying pulse repetition rate, laser fluence, and exposure time. The effects of the surface texture parameters, i.e., shape and spacing, on cell attachment, proliferation, and morphology of neonatal human dermal fibroblasts and mouse fibroblasts were studied. Cell attachment was the highest in the regions with cones at ∼4 μm spacing. As feature spacing increased, cell spreading decreased, and the fibroblasts became more circular, indicating a stress-mediated cell shrinkage. This study shows that UV laser ablation is a useful alternative to lithographic techniques to produce surface patterns for controlling cell attachment and growth on biomaterial surfaces.
In this study, we describe the attachment of biotin-functionalized beta-lactamase to different types of interfacial architectures. Generic biotin-NeutrAvidin binding matrices were assembled using biotin-terminated alkanethiol and poly (L-lysine)-g-poly (ethylene glycol) polymer. Quantitative comparisons were made between different matrices and binding strategies. In addition, the feasibility of regeneration was tested. Our results show that in general all matrices were well suited for the binding of the protein, although quantitative differences were observed and will be discussed. Furthermore, the results obtained by surface plasmon resonance spectrometer and optical waveguide measurements show excellent correlation. For all five matrices investigated, real time enzymatic activity assays of beta-lactamase were performed by a detection scheme that combines an affinity and a catalytic sensor. The results show that the surface-immobilized enzymes are stable and sufficiently active for highly sensitive catalytic activity measurements. The effect of surface immobilization on the catalytic activity of the enzyme is discussed.
With the ever-changing landscape of translational research, the medical device and pharmaceutical industries increasingly license technologies with the added value of clinical and/or pre-clinical data rather than those in earlier stages of development. Universities have the potential to fill the gap in product development from academic laboratories through enhanced student training and increased implementation of some development and manufacturing activities that are traditionally found only in the private sector. A development roadmap is described from initial product feasibility through commercialization in the context of efficient development practices. The specific challenges in the design and development of biomaterial-based medical devices are described in the context of this development path with an emphasis on unique challenges for academic laboratories.
Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex™, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex™ (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.
Interfacial force field (IFF) parameters for use with the CHARMM force field have been developed for interactions between peptides and high-density polyethylene (HDPE). Parameterization of the IFF was performed to achieve agreement between experimental and calculated adsorption
free energies of small TGTG–X–GTGT host–guest peptides (T = threonine, G = glycine, and X = variable amino-acid residue) on HDPE, with ±0.5 kcal/mol agreement. This IFF parameter set consists of tuned nonbonded parameters (i.e., partial charges and Lennard–Jones parameters) for use with an in-house-modified CHARMM molecular dynamic program that enables the use of an independent set of force field parameters to control molecular behavior at a solid–liquid interface. The R correlation coefficient between the simulated and experimental peptide
free energies increased from 0.00 for the standard CHARMM force field parameters to 0.88 for the tuned IFF parameters. Subsequent studies are planned to apply the tuned IFF parameter set for the simulation of protein adsorption behavior on an HDPE surface for comparison with experimental values of adsorbed protein orientation and conformation.
The simulation of the interactions of proteins with charged surfaces in a condensed-phase aqueous solution containing electrolytes using empirical force field based methods is predominantly governed by nonbonded interactions between the atoms of the protein, surface, and the solvent. Electrostatic effects represent the strongest type of these interactions and the type that is most difficult to accurately represent because of their long-range influence. While many different methods have been developed to represent electrostatic interactions, the particle mesh Ewald summation (PME) method is generally considered to be the most accurate one for calculating these effects. However, the PME method was designed for systems with three-dimensional (3D) periodicity, and not for interfacial systems such as the case of protein adsorption to a charged surface. Interfacial systems such as these have only two-dimensional periodicity, which may not be appropriate for treatment with PME due to the possibility that the presence of multiple charged image surfaces parallel to the primary simulation cell's surface, may introduce nonphysical effects on the behavior of the charged molecules in the system. In an effort to address this issue, the authors have conducted a set of nanosecond-scale molecular dynamics simulations to calculate the equilibrium distribution of Na(+) and Cl(-) ions near a charged surface using PME and a range of radial cutoff methods for treating electrostatic interactions, where the cutoffs prevent interaction with the periodic images of the system. The resulting ion concentration profiles were compared to one another and to a continuum analytical solution of the theoretical ion distribution obtained from the Poisson-Boltzmann equation. Their results show that the PME method does not introduce the suspected nonphysical effects in the ion distributions due to the 3D periodic images of the system, thus indicating that it is appropriate for use for this type of molecular simulation. Although their interest is motivated by protein-surface interactions, the conclusions are applicable for the treatment of electrostatics in other aqueous systems with two-dimensional periodicity.
When performing molecular dynamics simulations for a system with constrained (fixed) atoms, traditional isobaric algorithms (e.g., NPT simulation) often cannot be used. In addition, the calculation of the internal pressure of a system with fixed atoms may be highly inaccurate due to the nonphysical nature of the atomic constraints and difficulties in accurately defining the volume occupied by the unconstrained atoms in the system. The inability to properly set and control pressure can result in substantial problems for the accurate simulation of condensed-phase systems if the behavior of the system (e.g., peptide/protein adsorption) is sensitive to pressure. To address this issue, the authors have developed an approach to accurately determine the internal pressure for a system with constrained atoms. As the first step in this method, a periodically extendable portion of the mobile phase of the constrained system (e.g., the solvent atoms) is used to create a separate unconstrained system for which the pressure can be accurately calculated. This model system is then used to create a pressure calibration plot for an intensive local effective virial parameter for a small volume cross section or "slab" of the system. Using this calibration plot, the pressure of the constrained system can then be determined by calculating the virial parameter for a similarly sized slab of mobile atoms. In this article, the authors present the development of this method and demonstrate its application using the CHARMM molecular simulation program to characterize the adsorption behavior of a peptide in explicit water on a hydrophobic surface whose lattice spacing is maintained with atomic constraints. The free energy of adsorption for this system is shown to be dramatically influenced by pressure, thus emphasizing the importance of properly maintaining the pressure of the system for the accurate simulation of protein-surface interactions.
Characterisation of the electrostatic properties of dental enamel is important for understanding the interfacial processes that occur on a tooth surface and how these relate to the natural ability of our teeth to withstand chemical attack from the acids in many soft drinks. Whereas, the role of the mineral component of the tooth enamel in providing this resistance to acid erosion has been studied extensively, the influence of proteins that are also present within the structure is not well understood. In this paper, we report for the first time the use of double-layer force spectroscopy to directly measure electrostatic forces on as received and hydrazine-treated (deproteinated) enamel surfaces in solutions with different pH to determine how the enamel proteins influence acid erosion surface potential and surface charge of human dental enamel. The deproteination of the treated samples was confirmed by the loss of the amide bands (~1,300-1,700 cm(-1)) in the FTIR spectrum of the sample. The force characteristics observed were found to agree with the theory of electrical double layer interaction under the assumption of constant potential and allowed the surface charge per unit area to be determined for the two enamel surfaces. The values and, importantly, the sign of these adsorbed surface charges indicates that the protein content of dental enamel contributes significantly to the electrostatic double layer formation near the tooth surface and in doing so can buffer the apatite crystals against acid attack. Moreover, the electrostatic interactions within this layer are a driving factor for the mineral transfer from the tooth surface and the initial salivary pellicle formation.
Poly(N-isopropyl acrylamide) (pNIPAM) is one of the most popular stimulus-responsive polymers for research. It is especially of great interest in the field of tissue engineering. While it is known that the NIPAM monomer is toxic, there is little conclusive research on the cytotoxicity of the polymer. In this work, the relative biocompatibility of the NIPAM monomer, pNIPAM, and pNIPAM-coated substrates prepared using different polymerization (free radical and plasma polymerization) and deposition (spin coating and plasma polymerization) techniques was evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). Four different mammalian cell types (endothelial, epithelial, smooth muscle, and fibroblasts) were used for the cytotoxicity testing. The pNIPAM-coated surfaces were evaluated for their thermoresponse and surface chemistry using X-ray photoelectron spectroscopy and goniometry. We found that while cell viability on pNIPAM surfaces decreases when compared to controls, the viability also seems to be deposition type dependent, with sol-gel based pNIPAM surfaces being the least biocompatible. Long term experiments proved that all pNIPAM-coated surfaces were not cytotoxic to the four cell types evaluated in a direct contact test. Plating efficiency experiments did not show cytotoxicity. Cellular sensitivity to pNIPAM and to the NIPAM monomer varied depending on cell type. Endothelial cells consistently showed decreased viability after 48 hours of exposure to pNIPAM extracts and were more sensitive than the other cell lines to impurities in the polymer.
Thorough studies of protein interactions with stimulus responsive polymers are necessary to provide a better understanding of their applications in biosensors and biomaterials. In this study, protein behavior on a thermoresponsive polymer surface, plasma polymerized N-isopropyl acrylamide (ppNIPAM), is investigated using multiple characterization techniques above and below its lower critical solution temperature (LCST). Protein adsorption and binding affinity are probed using radiolabeled proteins. Protein activity is estimated by measuring the immunological activity of an antibody adsorbed onto ppNIPAM using surface plasmon resonance. Conformation/orientation of the proteins is probed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and principal component analysis (PCA) of the TOF-SIMS data. In this work, we find that at low protein solution concentrations, ppNIPAM-treated surfaces are low fouling below the LCST, but protein retentive above it. The protein adsorption isotherms demonstrate that apparent affinity between soluble protein molecules and the ppNIPAM surface are an order of magnitude lower at room temperature than at 37 degrees C. Although direct protein desorption is not observed in our study when the surface temperature drops below the LCST, the binding affinity of surface adsorbed protein with ppNIPAM is reduced, as judged by a detergent elution test. Furthermore, we demonstrated that proteins adsorbed onto ppNIPAM are functionally active, but the activity is better preserved at room temperature than 37 degrees C. The temperature dependent difference in protein activity as well as TOF-SIMS and PCA study suggest that proteins take different conformations/orientations after adsorption on ppNIPAM above and below the LCST.
The objective of this work was to compare poly(ethylene glycol) (PEG) and phosphorylcholine (PC) moieties as surface modifiers with respect to their ability to inhibit protein adsorption. Surfaces were prepared by graft polymerization of the methacrylate monomers oligo(ethylene glycol) methyl ether methacrylate (OEGMA, MW 300, PEG side chains of length n=4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC, MW 295). The grafted polymers thus contained short PEG chains and PC, respectively, as side groups. Grafting on silicon was carried out using surface-initiated atom transfer radical polymerization (ATRP). Graft density was controlled via the surface density of the ATRP initiator, and chain length of the grafts was controlled via the ratio of monomer to sacrificial initiator. The grafted surfaces were characterized by water contact angle, x-ray photoelectron spectroscopy, and atomic force microscopy. The effect of graft density and chain length on fibrinogen adsorption from buffer was investigated using radio labeling methods. Adsorption to both MPC- and OEGMA-grafted surfaces was found to decrease with increasing graft density and chain length. Adsorption on the MPC and OEGMA surfaces for a given chain length and density was essentially the same. Very low adsorption levels of the order of 7 ngcm(2) were seen on the most resistant surfaces. The effect of protein size on resistance to adsorption was studied using binary solutions of lysozyme (MW 14 600) and fibrinogen (MW 340 000). Adsorption levels in these experiments were also greatly reduced on the grafted surfaces compared to the control surfaces. It was concluded that at the lowest graft density, both proteins had unrestricted access to the substrate, and the relative affinities of the proteins for the substrate (higher affinity of fibrinogen) determined the composition of the layer. At the highest graft density also, where the adsorption of both proteins was very low, no preference for one or the other protein was evident, suggesting that adsorption did not involve penetration of the grafts and was occurring at the outer surface of the graft layer. It thus seems likely that preference among different proteins based on ability to penetrate the graft layer would occur, if at all, at a grafting density intermediate between 0.1 and 0.39 cm(2). Again the MPC and OEGMA surfaces behaved similarly. It is suggested that the main determinant of the protein resistance of these surfaces is the "water barrier layer" resulting from their hydrophilic character. In turn the efficacy of the water barrier depends on the monomer density in the graft layer.
Adhesion of red blood cells (RBCs) to endothelial cells (ECs) is usually insignificant but an enhanced adhesion has been observed in various diseases associated with vascular complications. This abnormal adhesion under pathological conditions such as sickle cell disease has been correlated with increased levels of various plasma proteins but the detailed underlying mechanism(s) remains unclear. Usually it is assumed that the proadhesive effects of plasma proteins originate from ligand interactions cross-linking receptors on adjacent cells, but explicit results detailing binding sites or receptors for some proteins (e.g., fibrinogen) on either RBC or EC surfaces that would support this model are missing. In this study, the authors tested whether there is an alternative mechanism. Their results demonstrate that dextran 2 MDa promotes the adhesion of normal RBCs to thrombin-activated ECs and that this effect becomes more pronounced with increasing thrombin concentration or with prolonged thrombin incubation time. It is concluded that depletion interaction originating from nonadsorbing macromolecules (i.e., dextran) can modulate the adhesion of red blood cells to thrombin-activated EC. This study thereby suggests macromolecular depletion as an alternative mechanism for the adhesion-promoting effects of nonadsorbing plasma proteins. These findings should not only aid in getting a better understanding of diseases associated with vascular complications but should also have many potential applications in biomedical or biotechnological areas that require the control of cell-cell or cell surface interactions.
The authors report a facile method for the selective immobilization of biomolecules onto a gold surface that was preactivated by a polymeric adlayer. The polymeric adlayer was designed to perform triple functions: high resistance to nonspecific protein adsorption, efficient surface anchoring, and subsequent covalent attachment of biomolecules. For this purpose, a random copolymer, poly(PEGMA-r-NAS), was synthesized by radical polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and N-acryloxysuccinimide (NAS). In the first step, the polymeric adlayer was formed onto amine-terminated self-assembled monolayers (SAMs) on gold through covalent bond formation between reactive N-hydroxysuccinimide (NHS) ester of the copolymer and the amine of the SAMs. In the second step, amine-bearing biotin as a model biomolecule was covalently attached onto the polymeric adlayer that still contained unreacted NHS esters. The degrees of the binding sensitivity for a target protein and the nonspecific binding for four model proteins on the biotinylated polymeric adlayer were examined by surface plasmon resonance spectroscopy. Finally, the specific immobilization of rhodamin (TRITC)-conjugated streptavidin on the biotinylated polymeric adlayer was achieved by a simple microcontact printing technique, resulting in well-defined patterns of the protein.
Platelets are considered to have important functions in inflammatory processes as key players in innate immunity. Toll like receptors (TLRs), expressed on platelets, recognize pathogen associated molecular patterns and trigger immune responses. Pathogens are able to adhere to human tissues and form biofilms which cause a continuous activation of the immune system. The authors aimed to investigate how immobilized Pam3CSK4 (a synthetic TLR2/1 agonist) and IgG, respectively, resembling a bacterial focus, affects adhesion and activation of platelets including release of two cytokines, regulated on activation normal T-cell expressed and secreted (RANTES) and macrophage migration inhibitory factor (MIF). The authors also aim to clarify the signaling downstream of TLR2/1 and FcγRII (IgG receptor) and the role of adenine nucleotides in this process. Biolayers of Pam3CSK4 and IgG, respectively, were confirmed by null-ellipsometry and contact angle measurements. Platelets were preincubated with signaling inhibitors for scr and Syk and antagonists for P2X1 or P2Y1 [adenosine triphosphate (ATP), adenosine diphosphate (ADP) receptors] prior to addition to the surfaces. The authors show that platelets adhere and spread on both Pam3CSK4- and IgG-coated surfaces and that this process is antagonized by scr and Syc inhibitors as well as P2X1 and P2Y antagonists. This suggests that Pam3CSK4 activated platelets utilize the same pathway as FcγRII. Moreover, the authors show that ATP-ligation of P2X1 is of importance for further platelet activation after TLR2/1-activation, and that P2Y12 is the prominent ADP-receptor involved in adhesion and spreading. RANTES and MIF were secreted over time from platelets adhering to the coated surfaces, but no MIF was released upon stimulation with soluble Pam3CSK4. These results clarify the importance of TLR2/1 and FcγRII in platelet adhesion and activation, and strengthen the role of platelets as an active player in sensing bacterial infections.
Surface ion equilibrium is hypothesized to play an important role in defining the interactions between foreign materials and biological systems. In this study, we compare two surfaces with respect to their ability to activate adhering platelets. One is a commonly used implant material TiO(2), which binds Ca(2+), and the other one is glass, which does not. We show, that in the presence of Ca(2+), TiO(2) acts as an agonist, activating adhering platelets and causing the expression on their surface of two well-known activation markers, CD62P (P-selectin) and CD63. On the contrary, in the absence of Ca(2+), platelets adhering on TiO(2) express only one of the two markers, CD63. Platelets adhering on glass, as well as platelets challenged with soluble agonists in solution, express both markers independently of whether Ca(2+) is present or not. The expression of CD62P and CD63 is indicative of the exocytosis of the so-called α- and dense granules, respectively. It is a normal response of platelets to activation. Differences in the expression profiles of these two markers point to differential regulation of the exocytosis of the two kinds of granules, confirming the recent notion that platelets can tune their microenvironment in a trigger-specific fashion.
A drop brought into contact with a nearby substrate can wet and spread against the substrate, forming a liquid bridge that exerts a capillary force. This force due to surface tension can be used to "grab" the substrate, pulling it toward the drop. "Wet" adhesion results from the parallel action of an array of small liquid bridges. The Florida palm beetle, Hemisphaerota cyanea, uses wet adhesion to defend itself against attacking predators by adhering to the palm leaf using an array of about 120 000 μm-sized liquid bridges. The beetle's survival depends on the strength of adhesion which, in turn, depends on how liquid bridges break. Individual bridges break when they go unstable, according to their response curves. However, the ultimate strength of an individual bridge depends on the class of disturbances to which it is subjected, and it has been speculated that the beetle may have some control over this class. The authors experimentally study families of liquid bridge equilibria for their breaking limits when subjected to constant-length (L) and constant-force (F) disturbances. While to control constant-L disturbances is straightforward, to apply and control constant-F disturbances on a liquid bridge requires more ingenuity. The authors introduce an apparatus with a lever-arm and a ball-bearing slide. The authors then compare our experimentally measured bridge response curves to the force trace from experiments on the beetle (prior literature) to infer the mode of beetle detachment. Under normal loads, the beetle detaches as a constant-L instability for smaller loads and as a constant-F instability for larger loads. The beetle's ability to adjust the type and magnitude of loading in real time is not only crucial to its survival but has implications for the design of various engineering devices.
We demonstrate a convenient chip platform for the addressable immobilization of protein-loaded vesicles on a microarray for parallelized, high-throughput analysis of lipid-protein systems. Self-sorting of the vesicles on the microarray was achieved through DNA bar coding of the vesicles and their hybridization to complementary strands, which are preimmobilized in defined array positions on the chip. Imaging surface plasmon resonance in ellipsometric mode was used to monitor vesicle immobilization, protein tethering, protein-protein interactions, and chip regeneration. The immobilization strategy proved highly specific and stable and presents a mild method for the anchoring of vesicles to predefined areas of a surface, while unspecific adsorption to both noncomplementary regions and background areas is nonexistent or, alternatively, undetectable. Furthermore, histidine-tagged receptors have been stably and functionally immobilized via bis-nitrilotriacetic acid chelators already present in the vesicle membranes. It was discovered though that online loading of proteins to immobilized vesicles leads to cross contamination of previously loaded vesicles and that it was necessary to load the vesicles offline in order to obtain pure protein populations on the vesicles. We have used this cross-binding effect to our benefit by coimmobilizing two receptor subunits in different ratios on the vesicle surface and successfully demonstrated ternary complex formation with their ligand. This approach is suitable for mechanistic studies of complex multicomponent analyses involving membrane-bound systems.
Collecting information at the interface between living cells and artificial substrates is exceedingly difficult. The extracellular matrix (ECM) mediates all cell-substrate interactions, and its ordered, fibrillar constituents are organized with nanometer precision. The proceedings at this interface are highly dynamic and delicate. In order to understand factors governing biocompatibility or its counterpart antifouling, it is necessary to probe this interface without disrupting labels or fixation and with sufficient temporal resolution. Here the authors combine nonlinear optical spectroscopy (sum-frequency-generation) and microscopy (second-harmonic-generation), fluorescence microscopy, and quartz crystal microgravimetry with dissipation monitoring in a strategy to elucidate molecular ordering processes in the ECM of living cells. Artificially (fibronectin and collagen I) and naturally ordered ECM fibrils (zebrafish, Danio rerio) were subjected to nonlinear optical analysis and were found to be clearly distinguishable from the background signals of diffusive proteins in the ECM. The initial steps of fibril deposition and ordering were observed in vitro as early as 1 h after cell seeding. The ability to follow the first steps of cell-substrate interactions in spite of the low amount of material present at this interface is expected to prove useful for the assessment of biomedical and environmental interfaces.
The response of fibroblast cells to periodically poled LiTaO3 ferroelectric crystals has been studied. While fibroblast cells do not show morphological differences on the two polarization directions, they show a tendency to avoid the field gradients that occur between polarization domains of the ferroelectric. The response to the field gradients is fully established after one hour, a time at which fibroblasts form their first focal contacts. If suspension cells, with a lower tendency to establish strong surface contacts are used, no influence of the field gradients is observed.
Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO(®) 9 with endogenously expressed eGFP, as SYTO(®) 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions.
For both, environmental and medical applications, the quantification of bacterial adhesion is of major importance to understand and support the development of new materials. For marine applications, the demand is driven by the quest for improved fouling-release coatings. To determine the attachment strength of bacteria to coatings, a microfluidic adhesion assay has been developed which allows probing at which critical wall shear stress bacteria are removed from the surface. Besides the experimental setup and the optimization of the assay, we measured adhesion of the marine bacterium Cobetia marina on a series of differently terminated self-assembled monolayers. The results showed that the adhesion strength of C. marina changes with surface chemistry. The difference in critical shear stress needed to remove bacteria can vary by more than one order of magnitude if a hydrophobic material is compared to an inert chemistry such as polyethylene glycol.
This study characterized human umbilical vein endothelial cell (HUVEC) adhesion, proliferation, and gene expression on bilayered polyelectrolyte coatings composed of an outermost layer of glycosaminoglycans (hyaluronan, heparin, or chondroitin sulfate), with an underlying layer of poly-L-lysine or chitosan. The proportion of cells that adhered to the various polyelectrolyte coatings after 1 and 2 h incubations was quantified by the WST-8 assay. Interchanging poly-L-lysine with chitosan resulted in significant differences in cellular adhesion to the outermost glycosaminoglycan layer after 1 h, but these differences became insignificant after 2 h. The proliferation of HUVEC on the various bilayered polyelectrolyte coatings over 10 days was characterized using the WST-8 assay. Regardless of whether the underlying layer was poly-L-lysine or chitosan, HUVEC proliferation on the hyaluronan outermost layer was significantly less than on heparin or chondroitin sulfate. Additionally, it was observed that there was more proliferation with poly-L-lysine as the underlying layer, compared to chitosan. Subsequently, real-time polymerase chain reaction was used to analyze the expression of seven genes related to adhesion, migration, and endothelial function (VWF, VEGFR, VEGFA, endoglin, integrin-α5, ICAM1, and ICAM2) by HUVEC cultured on the various bilayered polyelectrolyte coatings for 3 days. With poly-L-lysine as the underlying layer, biologically significant differences (greater than twofold) in the expression of VWF, VEGFR, VEGFA, endoglin, and ICAM1 were observed among the three glycosaminoglycans. With chitosan as the underlying layer, all three glycosaminoglycans displayed biologically significant differences in the expression of VWF and VEGFR compared to the chitosan control. CT-HA displayed the highest level of expression of VWF, whereas expression levels of VEGFR were almost similar among the three glycosaminoglycans.
The zeta potential of the motile spores of the green alga (seaweed) Ulva linza was quantified by video microscopy in combination with optical tweezers and determined to be -19.3+/-1.1 mV. The electrostatic component involved in the settlement and adhesion of spores was studied using electret surfaces consisting of PTFE and bearing different net charges. As the surface chemistry remains the same for differently charged surfaces, the experimental results isolate the influence of surface charge and thus electrostatic interactions. Ulva spores were demonstrated to have a reduced tendency to settle on negatively charged surfaces and when they did settle the adhesion strength of settled spores was lower than with neutral or positively charged surfaces. These observations can be ascribed to electrostatic interactions.
Understanding and controlling cell adhesion to biomaterials and synthetic materials are important issues in basic research and applied sciences. Supported lipid bilayers (SLBs) functionalized with cell adhesion peptides linked to lipid molecules are popular platforms of cell adhesion. In this paper, an alternative approach of peptide presentation is presented in which peptides are stereo-selectively linked to proteins self-assembling in a rigid two-dimensional (2D) matrix on SLBs. Annexin-A5 (Anx5) was used as prototype protein for its known properties of forming stable and rigid 2D matrices on lipid surfaces. Two types of Anx5-peptide complexes, containing either a RGD or an IKVAV sequence, were synthesized. The authors show that both Anx5-peptide complexes present the same properties of binding and 2D organization on lipid surfaces as Anx5, when investigated by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and transmission electron microscopy techniques. Anx5-RGD and Anx5-IKVAV 2D matrices were found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells, respectively. The influence of the surface density of exposed peptides on cell adhesion was investigated, showing that cells attach to Anx5-peptide matrices when the average distance between peptides is smaller than about 60 nm. This cell adhesion platform provides control of the orientation and density of cell ligands, opening interesting possibilities for future applications.
In this study, the effect of surface nanoscale roughness on fibrinogen adsorption and platelet adhesion was investigated. Nanorough silica surfaces with a low level of surface roughness (10 nm Rrms) were found to support the same level of fibrinogen adsorption as the planar silica surfaces, while nanorough silica surfaces with higher levels of surface roughness (15 nm Rrms) were found to support significantly less fibrinogen adsorption. All surfaces analyzed were found to support the same level of platelet adhesion; however, platelets were rounded in morphology on the nanorough silica surfaces while platelets were spread with a well-developed actin cytoskeleton on the planar silica. Unique quartz crystal microbalance with dissipation monitoring (QCM-D) responses was observed for the interactions between platelets and each of the surfaces. The QCM-D data indicated that platelets were more weakly attached to the nanorough silica surfaces compared with the planar silica. These data support the role of surface nanotopography in directing platelet-surface interactions even when the adsorbed fibrinogen layer is able to support the same level of platelet adhesion.
Inspired by the composition of the native extracellular matrix, biomimetic polyelectrolyte multilayers were assembled from polypeptides and the glycosaminoglycan chondroitin sulfate (CS). To investigate whether peptide conformation imposes an effect on the cell biological functions of osteoblasts, the secondary structure was analyzed by in situ infra-red and circular dichroism spectroscopy. Multilayers composed of polypeptides and CS reveal a predominantly random coiled conformation and impede osteoblast spreading. On the contrary, polypeptide chains in assemblies of poly-L-lysine and poly-L-glutamic acid (PGA) primarily adopt an intermolecular β sheet structure and reveal an increased area of spread, which consequently supports the proliferation of osteoblasts. When CS is replaced by PGA in mixed multilayers, we observe a structural rearrangement from random coils to β sheets with a concomitant improved cell response. We conclude that polypeptide conformation in biomimetic multilayer assemblies affects osteoblast response by altering the stiffness of the multilayer.
Force spectroscopy has been used to measure the adhesion of Saos-2 cells to a glass surface at different phases of the cell cycle. The cells were synchronized in three phases of the cell cycle: G(1), S, and G(2)M. Cells in these phases were compared with unsynchronized and native mitotic cells. Individual cells were attached to an atomic force microscope cantilever, brought into brief contact with the glass surface, and then pulled off again. The force-distance curves obtained allowed the work and maximum force of detachment as well as the number, amplitude, and position of discrete unbinding steps to be determined. A statistical analysis of the data showed that the number of binding proteins or protein complexes present at the cell surface and their binding properties remain similar throughout the cell cycle. This, despite the huge changes in cell morphology and adhesion that occur as the cells enter mitosis. These changes are rather associated with the changes in cytoskeletal organization, which can be quantified by force spectroscopy as changes in cell stiffness.
Protein resistance of self-assembled monolayers (SAMs) of hexa(ethylene glycols) (EG(6)) has previously been shown to be dependent on the alkoxyl end-group termination of the SAM, which determines wettability [S. Herrwerth, W. Eck, S. Reinhardt, and M. Grunze, J. Am. Chem. Soc. 125, 9359 (2003)]. In the present study, the same series of hexa(ethylene glycols) was used to examine the correlation between protein resistance and the settlement and adhesion of eukaryotic algal cells, viz., zoospores of the macroalga Ulva and cells of the diatom Navicula, which adhere to the substratum through the secretion of protein-containing glues. Results showed that the initial settlement of Ulva zoospores was highest on the hydrophilic EG(6)OH but that cells were only weakly adhered. The number of Ulva zoospores and Navicula cells firmly adhered to the SAMs systematically increased with decreasing wettability, as shown for the protein fibrinogen. The data are discussed in terms of hydration forces and surface charges in the SAMs.
Depletion of neuroproteins on the inner walls of storage tubes influences the accuracy of tests used for identification of various neurodegenerative disorders. In this paper, a strategy is described for surface modification of Eppendorf tubes leading to non-adhesive properties towards the recombinant human prion proteins (PrPrec(hum)). Tubes were pre-activated by helium plasma and grafted with three diverse coatings: pure poly(N-isopropylacrylamide) (PNIPAM), PNIPAM admixed with either neutral PEG(20)sorbitan monolaurate (PEG(20)) or positively charged cetyl trimethylammonium bromide (CTAB) at varying plasma activation times and polymer to surfactant ratios. New functionalized surfaces were analyzed by goniometry, streaming potential measurement and X-ray photoelectron spectroscopy, whereas the protein adhesion was monitored by enzyme linked immunosorbent assays and confocal microscopy. The mapping of PrPrec(hum) adhesion associated with surface analyses enabled us to determine that no or negligible depletion of PrPrec(hum) can be obtained by surfaces possessing basic component in the range between 50 and 60 mJ m(-2) and streaming potential ζ(7.4) ~ -50 mV.
Reflection interference contrast microscopy (RICM) allows the visualization of the cell's adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell's spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.
An attempt is made to estimate, via computer simulation of the force-distance relation, the free energy of adhesion between a phosphatidylethanolamine bilayer and an alkanethiolate self-assembled monolayer (SAM) in aqueous medium. The simulations are performed using the grand canonical Monte Carlo technique and atomistic force fields. The bilayer adhesion free energy is predicted to be -22 ± 3 mJ/m(2) (-1.4 ± 0.2 kcal/mol) on a hydrophilic carboxyl-terminated SAM and -1 ± 1 mJ/m(2) (-0.06 ± 0.06 kcal/mol) on a hydrophobic methyl-terminated SAM.
This review focuses on the surface modification of substrates with self-assembled monolayers (SAMs) and polymer brushes to tailor interactions with biological systems and to thereby enhance their performance in bioapplications. Surface modification of biomedical implants promotes improved biocompatibility and enhanced implant integration with the host. While SAMs of alkanethiols on gold substrates successfully prevent nonspecific protein adsorption in vitro and can further be modified to tether ligands to control in vitro cell adhesion, extracellular matrix assembly, and cellular differentiation, this model system suffers from lack of stability in vivo. To overcome this limitation, highly tuned polymer brushes have been used as more robust coatings on a greater variety of biologically relevant substrates, including titanium, the current orthopedic clinical standard. In order to improve implant-bone integration, the authors modified titanium implants with a robust SAM on which surface-initiated atom transfer radical polymerization was performed, yielding oligo(ethylene glycol) methacrylate brushes. These brushes afforded the ability to tether bioactive ligands, which effectively promoted bone cell differentiation in vitro and supported significantly better in vivo functional implant integration.
Nanotextured polymeric surfaces with inclined rods reveal highly anisotropic properties concerning wetting and adhesion. In this work, we report on the interaction of fibroblast cells with these highly anisotropic materials. The authors quantified removal of adherent cells from such surfaces by a laminar flow. The critical shear force needed for cell removal from the surface depends on the inclination direction. Based on electron microscopy cross sections we deduce that interactions of cellular filopodia extending into the nanotextured surface are causing the direction depending removal.
This article focuses on elucidating the key presentation features of neurotrophic ligands at polymer interfaces. Different biointerfacial configurations of the human neural cell adhesion molecule L1 were established on two-dimensional films and three-dimensional fibrous scaffolds of synthetic tyrosine-derived polycarbonate polymers and probed for surface concentrations, microscale organization, and effects on cultured primary neurons and neural stem cells. Underlying polymer substrates were modified with varying combinations of protein A and poly-D-lysine to modulate the immobilization and presentation of the Fc fusion fragment of the extracellular domain of L1 (L1-Fc). When presented as an oriented and multimeric configuration from protein A-pretreated polymers, L1-Fc significantly increased neurite outgrowth of rodent spinal cord neurons and cerebellar neurons as early as 24 h compared to the traditional presentation via adsorption onto surfaces treated with poly-D-lysine. Cultures of human neural progenitor cells screened on the L1-Fc/polymer biointerfaces showed significantly enhanced neuronal differentiation and neuritogenesis on all protein A oriented substrates. Notably, the highest degree of βIII-tubulin expression for cells in 3-D fibrous scaffolds were observed in protein A oriented substrates with PDL pretreatment, suggesting combined effects of cell attachment to polycationic charged substrates with subcellular topography along with L1-mediated adhesion mediating neuronal differentiation. Together, these findings highlight the promise of displays of multimeric neural adhesion ligands via biointerfacially engineered substrates to "cooperatively" enhance neuronal phenotypes on polymers of relevance to tissue engineering.
Substrates coated with specific bioactive ligands are important for tissue engineering, enabling the local presentation of extracellular stimulants at controlled positions and densities. In this study, we examined the cross-talk between integrin and epidermal growth factor (EGF) receptors following their interaction with surface-immobilized Arg-Gly-Asp (RGD) and EGF ligands, respectively. Surfaces of glass coverslips, modified with biotinylated silane-polyethylene glycol, were functionalized by either biotinylated RGD or EGF (or both) via the biotin-NeutrAvidin interaction. Fluorescent labeling of the adhering A431 epidermoid carcinoma cells for zyxin or actin indicated that EGF had a dual effect on focal adhesions (FA) and stress fibers: at low concentrations (0.1; 1 ng/ml), it stimulated their growth; whereas at higher concentrations, on surfaces with low to intermediate RGD densities, it induced their disassembly, leading to cell detachment. The EGF-dependent dissociation of FAs was, however, attenuated on higher RGD density surfaces. Simultaneous stimulation by both immobilized RGD and EGF suggest a strong synergy between integrin and EGFR signaling, in FA induction and cell spreading. A critical threshold level of EGF was required to induce significant variation in cell adhesion; beyond this critical density, the immobilized molecule had a considerably stronger effect on cell adhesion than did soluble EGF. The mechanisms underlying this synergy between the adhesion ligand and EGF are discussed.
In the present study, we investigated the hydrothermal treatment of titanium with divalent cation solutions and its effect in promoting the adhesion of gingival epithelial cells and fibroblasts in vitro. Gingival keratinocyte-like Sa3 cells or fibroblastic NIH3T3 cells were cultured for 1 h on experimental titanium plates hydrothermally-treated with CaCl(2) (Ca) or MgCl(2) (Mg) solution, or distilled water (DW). The number and adhesive strengths of attached cells on the substrata were then analyzed. The number of Sa3 cells adhering to the Ca- and Mg-treated plates was significantly larger than in the DW group, but the strength of this adhesion did not differ significantly between groups. In contrast, NIH3T3 cell adhesion number and strength were increased in both the Ca and Mg groups compared to the DW group. Fluorescent microscopic observation indicated that, in all groups, Sa3 had identical expression levels of integrin β4 and development of actin filaments, whereas NIH3T3 cells in the Ca and Mg groups displayed much stronger punctate cytoplasmic signals for vinculin and more bundle-shaped actin filaments than cells in the DW group. As a result, it was indicated that the hydrothermal treatment of titanium with Ca or Mg solution improved the integration of soft tissue cells with the substrata, which may facilitate the development of a soft tissue barrier around the implant.
Herein, the authors report and review polyelectrolyte complex
nanoparticles (NPs) loaded with zoledronate (ZOL) and simvastatin and their effects on bone cells. PEC NPs are intended for modification of bone substitute materials. For characterization, they can be solution casted on germanium
(Ge) substrates serving as analytically accessible model substrate. PEC NPs were generated by mixing poly(ethyleneimine) (PEI) either with linear cellulose sulfate (CS) or with branched dextransulfate (DS). Four important requirements for drug loaded PEC NPs and their films are addressed herein, which are the colloidal stability of PEC
dispersions (1), interfacial stability (2), cytocompatibility (3), and retarded drug release (4). Dynamic light scattering measurements (DLS) showed that both PEI/CS and PEI/DS PEC NP were obtained with hydrodynamic radii in the range of 35–170 nm and were colloidally stable up to several months. Transmission FTIR
spectroscopy evidenced that films of both systems were stable in contact to the release medium up to several days. ZOL-loaded PEI/CS nanoparticles, which were immobilized on an osteoblast-derived extracellular matrix, reduced significantly the resorption and the metabolic activity of human monocyte-derived osteoclasts. FTIR
spectroscopy at cast PEC/drug films at Ge substrates revealed retarded drug releases in comparison to the pure drug films.
The bone therapeutic drug zoledronate (ZOL) was loaded at and released by polyelectrolyte complex (PEC) particle films composed of either pure poly(ethyleneimine) (PEI) or maltose-modified poly(ethyleneimine) (PEI-M) and oppositely charged cellulose sulfate attached to model germanium (Ge) substrates by solution casting. Dispersions of colloidally stable polyelectrolyte complex (PEC) particles in the size range 11-141 nm were obtained by mixing PEI or PEI-M, CS and ZOL in defined stoichiometric ratios. TRANS-FTIR spectroscopy was used to determine the stability of the PEC films against detachment, in-situ-ATR-FTIR spectroscopy for the ZOL loss in the PEC film and UV-VIS spectroscopy for the ZOL enrichment of the release medium. Films of casted ZOL/CS/PEI-M or ZOL/CS/PEI particles were stable in contact to water, while films of the pure drug (ZOL) and of the binary systems ZOL/PEI-M or ZOL/PEI were not stable against detachment. Retarded releases of ZOL from various PEC films compared to the pure drug film were observed. The molecular weight of PEI showed a considerable effect on the initial burst (IB) of ZOL. No significant effect of the maltose modification of PEI-25 K on IB could be found. Generally, after one day the ZOL release process was finished for all measured ZOL/PEC samples and residual amounts of 0-30% were obtained. Surface adhesive drug loaded PEC particles are promising drug delivery systems to supply and release a defined amount of bone therapeutics and to functionalize bone substitution materials.
Micropatterned surfaces with cell adhesive areas, delimited by protein repellent microstructures, are in high demand for its potential use as relevant biological assays. This is not only because such surfaces allow directing cell growth in a spatially localized and restricted manner, but also because they can be used to elucidate basic cell growth and orientation mechanisms. Here, it is presented a laser-assisted micropatterning technique to fabricate large area microstructures of poly (ethylene glycol) hydrogel onto a cell adhesive surface: a biofunctional maleic anhydride copolymer. By varying photoinitiator, laser intensity, copolymer as well as the hydrogel layer thickness, the optimum conditions to produce high quality features were found. The suitability of these micropatterned substrates for bioassay applications was proved by cell adhesion studies. The introduced procedure could be used to prepare a broad range of microarrays for certain bioanalytical approaches and to create different types of biofunctional surfaces.
Plasma medicine is an emerging field where plasma physics is used for therapeutical applications. Temperature is an important factor to take into account with respect to the applications of plasma to biological systems. During the treatment, the tissue
temperature could increase to critical values. In this work, a model is presented, which is capable of predicting the skin
temperature during a treatment with a radio frequency driven plasma needle. The main gas was helium. To achieve this, a discharge model was coupled to a heat transfer and fluid flow model. The results provide maximum application times for different power depositions in order to avoid reaching critical skin
Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen egg-white lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems.
Although previous studies have demonstrated that TOF-SIMS is a powerful method for the characterization of adsorbed proteins due to its specificity and surface sensitivity, it was unclear from earlier work whether the differences between proteins observed on uniform flat surfaces were large enough to facilitate clear image contrast between similar proteins in small areas on topographically complex samples that are more typical of biological tissues. The goal of this study was to determine whether Bi(3) (+) could provide sufficiently high sensitivity to provide clear identification of the different proteins in an image. In this study, 10 μm polystyrene microspheres were adsorbed with one of three different proteins, human serum albumin (HSA), bovine serum albumin (BSA), and hemoglobin. Spheres coated with HSA were then mixed with spheres coated with either BSA (a very similar protein) or hemoglobin (a dramatically different protein), and deposited on silicon substrates. Fluorescent labeling was used to verify the SIMS results. With maximum autocorrelation factors (MAF) processing, images showed clear contrast between both the very different proteins (HSA and hemoglobin) and the very similar proteins (HSA and BSA). Similar results were obtained with and without the fluorescent labels. MAF images were calculated using both the full spectrum and only characteristic amino acid fragments. Although better image contrast was obtained using the full spectrum, differences between the spheres were still evident when only the amino acid fragments were included in the analysis, suggesting that we are truly observing differences between the proteins themselves. These results demonstrate that TOF-SIMS, with a Bi(3) (+) primary ion, is a powerful technique for characterizing interfacial proteins not only on large uniform surfaces, but also with high spatial resolution on the topographically complex samples typical in biological analysis.
Protein-surface interactions are crucial to the overall biocompatability of biomaterials, and are thought to be the impetus towards the adverse host responses such as blood coagulation and complement activation. Only a few studies hint at the ultra-low fouling potential of zwitterionic poly(carboxybetaine methacrylate) (PCBMA) grafted surfaces and, of those, very few systematically investigate their non-fouling behavior. In this work, single protein adsorption studies as well as protein adsorption from complex solutions (i.e. human plasma) were used to evaluate the non-fouling potential of PCBMA grafted silica wafers prepared by nitroxide-mediated free radical polymerization. PCBMAs used for surface grafting varied in charge separating spacer groups that influence the overall surface charges, and chain end-groups that influence the overall hydrophilicity, thereby, allows a better understanding of these effects towards the protein adsorption for these materials. In situ ellipsometry was used to quantify the adsorbed layer thickness and adsorption kinetics for the adsorption of four proteins from single protein buffer solutions, viz, lysozyme, α-lactalbumin, human serum albumin and fibrinogen. Total amount of protein adsorbed on surfaces differed as a function of surface properties and protein characteristics. Finally, immunoblots results showed that human plasma protein adsorption to these surfaces resulted, primarily, in the adsorption of human serum albumin, with total protein adsorbed amounts being the lowest for PCBMA-3 (TEMPO). It was apparent that surface charge and chain hydrophilicity directly influenced protein adsorption behavior of PCBMA systems and are promising materials for biomedical applications.
Lipid vesicles (liposomes) exhibit a wide range of behavior at inorganic (oxide) surfaces. A complete understanding of the vesicle-surface interactions, and of the ensuing transformations surface adsorbed liposomes undergo, has proven elusive. This is at least in part due to the large number of degrees of freedom of the system comprising vesicles with their molecular constituents, substrate surface, and electrolyte solution. The least investigated among these degrees of freedom are those intrinsic to the vesicles themselves, involving rearrangements of lipid molecules. In this study, the adsorption of two-component vesicles (phosphatidylcholine:phosphatidylserine) on titanium dioxide was investigated by dual polarization interferometry. Mixtures of these two lipids containing more than 20% of phosphatidylserine form supported bilayers on titania, with phosphatidylserine predominantly facing the surface of the oxide. The purpose of this investigation is to ascertain whether redistribution of phosphatidylserine occurs already in the adsorbing vesicles. Indeed, this was found to be the case. A possible mechanism of this process is discussed.