Excess fibrinogen adsorption to monolayers of mixed lipids.

Adsorption of fibrinogen to the monolayers of mixed lipids, dipalmitoyl phosphatidyl choline (DPPC) and eicosylamine (EA) was measured at a surface pressure of 20 mN/m by an in situ surface plasmon resonance technique. Pressure-area isotherms of DPPC+EA mixtures on water and buffer subphases indicated good lipid miscibility and some contraction of the monolayers at intermediate and higher surface pressures. Surface electric potential of the DPPC+EA monolayers showed excess values for intermediate DPPC:EA ratios. Fibrinogen adsorption and its adsorption rates from a dilute solution (0.03 mg/ml) were proportional to the fraction of EA in the monolayer indicating that protein binding was primarily driven by electrostatic interactions between positive EA charges in the monolayer and a net negative protein charge. At a higher protein concentration (0.06 mg/ml) both the fibrinogen adsorbed amount and its maximum adsorption rate showed excess values relative to the pure EA for 1:1, 2:1 and 3:1 DPPC+EA monolayers. This excess adsorption could be explained, in part, by the contraction of the monolayers with intermediate DPPC:EA ratios which resulted in an excess surface electric potential.

The effects of proteoglycan surface patterning on neuronal pathfinding.

Protein micropatterning techniques are increasingly applied in cell choice assays to investigate fundamental biological phenomena that contribute to the host response to implanted biomaterials, and to explore the effects of protein stability and biological activity on cell behavior for in vitro cell studies. In the area of neuronal regeneration the protein micropatterning and cell choice assays are used to improve our understanding of the mechanisms directing nervous system during development and regenerative failure in the central nervous system (CNS) wound healing environment. In these cell assays, protein micropatterns need to be characterized for protein stability, bioactivity, and spatial distribution and then correlated with observed mammalian cell behavior using appropriate model system for CNS development and repair. This review provides the background on protein micropatterning for cell choice assays and describes some novel patterns that were developed to interrogate neuronal adaptation to inhibitory signals encountered in CNS injuries.

Human Low Density Lipoprotein (LDL) and Human Serum Albumin (HSA) Co-Adsorption Onto the C18-Silica Gradient Surface.

Co-adsorption kinetics of human low density lipoprotein (LDL) and serum albumin (HSA) on hydrophilic/hydrophobic gradient silica surface were studied using Total Internal Reflection Fluorescence (TIRF) and autoradiography. Two experimental systems were examined: (1) fluorescein-labeled LDL (FITC-LDL) adsorption from a FITC-LDL + HSA solution mixture onto the octadecyldi-methylsilyl (C18)-silica gradient surface, and (2) the FITC-LDL adsorption onto the HSA pre-adsorbed on the C18-silica gradient surface. Experiments with fluorescein-labeled albumin (FITC-HSA) and unlabeled LDL have been performed in parallel. The adsorption kinetics of FITC-LDL onto the hydrophilic silica was found to be transport-limited and not affected by co-adsorption of HSA. A slower adsorption kinetics of lipoprotein onto the silica with pre-adsorbed HSA layer resulted from a slow appearance of LDL binding sites exposed by the process of HSA desorption. In the region of increasing surface density of C18 groups, the FITC-LDL adsorption rate fell below the transport-limited adsorption rate, except in the very early adsorption times. Pre-adsorption of HSA onto the C18-silica gradient region resulted in a significant decrease of both the FITC-LDL adsorption rate and adsorbed amount. The lowest FITC-LDL adsorption was found in the region of C18 self-assembled monolayer, where the pre-adsorbed HSA layer almost completely eliminated lipoprotein binding.

Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces.

Many research and commercial applications use a synthetic substrate which is seeded with cells in a serum-containing medium. The surface properties of the material influence the composition of the adsorbed protein layer, which subsequently regulates a variety of cell behaviors such as attachment, spreading, proliferation, migration, and differentiation. In this study, we examined the relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for MC3T3-E1 osteoblasts on glass surfaces modified with -SO(x), -NH(2), -N(+)(CH(3))(3), -SH, and -CH(3) terminal silanes. We also studied the relationship between cell spread area and migration rate for a variety of anchorage-dependent cell types on a model polymeric biomaterial, poly(acrylonitrile-vinylchloride). Our results indicated that MC3T3-E1 osteoblast behavior was surface chemistry dependent, and varied with individual functional groups rather than general surface properties such as wettability. In addition, cell migration rate was inversely related to cell spread area for MC3T3-E1 osteoblasts on a variety of silane-modified surfaces as well as for different anchorage-dependent cell types on a model polymeric biomaterial. Furthermore, the data revealed significant differences in migration rate among different cell types on a common polymeric substrate, suggesting that cell type-specific differences must be considered when using, selecting, or designing a substrate for research and therapeutic applications.

Atomic Force Microscopy and Wettability Study of Oxidized Patterns at the Surface of Polystyrene.

The surface properties of patterned surfaces made by a combination of photolithography and oxygen plasma treatment of polystyrene (PS) were investigated. PS and plasma-treated PS (PSox) were first characterized using X-ray photoelectron spectroscopy and the study of wetting dynamics (Wilhelmy plate method) in water and in solutions of different pH. The results indicated that the PSox surface may be viewed as covered with a polyelectrolyte-like gel, which swells depending on pH. It was then shown, using atomic force microscopy (AFM), that the adhesion force measured on PS with a silicon tip in water was higher compared with that measured on PSox. This feature allowed imaging of the oxidation patterns using the adhesion mapping mode. The origin of the pulloff force contrast, which could not be explained by combining Johnson-Kendall-Roberts theory and thermodynamic considerations, was attributed to repulsion between the tip and hydrated polymer chains present on the oxidized surface. Imaging was also performed in the lateral force mode, a higher friction being recorded on PS than on PSox. Copyright 1999 Academic Press.

Reflection interference contrast microscopy combined with scanning force microscopy verifies the nature of protein-ligand interaction force measurements.

The integration of a stand-alone scanning force microscope (SFM) scanner with a reflection interference contrast microscope (RICM) makes it possible to measure directly the separation distance between the SFM probe and the sample surface. The SFM-RICM combination, when applied to the force measurements between ligand-derivatized SFM probe and a protein receptor-derivatized surface, showed that the anomalous force discontinuities often observed for such interacting pairs were indeed a real behavior characteristic of a particular experimental configuration. Apart from small discrepancies due to transient damping, commercially available cantilevers did behave in an ideal mechanical fashion, thus indicating that protein-ligand unbinding events were occurring at distances much larger than their maximum extended length. This external verification of separation distance requires a closer examination of the physical events occurring upon detachment of the surfaces. An alternative interpretation of such force measurements is proposed here in which the protein and/or ligand immobilization chemistry is called into question.

Feasibility of measuring antigen-antibody interaction forces using a scanning force microscope.

The molecular affinity scanning force microscopy (MASFM) described in this study was developed in an effort to test the possibility of antigen-antibody binding measurement using force-separation distance profiles. The MASFM configuration was comprised of a spherical glass bead as an MASFM probe, to which the fluorescein antigen has been covalently attached, and a silicon dioxide-based substrate, to which the antifluorescyl IgG antibody was covalently bound. The bead was glued to the tip of a commercial SFM cantilever. Adhesion forces have been measured between two different specific antigen-antibody pairs and between nonspecific surfaces bearing only glycidoxypropylsilane immobilization chemistry. In force-separation (F-s) measurements, nonspecific forces displayed relatively few force discontinuities and mean adhesion forces lower than those found for specific antigen-antibody measurements. Force-separation profiles measured between specific antigen-antibody pairs showed many discontinuities and had higher mean forces. Positive controls revealed that the mean forces were slightly reduced by the addition of free ligand. The magnitude of mean forces did not correlate with the respective activation enthalpies of the proteins, as would be expected. At lower force values the force histograms for the specific pairs and for positive controls were indistinguishable. None of the force-separation data sets could fit a Poisson discrete-force model. This statistical analysis showed a large relative contribution from nonspecific interactions. It is concluded that the use of the large sphere as an SFM probe is counterproductive: while the large sphere does sample a larger number of specific interactions during each measurement, it also samples at the same time a large proportion of nonspecific forces. The presence of the nonspecific force contributions is likely due to the deformation of the polymerized GPS spacer layer which is thought to be delaminated from the surface upon the application of tension across the specific antigen-antibody bonds.

Adhesion mapping of chemically modified and poly(ethylene oxide)-grafted glass surfaces.

Two-dimensional mapping of the adhesion pull-off forces was used to study the origin of surface heterogeneity in the grafted poly(ethylene oxide) (PEO) layer. The variance of the pull-off forces measured over the µm-sized regions after each chemical step of modifying glass surfaces was taken to be a measure of the surface chemical heterogeneity. The attachment of γ-glycidoxypropyltrimethoxy silane (GPS) to glass decreased the pull-off forces relative to the clean glass and made the surface more uniform. The subsequent hydrolysis of the terminal epoxide groups resulted in a larger surface heterogeneity which was modeled by two populations of the terminal hydroxyl groups, each with its own distribution of adhesion forces and force variance. The activation of the hydroxyls with carbonyldiimmidazole (CDI) healed the surface and lowered its adhesion, however, the force variance remained rather large. Finally, the grafting of the α,ω-diamino poly(ethyleneoxide) chains to the CDI-activated glass largely eliminated adhesion except at a few discrete regions. The adhesion on the PEO grafted layer followed the Poisson distribution of the pull-off forces. With the exception of the glass surface, a correlation between the water contact angles and the mean pull-off forces measured with the Si(3)N(4) tip surfaces was found for all modified glass surfaces.

Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization.

Understanding the relationships between material surface properties, adsorbed proteins, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, we have prepared model surfaces with different functional groups to provide a range of surface wettability and charge. The cellular responses of attachment, spreading, and cytoskeletal organization have been studied following preadsorption of these surfaces with dilute serum, specific serum proteins, and individual components of the extracellular matrix. When preadsorbed with dilute serum, cell attachment, spreading, and cytoskeletal organization were significantly greater on hydrophilic surfaces relative to hydrophobic surfaces. Among the hydrophilic surfaces, differences in charge and wettability influenced cell attachment but not cell area, shape, or cytoskeletal organization. Moderately hydrophilic surfaces (20-40 degree water contact angle) promoted the highest levels of cell attachment. Preadsorption of the model surfaces with bovine serum albumin (BSA) resulted in a pattern of cell attachment very similar to that observed following preadsorption with dilute serum, suggesting an important role for BSA in regulating cell attachment to biomaterials exposed to complex biological media.

Tobacco mosaic virus adsorption on self-assembled and Langmuir-Blodgett monolayers studied by TIRF and SFM.

The adsorption of tobacco mosaic virus (TMV) on self-assembled and Langmuir-Blodgett monolayers was investigated using total internal reflection fluorescence (TIRF) spectroscopy and scanning force microscopy (SFM). Substrates were chosen to probe electrostatic, hydrophobic and surface fluidity effects on TMV adsorption. Positively charged and hydrophobic surfaces demonstrated similar initial rates of TMV adsorption; however, their respective surface TMV coverages differed greatly. Likewise, positively charged surfaces which differed primarily in surface fluidity exhibited similar adsorption rates for TMV, but different TMV surface coverages. In contrast, virus adsorption to negatively charged and zwitterionic substrates was negligible. To elucidate these differences in adsorption behavior, SFM was used to image the distribution and the aggregation state of adsorbed TMV.

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

A constant compliance force modulation technique for scanning force microscopy (SFM) imaging of polymer surface elasticity.

A new method of force modulation scanning force microscopy (SFM) imaging based on a constant compliance feedback loop is presented. The feedback adjusts the loading force applied by the SFM tip to the surface in order to maintain a constant compliance beneath the tip. The new method, constant compliance force modulation (CCFM), has the advantage of being able to quantify the loading force exerted by the tip onto the sample surface and thus to estimate the elastic modulus of the material probed by the SFM tip. Once the elastic modulus of one region is known, the elastic moduli of other surface regions can be estimated from the spatial map of loading forces using the Hertz model of deformation. Force vs. displacement measurements made on one surface locality could also be used to estimate the local modulus. Several model surfaces, including a rubber-toughened epoxy polymer blend which showed clearly resolved compliant rubber phases within the harder epoxy matrix, were analyzed with the CCFM technique to illustrate the method's application.

Human low density lipoprotein and human serum albumin adsorption onto model surfaces studied by total internal reflection fluorescence and scanning force microscopy.

The adsorption of low density lipoprotein (LDL) and human serum albumin (HSA) to model surfaces of different hydrophobicities has been studied using two, surface-sensitive, real-time, in situ techniques: total internal reflection fluorescence (TIRF) and scanning force microscopy (SFM). The model surfaces used were: (1) hydrophilic negatively charged silica (TIRF) and mica (SFM) surfaces, (2) hydrophobic octadecyldimethylsilyl-(ODS)-modified silica (TIRF) and ODS-modified oxidized silicon (SFM) surfaces and (3) amphiphilic ODS-silica gradient surfaces (TIRF). The kinetics of fluorescein isothiocyanate-LDL adsorption onto the ODS-silica gradient surface from FITC-LDL solution and from a solution mixture of LDL and HSA showed that a transport-limited process on the clean silica changed into an adsorption-limited process with increasing surface coverage of ODS chains. SFM analysis of the in situ adsorption of LDL on hydrophilic mica demonstrated a steady increase in surface coverage with time which was somewhat lower than determined by TIRF for FITC-LDL adsorption on silica. The adsorption behavior of a binary mixture of HSA and LDL suggested that lateral interactions between HSA and LDL affect the adsorption process. The diameter of LDL adsorbed on mica and ODS-modified silicon has been determined using SFM to be approximately 55 nm. Tetrameric LDL aggregates were observed on all of the surfaces in addition to some dimers and trimers. Imaging LDL and HSA adsorption on clean oxidized silicon surfaces using "contact mode' SFM techniques was hindered by probe manipulation of the proteins.

Protein adsorption on solid surfaces.

The research field of protein adsorption on surfaces appears to be as popular as ever. In the past year, several hundred published papers tackled problems ranging from fundamental aspects of protein surface interactions to applied problems of surface blood compatibility and protein surface immobilization. Although some parts of the protein adsorption process, such as kinetics and equilibrium interactions, can be accurately predicted, other aspects, such as the extent and the rate of protein conformational change, are still somewhat uncertain. The whole field is ripe for a comprehensive theory on protein adsorption.

Novel Method of Measuring Cantilever Deflection during an AFM Force Measurement.

A combination of a reflection interference contrast microscope (RICM) and the atomic force microscope (AFM) was used to monitor the cantilever-surface separation distance during force measurements using the streptavidin-biotin recognition pairs. The RICM showed that the cantilever loses contact with the surface before the final rupture of the adhesive bonds is measured by the AFM detection system. This finding suggests that the immobilization of biotin by physisorbed albumin and subsequent binding of streptavidin might have created a cross-linked protein network whose cohesion is tested by the AFM cantilever with the immobilized biotin ligands.

Effect of Tamm-Horsfall protein on calcium oxalate precipitation.

The effect of Tamm-Horsfall protein isolated from urine of healthy subjects on calcium oxalate precipitation was studied in model systems of precipitation. The study was performed using following conditions: concentrations of calcium chloride 10 mmol/l, sodium chloride 150 mmol/l, oxalic acid 300 mumol/l; pH 6.0, and temperature 310 K. The concentration of Tamm-Horsfall protein varied between 1-10 mg/l. The kinetics of calcium oxalate precipitation was observed by measuring the number and volume of particles in the suspension, and the precipitate composition by an optic microscope. In all the studied systems, the precipitate morphology corresponded to pure calcium oxalate monohydrate. Tamm-Horsfall protein was found to inhibit the growth of calcium oxalate monohydrate crystals and stimulate their aggregation in the given experimental conditions. Both effects were enhanced by increase in the concentrations of Tamm-Horsfall protein and were most pronounced at the concentration of Tamm-Horsfall protein of 10 mg/l.

Fluorescence assay for measuring lipid deposits on contact lens surfaces.

A simple fluorescence assay for estimating the propensity of the contact lens surface towards the uptake of lipids and formation of lipid deposits has been developed. The assay is based on the incubation of the contact lenses with an artificial tear-lipid mixture, staining of the lenses with a fluorescent lipid probe, Nile Red, and subsequent macroscopic imaging of the fluorescent lipid deposits on the contact lens surface with a thermoelectrically cooled charge-coupled device (CCD) camera. The results of the assay show that statistically significant differences in the formation of lipid deposits exist between the surfaces of different contact lens materials.

The desorption of ribonuclease A from charge density gradient surfaces studied by spatially-resolved total internal reflection fluorescence.

A quaternary amine surface gradient was prepared on fused silica by a three-step surface modification process. The gradient surface displayed a transition of surface charges along the gradient dimension from a net negative surface charge of silica to a net positive surface charge at the quaternary amine end. The gradient surface was characterized by X-ray photoelectron spectroscopy, ellipsometry, colloidal gold decoration, and dynamic contact angle measurements. It displayed an increased adhesion of negatively charged gold particles towards the quaternary amine end. The water contact angles also increased with the increased surface density of aminopropylsilyl groups. The desorption of ribonuclease A labeled with fluorescein-5-isothiocyanate (FITC-RNase) from the quaternary amine gradient surface was measured using spatially resolved total internal reflection fluorescence (TIRF) spectroscopy. The experimental FITC-RNase desorption results fitted exceptionally well to a two adsorbed protein populations model. A tentative assignment of the two adsorbed protein populations is proposed based on the effect of the ionic strength of the desorbing buffer. The faster desorption population interacted primarily with the quaternary amine gradient surface sites through electrostatic interactions. The slower desorption population interacted with the surface sites via hydrophobic and possibly some electrostatic interactions.