Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study

Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities.     

Interpretation of protein adsorption: surface-induced conformational changes

Protein adhesion plays a major role in determining the biocompatibility of materials. The first stage of implant integration is the adhesion of protein followed by cell attachment. Surface modification of implants (surface chemistry and topography) to induce and control protein and cell adhesion is currently of great interest. This communication presents data on protein adsorption (bovine serum albumin and fibrinogen) onto model hydrophobic (CH(3)) and hydrophilic (OH) surfaces, investigated using a quartz crystal microbalance (QCM) and grazing angle infrared spectroscopy. Our data suggest that albumin undergoes adsorption via a single step whereas fibrinogen adsorption is a more complex, multistage process. Albumin has a stronger affinity toward the CH(3) compared to OH terminated surface. In contrast, fibrinogen adheres more rapidly to both surfaces, having a slightly higher affinity toward the hydrophobic surface. Conformational assessment of the adsorbed proteins by grazing angle infrared spectroscopy (GA-FTIR) shows that after an initial 1 h incubation few further time-dependent changes are observed. Both proteins exhibited a less organized secondary structure upon adsorption onto a hydrophobic surface than onto a hydrophilic surface, with the effect observed greatest for albumin. This study demonstrates the ability of simple tailor-made monochemical surfaces to influence binding rates and conformation of bound proteins through protein-surface interactions. Current interest in biocompatible materials has focused on surface modifications to induce rapid healing, both of implants and for wound care products. This effect may also be of significance at the next stage of implant integration, as cell adhesion occurs through the surface protein layer.     

Poly(N-vinylpyrrolidone)-modified surfaces repel plasma protein adsorption

The present work aimed to study the interaction between plasma proteins and PVP-modified surfaces under more complex protein conditions.In the competitive adsorption of fibrinogen(Fg) and human serum albumin(HSA),the modified surfaces showed preferential adsorption of HSA.In 100%plasma,the amount of Fg adsorbed onto PVP-modified surfaces was as low as 10 ng/cm~2,suggesting the excellent protein resistance properties of the modified surfaces.In addition, immunoblots of proteins eluted from the modified surfaces after plasma contact confirmed that PVP-modified surfaces can repel most plasma proteins,especially proteins that play important roles in the process of blood coagulation.     

In deuterated water the unspecific adsorption of proteins is significantly slowed down: results of an SPR study using model organic surfaces

The control of unspecific adsorption of proteins to natural and technical surfaces plays an important role in biology and also for many applications. Organic model surfaces, e.g., self-assembled monolayers, are often used to identify fundamental surface and/or protein properties that rule protein adsorption. Some techniques involved in biointerface research require the use of heavy water, e.g. neutron scattering techniques. Also in NMR studies D(2)O is the solvent of choice when focusing on biomolecular and hydration dynamics. So far several studies have been concerned with the characterization of the unspecific adsorption of proteins from normal water buffers. In the present work, we report a comparison of the unspecific protein adsorption from normal and heavy water buffers. So far it has been assumed that the surface kinetic of the unspecific adsorption is unaffected by the substitution of water by D(2)O. However, for the four proteins investigated here, this assumption does not hold. The ratio k(H)/k(D) of the adsorption rate constants of the different buffer conditions describe the strength of the isotope effect. We have measured ratios between 1.0 and 2.6, indicating that the adsorption kinetics are strongly affected by a H(2)O-D(2)O substitution.     

Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry

Protein adsorption behavior is at the heart of many of today's research fields including biotechnology and materials science. With understanding of protein-surface interactions, control over the conformation and orientation of immobilized species may ultimately allow tailor-made surfaces to be generated. In this contribution protein-surface interactions have been examined with particular focus on surface curvature with and without surface chemistry effects. Silica spheres with diameters in the range 15-165 nm with both hydrophilic and hydrophobic surface chemistries have been used as model substrates. Two proteins differing in size and shape, bovine serum albumin (BSA) and bovine fibrinogen (Fg), have been used in model studies of protein binding with detailed secondary structure analysis being performed using infrared spectroscopy (IR) on surface-bound proteins. Although trends in binding affinity and saturation values were similar for both proteins, albumin is increasingly less ordered on larger substrates, while fibrinogen, in contrast, loses secondary structure to a greater extent when adsorbing onto particles with high surface curvature. These effects are compounded by surface chemistry, with both proteins becoming more denatured on hydrophobic surfaces. Both surface chemistry and topography play key roles in determining the structure of the bound proteins. A model of the binding characteristics of these two proteins onto surfaces having differing curvature and chemistry is presented. We propose that properties of an adsorbed protein layer may be guided through careful consideration of surface structure, allowing the fabrication of materials/surface coatings with tailored bioactivity.     

Surface enrichment of proteins at quartz/water interfaces: a neutron reflectivity study

Neutron reflectivity (NR) was used to study the adsorption of human serum albumin and human fibrinogen on quartz. The proteins were individually and sequentially adsorbed from heavy water and heavy water/methanol mixtures at pH 4 and 7.0. The technique allows for the subnanometer resolution of the adsorbed layer thickness and gross morphology. Under the conditions of our measurements we found that fibrinogen formed a distinct layer that we interpret as a mat of the protein three layers thick whereas albumin formed only diffuse layers. The adsorption pattern of the two proteins changed radically when one protein was adsorbed on top of the other (previously adsorbed). In general our measurements indicate that the adsorbed protein layers on quartz are rather loosely bound and that these layers, incorporating as much as 80% water, extend further into the bulk fluid than might have been expected.     

Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay

Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.     

Albumin and fibrinogen adsorption onto phosphatidylcholine monolayers investigated by Fourier transform infrared spectroscopy

Dipalmitoylphosphatidylcholine (DPPC) monolayers were deposited onto a germanium attenuated total reflectance (ATR) crystal using the Langmuir–Blodgett technique. The DPPC-coated crystal was then exposed to human serum albumin or human fibrinogen solutions while measuring the protein adsorption by recording FTIR spectra. The effect of the zwitterionic nature of the DPPC polar headgroup towards protein adsorption has been ascertained by exposing either the phospholipid headgroup or the acyl chains to the protein solution; this was possible by the use of a silanized or a bare germanium crystal. Calibration curves have been made to measure the protein surface concentrations. After 3 h, the albumin surface concentration on DPPC monolayers was about three times higher when the proteins were exposed to the lipid acyl chains instead of the polar headgroups (e.g. 3 vs. 1 μg cm⁻²). As for fibrinogen (FGN) adsorption, when the lipid polar headgroups were exposed to the protein solution, the FGN adsorption was low reaching a maximum value of 0.5 μg cm⁻². When interacting with the lipid acyl chains, the FGN adsorption reached a plateau at a value of 2.1 μg cm⁻² after 3 h. Clearly, both albumin and FGN showed a low tendency to adsorb on surfaces where the lipid polar headgroups are exposed toward the protein solution.     

Effect of surface composition on the adsorption of photosystem I onto alkanethiolate self-assembled monolayers on gold

We have used self-assembled monolayers (SAMs) prepared from omega-terminated alkanethiols on gold to generate model surfaces and examine the effect of surface composition on the adsorption of Photosystem I (PSI), stabilized in aqueous solution by Triton X-100. Triton-stabilized PSI adsorbs to high-energy surfaces prepared from HO- and HO2C-terminated alkanethiols but does not adsorb to low-energy surfaces. The inhibition of PSI adsorption at low-energy surfaces is consistent with the presence of a layer of Triton X-100 that adsorbs atop the hydrophobic SAM and presents a protein-resistant poly(ethylene glycol) (PEG) surface. While the presence of the PEG surface prevents the adsorption of PSI, the displacement of the inhibiting layer of Triton X-100 by dodecanol, a more active surfactant, greatly enhances the adsorption of PSI. This inhibiting effect by Triton X-100 can be extended to other protein systems such as bovine serum albumin.     

白蛋白原位复合的生物医用功能材料的研究(Ⅱ)──白蛋白的选择性吸附和血液相容性研究

采用¹²⁵I放射标记技术研究了血浆白蛋白和纤维蛋白原在聚甲基丙烯酸甲酯-接枝-十八烷基聚氧乙烯(PMMA-g-SPEO)、聚甲基丙烯酸甲酯-接枝-乙基聚氧乙烯(PMMA-g-EPEO)和聚甲基丙烯酸甲酯-甲基丙烯酸十八酯共聚物(PMMA-co-SMA)表面的竞争吸附行为.结果表明,十八烷基聚氧乙烯复合修饰的PMMA-g-SPEO可高选择性地形成白蛋白可逆吸附层,有效地阻抗血小板的粘附,延长材料的凝血时间,是一种理想的白蛋白原位复合的生物医用功能材料.     

Polymer surface reorientation after protein adsorption

Surface side-chain orientation changes of two polymers have been observed upon protein adsorption using sum frequency generation vibrational spectroscopy. Side-chain-deuterated poly(ethyl methacrylate) and poly(n-butyl methacrylate) were contacted with five protein solutions: albumin, fibrinogen, ubiquitin, cytochrome c, and lysozyme. The CD(3)/CD(2) symmetric stretch ratios of the surface polymer side chains in contact with these different media were compared to each other and to that of the polymer contacting air or phosphate buffered saline. The adsorption of different proteins to the surfaces resulted in polymer side-chain orientations slightly different from each other, with orientations between the air and buffer cases.     

聚(N-异丙基丙烯酰胺)改性硅表面与血浆蛋白质的相互作用

通过表面引发原子转移自由基聚合(ATRP)在硅表面接枝了聚(N-异丙基丙烯酰胺)(PNIPAAm)聚合物刷,并考察了PNIPAAm改性表面在单一蛋白质溶液以及血浆中与血浆蛋白质之间的相互作用.蛋白质吸附测试表明,与未改性的硅表面相比,改性后的表面对纤维蛋白原的吸附量大大降低,特别是在血浆中纤维蛋白原吸附量小于5ng/c...     

Characterization of fibrinogen adsorption onto glass microcapillary surfaces by ELISA

Adsorption of biomolecules onto microchannel surfaces remains a critical issue in microfluidic devices. This paper investigates the adsorption of fibrinogen on glass microcapillaries using an immunoassay method (ELISA) and X-ray photoelectron spectroscopy (XPS). Various adsorption conditions such as protein concentrations and incubation times, buffer pH, buffer ionic strengths and effects of flow are presented. ELISA is successfully demonstrated as a facile and robust technique to examine these phenomena. The highest adsorption level occurs near the isoelectric point of fibrinogen (pH 5.0) and low buffer ionic strengths (0-8 mM). Microchannel surface saturation was achieved at a fibrinogen solution concentration of approximately 50 microg ml(-1). Fibrinogen adsorption under flow was always higher than that seen in static systems. The importance of diffusion phenomena in microchannels on protein adsorption was demonstrated. ELISA experiments using fused silica and PEEK have also confirmed significant adsorption on these mass spectrometer transfer line materials.     

Surface modification on microfluidic devices with 2-methacryloyloxyethyl phosphorylcholine polymers for reducing unfavorable protein adsorption

Surface modification of polymer materials for preparing microfluidic devices including poly(dimethyl siloxane) (PDMS) was investigated with phospholipids polymers such as poly(2-methacryloyloxylethyl phosphorylcholine(MPC)-co-n-butyl methacrylate) (PMB) and poly(MPC-co-2-ethylhexyl methacrylate-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED). The hydrophilicity of every surface on the polymer materials modified with these MPC polymers increased and the value of zeta-potential became close to zero. The protein adsorption on the polymer materials with and without the surface modification was evaluated using a protein mixture of human plasma fibrinogen and serum albumin. Amount of proteins adsorbed on these polymeric materials showed significant reduction by the surface modification with the MPC polymers compared to the uncoated surfaces ranging from 56 to 90%. Furthermore, we successfully prepared PDMS-based microchannel which was modified by simple coating with the PMB and PMED. The modified microchannel also revealed a significant reduction of adsorption of serum albumin. We conclude that the MPC polymers are useful for reducing unfavorable protein adsorption on microfluidic devices.     

Influence of PEG architecture on protein adsorption and conformation

Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers with various grafting ratios were adsorbed to niobium pentoxide-coated silicon wafers and characterized before and after protein adsorption using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Three proteins of different sizes, myoglobin (16 kD), albumin (67 kD), and fibrinogen (340 kD), were studied. XPS was used to quantify the amount of protein adsorbed to the bare and PEGylated surfaces. ToF-SIMS and principal component analysis (PCA) were used to study protein conformational changes on these surfaces. The smallest protein, myoglobin, generally adsorbed in higher numbers than the much larger fibrinogen. Protein adsorption was lowest on the surfaces with the highest PEG chain surface density and increased as the PEG layer density decreased. The highest adsorption was found on lysine-coated and bare niobium surfaces. ToF-SIMS and PCA data evaluation provided further information on the degree of protein denaturation, which, for a particular protein, were found to decrease with increasing PEG surface density and increase with decreasing protein size.     

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

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

Adsorption-induced conformational changes of proteins onto ceramic particles: differential scanning calorimetry and FTIR analysis

Three model proteins, bovine serum albumin, hen's egg lysozyme and bovine serum fibrinogen, were adsorbed from aqueous solution onto finely dispersed ceramic particles, namely different kinds of alumina and hydroxyapatite particles. The influence of adsorption on protein secondary structure was investigated. The FTIR spectroscopic findings were compared with the results of DSC measurements. In almost all cases it was found that adsorption results in destabilisation and structural loss of the bound protein. A decrease in transition enthalpy is correlated with a loss in alpha-helical structure, which seems to be the most sensitive structure on adsorption-induced rearrangements. A total collapse of structure in the adsorbed proteins was not determined on any ceramic surface. Some residual structure is always retained. Structural changes in the D- or E-domains of fibrinogen could be independently observed by two different calorimetric signals. The two techniques applied in the present study -- micro-DSC and FTIR spectroscopy -- can be concluded to provide complementary information on adsorption-induced structural changes on both the molecular (thermal stability, overall structure) and the sub-molecular level (secondary structure).     

Poly(acrylic acid)-poly(ethylene glycol) layers on positively charged surface coatings: molecular structure, protein resistance, and application to single protein deposition

A new copolymer (PAA-PEG2000) has been designed, consisting of a negatively charged poly(acrylic acid) (PAA) backbone to which poly(ethylene glycol) (PEG) side chains with a molecular weight of about 2 kDa were grafted in a molecular ratio of 3:10. It readily adsorbs to positively charged surfaces and may be considered to be the anionic counterpart of PEG-grafted poly(l-lysine) (PLL-PEG), which was first described by Kenausis et al. and is widely used to render negatively charged surfaces protein-resistant. The synthesis of PAA-PEG2000 can be carried out in aqueous solution at room temperature and does not require any sophisticated techniques such as handling in an inert gas atmosphere. Using ellipsometry and infrared reflection absorption spectroscopy (IRRAS), the film structure has been carefully analyzed for copolymer adsorption onto three different positively charged surfaces, namely, thin layers of poly(allylamine) (PAH), poly(ethyleneimine) (PEI) and (3-aminopropyl)triethoxysilane (APTES). Besides the film thickness, the conformation of the PEG chains and their orientation with respect to the surface normal appear to be important parameters for the protein resistance of the films. Although PAA-PEG2000 adsorbed to PAH and PEI renders the surfaces inert, only partial protein resistance has been observed if the copolymer is deposited on APTES. In a model application, we have generated heterogeneous surfaces composed of isolated small Au nanoparticles (AuNP's) embedded in a protein-resistant layer of PAA-PEG2000 and demonstrated that the AuNP's can serve as adsorption sites for single protein species. In the future, these nanopatterned surfaces may be used for the investigation of isolated proteins.     

Anti-fouling chemistry of chiral monolayers: enhancing biofilm resistance on racemic surface

This work reports the resistance to protein adsorption and bacterial biofilm formation by chiral monolayers of polyol-terminated alkanethiols surrounding micrometer-sized patterns of methyl-terminated alkanethiols on gold films. We discover that patterned surfaces surrounded by chiral polyol monolayers can distinguish different stages of biofilm formation. After inoculation on the surfaces, bacteria first reversibly attached on the chiral polyol monolayers. Over time, the bacteria detached from the polyol surfaces, and attached on the hydrophobic micropatterns to form biofilms. Interestingly, while both enantiomers of gulitol- and mannonamide-terminated monolayer resisted adsorption of proteins (bovine serum albumin, lysozyme, and fibrinogen) and confined biofilms formed on the micropatterns, the monolayers formed by the racemic mixture of either pair of enantiomers exhibited stronger antifouling chemistry against both protein adsorption and biofilm formation than monolayers formed by one enantiomer alone. These results reveal the different chemistries that separate the different stages of biofilm formation, and the stereochemical influence on resisting biofoulings at a molecular-level.     

Conformational reorientation of immunoglobulin G during nonspecific interaction with surfaces.

To achieve a better understanding of the nonspecific adsorption process of proteins on solid surfaces, the mechanism of this interaction was investigated by a model system comprising the structurally flexible ("soft") protein goat anti-rabbit immunoglobulin G and a set of chemically defined surfaces. The thermodynamic properties of both protein and surfaces were derived from contact angle measurements by applying the Lifshitz-van der Waals acid-base approach, and the Gibbs free enthalpy of interaction was calculated. The protein shows two conformational states, one hydrophobic and the other hydrophilic. The interaction energy indicates that the hydrophobic conformation favorably adsorbs onto the surfaces. With real-time binding kinetics, measured by a supercritical angle fluorescence biosensor, we show that during the nonspecific adsorption the protein performs a reorientation in its three-dimensional amino acid structure from a hydrophilic to a hydrophobic molecular structure. Unlike the rates of adsorption and desorption, the transition rate is independent of the type of surface and only influenced by the structural reorganization of the protein.