Competitive protein adsorption on polysaccharide and hyaluronate modified surfaces

Adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) was measured on six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and Streptococcus zooepidemicus. Film thickness and surface morphology depended on the HA molecular weight and concentration. BSA coverage was enhanced on surfaces in competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of HA utilized. With changing bulk protein concentration from 20 to 40 μg ml^?1 for each species, Fg coverage on silicon increased by 4x, whereas both BSA and Fg adsorption on dextran and HA were far less dependent on protein bulk concentration.     

A quantitative and selective chromatography method for determining coverages of multiple proteins on surfaces

Competitive protein adsorption plays a key role in the surface hemocompatibility of biological implants. We describe a quantitative chromatography method to measure the coverage of multiple proteins physisorbed to surfaces. In this method adsorbed proteins are displaced by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and then analyzed by high performance liquid chromatography to separate and quantify the individual proteins, in this case bovine serum albumin (BSA) and bovine fibrinogen (Fg). CHAPS displaced over 95% of the adsorbed proteins and was easily removed from solution by dialysis. This method was tested by measuring the coverage of BSA, 66 kDa, and Fg, 340 kDa, simultaneously adsorbed from solutions with concentration of 20 microg/ml, on bare and dextranized silicon. Relative to silicon, the dextranized surfaces were found to strongly inhibit protein adsorption, decreasing BSA and Fg coverages by 76 and 60%, respectively.     

Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.     

Salivary protein adsorption onto hydroxyapatite and sds‐mediated elution studied by in situ ellipsometry

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na⁺, whereas Ca²⁺ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.     

原位椭圆偏振术研究牛血清清蛋白在固/液界面的吸附

用原位椭圆偏振术系统研究了硅片表面因素及缓冲液环境因素对牛血清清蛋白在固/液界面吸附的影响。在生理条件下,疏水表面与亲水表面相比BSA吸附量较大。随着硅片表面电荷密度增加,BSA吸附量增加。BSA吸附量当体溶液pH值等于BSA等电点时达到最大。而随着体溶液离子强度增加,BSA吸附量亦上升。实验结果提示:除了熵驱动作用之外,硅片表面与BSA分子及BSA分子之间的静电作用在BSA吸附中起着十分重要的作用。     

Emulsan, a tailorable biopolymer for controlled release

Microsphere hydrogels made with emulsan-alginate were used as carrier for the microencapsulation of blue dextran in order to study the effect of emulsan on the alginate bead stability. Blue dextran release studies indicated an increase of microsphere stability in presence of emulsan, as a coating agent. BSA adsorption by emulsan-alginate microspheres is also enhanced 40% compared to alginate alone. XPS studies confirm the presence of BSA adsorbed on emulsan microsphere surfaces. These results are in agreement with the equilibrium adsorption model of Freundlich. Studies of BSA adsorption using non-equilibrium Lagergren second-order and intraparticle models, are suggesting a complex mechanisms of protein adsorption by chemisorption and intraparticle diffusion. Also, enzymatic release of BSA from emulsan microspheres containing azo-BSA under physiological conditions is suggests the possibility of using microspheres as a depot for pre-proteins of medical interest.     

Selective adsorption of serum albumin on biomedical polyurethanes modified by a poly(ethylene oxide) coupling-polymer with cibacron blue (F3G-A) endgroups

A tri-block-coupling polymer, "PEO-MDI-PEO" ["poly(ethylene oxide)-4,4'-methylene diphenyl diisocyanate-poly(ethylene oxide)", abbreviated "MPEO"], was used to react with a triazine dye, Cibacron Blue F3G-A (ciba), in an alkaline environment. The product of this nucleophilic reaction was a penta-block-coupling polymer, "ciba-PEO-MDI-PEO-ciba" (abbreviated "cibaMPEO"). The cibaMPEO-modified poly(ether urethane) (PEU) surfaces were prepared by dip-coating and detected by XPS. The surface enrichment of both ciba endgroups and poly(ethylene oxide) spacer-arms was revealed. On the modified surfaces, bovine serum albumin (BSA)-adsorbing experiments were carried out, respectively, in the low and high BSA bulk-concentration solutions, and accordingly, the methods of radioactive (125)I-probe and ATR-FTIR were, respectively, employed for the characterization. The competitive adsorption of BSA and bovine serum fibrinogen (Fg) in the BSA-Fg binary solutions was also studied using a (125)I-probe, and through which the reversibly BSA-selective adsorption on cibaMPEO-modified PEU surfaces was confirmed. Finally, the improvement of blood-compatibility on the modified surfaces was verified by the plasma recalcification time (PRT) test.     

Protein resistance of dextran and dextran-poly(ethylene glycol) copolymer films

The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1–1.2 mg m^?2) of three molecular weights (10,000, 66,900, 400,000 g mol^?1) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ～5 to 0.5 mg m^?2 with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (～2 mg m^?2) indicating ternary adsorption of the smaller protein within the dextran layer.     

Protein resistance of dextran and dextran-poly(ethylene glycol) copolymer films

The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1-1.2 mg m(-2)) of three molecular weights (10,000, 66,900, 400,000 g mol(-1)) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ～5 to 0.5 mg m(-2) with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (～2 mg m(-2)) indicating ternary adsorption of the smaller protein within the dextran layer.     

Effect of Air Plasma Processing on the Adsorption Behaviour of Bovine Serum Albumin on Spin-Coated PMMA Surfaces

This paper reports the adsorption of Bovine Serum Albumin （BSA） onto Dielectric Barrier Discharge （DBD） processed Poly（methyl methacrylate） （PMMA） surfaces by a Quartz Crystal Microbalance with Dissipation monitoring （QCM-D） technique. The purpose is to study the influence of DBD processing on the nature and scale of BSA adsorption on PMMA surface in vitro. It was observed that DBD processing improves the surface wettability of PMMA film, a fact attributable to the changes in surface chemistry and topography. Exposure of the PMMA to Phosphate Buffed Saline （PBS） solution in the QCM-D system resulted in surface adsorption which reaches an equilibrium after about 30 minutes for pristine PMMA, and 90 minutes for processed PMMA surface. Subsequent injection of BSA in PBS indicated that the protein is immediately adsorbed onto the PMMA surface. It was revealed that adsorption behaviour of BSA on pristine PMMA differs from that on processed PMMA surface. A slower adsorption kinetics was observed for pristine PMMA surface, whilst a quick adsorption kinetics for processed PMMA. Moreover, the dissipation shift of protein adsorption suggested that BSA forms a more rigid structure on pristine PMMA surface that on processed surface. These data suggest that changes in wettability and attendant chemical properties and surface texture of the PMMA surface may play a significant role in BSA adsorption process.     

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.     

Optimisation of glass surfaces for optical immunosensors

The surfaces of glass sensor chips were modified with dextran to generate a layer protecting the sensor surface from unspecific protein binding and also serving as a matrix for covalent protein immobilisation. Dextran was coupled to the glass surface in different concentrations either covalently on amino-functionalised glass chips or via biotin-avidin binding. Unspecific binding of BSA was monitored with the grating coupler system, and was increasingly suppressed with increasing dextran concentrations. Using a solution with 100 mg/ml carboxymethylated dextran decreased the signals to approximately 2% of those obtained at an untreated glass chip. Antibodies were successfully immobilised in the dextran and binding to the corresponding Cy5-labelled antigen was repeatedly monitored using a fluorescence sensor system (total internal reflection fluorescence (TIRF)).     

The prevention of adsorption of interferents to radiolabelled protein by Tween 20

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.     

Human palatal saliva: adsorption behaviour and the role of low-molecular weight proteins

In situ ellipsometry was employed to study adsorption from human palatal saliva (HPalS) in terms of dependence on surface wettability and saliva concentration (相似文献   

Adsorption of avidin on microfabricated surfaces for protein biochip applications

The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radiofrequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that a simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

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

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

Apparent activation energies associated with protein dynamics on hydrophobic and hydrophilic surfaces

With the use of single-molecule total internal reflection fluorescence microscopy (TIRFM), the dynamics of bovine serum albumin (BSA) and human fibrinogen (Fg) at low concentrations were observed at the solid-aqueous interface as a function of temperature on hydrophobic trimethylsilane (TMS) and hydrophilic fused silica (FS) surfaces. Multiple dynamic modes and populations were observed and characterized by their surface residence times and squared-displacement distributions (surface diffusion). Characteristic desorption and diffusion rates for each population/mode were generally found to increase with temperature, and apparent activation energies were determined from Arrhenius analyses. The apparent activation energies of desorption and diffusion were typically higher on FS than on TMS surfaces, suggesting that protein desorption and mobility were hindered on hydrophilic surfaces due to favorable protein-surface and solvent-surface interactions. The diffusion of BSA on TMS appeared to be activationless for several populations, whereas diffusion on FS always exhibited an apparent activation energy. All activation energies were small in absolute terms (generally only a few kBT), suggesting that most adsorbed protein molecules are weakly bound and move and desorb readily under ambient conditions.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.