Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene
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We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins β-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PAA21-b-PAPEO14 (poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide)), with a negatively charged PAA block and a neutral PAPEO block and a positively charged homopolymer P2MVPI (poly(N-methyl 2-vinyl pyridinium iodide). In solution, these C3Ms partly disintegrate at salt concentrations between 50 and 100 mM NaCl. Adsorption of C3Ms and proteins has been studied with fixed-angle optical reflectometry, at salt concentrations ranging from 1 to 100 mM NaCl. In comparison with the adsorption of PAA21-b-PAPEO14 alone adsorption of C3Ms significantly increases the amount of PAA21-b-PAPEO14 on the surface. This results in a higher surface density of PEO chains. The stability of the C3M coatings and their influence on protein adsorption are determined by the composition and the stability of the C3Ms in solution. A C3M-PAPEO14/P2MVPI43 coating strongly suppresses the adsorption of all proteins on silica and polystyrene. The reduction of protein adsorption is the highest at 100 mM NaCl (>90%). The adsorbed C3M-PAPEO14/P2MVPI43 layer is partly removed from the surface upon exposure to an excess of β-lactoglobulin solution, due to formation of soluble aggregates consisting of β-lactoglobulin and P2MVPI43. In contrast, C3M-PAPEO14/P2MVPI228 which has a fivefold longer cationic block enhances adsorption of the negatively charged proteins on both surfaces at salt concentrations above 1 mM NaCl. A single PAA21-b-PAPEO14 layer causes only a moderate reduction of protein adsorption.
KeywordsGrafted block copolymer Complex coacervate core micelles Protein adsorption Silica Polystyrene
The aim of this work is to investigate the formation and stability of grafted block C3Ms in solution, their adsorption on silica and polystyrene, and the effect of the adsorbed C3M layer on protein adsorption.
Materials and methods
Sodium chloride (NaCl), sodium hydroxide (NaOH, 1 M), and hydrochloric acid (HCl, 1 M) were purchased from Sigma. All chemicals were used as received.
Silicon wafers (boron doped, orientation 100, and resistivity 7–15 Ω cm−1) were purchased from WaferNet, Inc., USA.
Quaternization of P2VP
P2VP43 and P2VP41-b-PEO204 were quaternized according to the following procedure: dissolved in 35 ml of N,N-dimethylmethanamide was 1 g of (co)polymer. Three milliliters of iodomethane were added, and the mixture was allowed to react for 48 h at 60 °C, under nitrogen gas flow and stirring. A second portion of 3 ml iodomethane was added 24 h after the beginning of the reaction. Subsequently, the quaternized polymer was precipitated with ether, filtered, washed several times with fresh portions of ether, and dried overnight in a vacuum oven at 48 °C. The degree of quaternization of P2MVPI43 was determined by elemental analysis to be approximately 89% . The degree of quaternization of P2MVPI41-b-PEO204 was verified by dynamic light scattering titration with PAA139 at pH 7. At stoichiometric charge ratio micelles are formed, and the position of the maximum intensity provides an estimation of the degree of quaternization.
Characterization of grafted block C3Ms in solution
Dynamic light scattering (DLS) titrations were performed in order to determine the PMC. During the titration, P2MVPI solutions were titrated into PAA21-b-PAPEO14 solution. The concentration of PAA21-b-PAPEO14 was 0.1 g/l, and the concentration of P2MVPI varied from 1 to 5 g/l. Polymer solutions were prepared in 10 mM NaCl. Titrations were performed using an ALV light scattering instrument equipped with an ALV_500 digital correlator and a 300 mW argon ion laser (532 nm), at 25 ± 0.5 °C and 90˚ detection angle. Decalin was used as a refractive index matching medium.
Zeta potentials of C3Ms in solution were measured with a Nanosizer (Nano ZS, Malvern Instruments). Samples at different mixing ratios (f-) of PAA21-b-PAPEO14 and P2MVPI were prepared in 10 mM NaCl solution. Conversion of the acrylic acid block to sodium acrylate was done by addition of a 1 M NaOH solution. The total polymer concentration was 0.5 g/l. Samples were measured 14 h after mixing to ensure equilibrium.
Preparation of protein solutions
Protein solutions were prepared prior to each experiment in MilliQ water with the addition of the required amount of NaCl. The pH was adjusted to 7 ± 0.1 with 1 and 0.1 M NaOH or HCl.
Preparation of the surfaces
Silica surfaces were prepared by heating silicon wafers at 1,000 °C for about 1.5 h. As a result, an oxide layer of 80–100 nm was formed. Prior to the measurements, oxidized wafers were cut into strips of approximately 1 × 5 cm2, immersed in a freshly prepared piranha solution (one part of 35% H2O2 and two parts of 95% H2SO4) for 2–3 min and rinsed with MilliQ water. Cleaned strips were stored in MilliQ water, in separate containers, to avoid contamination with dust.
Polystyrene (PS) surfaces were prepared according to the method described by Sieval et al. [23, 24] introducing slight modifications: strips of silica wafer were cleaned with a piranha solution, rinsed with MilliQ water, dried with acetone, and etched with 40% HF for 2–3 min to remove an oxide layer. Etched strips were immediately transferred into a solution of styrene in toluene (10%), and the solution was heated up to 100 °C. The surface polymerization reaction continued for about 1.5 h under nitrogen gas flux and reflux. After the reaction the strips were rinsed with fresh toluene several times to remove ungrafted polymer and finally left to dry in air. On these chemically grafted layers, an additional layer of polystyrene was spincoated (RDE-/SPINCOATER Motorcontroller, Eco Chemie B.V) from a solution in toluene (13 g/l). Coated strips were stored in MilliQ water. Surfaces used in experiments were not older than 3–4 days, and their quality was monitored with an optical (Olympus BX60 with Olympus DP70 camera) and a SEM microscope (JEOL SEM-6480LV).
The thickness of the silica and PS layers was determined by ellipsometry (SE 400, SENTECH Instruments GmbH, Germany). The complex refractive index of the silicon layer was set to n = 3.85, κ = 0.02, and the refractive indexes of silica and PS layer were 1.46 and 1.56, respectively. The thickness was measured at several positions of the strip, and an average of at least five measurements has been taken for calculation of the sensitivity factor used in reflectometry.
Determination of the refractive index increment (dn/dc)
Refractive index increments (dn/dc) of the polymers were determined with a differential refractive index detector (Shodex RI-71, Separations). Prior to the measurements, the instrument was calibrated with NaCl. C3Ms and polymer solutions were prepared and measured in 10 mM NaCl. Conversion to sodium acrylate was done by addition of a 1 M NaOH solution. Calibration and dn/dc values were determined based on at least five different concentrations of each sample.
The dn/dc of the adsorbed C3Ms cannot be measured directly but has to be estimated from the corresponding values measured in solution. The experimental dn/dc as measured in solution consists of two components: the dn/dc of polyelectrolytes forming the C3Ms and the dn/dc of the corresponding (expelled) counterions (salt). We assume that the adsorbed layer of C3Ms is homogeneous, but it is likely that this layer consists of sub-layers (complex coacervate layer, brush layer). The layer consists of a mixture of charged diblock copolymer and an oppositely charged homopolymer at stoichiometric charge ratio, assuming absence of excess salt. Therefore, the experimentally obtained value of dn/dc for the C3Ms in bulk has to be corrected for the salt expelled upon C3M formation before using it in calculations of the Q f values. The detailed calculation procedure is presented in Appendix 1.
Values of refractive index increments (dn/dc, [cubic centimeters per gram]) used for calculations
Results and discussion
Formation of grafted block C3Ms in solution
C3Ms are formed by the oppositely charged polyelectrolytes mixed at stoichiometric charge ratio. The neutral corona surrounding the complex coacervate core prevents the coacervate from growing to macroscopic dimensions. Thus, at f- = 0.5, electrically neutral particles are expected. Determination of the zeta potentials for different mixing ratios (f-) reveals that, in a solution at stoichiometric charge ratio (f- = 0.5), C3Ms are, within experimental error, electrically neutral. The dζ/df- values are almost identical for both systems and do not differ for negatively and positively charged aggregates (Fig. 4). For the short homopolymer, the isoelectric point corresponds well to the PMC, i.e., the maximum in the scattering intensity. However, for the long homopolymer, the maximum number of micelles is found if the aggregates carry an excess positive charge, i.e., at f- = 0.45. In this case, the maximum scattering intensity is more pronounced than for the short homopolymer. We relate this observation to the limited flexibility of the grafted block copolymer, resulting from the presence of the side chains. Apparently, due to the steric effect, the negatively charged PAA21-blocks are easier to access, i.e., can be more effectively neutralized, by the positively charged groups on short P2MVPI43 than by the charged groups of the long P2MVPI228. All C3Ms in this study were prepared at compositions corresponding to maximum in scattering intensity (PMC), i.e., at f- = 0.5 for C3M-PAPEO14/P2MVPI43 and at f- = 0.45 for C3M-PAPEO14/P2MVPI228.
The hydrodynamic radii of the C3M-PAPEO14/P2MVPI43 and C3M-PAPEO14/P2MVPI228, as determined with DLS titrations, are 105 and 85 nm, respectively, and do not vary significantly in the f- range between 0.4 and 0.6. The large hydrodynamic radii determined experimentally suggest that the discussed C3Ms are not regular micelles but rather aggregates consisting of small micelles. These small micelles aggregate due to hydrophobic interactions most probably originating from the hydrophobic end-group in the PAA21-b-PAPEO14 molecule (see Fig. 2). A more detailed discussion on the structure of these aggregates will be presented in a forthcoming paper.
Stability of grafted block C3Ms in a solution
Stability against salt
The stability of the C3Ms in solution is strongly dependent on the strength of the interactions between the polyelectrolytes in the coacervate core. At low salt concentration, electrostatic attraction between oppositely charged polyelectrolytes is strong, but, with increasing salt concentrations, electrostatic interactions are more and more screened. At salt concentrations higher than 50 mM NaCl, the scattering intensity of C3M-PAPEO14/P2MVPI43 in solution decreases, and the observed hydrodynamic radii of C3Ms increase to values above 250 nm. Above 200 mM NaCl, the scattering intensity of C3M-PAPEO14/P2MVPI43 stabilizes at 60% of its value at 1 mM NaCl. A similar decrease in scattering intensity and increase in the hydrodynamic radii is observed for C3M-PAPEO14/P2MVPI228 at salt concentrations above 100 mM NaCl. Above 400 mM NaCl, the scattering intensity of C3M-PAPEO14/P2MVPI228 stabilizes at 40% of its initial value. Thus, grafted block C3Ms are sensitive to salt concentration and partly disintegrate beyond 50–100 mM NaCl.
Stability against proteins
DLS titrations show that β-lac forms aggregates with the positively charged P2MVPI irrespective of their length (Fig. 5a). Here, f- is defined as [protein-]/([protein-] + [P2MVPI+]) and [protein-]/([protein-] + [PAA-]) for titrations of P2MVPI groups and PAA groups, respectively. With excess P2MVPI43, aggregates of β-lac remain stable, whereas aggregates formed with P2MVPI228 disintegrate if an excess of homopolymer is added, i.e., f- < 0.5. With the positively charged block copolymer (P2MVPI41-b-PEO204), reduced scattering intensity (I/c) is very low and formation of aggregates is prevented by stabilization due to the neutral PEO block. The negatively charged copolymer seems to have no interaction with β-lac. Aggregates are also formed between the long homopolymer, P2MVPI228, and BSA (Fig. 5b). With an excess of homopolymer, these aggregates remain stable between f- = 0.4 and 0.8. However, the scattering intensity is much lower for aggregates formed between BSA and the short homopolymer, P2MVPI43. This may be due to the low charge density on the protein surface [26, 27, 28]. Under the experimental conditions, BSA was assumed to carry 13 negative charges per molecule  and β-lac, 13 negative charges per dimer . Considering the difference in size of both proteins, it implies that BSA has a lower density of negative charges and may require a longer polycation to form aggregates. Contrary to β-lac, for BSA, a substantial scattering intensity around f- = 0.5 is also found in mixtures with P2MVPI41-b-PEO204 and PAA21-b-PAPEO14, indicating formation of aggregates.
Adsorption of grafted block copolymer and grafted block C3Ms on silica and polystyrene
The adsorbed amount of grafted block C3Ms on silica and polystyrene is much higher than the adsorption of the single grafted block copolymer. The adsorption properties of the micelles with short and long homopolymer differ significantly. Initial adsorption is fast and then levels off to reach a relatively stable level within 1 or 2 h. Upon rinsing with solvent, indicated by arrows in Fig. 6, any reversibly adsorbed fraction is removed. The adsorbed amount of C3Ms with the long homopolymer is much lower than of C3Ms with the short homopolymer. The reversibly adsorbed fraction of micelles is much smaller for C3M-PAPEO14/P2MVPI228 (5-10%) on both silica and polystyrene. The adsorbed amounts of micelles with the long homopolymer on silica and polystyrene are almost equal. It is likely that for the long homopolymer C3Ms adsorb primarily according to a mechanism proposed by van der Burgh et al.  and Voets et al. . C3Ms are expected to unfold upon adsorption in a way that the coacervate core adsorbs directly on the surface, and the neutral corona forms a brush layer on top of the coacervate layer and do not disintegrate during the adsorption process. Base on these assumptions, the thickness of an unfolded C3M layer is roughly half the diameter of a micelle, i.e., half of the thickness of micelles which do not unfold. Unfolding of C3Ms on solid surfaces is, however, governed by a subtle balance between the stability of the micelles and the wetting properties of the surface, core, and corona . The differences in the adsorbed amounts suggest that the micelles with the short homopolymer are more stable against unfolding than the micelles with the long homopolymer. It seems that, on silica, the micelles with the short homopolymer initially do not unfold and unfold only partly after rinsing with solvent. Possibly, the difference in adsorbed amounts after rinsing with solvent can be attributed to an excess of the positive charges on C3Ms with long homopolymer. However, the wetting properties of polystyrene seem to fully suppress unfolding of C3M-PAPEO14/P2MVPI43 resulting in an adsorbed amount (after rinsing) being twice as high as for C3M-PAPEO14/P2MVPI228.
PAA21-b-PAPEO14 is a bulky block copolymer. In addition to a charged PAA block, a neutral block with grafted PEO chains, it contains also a small hydrophobic end-group (-S-CS-S-C12H25, see Fig. 2) that originates from the initiator used to synthesize this polymer . This hydrophobic “sticker’” influences the structure of the C3Ms. The coacervate core and neutral corona may not be as well-defined as in typical C3Ms containing only linear polyelectrolytes resulting in relatively big micellar sizes. As a consequence, also the stability of the C3Ms in solution may be altered and the structure of the C3M layer formed upon adsorption of the micelles at the solid–liquid interface may differ from the expected one [4, 10]. Due to an additional aggregation mechanism, the adsorbed amounts of grafted block C3Ms are higher than for regular C3Ms with linear PEO blocks, described in our previous paper . The structure of the adsorbed C3M layer determines the formation and the density and, therewith, the functionality of the polymer brush.
Stability of the C3M layers against an excess of protein
Exposure of the surface-coated with C3M-PAPEO14/P2MVPI43 to a β-lac solution leads to a reduction of the total adsorbed mass (Fig. 8a, b). This effect is much stronger on polystyrene than on silica and depends on the salt concentration. An initial increase in the adsorbed amount upon addition of β-lac corresponds to an accumulation of the protein on the adsorbed C3M layer. Immediately after this increase, the adsorbed amount decreases to a level indicating partial removal of the C3M layer from the surface.
With the following simple experiment, we confirmed that β-lac is most probably not replacing P2MVPI43 in the adsorbed layer but forms soluble aggregates with the homopolymer. As is discussed below, an adsorbed C3M-PAPEO14/P2MVPI43 layer completely suppresses Lsz adsorption on coated surfaces. If β-lac was present in this layer, one would expect an increase in Lsz adsorption due to the electrostatic attraction between the two proteins. During the experiment C3M-PAPEO14/P2MVPI43 was exposed to β-lac and subsequently to Lsz solution. No significant adsorption of Lsz was observed.
On both silica and polystyrene at 10 mM NaCl, we observe a significant reduction of the adsorbed mass but at 1 and 100 mM NaCl such effects were absent. These observations may be related to the strength of interaction between the block copolymer and the homopolymer in the C3Ms. At 1 mM NaCl, electrostatic attraction between P2MVPI43 and PAA21-b-PAPEO14 is strong, and the coacervate part of C3M is more glass-like . Micelles in a solution are stable and form a stable layer upon adsorption at the surfaces. Thus, complexes of β-lac and P2MVPI43 are not formed. As the salt concentration increases, attraction between micellar components weakens. An adsorbed C3M layer may be partly penetrated by small molecules, like β-lac. Soluble complexes of β-lac and P2MVPI43 are formed. At salt concentrations above 50 mM NaCl, attraction between oppositely charged polyelectrolytes is weak, and we observe that the C3Ms partly disintegrate in solution. Under these conditions, attraction between β-lac and the oppositely charged polyelectrolyte is weak as well, and they are less likely to aggregate. A similar pattern was observed on silica.
The differences in the amount of mass removed by β-lac from silica and polystyrene may be related to the structure of the adsorbed layer and the size of the protein. Above, we have argued that, on silica, C3Ms unfold, and the P2MVPI43 is in contact with the surface, whereas on polystyrene C3Ms do not unfold, and the short P2MVPI has no direct interaction with the solid surface, and it will be more susceptible to erosion due to complexation with β-lac by penetration through an imperfect brush layer. However, the protein may only partly penetrate the adsorbed layer due to its dimensions, which limits the erosion of the coating.
Stability of the C3Ms layer against an excess of block copolymer
The reduction of the adsorbed amount of C3M-PAPEO14/P2MVPI43 upon rinsing with a PAA21-b-PAPEO14 solution could be explained by formation of SCPs consisting of P2MVPI43 and PAA21-b-PAPEO14. As shown in Figs. 10a and 11a, it is not possible to restore the coating by simple re-adsorption of P2MVPI43 on the remaining layer. Subsequent exposure to PAA21-b-PAPEO14 did not result in increased adsorbed mass, i.e., we did not observe the formation of polyelectrolyte multilayers.
The coating can be restored by re-adsorption of C3M-PAPEO14/P2MVPI43 and removed again upon exposure to PAA21-b-PAPEO14 (Figs. 10b and 11b). This indicates that the layer remaining on the surface is electrically neutral.
The amount of the coating remaining on the surface depends on the properties of this surface. The negatively charged silica adsorbs positively charged P2MVPI43. To ensure electroneutrality, corresponding amount of negatively charged PAA21-b-PAPEO14 is bonded to this surface. Polystyrene has strong affinity to the hydrophobic tail of PAA21-b-PAPEO14, resulting in the accumulation of the negative charge on the surface. As on silica, to ensure electroneutrality, a corresponding amount of the positively charged P2MVPI43 is adsorbed on the surface. These results suggest that, under discussed conditions, the interactions between the C3Ms components in the adsorbed layer are relatively weak. Upon rinsing with a PAA21-b-PAPEO14 solution, PAA21-b-PAPEO14 penetrates the adsorbed layer and removes its components.
Effect of grafted block C3M coatings on protein adsorption
From the results in Figs. 14 and 15, it follows that the properties of the coating with the long homopolymer, P2MVPI228, are very different from the properties of the coating with the short homopolymer, P2MVPI43. On a C3M-PAPEO14/P2MVPI228 layer, the adsorption of the negatively charged proteins, β-lac and BSA, is enhanced by the coating (negative values of the reduction factor) instead of being suppressed, at salt concentrations above 10 mM NaCl. Adsorption of positively charged Lsz is strongly suppressed. The grafted block C3M-PAPEO14/P2MVPI228 micelles are stable in solution at salt concentrations below 100 mM NaCl and adsorb irreversibly on silica and polystyrene. The results suggest that these micelles do not have a well-defined core-corona structure and that upon their adsorption on the surfaces, they do not form a well-developed brush but rather, the positively charged polyelectrolyte is exposed to the solution. This was confirmed by zeta potential measurements (see Fig. 4b). This effect is much more pronounced on hydrophobic polystyrene than on hydrophilic silica.
An excellent reduction of protein adsorption on silica and polystyrene coated with micelles containing the short homopolymer, C3M-PAPEO14/P2MVPI43, is obtained both for β-lac and Lsz. At 1 and 10 mM NaCl, adsorption of the negatively charged BSA is only weakly repelled from the surface. At 100 mM NaCl, adsorption of all proteins on both surfaces was suppressed by more than 90%. These observations suggest that the coating does not completely screen the interactions between surface and proteins at low salt concentrations, and BSA can still adapt to the surface. As discussed above, these phenomena are independent of the nature of the native surface. We observed that, at salt concentrations above 50 mM NaCl, C3M-PAPEO14/P2MVPI43 partly disintegrates in solution. Results presented in Fig. 15 confirm that, at 100 mM NaCl, a fully functional brush layer is formed upon their adsorption on silica and polystyrene.
Coatings consisting of PAA21-b-PAPEO14 only show limited resistance towards protein adsorption. This may be related to insufficient surface coverage by the block copolymer and, as a consequence, to a low density of PEO chains. As mentioned before (see Fig. 7), adsorption of grafted block C3Ms results in higher amounts of adsorbed PAA21-b-PAPEO21 than adsorption of single PAA21-b-PAPEO14 and thus in a higher density of the PEO chains on the surface. Higher density of PEO chains is expected to result in more effective suppression of protein adsorption. However, results presented for C3M-PAPEO14/P2MVPI228 are not in agreement with this expectation. Our results show that the structure of the adsorbed C3Ms layer is influenced by the length of the polyelectrolyte blocks and does not allow for the exposure of all PEO chains to the solution.
Grafted block complex coacervate core micelles are formed upon mixing PAA21-b-PAPEO14 with P2MVPI at stoichiometric charge ratios. Their stability in solution strongly depends on the block length of the homopolymer and the salt concentration. At pH 7, C3Ms containing the short homopolymer, P2MVPI43, partly disintegrate in solution at salt concentrations above 50 mM NaCl. The micelles with the long homopolymer, P2MVPI228, partly disintegrate at salt concentrations above 100 mM NaCl. Upon adsorption at a solid–liquid interface, grafted block C3Ms form a layer that suppresses adsorption of proteins. The effectiveness of this coating depends on the charge of the aggregates, the length of the homopolymer block, and the salt concentration. The adsorption of grafted block C3Ms results in a higher density of neutral PEO chains on the surface than the adsorption of the single grafted block copolymer, PAA21-b-PAPEO14. At 100 mM NaCl, the C3M-PAPEO14/P2MVPI43 layer reduces protein adsorption on silica and polystyrene by more than 90%. However, the C3M-PAPEO14/P2MVPI228 layer enhances the adsorption of the negatively charged proteins at salt concentrations above 1 mM NaCl. It suggests that adsorbed C3M-PAPEO14/P2MVPI228 do not form a well-structured bilayer with a coacervate layer attached to the surface and a PEO brush layer exposed towards the solution, but that the positively charged blocks are also penetrating the brush layer. These findings were confirmed by zeta potential measurements which show an excess of positive charge on C3M-PAPEO14/P2MVPI228 while C3M-PAPEO14/P2MVPI43 were found to be nearly neutral. The C3M-PAPEO14/P2MVPI43 layer may be partly destroyed upon exposure to β-lac and to PAA21-b-PAPEO14. At 10 mM NaCl, these molecules form soluble aggregates with the polyelectrolytes extracted from the adsorbed C3Ms layer. This results in a significant decrease in the adsorbed mass on the surface.
A well-developed PEO brush layer is not only required to effectively reduce protein adsorption but is also crucial to protect the complex coacervate layer from being solubilized by supplying an excess of block copolymer or protein.
This work was performed in the TTIW-cooperation framework of Wetsus, Centre of Excellence for Sustainable Water Technology (www.wetsus.nl). Wetsus is funded by the Dutch Ministry of Economic Affairs, the European Union Regional Development Fund, the Province of Fryslân, the City of Leeuwarden and the EZ/Kompas program of the “Samenwerkingsverband Noord-Nederland”. The authors thank the participants of the research theme “Biofouling” for the discussions and their financial support.
C.D. thanks the Politique Scientifique Fédérale in the frame of the Interuniversity Attraction Pôles Programme (PAI VI/27): Supramolecular Chemistry and Supramolecular Catalysis, and the National Funds for Scientific Research (F.R.S.-FNRS) for financial support.
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- 11.Pasche S, De Paul SM, Voros J, Spencer ND, Textor M (2003) Poly(l-lysine)-graft-poly(ethylene glycol) assembled monolayers on niobium oxide surfaces: a quantitative study of the influence of polymer interfacial architecture on resistance to protein adsorption by ToF-SIMS and in situ OWLS. Langmuir 19(22):9216–9225CrossRefGoogle Scholar
- 20.Bachmann L, Schmittfumian WW, Hammel R, Lederer K (1975) Size and shape of fibrinogen .1. Electron-microscopy of hydrated molecule. Makromol Chem-Macromol Chem Phys 176(9):2603–2618Google Scholar
- 27.Kabanov VA, Evdakov VP, Mustafaev MI, Antipina AD (1977) Cooperative bonding of serum-albumin by quaternized poly-4-vinylpyridines and structure of complexes formed. Mol Biol 11(3):443–454Google Scholar