Sorption of Proteins onto Porous Single-Component Poly(vinyl amine

Biomacromolecules 2010, 11, 787–796

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Sorption of Proteins onto Porous Single-Component Poly(vinyl amine) Multilayer Thin Films Ecaterina Stela Dragan,* Florin Bucatariu, and Gabriela Hitruc “Petru Poni” Institute of Macromolecular Chemistry, Aleea Grigore Ghica Voda 41 A, RO-700487 Iasi, Romania Received December 9, 2009; Revised Manuscript Received January 22, 2010

Porous multilayer thin films consisting solely of cross-linked poly(vinyl amine) ((PVAm)n) were generated by a selective cross-linking of the PVAm layers in the [PVAm/poly(acrylic acid) (PAA)]n thin films, followed by the removal of PAA. A regular increase in the (PVAm/PAA)n multilayer onto silica particles was observed by potentiometric titration when PVAm with a low molar mass (Mw ) 15 000 g mol-1) was used in the construction of LbL thin films. The amount of human serum albumin (HSA) loaded on the single-component (PVAm)5 thin film was ∼32 mg HSA/g of hybrid when PVAm with a molar mass of 15 000 g mol-1 was used compared with the single-component film prepared with PVAm with a molar mass of 340 000 g mol-1 when the amount of HSA loaded was 15.5 mg HSA/g of hybrid. The sorption modalities of HSA and bovine serum albumin (BSA) onto the single-component thin films deposited on silicon wafers as a function of the number of PVAm layers was investigated by atomic force microscopy and contact angle measurements. The decrease in the film roughness and the increase in the film wettability after the loading of both proteins showed the protein was mainly absorbed into the porous (PVAm)n thin film.

I. Introduction Layer-by-layer (LbL) deposition of polyelectrolytes and biological species like DNA and proteins, introduced by Decher,1,2 is perhaps the most widely used technique to tailor surfaces with controlled biological properties, such as enzymatic activity,3–7 immunosensing,8 nanoreactors,9,10 biosensors,11,12 and controlled release of entrapped species.13–19 Controlled release of drugs from LbL thin films, either with embedded dyes as models for drugs15 or with so-called “prodrugs” (drug attached to a biocompatible polyanion by a labile linkage),16 has been promoted for the creation of more efficient controlled delivery systems. DNA has been entrapped in the LbL thin films either as polyplex (soluble interpolyelectrolyte complex) formed with a synthetic polycation, which has been used in the multilayer construction by alternation with a biocompatible polyanion,17 embedded into polyelectrolyte multilayer films,18 or alternately adsorbed with polycations to get multilayer thin films.19 The release of DNA by the erosion (deconstruction) of the LbL thin films could have a significant impact on the development of localized gene therapies.19 The preservation of protein structure in the “electrostatic cage” formed by polyelectrolytes motivated the use of proteins for biological functionalization of polyelectrolyte multilayers.20 For this purpose, proteins are embedded in or adsorbed onto polyelectrolyte multilayers, with further stabilization gained through crosslinking. The adsorption of charged proteins onto multilayer-coated surfaces is a complex phenomenon widely studied so far either to reduce the fouling process or to increase the adsorption of proteins.21–25 Even if various interactions contribute to the adsorption/rejection of proteins on the LbL thin films, it is generally accepted that the surface charge plays a major role.21,22,24 Therefore, only a monolayer of protein has been * To whom correspondence should be addressed. Tel: +40.232217454. Fax: +40.232211299. E-mail: [email protected].

found on the negatively charged film surface, whereas, on the positively charged surface, the thickness of the adsorbed protein film increased up to several times the largest dimension of the native protein molecule.21,22 The influence of the surface charge on the secondary structure of the adsorbed protein has also been investigated, and it has been found that the structural changes were larger when the surface charge and protein charge were opposite.20 However, the film morphology, which is strongly correlated with the conditions of film construction, would have a definite influence on the protein adsorption/diffusion into the LbL thin films. One of the most interesting categories of building blocks for the construction of LbL thin films with desired properties is that of weak polyelectrolytes because they allow a fine control of the layer pair thickness, surface roughness, and film morphology by their complex behavior over a wide range of pH. Normally, there is a fine balance between electrostatics26–30 and hydrogen bondings,31–34 their role in the LbL film growth and stability being determined by the local microenvironment. Recently, we have reported on reactive polyelectrolyte multilayers generated by the LbL deposition of two weak polyelectrolytes, poly(vinyl amine) (PVAm) and poly(acrylic acid) (PAA), onto silica microparticles, the LbL films being stabilized by a heat-induced reaction with the formation of amide bonds. The hybrid consisting of silica particles coated with cross-linked (PVAm/PAA)n multilayer, having enough reactive amino groups on the surface, was subsequently decorated with short peptide brushes of S-benzyl-L-cysteine.35 Nanoporous and microporous LbL thin films have been prepared from weak polyelectrolytes,33,34,36–38 and their possible applications as biomaterials have been widely explored.33,38 Saltinduced structural changes34 and heat-induced formation of amide bonds36 have been used to generate and to lock in the porosity in LbL thin films. Caruso et al. prepared nanoporous thin films via polyelectrolyte templating, where two components, PAA and poly(allylamine hydrochloride), were interconnected

10.1021/bm9014057  2010 American Chemical Society Published on Web 02/15/2010

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Chart 1.

Structures of the Polyelectrolytes Used in This Study

by amide bonds, and the third polyelectrolyte used as template, poly(4-vinylpyridine), has been removed after the film construction.32 We have recently reported on a novel strategy to design porous thin films, which consists of the alternate deposition of PVAm and PAA onto silica microparticles or silicon wafers, followed by: (1) a selective chemical cross-linking of polycation layers and (2) the removal of PAA, a single-component crosslinked multilayer of (PVAm)n remaining at the end.39 The main parameters that control the film properties and the interaction of the human serum albumin (HSA) and bovine serum albumin (BSA) with the single-component (PVAm)n thin films are investigated in this work. To the best of our knowledge, this is the first extended study on the formation of porous singlecomponent (PVAm)n thin films and on the sorption of proteins onto these films as a function of the conditions of the doublecomponent multilayer construction. The chemical structures of polyelectrolytes used in this work are presented in Chart 1. Film construction and stability were examined by potentiometric titration when silica microparticles were used as substrate and by atomic force microscopy (AFM) when the LbL films were deposited on silicon wafers. The protein sorption on the single-component multilayers was followed by UV-vis spectroscopy when the (PVAm)n films were deposited on silica microparticles and by AFM and contact angle measurements when the films were deposited on silicon wafers.

II. Materials and Methods II.1. Materials. The PVAm samples with molar masses of 15 000 and 340 000 g mol-1 were provided by BASF (Ludwigshafen, Germany). The degree of hydrolysis of both samples was ∼95 mol %. PAA samples with molar masses of 11 000 and 57 000 g mol-1 were synthesized and characterized according to ref 40 with Mw/Mn of 1.13 and 1.9, respectively. Epichlorohydrin (ECH) analytical grade (SigmaAldrich, Germany) was used as received. HSA with 96-99% purity and phosphate-buffered salt (PBS) purchased from Sigma-Aldrich, Germany, were used without any further purification. BSA from Fluka, with protein content >95% and ∼3% water, was used as received. Aqueous solutions of PVAm and PAA in distilled water, both with concentrations of 5 × 10-3 mol repeat unit L-1, were prepared for multilayer deposition. Quantitative determination of the charged groups in solution was performed by polyelectrolyte titration with a particle charge detector PCD 03, Mu¨tek GmbH, Herrsching, Germany, using either poly(sodium ethylenesulfonate) or poly(diallyldimethylammonium chloride), with a concentration of 10-3 mol L-1, in dependence on the nature of charges. Kieselgel 60 (Merck, Darmstadt, Germany) having the main diameter of the silica microparticles ranging between 15 and 40 µm, with a maximum of the pore diameters in the range of 4-6 nm, has been used after the activation with NaHCO3 aqueous

Dragan et al. solution, with a concentration of 1%, followed by washing with distilled water to neutral pH. Silicon wafer substrates were carefully cleaned in two steps: (1) in Piranha solution (70:30% v/v sulfuric acid/hydrogen peroxide) (Caution! Piranha solution is highly corrosiVe and extreme care should be taken when handling it), followed by intensive rinsing with distilled water and (2) with the mixture NH4OH/H2O2/distilled water (1:1:1) at 70 °C, in an ultrasonic bath, and finally intensive rinsing with distilled water. II.2. Construction of (PVAm/PAA)n Multilayers. Two strategies were used for the (PVAm/PAA)n multilayer buildup onto silica microparticles. According to the first strategy, both polyions were adsorbed from salt-free aqueous solutions at the pH corresponding to the solution of each polyion, that is, 9.5 for PVAm, this being the pH of PVAm at a concentration of 5 × 10-3 mol L-1, and 3.5 for PAA, this being the pH of PAA at a concentration of 5 × 10-3 mol L-1. The pH of water used in washing steps was adjusted at 3.5 for washing after the PVAm adsorption steps, and at 9.5 for washing after the PAA adsorption steps to remove the weakly adsorbed polyions. Samples of 3 g of silica were suspended in 150 mL of a 5 × 10-3 mol L-1 PVAm salt-free aqueous solution. During the adsorption process, over 1 h, the suspension was gently shaken at room temperature. The modified particles were washed three times with water at pH 3.5 to rinse the weakly adsorbed PVAm chains from the surface of silica particles. For the second adsorption step, the PVAm/silica particles were suspended in 150 mL of a 5 × 10-3 mol L-1 PAA salt-free aqueous solution. After 1 h, the hybrid particles were washed three times with water at pH 9.5 to rinse the weakly adsorbed PAA chains. The adsorption and washing steps were repeated until the desired number of layers had been deposited. According to the second strategy, a constant pH of 5.0 was kept in both the polyions adsorption steps and in the washing steps. The pairs of polyelectrolytes, their molar masses, and pH values used in the adsorption and washing steps are listed in Table 1. The multilayer construction on silicon wafers was performed under the same conditions as those used for the multilayer deposition on silica microparticles, only according to the first strategy. II.3. Post-Construction Treatments of the (PVAm/PAA)n Thin Films. The selective cross-linking of the PVAm in the multilayer was performed with ECH at a ratio of (5 mg m-2) × n, where n is the number of PVAm layers in the film. The surface of silica particles has been estimated by approximation of silica particles with spheres according to ref 39. The silica particles (3 g) having (PVAm/PAA)n thin films deposited on the surface were suspended in 20 mL of water, and after that, a 0.6 × n mL of solution of ECH in water/acetone 1:1 (v/v) with a concentration of 10 mg mL-1 was added drop-by-drop with agitation; the cross-linking reaction was carried out for 3 h, at room temperature. The silicon wafers with a surface of ∼1 cm2 having (PVAm/PAA)3.5, (PVAm/PAA)5.5, and (PVAm/PAA)7.5 multilayer thin films deposited were immersed, separately, in 4 mL of water, and then 20, 30, and 40 µL of ECH in water/acetone 1:1 (v/v), with a concentration of 0.1 mg mL-1, was added drop-by-drop with agitation at room temperature for 3 h. Silica particles and silicon wafers covered with (PVAm/PAA)n multilayers thin films were washed three times with water and dried under vacuum at room temperature. As far as the AFM technique was concerned, freeze-drying technique was also applied, the drying effect on the film architecture leveled off, and the film morphology was thus preserved.

Table 1. Combinations of Polyion Pairs Used in the Construction of LbL Multilayer Films on Silica Microparticles series

PVAm, Mw (g mol-1)

PAA, Mv (g mol-1)

A B C D

15 000 (low) 340 000 (high) 340 000 (high) 340 000 (high)

57 000 (high) 11 000 (low) 57 000 (high) 11 000 (low)

pH of water in the washing steps 3.5 3.5 3.5 5.0

after after after after

the adsorption of PVAm; 9.5 after the adsorption of PAA the adsorption of PVAm; 9.5 after the adsorption of PAA the adsorption of PVAm; 9.5 after the adsorption of PAA every adsorbed layer

Sorption of Proteins on PVAm Multilayers

Figure 1. Removal of PAA from the cross-linked (PVAm/PAA)4.5 thin film deposited on silica particles at room temperature and pH 9.0.

After cross-linking, we removed the PAA by shaking the silica particles and silicon wafers in water at pH 11 and 60 °C until PAA was completely removed, followed by rinsing with water at neutral pH. Quantitative determination of PAA in the extraction environment was performed by polyelectrolyte titration with the particle charge detector PCD 03, Mu¨tek GmbH, Germany. The time and the number of extraction steps depended on the number of PAA layers. Herein we include some results obtained on series C (Table 1), which demonstrate that the optimum conditions for the removal of PAA have been established step-by-step. We have started the extraction of PAA from the multilayers deposited on silica particles at pH 9 and at room temperature, and the amount of PAA removed under these conditions is plotted as a function of time in Figure 1. As can be observed, the time necessary to remove PAA from the (PVAm/PAA)4.5 multilayer under these conditions was very long (>45 h). Therefore, the pH and temperature of the extraction solution were gradually increased, with the fastest removal of PAA being found at pH 11 and 60 °C. These conditions were kept for all extraction steps; only the extraction duration depended on the number of PAA layers. II.4. Characterization of Multilayer Thin Films before and after the Removal of PAA. Potentiometric titration of the silica microparticles as a function of the number of adsorbed polyion layers was performed with the particle charge detector PCD 03, Mu¨tek GmbH, Herrsching, Germany. The topography of the multilayers (PVAm/ PAA)n, before and after the removal of PAA and also after the sorption of proteins, was examined with a SPM Solver PRO-M AFM (NT-MTD Co. Zelenograd, Moscow, Russia). For this purpose, the polyelectrolytes were alternately deposited on silicon wafers under conditions similar to those used for the multilayer deposition onto silica microparticles. Before the AFM measurements, silicon wafers were either dried under vacuum for 48 h at room temperature or freeze-dried when the


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preservation of film morphology was of interest. Images were taken in tapping mode at room temperature. All images were acquired using a high-resolution no contact “Golden” silicon NSG10/Au/50 cantilever with Au conductive coating. The images were examined by means of the Nova 9.09 Software and by the scanning probe microscope software, Gwyddion 2.15, to measure the surface roughness parameters. The contact angle values were measured using an EASYDROP ¨ SS) equipped with DSA 1 evaluation Shape Analysis System (KRU software.41 Water drop was placed on the polymeric film located on a moveable sample table. The drop was illuminated from one side, and a video camera at the opposite side records an image of the drop. This image was transferred to a computer equipped with a video-digitizer board (frame-grabber). The DSA 1 software contains time-proven tools for analyzing the drop image to calculate the contact angle values. The contact angle was measured on static drops with distilled water. II.5. Sorption of Proteins onto Cross-Linked (PVAm)n Films. The protein sorption was performed in PBS solution at pH 7.4, the protein concentration being 1 g L-1. For each experiment on the crosslinked (PVAm)n films deposited onto silica particles, 200 mg of substrate was suspended in 30 mL protein solution. After 4 h at room temperature, the concentration of supernatant was determined by UV-vis spectroscopy at 278 nm by using a SPECORD 200 Analytic Jena apparatus. Three measurements were averaged. For protein sorption onto the cross-linked (PVAm)n films deposited on silicon wafers, each wafer was immersed in 5 mL of HSA solution for 4 h at room temperature. After that, all wafers were washed three times with distilled water and freeze-dried to preserve the film morphology.

III. Results and Discussion III.1. Construction and Characterization of CrossLinked (PVAm)n Multilayers. The schematic representation of the (PVAm/PAA)n multilayer buildup and the generation of the cross-linked single-component PVAm multilayers onto silica microparticles, according to the first strategy (Materials and Methods section), is illustrated in Scheme 1. The pH of the polyelectrolyte solutions used in the construction of LbL multilayers was 9.5 for the PVAm solution and 3.5 for PAA solution, that is, the pH corresponding to the concentration of 5 × 10-3 mol · L-1 of each polyion. Under these conditions, the main driving force in the deposition of the next polyelectrolyte layer is hydrogen bonding, polyelectrolyte chains being in a coiled conformation. To remove the excess of polyelectrolyte, the pH in the washing steps was changed, being the same as that of the next layer of polyelectrolyte to be adsorbed. Therefore,

Scheme 1. Idealized Schematic Representation of the Generation of Porous Cross-Linked (PVAm)n Thin Films Deposited onto Silica Particlesa

a For the sake of clarity, the schematic representation does not consider the interaction between the different layers; therefore, the porosity of the final (PVAm)n/silica hybrid appears to be too large.

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Dragan et al. Table 2. Adsorbed Amount of HSA as a Function of Extraction Conditions of PAA from (PVAm/PAA)4.5/Silica Hybrid

Figure 2. Potentiometric titration of (a) hybrid particles consisting of LbL multilayers constructed on silica microparticles with low PVAm and high PAA and (b) one layer of low PVAm adsorbed onto silica, dicomponent cross-linked multilayer thin film deposited on silica, and cross-linked single-component (PVAm)5 on silica particles.

Figure 3. Potentiometric titration of hybrid particles consisting of LbL multilayers constructed onto silica particles with high PVAm and high PAA (series C, Table 1).

the surface of the last adsorbed layer will mainly contain ionized groups, being still able to interact with the next layer of polyelectrolyte by hydrogen bonds.42 The film construction on silicon wafers was carried out according to the same protocol. To gain information on the influence of PVAm molar mass on the LbL multilayer construction and stability, the potentiometric titration after each adsorbed layer (PVAm or PAA) on silica microparticles was performed, and the results obtained for the multilayer buildup according to the first strategy are summarized in Figures 2 and 3.

conditions for PAA removal

pH 10, 25 °C, 24 h

pH 10, 60 °C, 2 h

pH 11, 60 °C, 3 h

loaded HSA (mg/g)

8.02

16.51

31.87

The potential, Ψ, of the (PVAm/PAA)n/silica particles as a function of solution pH, when PVAm with a molar mass of 15 000 g mol-1 and PAA with molar mass of 57 000 g mol-1 were used in the construction of LbL thin films, are plotted in Figure 2a. A positive potential Ψ can be observed in the range of pH 3-9 when the first PVAm layer is adsorbed onto the silica particles. The point-of-zero charge (pzc ) pH|Ψ)0) of these hybrid particles was located at ∼9.2. After the adsorption of the first layer of PAA onto the PVAm/ silica hybrid, the pzc of (PVAm/PAA)/silica hybrid particles shifted to 3.8. The next layer of PVAm shifted the pzc into the basic range (pzc ) 9), whereas the next layer of PAA again shifted the pzc to the acidic range. This behavior demonstrates that the last adsorbed polyelectrolyte layer, polycation or polyanion, strongly determined the surface Brønsted acidity of the hybrid particles. The values of pzc around 9 and 4 when the last layer adsorbed was PVAm and PAA, respectively, suggest a constant and regular increase in the multilayer onto the surface of silica particles. Figure 2b shows that the pzc of PVAm/silica, (PVAm/ PAA)4.5-ECH/silica, and the (PVAm)5/silica hybrid particles were nearly identical (pzc ) 9.2) because the last layer adsorbed was the polycation. However, the values of the potential Ψ corresponding to the plateau region, located in the acidic range, were different. Therefore, the silica particles covered with one layer of PVAm had a potential of plateau region at nearly 600 mV, and this value was higher than the potential of cross-linked hybrid particles (PVAm/PAA)4.5-ECH/silica because the polyanion locked in the cross-linked multilayer decreased the potential. The potential of (PVAm)5/silica hybrid particles in the plateau region was 750 mV, higher than the potential of PVAm/silica, because the five polycation layers significantly reduced the influence of silica surface on the potential Ψ. A different behavior was observed when high PVAm and high PAA were used as polyions (series C, Table 1) in the construction of LbL thin films on silica microparticles (Figure 3). Therefore, the pzc values of (PVAm/PAA)n/silica hybrids having PVAm as the last layer adsorbed decreased from 9.2 to 7.5, 6.6, and 6.8 when one, two, four, or five polycation layers were adsorbed. When PAA was the last layer adsorbed, the pzc values were 5.2 and 3.9 for two and three PAA layers, respectively. This irregular variation of pzc values shows that PVAm with a very high molar mass is not suitable for a regular cover of silica microparticles with LbL thin films. It seems that the removal of large coils, which did not establish enough hydrogen bonds with the film surface, was more probable in this case. III.2. Sorption of Proteins onto Cross-Linked (PVAm)n Multilayers. To demonstrate the protein sorption properties of the cross-linked (PVAm)n thin films deposited either on silica microparticles or silicon wafers, two proteins have been selected, HSA and BSA, both being widely used in protein adsorption tests on multilayer-coated surfaces.21–23 To demonstrate that the level of the PAA removal is very important for the amount of loaded protein, some results concerning the amount of HSA sorbed by the (PVAm)5/silica hybrids generated from series A (Table 1) were presented in Table 2.


Figure 4. Amount of HSA adsorbed onto the cross-linked singlecomponent (PVAm)5 multilayer film deposited onto silica microparticles as a function of the polyion pair used in the LbL thin film construction: (A) low PVAm/high PAA; (B) high PVAm/low PAA; (C) high PVAm/high PAA; (D1) high PVAm/low PAA, (PVAm)8; and (D2) high PVAm/low PAA, (PVAm)11. The averages and the standard deviations were calculated from three independent measurements.

As can be seen from Table 2, the amount of loaded protein increased with the increase in pH and temperature at the extraction step of PAA, the highest amount of HSA being found when PAA was removed at pH 11 and 60 °C, conditions recommended in the Materials and Methods section. Figure 4 illustrates the influence of the molar mass of polyions used as building blocks in the LbL multilayer construction and of the pH used in different steps of the multilayer deposition on the amount of HSA loaded on the cross-linked (PVAm)5 thin film deposited onto silica microparticles. As Figure 4 shows, the amount of HSA sorbed onto the single-component multilayer thin film was influenced by the molar mass of the polyion pairs used in the LbL multilayer construction. The highest amount of protein has been loaded on the cross-linked (PVAm)5 film obtained by the removal of PAA from the (PVAm/PAA)4.5 multilayer constructed with low PVAm and high PAA (series A, Table 1). This result was correlated with the regular increase in the (PVAm/PAA)n thin film shown by potentiometric titration (Figure 2) for this polyion pair. When high PVAm and high PAA were used for the (PVAm/PAA)4.5 multilayer construction (series C, Table 1), the lowest amount of HSA was found, in agreement with the results of polyelectrolyte titration (Figure 3). Concerning the protein sorption on the porous (PVAm)n thin films resulted from the (PVAm/PAA)n films prepared with high PVAm and low PAA (series D, Table 1), constructed with the same polyion pair like series B but at a constant pH


of 5.0 in both the polyion deposition and washing steps (the second strategy), the decrease in the amount of sorbed HSA with the increase in PVAm layers was found; this behavior supports the lower stability of the multilayer constructed with high PVAm. However, the amount of protein loaded on the cross-linked (PVAm)8 film was a little higher than that found in series B, constructed according to the first strategy (Materials and Methods section). The results presented in Figure 4 do not give clear information on the sorption modalities, adsorption, or absorption.22 To discriminate which process is the most probable, we roughly calculated the amount of protein that could be adsorbed in a monolayer on the film surface. Therefore, the volume of HSA molecule assumed as an ellipsoid, at pH 7.4, is 12 × 2.7 × 2.7 nm3.21 On the basis of the assumption that the protein molecules were preferentially adsorbed with their major axis perpendicular to the adsorbing surface,44 the occupied area of one single HSA adsorbed molecule is 5.72 nm2 (adsorption in “side-on” conformation21). the maximum number of HSA molecules adsorbed on 1 cm2, in this case, would be 17.48 × 1012 molecules. Taking into account the molecular weight of protein molecule, which is 66 000 g mol-1, the coverage of silica surface by the protein, as monolayer, would be 19.1 mg m-2. Because the external surface of silica microparticles was approximated to be 0.4 m2 g-1,39 a loading of 7.64 mg g-1 resulted. For the protein adsorption in “end-on” conformation,21 the calculated loading was 1.72 mg g-1. Even if this calculation was very rough, the value obtained based on the adsorption in the “side-on” conformation is almost the same as that experimental value found from the adsorption isotherm of HSA on one layer of PVAm deposited on silica particles.45 Because the maximum adsorbed amount of HSA found for series A was ∼32 mg g-1, at least two modalities of sorption could be assumed, according to Scheme 2: either a multilayer of protein molecules adsorbed on the film surface (a) or the protein absorption into the bulk of the porous cross-linked film (b). To gain information on the most probable sorption process, we have investigated the surface morphology of the multilayer films, before and after the protein sorption, by tapping-mode AFM. Figures 5 and 6 illustrate the topography of the crosslinked (PVAm)n multilayer thin films deposited on silicon wafers, generated after the removal of PAA, and of the same films after the sorption of HSA on the single-component (PVAm)n multilayer thin films as a function of the number of PVAm layers. Calculation of surface properties could be a key factor in comparing the topography before and after the adsorption of protein. Surface characteristics (root-mean-square roughness, Sq, average roughness, Sa, average height, HA, average diameter,

Scheme 2. Schematic Representation of the Interaction between Protein and Cross-Linked Monocomponent Porous (PVAm)n Films Deposited onto Silica Particlesa

a

(a) Apparent multilayers; (b) absorption into the film.

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Figure 5. Height AFM images of the cross-linked single-component (PVAm)4 films deposited on the silicon wafers: 2D (a) before and (b) after the adsorption of HSA; the insets show the 3D height images of selected area of the same samples, scan size was 2 × 2 µm2 in all images. Height profiles generated along the horizontal lines (c) before and (d) after the protein adsorption and (e,f) the height histograms of the same samples.

ADP, and height, HHP) evaluated for the films having up to 8 PVAm layers are summarized in Table 3. As Table 3 shows, Sq, Sa, ADP, and HHP of the singlecomponent (PVAm)n thin films, decreased with the increase in the number of layers, Sa, for example, being 5.04 nm for the film having (PVAm)4 and 1.97 nm for the film having (PVAm)8. The decrease in the film roughness was attributed to the regular coverage of the surface, the film getting smoother with the increase in the PVAm layer number. (See Figures 5 and 6.) The decrease in all selected parameters after the HSA adsorption, irrespective of the number of PVAm layers, which constitute the monocomponent thin film, can be associated with the sorption of protein mainly inside the film.22 Also, the images

of height profiles on the cross-section show that the distance hills-to-grooves in the case of (PVAm)4 ranges from 25 nm before protein adsorption to 12 nm after the adsorption of HSA, which demonstrates that the film surface was getting smoother. The surface modification is also obvious from 3D images (Figure 5 and 6). The increase in the film surface homogeneity after the sorption of protein is well illustrated by histograms, which show that the majority of the highest points on the surface are situated between 7 and 15 nm after the protein loading on the films having six and eight PVAm layers. The processing of AFM images and the calculation of the surface texture parameters by the Nova 9.09 Software gave some characteristics of the pores, which are summarized in Table 4.



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Figure 6. Height AFM images of the cross-linked single-component (PVAm)8 films deposited on silicon wafers: 2D images (a) before and (b) after the adsorption of HSA; the insets show the 3D height images of selected area of the same samples, scan size was 2 × 2 µm2 in all images. Height profiles generated along the horizontal lines (c) before and (d) after the protein adsorption and (e,f) the height histograms of the same samples. Table 3. Characteristics of Surface Roughness Parameters of the (PVAm)n Thin Films Including Root-Mean-Square Roughness (Sq), Average Roughness (Sa), and Average Height (AH) from the Histograms and Average Diameter (ADP) and Height (HHP) from the Height Profiles before and after the Adsorption of HSAa sample

Figure Sq

(PVAm)4 5 (PVAm)4 + HSA (PVAm)6 not included here (PVAm)6 + HSA (PVAm)8 6 (PVAm)8 + HSA a

6.35 6.03 4.38 2.27 2.64 2.10

AFM data (nm) Sa AH ADP HHP 5.04 4.64 3.06 1.59 1.97 1.53

13.47 14.92 10.35 6.53 17.63 10.92

100 60 110 50 120 90

25 12 30 10 14 11

Scanned area 2 × 2 µm2.

Even if the values presented in Table 4 are statistical results given by the software, the characteristics of pores such as area, volume, perimeter, and length could be somehow correlated with the status of the film before or after the protein sorption. The decrease in almost all of these parameters was observed after

the sorption of HSA, and this demonstrates that the protein has been loaded into the porous film, which acts as a sponge for protein. The AFM images in phase of the (PVAm)n after the sorption of HSA were obtained by the measurement of the differences between the cantilever amplitude oscillations in contact with different phases on the surface. Figure 7 shows a much smoother surface of the film containing (PVAm)8 after the sorption of HSA compared with that of the film containing (PVAm)4, the differences in color being very small in the first case; that is, the protein molecules are not on the film surface but inside the film, thus supporting the sorption modality (b) in Scheme 2. The AFM information was supported by the contact angle measurements before and after the sorption of HSA as a function of the number of PVAm layers, the results being summarized in Table 5. The decrease in the contact angle of 6-8° when the protein has been loaded on the films containing six and eight layers of PVAm shows the increase in the hydrophilicity as it was

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Table 4. Pore Characteristics Including the Area, Volume, Perimeter, and Length of the Pores on the Surfacea film description

number of pores collected

area (µm2)

volume (µm3)

perimeter (µm)

length (µm)

(PVAm)4 (PVAm)4 + HSA (PVAm)6 (PVAm)6 + HSA (PVAm)8 (PVAm)8 + HSA

70 138 58 131 40 101

0.025 0.010 0.015 0.005 0.021 0.017

0.202 0.13 0.116 0.063 0.353 0.141

1.745 0.647 0.524 0.184 0.6 0.53

0.082 0.102 0.172 0.133 0.176 0.114

a

Scanned area 2 × 2 µm2.

Figure 7. AFM phase images of two (PVAm)n films after the adsorption of HSA; scan size was 1 × 1 µm2. Table 5. Contact Angle and Standard Deviation of the Single-Component Multilayer Thin Films before and after the Adsorption of HSA as a Function of the Number of PVAm Layers n

(PVAm)n

(PVAm)n + HSA vacuum-dried

(PVAm)n + HSA freeze-dried

4 6 8

88.8 ( 1.0 95.3 ( 1.2 95.0 ( 0.2

87.0 ( 0.4 85.6 ( 0.7 87.3 ( 0.8

85.5 ( 0.4 85.1 ( 0.7 89.2 ( 0.1

Table 6. Contact Angle and Standard Deviation of the (PVAm/ PAA)n Thin Films Deposited on Silicon Wafers and of the Single-Component (PVAm)n Thin Films before and after the Adsorption of BSA as a Function of the Number of PVAm Layers n

(PVAm/PAA)n-0.5

(PVAm)n

(PVAm)n + BSA

4 6 8

90.0 ( 1.6 89.6 ( 0.2 89.7 ( 0.9

88.8 ( 1.0 95.3 ( 1.2 95.0 ( 0.2

70.5 ( 2.0 74.4 ( 0.5 77.8 ( 0.9

observed when HSA had been adsorbed on other surfaces.22,43 The increase in the film hydrophylicity shows that the adsorption of protein occurred inside the porous single-component film, according to Scheme 2. The smaller difference between the contact angle of the film before and after the adsorption of protein when the film consisted of only four layers of PVAm deposited on silicon wafers shows that the homogeneity of the film was not high enough, as was already observed from the AFM images in both height (Figure 5) and phase (Figure 7). The drying procedure of the film influenced the adsorption of protein mainly for the film with the lowest number of PVAm layers. The sorption of BSA on the cross-linked (PVAm)n films deposited on silicon wafers as a function of the number of polycation layers has been investigated by AFM and contact angle measurements. Figure 8 presents the height AFM images of three (PVAm)n films after the sorption of BSA, and the characteristics of film surfaces, average roughness, Ra, and average height, ha, given by the Gwyddion 2.15 Software, are also included.

As can be seen, the values of Ra and ha decreased with the increase in the number of PVAm layers. As in the case of the sorption of HSA, the decrease in Ra and ha with the increase in the polycation layers, after the interaction with BSA, has been attributed to the protein absorption inside the film. The values of contact angles and standard deviations measured on the films after the sorption of BSA compared with those of the dicomponent LbL film and the single-component film, as a function of the number of PVAm layers, are summarized in Table 6. As Table 6 shows, the contact angle of the dicomponent films, that is, the films before the removal of PAA, all having PVAm as the last layer adsorbed, were independent of the number of layers, being ∼90°. After the interaction with protein, the contact angle decreased, and the hydrophilicity of the film increased even more than in the case of HSA sorption (Table 5), supporting the same mechanism of interaction between the porous film and protein (Scheme 2). Even if HSA and BSA are ∼80% homologous with their molecular weights and isoelectric points being very similar, significant differences have been observed at their adsorption at silica-titania surfaces.44 The differences in the wettability of the film surface after the protein sorption on the porous cross-linked multilayer film, that is, the lower values of contact angles after the sorption of BSA compared with HSA, could be attributed to the increase in the density of hydrophilic groups on the film surface as a consequence of the higher probability of hydrophobic interactions between BSA and the porous multilayer film.

IV. Conclusions Herein we have described the preparation of cross-linked single-component multilayer thin films based on PVAm, which showed a high absorption for proteins like HSA and BSA. The PVAm molar mass strongly influenced the construction of the (PVAm/PAA) multilayer, and the protein loading onto the porous single-component (PVAm)n resulted after the removal



795

Figure 8. Height AFM images of the cross-linked single-component (PVAm)n films deposited on the silicon wafers after the adsorption of BSA: (a) n ) 4, (b) n ) 6, and (c) n ) 8; scan size was 1 × 1 µm2 in all images. On the right, the height profiles generated along the horizontal lines for the same samples are shown.

of PAA. A regular increase in the film on silica particles was found by the potentiometric titration when PVAm with a low molar mass (15 000 g mol-1) was used in the construction of the precursor (PVAm/PAA) multilayer, the highest amount of HSA being found on the porous single-component (PVAm)n deposited on silica particles. By AFM and contact angle measurements on the films deposited on silicon wafers, we have demonstrated that the sorption of proteins occurred inside the porous cross-linked single-component film as much as the number of PVAm layers was higher. Therefore, the surface roughness decreased and the hydrophilicity increased after the sorption of both proteins, which recommends these singlecomponent films as potential reservoirs for proteins

Acknowledgment. We gratefully acknowledge the financial support of this research from the Exploratory Research Project ID_981/2009.

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BM9014057

Sorption of Proteins onto Porous Single-Component Poly(vinyl amine

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