Sorption of Proteins onto Porous Single-Component Poly(vinyl amine) Multilayer Thin Films

Sorption of Proteins onto Porous Single-Component Poly(vinylamine) 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 aselective cross-linking of the PVAm layers in the [PVAm/poly(acrylic acid) (PAA)]n thin films, followed by theremoval of PAA. A regular increase in the (PVAm/PAA)n multilayer onto silica particles was observed bypotentiometric titration when PVAm with a low molar mass (Mw ) 15 000 g mol-1) was used in the constructionof LbL thin films. The amount of human serum albumin (HSA) loaded on the single-component (PVAm)5 thinfilm was ∼32 mg HSA/g of hybrid when PVAm with a molar mass of 15 000 g mol-1 was used compared withthe single-component film prepared with PVAm with a molar mass of 340 000 g mol-1 when the amount of HSAloaded was 15.5 mg HSA/g of hybrid. The sorption modalities of HSA and bovine serum albumin (BSA) ontothe single-component thin films deposited on silicon wafers as a function of the number of PVAm layers wasinvestigated by atomic force microscopy and contact angle measurements. The decrease in the film roughnessand the increase in the film wettability after the loading of both proteins showed the protein was mainly absorbedinto the porous (PVAm)n thin film.

I. Introduction

Layer-by-layer (LbL) deposition of polyelectrolytes andbiological species like DNA and proteins, introduced byDecher,1,2 is perhaps the most widely used technique to tailorsurfaces with controlled biological properties, such as enzymaticactivity,3–7 immunosensing,8 nanoreactors,9,10 biosensors,11,12

and controlled release of entrapped species.13–19 Controlledrelease of drugs from LbL thin films, either with embedded dyesas models for drugs15 or with so-called “prodrugs” (drugattached to a biocompatible polyanion by a labile linkage),16

has been promoted for the creation of more efficient controlleddelivery systems. DNA has been entrapped in the LbL thin filmseither as polyplex (soluble interpolyelectrolyte complex) formedwith a synthetic polycation, which has been used in themultilayer construction by alternation with a biocompatiblepolyanion,17 embedded into polyelectrolyte multilayer films,18

or alternately adsorbed with polycations to get multilayer thinfilms.19 The release of DNA by the erosion (deconstruction) ofthe LbL thin films could have a significant impact on thedevelopment of localized gene therapies.19 The preservation ofprotein structure in the “electrostatic cage” formed by poly-electrolytes motivated the use of proteins for biological func-tionalization of polyelectrolyte multilayers.20 For this purpose,proteins are embedded in or adsorbed onto polyelectrolytemultilayers, with further stabilization gained through cross-linking.

The adsorption of charged proteins onto multilayer-coatedsurfaces is a complex phenomenon widely studied so far eitherto reduce the fouling process or to increase the adsorption ofproteins.21–25 Even if various interactions contribute to theadsorption/rejection of proteins on the LbL thin films, it isgenerally accepted that the surface charge plays a majorrole.21,22,24 Therefore, only a monolayer of protein has been

found on the negatively charged film surface, whereas, on thepositively charged surface, the thickness of the adsorbed proteinfilm increased up to several times the largest dimension of thenative protein molecule.21,22 The influence of the surface chargeon the secondary structure of the adsorbed protein has also beeninvestigated, and it has been found that the structural changeswere larger when the surface charge and protein charge wereopposite.20 However, the film morphology, which is stronglycorrelated with the conditions of film construction, would havea definite influence on the protein adsorption/diffusion into theLbL thin films.

One of the most interesting categories of building blocks forthe construction of LbL thin films with desired properties isthat of weak polyelectrolytes because they allow a fine controlof the layer pair thickness, surface roughness, and film morphol-ogy by their complex behavior over a wide range of pH.Normally, there is a fine balance between electrostatics26–30 andhydrogen bondings,31–34 their role in the LbL film growth andstability being determined by the local microenvironment.Recently, we have reported on reactive polyelectrolyte multi-layers generated by the LbL deposition of two weak polyelec-trolytes, poly(vinyl amine) (PVAm) and poly(acrylic acid)(PAA), onto silica microparticles, the LbL films being stabilizedby 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 groupson the surface, was subsequently decorated with short peptidebrushes of S-benzyl-L-cysteine.35

Nanoporous and microporous LbL thin films have beenprepared from weak polyelectrolytes,33,34,36–38 and their possibleapplications as biomaterials have been widely explored.33,38 Salt-induced structural changes34 and heat-induced formation ofamide bonds36 have been used to generate and to lock in theporosity in LbL thin films. Caruso et al. prepared nanoporousthin films via polyelectrolyte templating, where two components,PAA and poly(allylamine hydrochloride), were interconnected

* To whom correspondence should be addressed. Tel: +40.232217454.Fax: +40.232211299. E-mail: [email protected].

Biomacromolecules 2010, 11, 787–796 787

10.1021/bm9014057 2010 American Chemical SocietyPublished on Web 02/15/2010

by amide bonds, and the third polyelectrolyte used as template,poly(4-vinylpyridine), has been removed after the film construc-tion.32 We have recently reported on a novel strategy to designporous thin films, which consists of the alternate deposition ofPVAm and PAA onto silica microparticles or silicon wafers,followed by: (1) a selective chemical cross-linking of polycationlayers and (2) the removal of PAA, a single-component cross-linked multilayer of (PVAm)n remaining at the end.39 The mainparameters that control the film properties and the interactionof the human serum albumin (HSA) and bovine serum albumin(BSA) with the single-component (PVAm)n thin films areinvestigated in this work. To the best of our knowledge, this isthe first extended study on the formation of porous single-component (PVAm)n thin films and on the sorption of proteinsonto these films as a function of the conditions of the double-component multilayer construction.

The chemical structures of polyelectrolytes used in this workare presented in Chart 1. Film construction and stability wereexamined by potentiometric titration when silica microparticleswere used as substrate and by atomic force microscopy (AFM)when the LbL films were deposited on silicon wafers. Theprotein sorption on the single-component multilayers wasfollowed by UV-vis spectroscopy when the (PVAm)n filmswere deposited on silica microparticles and by AFM and contactangle measurements when the films were deposited on siliconwafers.

II. Materials and Methods

II.1. Materials. The PVAm samples with molar masses of 15 000and 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 weresynthesized and characterized according to ref 40 with Mw/Mn of 1.13and 1.9, respectively. Epichlorohydrin (ECH) analytical grade (Sigma-Aldrich, Germany) was used as received. HSA with 96-99% purityand 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 withconcentrations of 5 × 10-3 mol repeat unit L-1, were prepared formultilayer deposition. Quantitative determination of the charged groupsin solution was performed by polyelectrolyte titration with a particlecharge detector PCD 03, Mutek GmbH, Herrsching, Germany, usingeither poly(sodium ethylenesulfonate) or poly(diallyldimethylammo-nium chloride), with a concentration of 10-3 mol L-1, in dependenceon the nature of charges. Kieselgel 60 (Merck, Darmstadt, Germany)having the main diameter of the silica microparticles ranging between15 and 40 µm, with a maximum of the pore diameters in the range of4-6 nm, has been used after the activation with NaHCO3 aqueous

solution, with a concentration of 1%, followed by washing with distilledwater to neutral pH. Silicon wafer substrates were carefully cleaned intwo steps: (1) in Piranha solution (70:30% v/v sulfuric acid/hydrogenperoxide) (Caution! Piranha solution is highly corrosiVe and extremecare should be taken when handling it), followed by intensive rinsingwith distilled water and (2) with the mixture NH4OH/H2O2/distilledwater (1:1:1) at 70 °C, in an ultrasonic bath, and finally intensive rinsingwith distilled water.

II.2. Construction of (PVAm/PAA)n Multilayers. Two strategieswere used for the (PVAm/PAA)n multilayer buildup onto silicamicroparticles. According to the first strategy, both polyions wereadsorbed from salt-free aqueous solutions at the pH corresponding tothe solution of each polyion, that is, 9.5 for PVAm, this being the pHof 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. ThepH of water used in washing steps was adjusted at 3.5 for washingafter the PVAm adsorption steps, and at 9.5 for washing after the PAAadsorption steps to remove the weakly adsorbed polyions. Samples of3 g of silica were suspended in 150 mL of a 5 × 10-3 mol L-1 PVAmsalt-free aqueous solution. During the adsorption process, over 1 h,the suspension was gently shaken at room temperature. The modifiedparticles were washed three times with water at pH 3.5 to rinse theweakly adsorbed PVAm chains from the surface of silica particles. Forthe second adsorption step, the PVAm/silica particles were suspendedin 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 atpH 9.5 to rinse the weakly adsorbed PAA chains. The adsorption andwashing steps were repeated until the desired number of layers hadbeen deposited.

According to the second strategy, a constant pH of 5.0 was kept inboth the polyions adsorption steps and in the washing steps.

The pairs of polyelectrolytes, their molar masses, and pH valuesused in the adsorption and washing steps are listed in Table 1.

The multilayer construction on silicon wafers was performed underthe same conditions as those used for the multilayer deposition on silicamicroparticles, 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 multilayerwas performed with ECH at a ratio of (5 mg m-2) × n, where n is thenumber of PVAm layers in the film. The surface of silica particles hasbeen estimated by approximation of silica particles with spheresaccording 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-dropwith agitation; the cross-linking reaction was carried out for 3 h, atroom 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 depositedwere immersed, separately, in 4 mL of water, and then 20, 30, and 40µL of ECH in water/acetone 1:1 (v/v), with a concentration of 0.1 mgmL-1, was added drop-by-drop with agitation at room temperature for3 h. Silica particles and silicon wafers covered with (PVAm/PAA)n

multilayers thin films were washed three times with water and driedunder vacuum at room temperature. As far as the AFM technique wasconcerned, freeze-drying technique was also applied, the drying effecton the film architecture leveled off, and the film morphology was thuspreserved.

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) pH of water in the washing steps

A 15 000 (low) 57 000 (high) 3.5 after the adsorption of PVAm; 9.5 after the adsorption of PAAB 340 000 (high) 11 000 (low) 3.5 after the adsorption of PVAm; 9.5 after the adsorption of PAAC 340 000 (high) 57 000 (high) 3.5 after the adsorption of PVAm; 9.5 after the adsorption of PAAD 340 000 (high) 11 000 (low) 5.0 after every adsorbed layer

Chart 1. Structures of the Polyelectrolytes Used in This Study

788 Biomacromolecules, Vol. 11, No. 3, 2010 Dragan et al.

After cross-linking, we removed the PAA by shaking the silicaparticles and silicon wafers in water at pH 11 and 60 °C until PAAwas completely removed, followed by rinsing with water at neutralpH. Quantitative determination of PAA in the extraction environmentwas performed by polyelectrolyte titration with the particle chargedetector PCD 03, Mutek GmbH, Germany. The time and the numberof extraction steps depended on the number of PAA layers. Herein weinclude some results obtained on series C (Table 1), which demonstratethat the optimum conditions for the removal of PAA have beenestablished step-by-step. We have started the extraction of PAA fromthe multilayers deposited on silica particles at pH 9 and at roomtemperature, and the amount of PAA removed under these conditionsis 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 (>45h). Therefore, the pH and temperature of the extraction solution weregradually increased, with the fastest removal of PAA being found atpH 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 andafter the Removal of PAA. Potentiometric titration of the silicamicroparticles as a function of the number of adsorbed polyion layerswas performed with the particle charge detector PCD 03, Mutek GmbH,Herrsching, Germany. The topography of the multilayers (PVAm/PAA)n, before and after the removal of PAA and also after the sorptionof proteins, was examined with a SPM Solver PRO-M AFM (NT-MTDCo. Zelenograd, Moscow, Russia). For this purpose, the polyelectrolyteswere alternately deposited on silicon wafers under conditions similarto those used for the multilayer deposition onto silica microparticles.Before the AFM measurements, silicon wafers were either dried undervacuum for 48 h at room temperature or freeze-dried when the

preservation of film morphology was of interest. Images were taken intapping mode at room temperature. All images were acquired using ahigh-resolution no contact “Golden” silicon NSG10/Au/50 cantileverwith Au conductive coating. The images were examined by means ofthe 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 EASYDROPShape Analysis System (KRUSS) equipped with DSA 1 evaluationsoftware.41 Water drop was placed on the polymeric film located on amoveable sample table. The drop was illuminated from one side, anda video camera at the opposite side records an image of the drop. Thisimage was transferred to a computer equipped with a video-digitizerboard (frame-grabber). The DSA 1 software contains time-proven toolsfor analyzing the drop image to calculate the contact angle values. Thecontact 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 cross-linked (PVAm)n films deposited onto silica particles, 200 mg ofsubstrate was suspended in 30 mL protein solution. After 4 h at roomtemperature, the concentration of supernatant was determined byUV-vis spectroscopy at 278 nm by using a SPECORD 200 AnalyticJena apparatus. Three measurements were averaged. For protein sorptiononto the cross-linked (PVAm)n films deposited on silicon wafers, eachwafer was immersed in 5 mL of HSA solution for 4 h at roomtemperature. After that, all wafers were washed three times with distilledwater and freeze-dried to preserve the film morphology.

III. Results and Discussion

III.1. Construction and Characterization of Cross-Linked (PVAm)n Multilayers. The schematic representationof the (PVAm/PAA)n multilayer buildup and the generation ofthe cross-linked single-component PVAm multilayers onto silicamicroparticles, according to the first strategy (Materials andMethods section), is illustrated in Scheme 1.

The pH of the polyelectrolyte solutions used in theconstruction of LbL multilayers was 9.5 for the PVAmsolution and 3.5 for PAA solution, that is, the pH corre-sponding to the concentration of 5 × 10-3 mol ·L-1 of eachpolyion. Under these conditions, the main driving force inthe deposition of the next polyelectrolyte layer is hydrogenbonding, polyelectrolyte chains being in a coiled conforma-tion. To remove the excess of polyelectrolyte, the pH inthe washing steps was changed, being the same as that ofthe next layer of polyelectrolyte to be adsorbed. Therefore,

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

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

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.

Sorption of Proteins on PVAm Multilayers Biomacromolecules, Vol. 11, No. 3, 2010 789

the surface of the last adsorbed layer will mainly containionized groups, being still able to interact with the next layerof polyelectrolyte by hydrogen bonds.42 The film constructionon silicon wafers was carried out according to the sameprotocol. To gain information on the influence of PVAmmolar mass on the LbL multilayer construction and stability,the potentiometric titration after each adsorbed layer (PVAmor PAA) on silica microparticles was performed, and theresults obtained for the multilayer buildup according to thefirst strategy are summarized in Figures 2 and 3.

The potential, Ψ, of the (PVAm/PAA)n/silica particles asa function of solution pH, when PVAm with a molar massof 15 000 g mol-1 and PAA with molar mass of 57 000 gmol-1 were used in the construction of LbL thin films, areplotted in Figure 2a. A positive potential Ψ can be observedin the range of pH 3-9 when the first PVAm layer isadsorbed 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 particlesshifted to 3.8. The next layer of PVAm shifted the pzc intothe basic range (pzc ) 9), whereas the next layer of PAAagain shifted the pzc to the acidic range. This behaviordemonstrates that the last adsorbed polyelectrolyte layer,polycation or polyanion, strongly determined the surfaceBrønsted acidity of the hybrid particles. The values of pzcaround 9 and 4 when the last layer adsorbed was PVAm andPAA, respectively, suggest a constant and regular increasein 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 particleswere nearly identical (pzc ) 9.2) because the last layer adsorbedwas the polycation. However, the values of the potential Ψcorresponding to the plateau region, located in the acidic range,were different. Therefore, the silica particles covered with onelayer of PVAm had a potential of plateau region at nearly 600mV, and this value was higher than the potential of cross-linkedhybrid particles (PVAm/PAA)4.5-ECH/silica because the polya-nion locked in the cross-linked multilayer decreased thepotential. The potential of (PVAm)5/silica hybrid particles inthe plateau region was 750 mV, higher than the potential ofPVAm/silica, because the five polycation layers significantlyreduced the influence of silica surface on the potential Ψ.

A different behavior was observed when high PVAm andhigh PAA were used as polyions (series C, Table 1) in theconstruction of LbL thin films on silica microparticles (Figure3).

Therefore, the pzc values of (PVAm/PAA)n/silica hybridshaving PVAm as the last layer adsorbed decreased from 9.2 to7.5, 6.6, and 6.8 when one, two, four, or five polycation layerswere adsorbed. When PAA was the last layer adsorbed, the pzcvalues were 5.2 and 3.9 for two and three PAA layers,respectively. This irregular variation of pzc values shows thatPVAm with a very high molar mass is not suitable for a regularcover of silica microparticles with LbL thin films. It seems thatthe removal of large coils, which did not establish enoughhydrogen bonds with the film surface, was more probable inthis case.

III.2. Sorption of Proteins onto Cross-Linked (PVAm)n

Multilayers. To demonstrate the protein sorption properties ofthe cross-linked (PVAm)n thin films deposited either on silicamicroparticles or silicon wafers, two proteins have been selected,HSA and BSA, both being widely used in protein adsorptiontests on multilayer-coated surfaces.21–23 To demonstrate that thelevel of the PAA removal is very important for the amount ofloaded protein, some results concerning the amount of HSAsorbed by the (PVAm)5/silica hybrids generated from series A(Table 1) were presented in Table 2.

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

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

Table 2. Adsorbed Amount of HSA as a Function of ExtractionConditions of PAA from (PVAm/PAA)4.5/Silica Hybrid

conditions forPAA 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


As can be seen from Table 2, the amount of loaded proteinincreased with the increase in pH and temperature at theextraction step of PAA, the highest amount of HSA being foundwhen PAA was removed at pH 11 and 60 °C, conditionsrecommended in the Materials and Methods section.

Figure 4 illustrates the influence of the molar mass of polyionsused as building blocks in the LbL multilayer construction andof the pH used in different steps of the multilayer depositionon 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 themolar mass of the polyion pairs used in the LbL multilayerconstruction. The highest amount of protein has been loadedon the cross-linked (PVAm)5 film obtained by the removalof PAA from the (PVAm/PAA)4.5 multilayer constructed withlow PVAm and high PAA (series A, Table 1). This resultwas correlated with the regular increase in the (PVAm/PAA)n

thin film shown by potentiometric titration (Figure 2) for thispolyion pair. When high PVAm and high PAA were usedfor the (PVAm/PAA)4.5 multilayer construction (series C,Table 1), the lowest amount of HSA was found, in agreementwith the results of polyelectrolyte titration (Figure 3).Concerning the protein sorption on the porous (PVAm)n thinfilms resulted from the (PVAm/PAA)n films prepared withhigh PVAm and low PAA (series D, Table 1), constructedwith the same polyion pair like series B but at a constant pH

of 5.0 in both the polyion deposition and washing steps (thesecond strategy), the decrease in the amount of sorbed HSAwith the increase in PVAm layers was found; this behaviorsupports the lower stability of the multilayer constructed withhigh PVAm. However, the amount of protein loaded on thecross-linked (PVAm)8 film was a little higher than that foundin series B, constructed according to the first strategy(Materials and Methods section).

The results presented in Figure 4 do not give clear informationon the sorption modalities, adsorption, or absorption.22 Todiscriminate which process is the most probable, we roughlycalculated the amount of protein that could be adsorbed in amonolayer on the film surface. Therefore, the volume of HSAmolecule assumed as an ellipsoid, at pH 7.4, is 12 × 2.7 × 2.7nm3.21 On the basis of the assumption that the protein moleculeswere preferentially adsorbed with their major axis perpendicularto the adsorbing surface,44 the occupied area of one single HSAadsorbed molecule is 5.72 nm2 (adsorption in “side-on” con-formation21). the maximum number of HSA molecules adsorbedon 1 cm2, in this case, would be 17.48 × 1012 molecules. Takinginto account the molecular weight of protein molecule, whichis 66 000 g mol-1, the coverage of silica surface by the protein,as monolayer, would be 19.1 mg m-2. Because the externalsurface of silica microparticles was approximated to be 0.4 m2

g-1,39 a loading of 7.64 mg g-1 resulted. For the proteinadsorption in “end-on” conformation,21 the calculated loadingwas 1.72 mg g-1. Even if this calculation was very rough, thevalue obtained based on the adsorption in the “side-on”conformation is almost the same as that experimental valuefound from the adsorption isotherm of HSA on one layer ofPVAm deposited on silica particles.45 Because the maximumadsorbed amount of HSA found for series A was ∼32 mg g-1,at least two modalities of sorption could be assumed, accordingto Scheme 2: either a multilayer of protein molecules adsorbedon the film surface (a) or the protein absorption into the bulkof the porous cross-linked film (b).

To gain information on the most probable sorption process,we have investigated the surface morphology of the multilayerfilms, before and after the protein sorption, by tapping-modeAFM. Figures 5 and 6 illustrate the topography of the cross-linked (PVAm)n multilayer thin films deposited on siliconwafers, generated after the removal of PAA, and of the samefilms after the sorption of HSA on the single-component(PVAm)n multilayer thin films as a function of the number ofPVAm layers.

Calculation of surface properties could be a key factor incomparing the topography before and after the adsorption ofprotein. Surface characteristics (root-mean-square roughness, Sq,average roughness, Sa, average height, HA, average diameter,

Figure 4. Amount of HSA adsorbed onto the cross-linked single-component (PVAm)5 multilayer film deposited onto silica micro-particles as a function of the polyion pair used in the LbL thin filmconstruction: (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 thestandard deviations were calculated from three independentmeasurements.

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

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


ADP, and height, HHP) evaluated for the films having up to 8PVAm layers are summarized in Table 3.

As Table 3 shows, Sq, Sa, ADP, and HHP of the single-component (PVAm)n thin films, decreased with the increase inthe number of layers, Sa, for example, being 5.04 nm for thefilm having (PVAm)4 and 1.97 nm for the film having (PVAm)8.The decrease in the film roughness was attributed to the regularcoverage of the surface, the film getting smoother with theincrease 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 constitutethe monocomponent thin film, can be associated with thesorption of protein mainly inside the film.22 Also, the images

of height profiles on the cross-section show that the distancehills-to-grooves in the case of (PVAm)4 ranges from 25 nmbefore 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 (Figure5 and 6). The increase in the film surface homogeneity afterthe sorption of protein is well illustrated by histograms, whichshow that the majority of the highest points on the surface aresituated between 7 and 15 nm after the protein loading on thefilms having six and eight PVAm layers.

The processing of AFM images and the calculation of thesurface texture parameters by the Nova 9.09 Software gave somecharacteristics of the pores, which are summarized in Table 4.

Figure 5. Height AFM images of the cross-linked single-component (PVAm)4 films deposited on the silicon wafers: 2D (a) before and (b) afterthe 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 samesamples.


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

the sorption of HSA, and this demonstrates that the protein hasbeen loaded into the porous film, which acts as a sponge forprotein.

The AFM images in phase of the (PVAm)n after the sorptionof HSA were obtained by the measurement of the differencesbetween the cantilever amplitude oscillations in contact withdifferent phases on the surface. Figure 7 shows a much smoothersurface of the film containing (PVAm)8 after the sorption ofHSA compared with that of the film containing (PVAm)4, thedifferences in color being very small in the first case; that is,the protein molecules are not on the film surface but inside thefilm, thus supporting the sorption modality (b) in Scheme 2.

The AFM information was supported by the contact anglemeasurements before and after the sorption of HSA as a functionof the number of PVAm layers, the results being summarizedin Table 5.

The decrease in the contact angle of 6-8° when the proteinhas been loaded on the films containing six and eight layers ofPVAm shows the increase in the hydrophilicity as it was

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 samesamples.

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 theHistograms and Average Diameter (ADP) and Height (HHP) fromthe Height Profiles before and after the Adsorption of HSAa

sample Figure AFM data (nm)Sq Sa AH ADP HHP

(PVAm)4 5 6.35 5.04 13.47 100 25(PVAm)4 + HSA 6.03 4.64 14.92 60 12(PVAm)6 not included here 4.38 3.06 10.35 110 30(PVAm)6 + HSA 2.27 1.59 6.53 50 10(PVAm)8 6 2.64 1.97 17.63 120 14(PVAm)8 + HSA 2.10 1.53 10.92 90 11

a Scanned area 2 × 2 µm2.


observed when HSA had been adsorbed on other surfaces.22,43

The increase in the film hydrophylicity shows that the adsorptionof protein occurred inside the porous single-component film,according to Scheme 2.

The smaller difference between the contact angle of the filmbefore and after the adsorption of protein when the film consistedof only four layers of PVAm deposited on silicon wafers showsthat the homogeneity of the film was not high enough, as wasalready observed from the AFM images in both height (Figure5) and phase (Figure 7). The drying procedure of the filminfluenced the adsorption of protein mainly for the film withthe lowest number of PVAm layers.

The sorption of BSA on the cross-linked (PVAm)n filmsdeposited on silicon wafers as a function of the number ofpolycation layers has been investigated by AFM and contactangle measurements. Figure 8 presents the height AFM imagesof three (PVAm)n films after the sorption of BSA, and thecharacteristics of film surfaces, average roughness, Ra, andaverage height, ha, given by the Gwyddion 2.15 Software, arealso included.

As can be seen, the values of Ra and ha decreased with theincrease in the number of PVAm layers. As in the case of thesorption of HSA, the decrease in Ra and ha with the increase inthe polycation layers, after the interaction with BSA, has beenattributed to the protein absorption inside the film.

The values of contact angles and standard deviations mea-sured on the films after the sorption of BSA compared withthose of the dicomponent LbL film and the single-componentfilm, as a function of the number of PVAm layers, aresummarized 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 PVAmas the last layer adsorbed, were independent of the number oflayers, being ∼90°. After the interaction with protein, the contactangle decreased, and the hydrophilicity of the film increasedeven more than in the case of HSA sorption (Table 5),supporting the same mechanism of interaction between theporous film and protein (Scheme 2). Even if HSA and BSA are∼80% homologous with their molecular weights and isoelectricpoints being very similar, significant differences have beenobserved at their adsorption at silica-titania surfaces.44 Thedifferences in the wettability of the film surface after the proteinsorption on the porous cross-linked multilayer film, that is, thelower values of contact angles after the sorption of BSAcompared with HSA, could be attributed to the increase in thedensity of hydrophilic groups on the film surface as a conse-quence of the higher probability of hydrophobic interactionsbetween BSA and the porous multilayer film.

IV. Conclusions

Herein we have described the preparation of cross-linkedsingle-component multilayer thin films based on PVAm, whichshowed a high absorption for proteins like HSA and BSA. ThePVAm molar mass strongly influenced the construction ofthe (PVAm/PAA) multilayer, and the protein loading onto theporous single-component (PVAm)n resulted after the removal

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 70 0.025 0.202 1.745 0.082(PVAm)4 + HSA 138 0.010 0.13 0.647 0.102(PVAm)6 58 0.015 0.116 0.524 0.172(PVAm)6 + HSA 131 0.005 0.063 0.184 0.133(PVAm)8 40 0.021 0.353 0.6 0.176(PVAm)8 + HSA 101 0.017 0.141 0.53 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 theSingle-Component Multilayer Thin Films before and after theAdsorption of HSA as a Function of the Number of PVAm Layers

n (PVAm)n (PVAm)n + HSA (PVAm)n + HSAvacuum-dried freeze-dried

4 88.8 ( 1.0 87.0 ( 0.4 85.5 ( 0.46 95.3 ( 1.2 85.6 ( 0.7 85.1 ( 0.78 95.0 ( 0.2 87.3 ( 0.8 89.2 ( 0.1

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

n (PVAm/PAA)n-0.5 (PVAm)n (PVAm)n + BSA

4 90.0 ( 1.6 88.8 ( 1.0 70.5 ( 2.06 89.6 ( 0.2 95.3 ( 1.2 74.4 ( 0.58 89.7 ( 0.9 95.0 ( 0.2 77.8 ( 0.9


of PAA. A regular increase in the film on silica particles wasfound by the potentiometric titration when PVAm with a lowmolar mass (15 000 g mol-1) was used in the construction ofthe precursor (PVAm/PAA) multilayer, the highest amount ofHSA being found on the porous single-component (PVAm)n

deposited on silica particles. By AFM and contact angle mea-surements on the films deposited on silicon wafers, we havedemonstrated that the sorption of proteins occurred inside theporous cross-linked single-component film as much as thenumber of PVAm layers was higher. Therefore, the sur-face roughness decreased and the hydrophilicity increased afterthe sorption of both proteins, which recommends these single-component films as potential reservoirs for proteins

Acknowledgment. We gratefully acknowledge the financialsupport of this research from the Exploratory Research ProjectID_981/2009.

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Sorption of Proteins onto Porous Single-Component Poly(vinyl amine) Multilayer Thin Films

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