PAH

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J. Phys. Chem. B 2008, 112, 6322–6330

Granular Structure of Self-Assembled PAA/PAH and PSS/PAH Nascent Films Imaged in situ by LC-AFM Ferdinando Tristan,† J.-Luis Menchaca,§ Fre´de´ric Cuisinier,| and Elıás Pe´rez*,‡ CIEP/Facultad de Ciencias Quı´micas, Instituto de Fı´sica, UniVersidad Autońoma de San Luis Potosı´, AlVaro Obregoń 64, 78000 San Luis Potosı´, Me´xico, Unidad Acade´mica de Fı´sica, UniVersidad Autońoma de Zacatecas, Calzada Solidaridad esq. Paseo a la Bufa s/n, 98060 Zacatecas, Me´xico, and BioNano, UniVersite´ Montpellier 1, 545 AV. du Professeur Jean-Louis Viala, 34193 Montpellier, France ReceiVed: October 21, 2007; ReVised Manuscript ReceiVed: February 1, 2008

Liquid cell atomic force microscopy (LC-AFM) is used to image self-assembled polyelectrolyte films eliminating any drying effects on the film structure. Weak/weak and strong/weak polyelectrolyte films are formed by the alternated deposition of poly(acrylic acid) [PAA]/poly(allylamine hydrochloride) [PAH], and poly(sodium 4-styrene sulfonate) [PSS]/PAH, respectively, forming a granular surface structure. Number and area of grains (GN, GA) are used to characterize the surface of these films during their build up process. We show that hydrophilic PAA increases GA and decreases GN, while these parameters follow an opposite behavior with PSS. In both cases, GA and GN always have a simple inverse relationship, and then grain surface coverage (GS ) GNGA) is nearly constant and independent of polyelectrolyte nature and the substrates used here, but also in the published data as well. The drying of the weak/weak film was also imaged after natural and forced solvent evaporation, and the surface structure is strongly affected, although the GS values keep roughly the same value found for wet films. The set of these results indicates that GS may be considered as a constant parameter during the build-up for the self-nascent assembled polyelectrolytes. The granular structure is still maintained after glucose oxidase adsorption on these films with comparable GS values. 1. Introduction Self-assembled polyelectrolyte multilayer films were first introduced by Decher et al. in 1992.1 Since then, polyelectrolyte multilayer films have been extensively used as support for synthetic materials (metal2 or polymer3 nanoparticles, clays,4 and carbon nanotubes5) or biomolecules (proteins,6 nucleic acids,7 polysaccharides,8 and among others) in different fields of material science. The surface structure of polyelectrolyte films is an important issue in this domain because it may define the conditions for the material deposition onto these films. The final structure of polyelectrolyte films is dependent on several parameters, such as polyelectrolyte molecular weight,9 ionic strength,10 pH of the solution,11 and also on the preparation methods.12 Numerous studies have been performed to elucidate the basic mechanism of the multilayer formation and its bulk structure. Techniques such as ellipsometry, X-ray reflectivity, scanning force microscopy, and so forth have been used to this end.13 However, the surface structure of these films has received relatively less attention. Atomic force microscopy (AFM) is a straightforward technique to observe the polyelectrolyte film surface, and it has been used in two contrasted conditions: when the film is prepared ex-situ and imaged after a drying process14 and when the film is built up and imaged in situ in a liquid cell of AFM,15,16 or even in a liquid droplet.17 These methods have produced images of granular surface structure for poly(sodium 4-styrene sul* Corresponding author. Phone: (+52) (444) 8.26.23.62 ext. 134. Fax: (+52) (444) 8.13.38.74. E-mail: [email protected]. † CIEP/Facultad de Ciencias Quı´micas, Universidad Auto ´ noma de San Luis Potosı´. ‡ Instituto de Fı´sica, Universidad Auto ´ noma de San Luis Potosı´. § Universidad Auto ´ noma de Zacatecas. | Universite ´ Montpellier 1.

fonate) [PSS]/poly(allylamine hydrochloride) [PAH], which have been also imaged when a high number of depositions has been reached17 and when nitrogen flow dries the polyelectrolyte film surface.14 In the last case, drying effects have been evident because polymer grains are aligned with the drying flow. The reported images reveal a dependence of the surface structure on the experimental conditions. An important dependence on the nature of polyelectrolytes is also expected, as occurs with the bulk structure,11,18 i.e., the films are characterized by a thick linear growth with the deposited layers for a strong/weak polyelectrolyte pair, such as PSS/PAH,19 and an exponential growth regime for films formed by weak polyelectrolytes.20 This nonlinear growth regime may be also reflected on the final surface structure of the films, as well as on their surface physical properties.21 In this work, the granular structure of the weak/weak PAA/ PAH self-assembled films is analyzed in situ by LC-AFM and compared with the corresponding strong/weak PSS/PAH ones during their build up. Liquid cell AFM (LC-AFM) is a suitable technique for these purposes because it eliminates any drying effect on the morphology of the polyelectrolyte film during the self-assembly process. This technique has shown its utility, showing the granular and ring-like surface structures of polyelectrolyte films.15,16 This study is addressed to first depositions of the polyelectrolytes, where the conditions of the polyelectrolyte absorption imposed by the substrate may be important, and when the self-assembly process begins. Surface effect is tested using silicon wafer and mica. The drying effects were also observed for a weak/weak film. The number and area of grains characterize the surface structure. They also define the granular surface coverage that seems to be a constant quantity, even after glucose oxidase adsorption.

10.1021/jp710195e CCC: $40.75  2008 American Chemical Society Published on Web 04/29/2008

Self-assembled PAA/PAH and PSS/PAH Nascent Films

J. Phys. Chem. B, Vol. 112, No. 20, 2008 6323

2. Materials and Methods Anionic poly(acrylic acid) [PAA], Mw ∼70 kDa in 35% aqueous solution (Sigma), anionic poly(sodium 4-styrene sulfonate) [PSS], Mw ∼70 kDa (ACROS Organics), cationic poly(allylamine hydrochloride) [PAH], Mw ∼70 kDa (AlfaAesar), and cationic poly(ethyleneimine) [PEI] Mw ∼50-60 kDa in 50% aqueous solution (ACROS Organics) were used as received. All of the polyelectrolyte solutions were prepared at pH 7.4 in MES-TRIS-NaCl buffer solution using Millipore water (17.6 MΩ cm). Concentrations of 2-(N-morpholino)ethanesulfonic acid (MES) and Tris(hydroxymethyl)aminomethane (TRIS) were both of 25 mM and NaCl of 100 mM (all from Sigma). The concentration of the polyelectrolyte solutions was 1 mg/mL. The substrates were silicon wafers [1 0 0] (SW, Wacker-Chemie) and freshly cleaved mica (M, MTO 220, Steren). SW was cleaned as follows: 20 min in 2% alkaline detergent solution at 80 °C, and 20 min in piranha solution (50% sulfuric acid and 50% hydrogen peroxide). They were cleaned after each step with deionized water at 80 °C for 20 min. The procedure used to assemble the polyelectrolyte films inside the LC-AFM is described elsewhere.15 Nanoscope III AFM (Digital Instruments) was used with silicon nitride tips (NCH-R, MLCT-AUHW-Park Scientific). The polyelectrolyte injection and adsorption on the surface was done as follows: PEI always came first and once, followed by alternated injection of polyanions and polycations. PEI is adsorbed on the surface by electrical and Van der Waals forces, and it allows the subsequent polyelectrolyte adsorptions. Once the polyelectrolyte solution was injected, it remained inside the LC-AFM for 20 min. A rinsing step was done flowing approximately five times 1 mL of MES-TRIS-NaCl buffer solution through the cell between each polyelectrolyte injection. The MES-TRIS-NaCl buffer volume used for rinsing was approximately 180 times the volume of LC-AFM (about 0.028 mL). This was an efficient way to rinse the film. Images were taken 15 min after the last buffer injection. The polyelectrolyte films were imaged from the first layer of PAA, over a first PEI layer until the four bilayers PAH/PAA, and the last film is denoted as PEI[PAA/ PAH]4; the same process was followed for the PSS/PAH imaged films, from the first PSS deposition until the last PAH deposition in the so denoted PEI[PSS/PAH]4 film. Glucose oxidase (GOX) from Aspergillus niger (VII-S type; Sigma) with Mw ≈160 kDa was used in this study at 0.4 mg/mL in the same buffer solution, and it was deposited at the top of the PEI[PAA/PAH]4 and PEI[PSS/PAH]4 films. The PEI[PAA/PAH]4 dried films were first prepared in situ, then dried under a N2 flux for two minutes or left overnight in room conditions, and again placed and hydrated by the buffer solution in the LC-AFM. In all cases, height images were captured in LC-AFM contact mode. The images were taken at a scan rate of 1 Hz with a resolution of 512 × 512 pixels. Images of 5 × 5 and 2.5 × 2.5 µm2 were obtained. The last images were used to show the surface structures and the former ones to calculate parameters of the film such as the roughness (Rms), the area of grains (GA) and number of grains (GN) using Nanoscope Software (version 5.12r2). The threshold limit to calculate the grain parameters was half of the correlation depth. The granular surface coverage (GS) and its surface coverage fraction (fGS) were calculated from GS ) GNGA, and fGS ) GS/A, respectively, where A is the analyzed area, 5 × 5 µm2 in this case. Polyelectrolyte film thickness was determined by a commercial optical waveguide lightmode spectroscopy system (OWLS 110, MicroVacuum) with the same experimental

Figure 1. Thickness growth behavior for the PEI[PSS/PAH]4 and PEI[PAA/PAH]4 films, obtained by OWLS after sequential polyanion or polycation adsorption. PEI deposition is not reported here. The experimental conditions were pH 7.4, ionic strength of 0.1 M NaCl, and polyelectrolyte concentration 1 mg/mL, which were the same for the films imaged by LC-AFM.

conditions as those of the LC-AFM experiments. OWLS consists of a He-Ne laser (λ ) 632.8 nm) and OW 2400 sensors (16 mm × 48 mm, MicroVacuum). Sensors were first cleaned using a 10 mM SDS solution followed by a 0.1 N HCl solution and finally washed with deionized water for 20 min at 80 °C. During the experiments, the solution was flushed through the cell at a constant flow rate (0.3 mL/min) using an injection system until equilibrium was reached (less than 10-5 variation on absolute values of refractive index in the electric [NTE] and magnetic [NTM] modes). After a stable baseline was obtained, the construction of the polyelectrolyte film was performed as follows: 200 µL of polyelectrolyte solution were injected in the cell through the injection port using a microsyringe. NTE and NTM values increased and reached a plateau after about 20 min. When a stable adsorption signal was obtained, buffer flow was restarted for about 20 min to rinse the excess polyelectrolyte solution from the cell. This procedure was repeated alternating cationic and anionic polyelectrolytes until the desired multilayer film was completed. The analysis of the obtained data was done using the Biosense 2.2 sofware to calculate the thickness of the films.22 3. Results and Discussion Film Thickness and Granular Structure. The polyelectrolyte film thickness obtained by OWLS during the PEI[PAA/ PAH]4 and PEI[PSS/PAH]4 film formation is shown in Figure 1. While the first film has a nonlinear growth regime, the second one has a clear linear behavior with the layer number. This difference in the regime of growth for thickness is only due to the substitution of the polyanion during the self-assembly of the film, although both are built up at the same experimental conditions. This behavior is associated to the polyelectrolytes structure in the bulk solution. However, surface structure may also reveal some difference and give us information about the autoassembly process during the build up of these films. LC-AFM images of these films on silicon wafer (SW) are shown in Figures 2 and 3, where PEI[PAA/PAH]4 and PEI[PSS/ PAH]4 films are imaged during their construction step by step. Figure 2, corresponding to PEI[PAA/PAH]4 film sequence, shows that after one layer a granular structure is clearly identified on the surface. This granular structure is formed by polyelec-

6324 J. Phys. Chem. B, Vol. 112, No. 20, 2008

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Figure 2. PEI[PAA/PAH]4 build up on silicon wafer (SW) by LC-AFM. The deposited polyelectrolyte is indicated above the corresponding image as PSS1, PAH1, and so on, until PAH4. The granular structure is clearly observed. (AFM images of 2.5 × 2.5 µm2; height ) 80 nm.)

trolyte complexes on the surface, and every grain in the structure is formed by several cationic and anionic polyelectrolyte chains.23 These grains have a more defined globular shape when the PAH polyelectrolyte layer is adsorbed during the process. In the images, void spaces can be identified among the grains observed. In some cases, the structures observed on the surface seem to be formed by two or more smaller grains obtaining larger structures by coalescence. Fujita et al. have studied the same PEI[PAA/PAH]4 film layer by layer, using different pH conditions and under dry conditions. They reported two different structures for the PAA (pH 7.5)/PAH (pH 3.5) multilayer:24 the first one is an island-like structure when the number of PAA/ PAH bilayers is less than four, and the second one is like a cobweb when this number of bilayers is more than 4.5. In both cases, the structures are different from the structure shown in Figure 2. It is clear that the grain structure formed on the surface

by PAA and PAH may be dependent on the pH conditions because the weak nature of these polyelectrolytes, but it may also have a contribution produced by the drying process. LC-AFM images of PEI[PSS/PAH]4 film on SW are shown in Figure 3. As in the previous cases, a granular structure was also formed during the assembly of the film. We can observe that when PSS is deposited new grains are formed on surface. These grains have a well defined shape, and they are uniformly distributed on the surface. PSS is well-known to be a strongly charged polyelectrolyte; this means that its charge density is high and that it is less dependent on the pH of the surrounding media. However, PSS also has a very important hydrophobic contribution coming from the aromatic ring of its chemical structure. Both characteristics are involved in the formation of the grain structures observed in LC-AFM. After PSS is adsorbed on the film, this polyelectrolyte wraps the PAH chains, forming



Figure 3. Same as described in Figure 2 but for the PEI[PSS/PAH]4 film. Again granular structure is present but a homogeneous film is obtained with this system. (AFM images of 2.5 × 2.5 µm2; height ) 30 nm.)

new grains on the surface. These grains remain separated from each other because of the high charge density of the PSS. When a new PAH layer is deposited on this film, the grains increase in size, and their number decreases on the surface. Although the grain shape is kept on the surface, it is clear that some of the grains have coalesced. This fact could be explained in terms of the weak nature of PAH (less charge density than PSS) and that is less hydrophobic than the PSS. Both characteristics allow more interaction between small grains favoring the coalescence in bigger grains on the surface. However, the more hydrophobic behavior and charge effects of the PSS maintain the globular shape of the grains after PAH adsorption; this may be the main reason that the size of each grain is increased by addition of the PAH, but the distribution on the surface is not extremely affected.

The size of the PEI[PAA/PAH]4 grains is also bigger than those obtained in the PEI[PSS/PAH]4 grains. The only difference between them is the substitution of the anionic polyelectrolyte. It is accepted that the formation of the granular structure is due to the conformational rearrangements during the adsorption process because adsorbed polyelectrolytes in the first steps of the film favor the scrambled-egg complexes (grains) that present partial shrinkage, exposing part of the surface of the substrate and leaving void spaces on it.25 However, the shrinkage of the grains may be promoted for long-range interactions such as electrostatic forces, and short-range interaction such as hydrophobic forces are also involved. Substrate Deposition Effects. In order to know the effect of the surface on the structure formed during the assembly of the PEI[PAA/PAH]4 film, the film was also assembled on mica.


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Figure 4. Same as described in Figure 2 but the PEI[PAA/PAH]4 film is built up on mica, a higher charged surface than SW. (AFM images of 2.5 × 2.5 µm2; height ) 50 nm.)

The series of images of PEI[PAA/PAH]4 sequence on mica are shown in Figure 4, which are compared with images of the same film but deposited on SW of Figure 2. The complexes formed on mica are plainer than those structures obtained on the silicon substrate. This means that interactions among the opposite charged polyelectrolyte chains are stronger than those interactions established between the polyelectrolyte chains and the SW surface. Interactions between polyelectrolyte chains and the mica surface seem to be stronger. This behavior is in agreement with the higher charge density of the mica compared with that of the silicon wafer.26,27 Dried Films. The PEI[PAA/PAH]4 dried films are shown in Figure 5A and B. The initial in situ granular structure can be observed in Figure 2 labeled as PAH4. The differences in each film are evident from these three images. When the PEI[PAA/ PAH]4 film was dried under environmental conditions, Figure

5A, the film collapses, and the structures on the surface shrink as a result of the water remotion,21,25 leading to the formation of new coalesced grains. This surface structure is similar to a cobweb, which was widely reported.24 The drying effects are more dramatic when N2 flow is used to dry the PEI[PAA/PAH]4 film (Figure 5B. In this case, the in situ granular structure has totally disappeared from the surface, obtaining a film with welldefined porous structures on the surface. A previous report has already shown that films dried under environmental conditions are smoother than those dried under N2 flow.28 Granular Parameters. Additional information about the surface structure on PEI[PAA/PAH]4 and PEI[PSS/PAH]4 films during their formation can be found through the GA, GN, and Rms parameters obtained after image analysis. These quantities are reported in Figure 6, and the general behavior of the grain structures discussed above is confirmed. Moreover, we can



Figure 5. Drying effects on the PEI[PAA/PAH]4 on SW after (A) environmental and (B) nitrogen flow drying. These images can be compared with PAH4 of Figure 2. (AFM images of 2.5 × 2.5 µm2; height ) 80 nm.)

observe that GN for the PEI[PAA/PAH]4 film on SW has a global tendency to decrease when the number of layers increases, but locally, there are small but clear increases in GN every time PAH is deposited on the film. This suggests that PAH is forming new small polyelelectrolyte grains after its deposition. This means that it promotes the shrinkage of the grain structure. However, when a new PAA layer is added to this film, these structures are covered by the polyanion promoting the coalescence and decreasing the grain number on the surface due to a relaxation process. Coalescence seems to be the most important mechanism during film formation in the PEI[PAA/PAH]4 system, producing bigger grains while layer deposition occurs. However, the PEI[PSS/PAH]4 film on SW has a different behavior for GN. When the number of depositions increases, GN tends to increase as well. This behavior reaches an oscillating plateau after a few depositions. These fluctuations are a consequence of PAH and PSS depositions on the film. When PAH is deposited on the film, GN decreases but if PSS is deposited on the film, GN increases. According to previous reports for the PSS/PAH film, observed by AFM after drying, a constant increase in the number of grains versus layer deposition was observed in the early stages of the formation of the film; this behavior could be due to different experimental conditions.29 In this case, PSS is forming domains on the surface because of its hydrophobic behavior and higher charge density, promoting the shrinkage of the grain structures on the silicon surface. In this system, PAH is the one that covers the structures and promotes the coalescence of structures. However, coalescence is less important in this system because after PSS adsorption, almost the same number of grains is formed again, and their distribution on the surface is almost the same. This

Figure 6. (A) Number of grains (GN), (B) area of grains (GA), and (C) roughness (Rms) analysis for the PEI[PSS/PAH]4 and PEI[PAA/ PAH]4 films on silicon wafer and mica substrate from 5 × 5 µm2 AFM images.

means that hydrophobic behavior and density of charge of PSS is regulating the morphology of the film on the surface. This was also suggested by AFM observation in drying conditions.30 Figure 6B shows the behavior for the GA values for these systems. The GA values for the PEI[PAA/PAH]4 film on SW tend to increase when the number of depositions increases. This indicates that every new polyelectrolyte deposition increases the size of the grains on the surface, supporting the idea that coalescence is the most important phenomenon involved in the


Figure 7. Number of grains (GN) versus area of grain (GA) for the polyelectrolyte films presented here including the dried ones. Independently on the polyelectrolyte nature and surface, these quantities follow a simple inverse relationship. Two zones are clearly identified: (A) corresponding to the strong/weak and (B) weak/weak polyelectrolyte interactions.

morphology of the PEI[PAA/PAH]4 film. Coalescence of granular structures, even when it exists, is less important in the morphology of the PEI[PSS/PAH]4 film. Figure 6C shows the behavior of Rms with each polyelectrolyte deposition. It is clear that the PEI[PAA/PAH]4 film is less smooth than the PEI[PSS/ PAH]4 film. However, both films on SW exhibit the same tendency, and after a few polyelectrolyte depositions, the Rms values in both systems reach a plateau probably because of steady behavior. These general behaviors of granular parameters would then relate the surface structure of these films with the thickness behavior shown in Figure 1. Exponential behavior of the PEI[PAA/PAH]4 film may be related to a nonuniform relative big grain distribution, while linear behavior shown by the PEI[PSS/PAH]4 film is formed by smaller grains that are uniformly distributed. The values of GN and Rms for the film assembled on mica are lower than those for the film assembled on SW (see Figure 6A and C), and the GA values for films built up on mica are higher than those for film built up on SW. This indicates that bigger grains with higher area are formed on mica compared to SW. GN and GA follow a similar trend for film on mica; however, the increases and decreases for these quantities after every cationic or anionic polyelectrolyte adsorption are not observed. Complexes are formed on both substrates, but on mica, the interactions are higher, producing a smoother surface (lower Rms values) and bigger values of GA. This is certainly due to the higher charge density of mica. Granular Surface Cover. Granular surface coverage (GS), defined as GS ) GAGN, is formed by the two parameters that follow in general an opposite tendency. We have shown that when one of them increases, the other decreases. This is confirmed by plotting GN versus GA in Figure 7. These quantities follow, in fact, a simple lineal relationship with the slope close to -1 (an average value of -0.99 in this case) in a log-log plot, in all the systems studied here independently of the polyelectrolytes or substrates used here. That implies that the granular surface coverage is constant for these films, but also there is simple relationship between these granular parameters. The simple inverse relationship between GN and GA defines them as conjugated quantities. Therefore, GS has the tendency to be a constant during the self-assembling of the polyelectrolyte films. Additionally, two well-defined zones are identified in Figure 7.

Tristan et al.

Figure 8. Granular surface coverage fraction (fGS ) GS/A) of the films studied here. The average of this parameter is almost constant (within the experimental error) for the different multilayers.

Figure 9. An inverse relationship is also observed for the number of grains (GN) and area of grain (GA) of polyelectrolyte films prepared at different pH values; the data were generated from images reported in ref 15.

The PEI[PSS/PAH]4 films are located in the zone of large GN and small GA, zone A in Figure 7, and the PEI[PAA/PAH]4 film (on silicon or mica substrates) is located in the zone of large GA and small GN, zone B in Figure 7. Zone A is related with the existence of stronger interactions between polyelectrolytes due to the high charge of PSS and zone B with weak interactions established between these polyelectrolytes because of the low electric charge of PAA. The granular surface coverage fraction (fGS ) GS/A) is also reported in Figure 8 for these films. Although the zigzag behavior due to the different interactions of polycation and polyanion present, the value for the PEI[PSS/PAH]4 film on SW is 0.184 ( 0.01, for the PEI[PAA/PAH]4 film on SW 0.184 ( 0.02, and for the PEI[PAA/PAH]4 on mica substrate 0.172 ( 0.02. The two first values are identical, within data dispersion, and the third one is slightly lower than the value defined by the PEI[PAA/PAH]4 films, less than 5%, indicating an almost equal value for these films, as suggested by Figure 7. However, this surface coverage fraction is much smaller than the maximum surface coverage fraction for noninteractive particles (0.547), indicating repulsion between grains due to the electrostatic charge of the aggregate forming the grain. All of the grains in the surface are equally charged, leading to repulsive interactions



Figure 10. The adsorption of glucose oxidase (GOX) on (A) PEI[PAA/PAH]4 and (B) PEI[PSS/PAH]4 films also presents a granular structure with fGS ) GS/A close to the values found for the polyelectrolyte films.

among them. Figures 7 and 8 resume the experiments formed by weak/strong on SW and weak/weak polyelectrolytes on SW and mica studied here. Because of the importance of the electrostatic interaction in the build up of such films, these results suggest that the surface structure is driven by the electrostatic conditions imposed by the buffer solution; in this case, the pH and ionic strength were the same for all of these films. Thus, the buffer solution effectively should play an important role in the polyelectrolyte self-assembling process. This hypothesis should be tested for films made in other experimental conditions. The films reported in ref 15 can be analyzed in this perspective. These films were prepared with the same buffer but at different pH values (3.5, 6.8 and 10.5) and on glass substrate. GN versus GA for these films in a log-log plot is shown in Figure 9. The slopes are again close to -1 for these data, and the average slope values were -1.1, -1.05, -0.96 for pH values 3.5, 6.8, and 10.8, respectively. This shows the generality of our first observations for polyelectrolyte films prepared in a wide range of pH values. Additionally, PEI[PAA/PAH]4 films environmentally and N2 dried are also reported in Figures 7 and 8. The corresponding points are located in zone A of figure 7, showing that after water loss the structure collapses and the interactions between weak polyelectrolytes seem to increase. Both phenomena could be explained in terms of short-range interactions, especially the hydrophobic ones established mainly among the backbones of the polyelectrolytes after the drying process, which produces shrinkage on the grain structures. The shrinkage produces a new arrangement on the surface structure when the film is placed inside the LC-AFM and hydrated with buffer solution. After the analysis of GA and GN, the values are very close to the line plotted for the in situ films in Figure 7, indicating a common behavior for all these films established by the experimental conditions and especially by the buffer solution where these films were imaged. A last question is raised concerning the conditions that impose these surfaces to be used as support for specific materials. The adsorption of a protein on their surfaces is a particular case. We tested the adsorption of the glucose oxidase (GOX) as a protein model on these PEI[PAA/PAH]4 and PEI[PSS/PAH]4 films. In both cases, GOX adsorption is observed as shown in Figure 10. However, GOX adsorption is also related to electrical charge of the polyelectrolytes, the nature of the polyelectrolytes, and on the surface structure of polyelectrolytes. The presence of PAH in the last layer of the films produces a positively charged surface in these cases, and the nature of the hydrophobe of PSS or hydrophile of PAA is apparently not important. Further analyses are necessary to elucidate the main factor for

the GOX adsorption on these films. In the context of the present work, it is important to point out that the GOX is also adsorbed as agglomerates on these surfaces, which is far from a homogeneous film as sometimes supposed. Indeed, the fGS values are around 0.20 in both cases, which are close to the values found for these films before GOX adsorption. Granular structure may be also a dominant structure for the materials deposed on top of these polyelectrolytes films. 4. Conclusions Granular surface structure of PEI[PAA/PAH]4 and PEI[PSS/ PAH]4 sequential films on silicon wafer were analyzed in situ, avoiding any drying effects, by LC-AFM. The number and area of grains (GN, GA) were defined in order to quantify the surface structures. The main differences between these films were produced by the substitution of the weak anionic hydrophilic polyelectrolyte (PAA) by a strong anionic with higher hydrophobic polyelectrolyte (PSS). Electrostatic interactions during the film self-assembly between the two polyanions and the weak cationic polyelectrolyte (PAH) have shown that while the PSS is promoting the formation of small grains on the surface, the PAA is forming bigger grains. This means that PSS is wrapping up the existing complexes on the surface and splits them with every deposition. However, the PAA is promoting coalescence on the complexes existing on the surface, forming new grains with bigger size. These granular structures may have a direct relationship with the exponential and linear and thickness growth of these systems. Additionally, PEI[PAA/PAH]4 films on mica are smoother than films on silicon wafer as a possible consequence of the high charge density of this surface. An important result in this study is that the granular surface coverage measured during all of the polyelectrolyte depositions is practically constant and independent of the polyelectrolytes or surfaces used here. This finding is a direct consequence of the simple inverse relationship that follows the number and area of grains. The only experimental condition in common during polyelectrolyte film construction of these films was the surrounding environment imposed by buffer solution; this interaction seems to regulate the surface coverage by the polyelectrolytes and the glucose oxidase deposited on their surface, even for the drying films that are hydrated before observation in LCAFM. The drying processes practiced on the PEI[PAA/PAH]4 film produce different surface structures compared with its original structure in the LC-AFM, indicating an evident dependence on the drying process. The simple inverse relationship was also found for polyelectrolyte films built up at different pH values reported in ref 15.

6330 J. Phys. Chem. B, Vol. 112, No. 20, 2008 Acknowledgment. This work was supported by CONACYT through projects SEP-2004-C01-45951 and 47611, PROMEP, and the Mexico-France program SEP-CONACYT-ANUIESECOS, project M06P01. We are grateful to J. Ruiz from IFUASLP, Mexico, and G. Ladam from La2B, France, for the use of the LC-AFM and the OWLS, respectively, and to M. L. Gonzalez (UASLP) for technical support. J.-L. Menchaca thanks CONACYT for support through the retention program (51469). References and Notes (1) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831. (2) Lvov, Y.; Munge, B.; Giraldo, O.; Ichinose, I.; Suib, S. L.; Rusling, J. F. Langmuir 2000, 16, 8850. (3) Bliznyuk, V. N.; Campbell, A.; Tsukruk, V. V. In Organic Thin Films: Structure and Applications; Frank, C. W., Ed.; ACS Symposium Series 695; American Chemical Society: Washington, DC, 1998; p 220. (4) Ferguson, G. S.; Rouse, J. H.; MacNeill, B. A. Abstracts of Papers of the American Chemical Society 2000, 219, U558-U558302-COLL. Struth, B.; Eckle, M.; Decher, G.; Oeser, R.; Simon, P.; Schubert, D. W.; Schmitt, J. Eur. Phys. J. E 2001, 6, 351. (5) Mamedov, A. A.; Kotov, N. A.; Prato, M.; Guldi, D. M.; Wicksted, J. P.; Hirsch, A. Nat. Mater. 2002, 1, 190. (6) Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. Biomacromolecules 2000, 1, 674. (7) Lvov, Y.; Decher, G.; Sukhorokov, G. Macromolecules 1993, 26, 5396. (8) Schoeler, B.; Delorme, N.; Doench, I.; Sukhorukov, G. B.; Fery, A.; Glinel, K. Biomacromolecules 2006, 7, 2065. (9) Kolarik, L.; Furlong, D. N.; Joy, H.; Struijk, C.; Rowe, R. Langmuir 1999, 15, 8265. (10) Steitz, R.; Leiner, V.; Siebrecht, R.; von Klitzing, R. Colloids Surf., A 2000, 163, 63. (11) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213.

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