Formation of Multigradient Porous Surfaces for Selective Bacterial

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Formation of Multigradient Porous Surfaces for Selective Bacterial Entrapment Alberto S. de León,† Adolfo del Campo,‡ Aitziber L. Cortajarena,§ Marta Fernández-García,† Alexandra Muñoz-Bonilla,*,† and Juan Rodríguez-Hernández*,† †

Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), C/Juan de la Cierva 3, 28006-Madrid, Spain Instituto de Cerámica y Vidrio (ICV-CSIC), C/Kelsen 5, 28049-Madrid, Spain § Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia), Cantoblanco, and CNB-CSIC-IMDEA Nanociencia Associated Unit “Unidad de Nanobiotecnología”, 28049-Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Herein we describe the preparation of multigradient porous platforms by using the breath figures approach. In a single and straightforward step, we prepared porous surfaces in which three different parameters vary gradually from the edge of the sample to the center in a radial manner. Thus, we evidenced the gradual variation of the pore size and the shape of the pores that can be varied, depending on the sample concentration, but also depending on their radial position within the same sample. In addition, we succeeded in the control over the chemical composition inside and outside the pores as well as the variation of the concentration of block copolymer inside the pores as a function of their radial position. Moreover, the chemical composition and the variable cavity size of porous surfaces have been evaluated to analyze the influence of these variables on the selective bacterial immobilization. To the best of our knowledge this is the first example in which, by using a simple one-step strategy, a multigradient surface can be obtained. These initial results can be the base to construct platforms for selective immobilization and isolation of bacteria.

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A large number of procedures3 have been described to create both molecular and macromolecular gradients.18−26 Surfaces with gradual polymer properties have been obtained using a large variety of approaches. Chemically gradient surfaces have been prepared by controlled gradual surface modification approaches, such as hydrolysis reactions,27 by radio frequency plasma28 or corona discharge.29 Equally, fine-tuning of the polymerization parameters leads to surfaces with macromolecular gradients. The approaches concerning the use of polymerization steps generally involve grafting-from procedures. Examples of the use of this approach include the immersion of substrates into polymerization media,30 the preparation of random copolymer brushes by steadily varying the monomer composition,31,32 the preparation of statistical copolymers by the microfluidic mixing of two monomers followed by chamber filling method,33 the use of the solution draining method for preparing polymer brushes,34 the formation of a molecular gradient of an initiator on a substrate followed by grafting-from polymerization,35,36 or the formation of opposite grafting density counter gradients of two polymers formed by sequential grafting from two different set of

INTRODUCTION Gradient surfaces (GS) consist of interfaces in which a particular characteristic gradually varies as a function of their position between two extremes.1−4 Surface gradients are characterized by a different number of attributes that indicate directionality (orthogonal, radial, triangular, etc.), type (chemical, mechanical, topographical), dimensionality (1D, 2D, 3D), gradient length scale (narrow or broad), and time dependency (responsive, dynamic, etc.).5 Among the characteristics varied it is worth mentioning, among others, the chemical composition, the topography, or even the mechanical properties. The interest in the preparation of gradient surfaces, which constitutes a particular case of surface patterning, relies on their potential uses.1 Gradient surfaces have been employed in combinatorial chemistry6 and in biomedical applications,7 including the discovery of drugs, to analyze cell−substrate interaction phenomena8 and cell culture systems,9−12 or to prepare biological devices by using microfluidic systems.13 A variation of the chemical contrast can be employed as a reference tool for calibrating image contrast in surface nanometrology.14 Finally, GS are also useful for the development of gradient polymeric sensor materials,15 to create polymer libraries,16 or to control the deformation and damage resistance.17 © XXXX American Chemical Society

Received: June 5, 2014 Revised: July 23, 2014

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dx.doi.org/10.1021/bm500824d | Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Evaporation of a polymer solution droplet placed on a solid support. The evaporation occurs from the edge to the center and thus a radial drying gradient is observed. As a consequence the water vapor condensation takes place to a larger extent toward the center of the droplet thus leading to an increase of the pore diameter.

initiators.37,38 Nevertheless, grafting-onto methods in conjunction with temperature gradient heating of the substrate have been investigated as well.39 Gradient surfaces with variable topography have also been reported. These have been prepared using polystyrene microspheres40 or gradually varying either the molecular weight on solid substrates34 or the grafting density of the polymer chains, thus, leading to chains able to form either mushroom or brush topographies.36,37 The approaches depicted above allow us to control either surface pattern or surface composition. They require sophisticated techniques and, in some cases, the use of expensive equipment, as illustrated for the case of gradients formed using microfluidic systems. In contrast, herein we will describe a straightforward and cheap alternative in order to control simultaneously chemical composition, size, and shape of the surface. For this purpose we will employ the breath figures (BF) approach41−52 to create porous interfaces with variable average pore size and chemical composition. As will be depicted, the choice of the preparation conditions and, among others, the solvent employed are crucial to produce broad or narrow gradients. In addition, the use of blends of a polymer matrix (polystyrene, in this particular case) and a block copolymer allow us to simultaneously vary the surface chemical composition. Moreover, we will illustrate how the porous interfaces with variable pore size and chemical composition can direct the microorganism immobilization inspired by recent studies that have demonstrated that the pore diameter plays a key role on

such process as cell differentiation.53 In this study we will use a particular bacterial strain, more precisely, Staphylococcus aureus, which is one of the most common pathogenic bacteria that cause disease in humans. In addition, S. aureus bacteria are spherial shape with about 1 μm in diameter size, thus, in the range of the pore dimension of surface and adequate to evaluate their surface immobilization as a function of the pore diameter. Therefore, we will evidence the role of the surface features on the retention of bacteria.

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EXPERIMENTAL SECTION

Materials. The hydrophobic poly(2,3,4,5,6-pentafluorostyrene)-bpolystyrene (PS5F21-b-PS31) and amphiphilic block copolymers polystyrene-b-poly[poly(ethylene glycol) methyl ether methacrylate] with two different compositions, that is, PS45-b-P(PEGMA300)34 and PS40-b-P(PEGMA300)48 (copolymer is labeled with the degree of polymerization of each block) were synthesized via atom transfer radical polymerization (ATRP) as previously reported.54,55 High molecular weight polystyrene (Aldrich, Mw = 2.50 × 105g/mol) was used as polymeric matrix. Tetrahydrofuran (THF), chloroform (CHCl3) and carbon disulfide (CS2) were purchased from Scharlau. Round glass coverslips of 12 mm diameter were obtained from Ted Pella, Inc. Film Preparation. Different blends were studied varying the type and the proportion of the block copolymer (10 and 20 wt %) and linear polystyrene (90 and 80 wt %) and total concentration of polymer in the solution (15, 30, 45, and 60 mg/mL). Films were prepared from these solutions by casting onto glass wafers at room temperature under controlled humidity inside of a closed chamber. Measurements. Scanning electron microscopy (SEM) micrographs were taken using a Philips XL30 with an acceleration voltage of B


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Figure 2. SEM images of the PS polymer surfaces obtained using two different solvents, CHCl3 (A−D) and THF (E−H), as a function of the radial position. Whereas CHCl3 produces a rather narrow gradient, the use of THF did not prevent the coagulation, thus, leading to heterogeneous surfaces.

and also chemical composition.45,48,54 This approach consists on the evaporation of a polymer solution using a volatile solvent under a moist atmosphere. As a consequence of the solvent evaporation, the temperature of the solvent/air interface decreases and water vapor starts to condense. The water vapor condensation leads to the formation of water droplets that increase in diameter during the solvent evaporation. Upon removal of both solvent and the condensed water droplets, porous interfaces can be easily obtained. As has been extensively reported, among others, the polymer concentration, the humidity, and the solvent employed have been demonstrated to largely influence the pore characteristics. Herein we study the evaporation process using different solvents (CHCl3, CS2, and THF) and different polymeric systems (polystyrene or polystyrene/block copolymer blends) to prepare porous surfaces with a gradient pore size. Equally, as will be analyzed in detail the incorporation of additives, in this case, block copolymers with different functional groups (hydrophilic and hydrophobic) will provide the possibility not only to modify the chemical composition of the films but also to produce an additional gradient in the pore composition. Finally, in some cases, convection and the drying front induce deformation of the pores, thus, leading to ellipsoidal shapes. Gradual Size Pore Variation by Using Breath Figures. When a polymer solution droplet using a volatile solvent is placed on a support, the solution starts to evaporate and a film is formed from the edge to the center of the droplet, as

25 kV. The samples were coated with gold−palladium (80/20) prior to scanning. The topography, chemical composition and distribution of the different components on the polymeric films were determined by confocal Raman microspectroscopy integrated with atomic force microscopy (AFM) on a CRM-Alpha 300 RA microscope (WITec, Ulm, Germany) equipped with Nd:YAG dye later (maximum power output of 50 mW power at 532 nm). The analysis of the pore size (average diameter), pore size distribution and spacing between pores were performed using the image analysis software (ImageJ, http://rsb.info.nih.gov/ij/). Bacteria Immobilization. Staphylococcus aureus strain RN4220 carrying the plasmid pCN57 for GFP expression (from Iñigo Lasa Lab at UPNA-CSIC) was grown overnight at 37 °C in Luria−Bertani (LB) media with erythromycin (10 μg/mL). The cells were centrifuged and washed three times in PBS buffer (150 mM NaCl, 50 mM Naphosphate pH 7.4). The solution was adjusted to a fixed cell concentration that corresponds to an optical density (OD) at 600 nm of 1.0. The patterned polymeric surfaces were incubated during 1 h with a bacterial suspension at OD = 1.0 in PBS buffer with 0.05% v/v Tween 20. After incubation the surfaces were thoroughly washed with PBS. Fluorescence microscopy for bacteria imaging was performed using a Leica DMI-3000-B fluorescence microscope. Images were acquired using different magnifications (10×, 20×, 40×, and 60×) and the corresponding set of filters for imaging green fluorescence and bright field.

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DISCUSSION The BFs approach has been largely employed to prepare porous surfaces with controlled pore diameters, distribution C


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Figure 3. (Left) Average pore diameter vs radial position obtained from different solvents and (right) SEM images of the porous films from a PS/ PS45-b-P(PEGMA300)34 (90/10 wt %) blend using 70% RH, a polymer concentration of (30 mg/mL) and at room temperature.

improve the gradient and the droplet stability against coagulation and additionally modify the chemical composition of the films, poly(2,3,4,5,6-pentafluorostyrene)-b-polystyrene (PS5F21-b-PS31) and amphiphilic polystyrene-b-poly[poly(ethylene glycol) methyl ether methacrylate] (PS-b-P(PEGMA300)) block copolymers were incorporated into the solution. The synthesis of these two block copolymers has been previously described.54,55 Therefore, for this study, we explored blends of a high molecular weight PS with two different block copolymers, that is, either PS5F-b-PS or PS-b-P(PEGMA300). Moreover, for the case of the amphiphilic PS-b-P(PEGMA300) block copolymer, we studied two different block copolymers with variable composition, PS 45 -b-P(PEGMA300) 34 and PS 40 -b-P(PEGMA300)48, which are insoluble and soluble in aqueous solution, respectively. Hence, the blend compositions used in this study are formed by 10 or 20 wt % of block copolymer and 90 or 80 wt % of PS, respectively. All other parameters, including temperature and relative humidity, were maintained constant. The incorporation of block copolymers was crucial to prevent coagulation, thus, improving significantly the homogeneity of the porous surface. As an example, in Figure 3 are depicted the average pore diameters for films prepared from the blend PS/PS45-b-P(PEGMA300)34 (90/10 wt %) using different solvents. In this case, no coagulation could be observed maintaining the same conditions as those employed for the film containing only high molecular weight PS. Moreover, as expected, THF seems to be the solvent that produces the larger gradients. Whereas for highly volatile solvents (chloroform or CS2) the gradient is exclusively formed in the region closed to the edge, THF forms surfaces with a constant increase of pore sizes toward the center of the sample.

depicted in Figure 1. If we consider a circular droplet shape, we will thus observe a radial distribution. More interestingly, when the evaporation takes place in a moist environment (as depicted above) the water vapor condenses during the solvent evaporation. As a consequence, water vapor condensation occurs to a larger extent from the edge to the center of the droplet. As is schematically shown in Figure 1 and as will be discussed in detail, this effect produces films with variable pore sizes that increase from the edge to the center as a result of the longer evaporation (and therefore water condensation) times. The first series of experiments to study the gradient formed in films obtained by the breath figures approach were carried out using high molecular weight linear polystyrene. A droplet of a polystyrene solution (30 mg/mL) either in THF or CHCl3 was cast on a glass support (previously cleaned) under a 70% relative humidity (RH) moist atmosphere. Upon complete solvent and condensed water droplets evaporation, the films were studied by SEM. The images obtained as a function of the radial position for films formed using either THF or CHCl3 are illustrated in Figure 2. The films obtained using CHCl3 lead to a very narrow gradient in diameter pore size (varying between ∼3.34 and ∼3.25 μm) as a consequence of the fast evaporation. Only on the edge of the films, a significant decrease was observed. For this reason, THF with higher boiling point has been selected to enlarge the gradient (broad gradient). In the case of THF the increase in pore size is also observed focusing on the areas close to the edge. In this case, large and heterogeneous pores were produced close to the center of the films as a consequence of longer evaporation times that lead to coagulation. Thus, although a broad gradient is obtained under these conditions using THF, the coagulation process should be avoided to achieve a regular gradient. Coagulation in BFs has been prevented using functional copolymers, typically block copolymers that stabilize the condensed water droplet.44,48,54,56 Thus, to simultaneously D


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Figure 4. Variation of the pore size as a function of the radial distance to the edge for blends containing PS45-b-P(PEGMA300)34 and PS and having either 10 or 20 wt % of PS45-b-P(PEGMA300)34 and 90 or 80 wt % of PS. The cross section and the pore depth variation as a function of the radial distance to the edge for blends with 10 wt % of block copolymer.

As aforementioned, this phenomenon is probably related to the higher boiling temperature of THF compared with both CHCl3 and CS2, and consequently longer evaporation times. Since THF leads to the higher gradient, this solvent was then selected for the following experiments in spite of losing, at least to some extent the surface order. Figure 4 (see also Supporting Information, Figures S1 and S2) contains the SEM images of the surfaces obtained from the different blends containing either 10 or 20 wt % of block copolymer within the blend. In all cases and independently of the blend composition or the amount of block copolymer employed, the average pore diameter increases from the edge to the center. In Figure 4 is also shown the cross section of films with 10 wt % of block copolymers and is easy to see the decrease in pore depth from the center to the edge, in correlation with the pore size trend. Depending on the type of block copolymer, that is, amphiphilic or double hydrophobic, the average pore diameters significantly differ for a particular radial position (Figure 5). The hydrophobic block copolymer produces less condensation and leads to smaller pores than the amphiphilic block copolymers. As observed here and in agreement with precedent studies, the presence of hydrophilic moieties favors the water condensation, thus, leading to higher pore sizes.44,48,56,57 The variation of the block copolymer concentration using either the hydrophobic or the amphiphilic did not play a significant role on the average pore diameter. As mentioned above, in principle, an increase in the amount of hydrophilic block copolymer within the blend should favor the water condensation and therefore an increase of the pore size is expected. The average pore size seems to be similar for the same block copolymer blend independently of the amount of block copolymer employed in the mixture. However, it is possible to detect significant variation on the number of pores

Figure 5. Comparison of the average pore diameter as a function of the radial position obtained depending on the block copolymer and its amount on the blend using THF as solvent. The data shown correspond to the average values for the different areas of the gradient films.

formed. The number of pores at the surface is related to surface area occupied by pores/total surface area (SPC: surface pore coverage). The values of the parameter f ( f = 1 − SPC) for the different blends are depicted in Figure 6. As can be seen in the graph, the number of pores increases (low f values) for high content of the copolymers, 20 wt %. In addition, the f value that is related to the surface out of the pores decreases with the hydrophilicity of the block copolymer employed. E


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ε=

⎛ d2 ⎞ ⎜1 − 2 ⎟ ⎝ D ⎠

where d is the semiminor axis and D is the semimajor axis. As can be observed, ε clearly depends of the radial position and varied from a rather elliptical shape of the pores at the edge of the film to spherical toward the center of the sample. In order to, at least to some extent, explain this observation, we have to resort the breath figures mechanism proposed by Karthaus et al.58 These authors proposed a breath figures formation by water vapor condensation during the solvent evaporation but specified that at the edge of the sample a convection of the water droplets takes place rearranging the water droplets. Convection movements may be the cause for the pore deformation in solution that is rapidly frozen when the solvent is completely evaporated. The concentration of the polymer solution has been also evidenced to play a key role on the pore formation. An increase of the concentration leads to a higher viscosity and reduces the mobility at the surface and therefore the pore size. The behavior observed for the blend composed of 90/10 PS/PS45-bP(PEGMA300)34 is depicted in Figure 8. As expected a radial pore size distribution is observed. Moreover, an increase of the concentration affords pores with lower diameters. Notwithstanding, the polymer concentration significantly affects the aspect ratio of the pores. An increase of the concentration leads to rather spherical pores while a decrease of the concentration produces elongated pores. Variation of the Chemical Composition within the Pore as a Function of Its Radial Position. Size and pore

Figure 6. Relative surface occupied by pores versus total surface for films prepared from blends having 10 and 20 wt % of block copolymer (conditions: 70% RH, room temperature, polymer concentration 30 mg/mL).

Shape of the Pores: Spherical to Ellipsoidal. Apart from the gradient in size observed from Figure 4, we can observe that in some of the images the pores formed are not completely spherical but rather ellipsoidal. This interesting observation is particularly clear in the pores formed at the edge of the film. In Figure 7 is depicted the aspect ratio or eccentricity (ε) of the pores observed for each particular radial position. The eccentricity is defined as follows:

Figure 7. (A) Mechanism of water droplet arrangement during the solvent evaporation. (B) Aspect ratio of the pores given by ε as a function of the radial position for films prepared from blends containing 10 wt % of block copolymers. F


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Figure 8. (A) Average pore diameter versus position within the dried droplet for different polymer concentrations: blend 90/10 PS/PS45-bP(PEGMA300)34 70% RH; (B) Variation of the eccentricity (ε) as a function of the concentration in two different positions r = 3R/4 and r = R/4.

shape have been demonstrated to vary within the film as a consequence of the evaporation from the edge to the center. Since, the films are formed from blends of two components, herein we will attempt to analyze the chemical composition of the film at different positions. For this purpose we will employ Raman confocal microscopy. In previous contributions our group has made use of this technique to study the spatial distribution of the different blend components, corroborating that the amphiphilic copolymers is located mainly within the

cavities as expected from the breath figures mechanism. Figure 9A depicts the Raman image of single pores obtained at different positions for the films prepared form the blend containing 10 wt % of the water-soluble PS 40 -b-P(PEGMA300)48 and 90 wt % of PS, indicating the presence of the amphiphilic copolymer inside of the pore (blue region). Moreover, the decrease of the blue color intensity from the edge to the center of the sample reveals the gradual variation on chemical composition of the pore. This effect is also shown in G


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Figure 9. Variation of the chemical composition of the pores as a function of the position. (A) Raman image of a single pore, indicating the presence of PS40-b-P(PEGMA300)48 inside of the pore (blue color that corresponds to the high intensity of the 1735 cm−1 attributed to the carbonyl groups of the P(PEGMA300)). Outside the pore, the film is mainly composed of PS (red corresponds to high intensity of the 1012 cm−1, attributed to the ring breathing mode of the polystyrene). Moreover, the amount of P(PEGMA300) within the pore decreased from the edge to the center of the film. (B) Raman spectra obtained at different positions. Variation of the intensity of the P(PEGMA300) signal at 1740 and at 1510 cm−1. (C) Intensity of the P(PEGMA300) signal, 1510 cm−1, as a function of the radial position within the pores for both PS45-b-P(PEGMA300)34 (red) and PS40-bP(PEGMA300)48 (black) block copolymers.

amount of block copolymer available for the pores formed at the center of the sample. Selective Bacterial Immobilization on Gradient Platforms Based on Variable Surface Topography and Chemistry. The generated surfaces with a defined gradient in pore size as a function of the radial distance to the edge are hypothesized to potentially act as platforms for cellular selectivity. In this experiment we tested the ability of the surfaces to trap/repel bacteria based both on the pore sizes and on the pore composition. For this purpose, we have incubated pore gradient surfaces constructed using either PS40-b-P(PEGMA300)48 or PS45-bP(PEGMA300)34 (10 wt %, 70% RH) with fluorescent spherical-shaped bacteria, washed the surfaces and imaged the adsorption of the bacteria by fluorescence microscopy. As described above, the water-soluble copolymer PS40-b-P(PEGMA300)48 conducts to a porous surface with a gradient not only in pore size but also in composition. The pores closed to the edge are highly enriched in copolymers containing poly(ethylene glycol) segments that avoid the adhesion of bacteria. In contrast, the PS45-b-P(PEGMA300)34 generates surfaces where the pores are homogeneously and poorly enriched in poly(ethylene glycol). Figure 10 shows the fluorescence images of both porous surfaces with either PS40b-P(PEGMA300)48 (A) or PS45-b-P(PEGMA300)34 (B) at different radial distances. In Figure 10A it is observed that there is a low amount of bacteria immobilized on the surface which,

Figure 9B where are depicted the Raman spectra obtained at the edge of the pore at different positions for the films prepared from the same blend. The intensity of the two characteristic signals at 1510 and 1740 cm−1 provided by the P(PEGMA300) block segment clearly decreases from the edge to the center indicating an enrichment in block copolymer toward the edge of the film. To determine if the composition of the block copolymer plays a key role on the formation of the compositional gradient, these results were compared with those obtained in case of the water insoluble copolymer PS45-bP(PEGMA300)34. In Figure 9C are represented the ratio between the following two areas: on the one hand the area corresponding to a region with peaks attributed to both PEGMA and PS (1425−1522 cm−1). On the other hand, a region exclusively associated with PS peak (1542−1663 cm−1). This analysis has been made for blends having the same amount of block copolymer, that is, 10 wt % and for the two amphiphilic block copolymers employed (water-soluble PS40-bP(PEGMA300)48 and water-insoluble PS45-b-P(PEGMA300)34. As evidenced in this figure, the gradual variation in composition is exclusive of the PS40-b-P(PEGMA300)48 block copolymer. Most probably this phenomenon can be explained as follows: during the water condensation, the soluble block copolymer has a larger tendency toward the water droplets than the insoluble block copolymer. As a consequence of the rapid migration of the soluble block copolymer to the condensed water, it enriches the water droplets formed at the edge while decrease the H


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Figure 10. Adhesion of fluorescent S. aureus bacteria on (A) PS40-b-P(PEGMA300)48 and (B) PS45-b-P(PEGMA300)34 gradient surfaces. Differential immobilization of bacteria on the porous films as a function of the radial distance to the edge of the film. The images are acquired with a 10× objective. The panel on the right shows an image of the r = 3R/4 area at 40× magnification, where is clear the presence of individual bacteria in the pores.

corona region of high bacteria population in the film. Toward the center of the sample where the pores are larger it can be observed a decrease in bacteria cell density. This could be due to the larger sizes of the pores with respect to the bacteria size. For bacteria to get entrapped the pores need to be sufficiently big for the bacteria to get in and sufficiently small to generate a confined environment where bacteria stay bound even after extensive washes. These results are in agreement with other studies in which is described that when the surface features are similar in dimension to the microbial size, cell retention may be favored.59 In summary, we have shown the great potential of surfaces displaying gradient in pore size, shapes and composition as platforms for selective bacteria trapping evidencing both the role of the pore size and the functionality in order to direct the surface immobilization.

in addition, is randomly distributed at the surface independently of the pore size. On the contrary, the low magnification images in Figure 10B clearly show that within the different topographical areas in the surfaces there is a clear difference in the bacteria immobilization density with a region showing high population density of adhered bacteria. Thus, both chemical composition and pore size appear to play a key role on the bacterial immobilization. In the surface containing PS40-bP(PEGMA300)48, Figure 10A, the presence of a larger amount of PEGMA block in regions closed to the edge prevents the bacterial immobilization while the adhesion in positions near the center of the film, r = 0 or r = R/2, is reduced by a matter of pore size. In the later surface with PS45-b-P(PEGMA300)34 where the amount of PEGMA at the surface is rather low, as demonstrated by Raman microscopy, the immobilization of the bacteria is not prevented by chemical composition. Moreover, the immobilization depends to a large extent on the pore size. Hence, at the edge of the films where the sizes of the pores are largely below 1 μm, no presence of bacteria can be observed, which perfectly fits with the results obtained for flat PS surfaces (data not shown). When the size of pores increases in the radial region r = 3R/4, a high density of bacteria’s immobilized within the pores is observed. The higher magnification image on the right panel shows individual bacterium in many of the pores. In this region of the film the pores have a diameter of about 2 μm, therefore are perfect cavities to host S. aureus bacteria with a diameter of 1−1.3 μm. Around this area, where the pore diameter is close to the bacteria diameter, there is a circular

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CONCLUSIONS In this manuscript we demonstrated how the breath figures approach can be employed to prepare multigradient platforms. In a single and straightforward step we prepared porous surfaces in which three different parameters vary gradually from the edge of the sample to the center. First of all, we evidenced the gradual variation of the pore size. Since the film formation by casting dries from the edge to the center, pores formed at the periphery of the sample are smaller in size compared to those obtained in the center of the sample. I


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Second, the shape of the pores can be varied depending on the sample concentration but also depending on their radial position within the same sample. By measuring the eccentricity of the pores we have shown that close to the film edge and most probably, according to Karthaus et al.58 due to convection occurring principally at the edge of the films, the pores are rather elliptical. When approaching to the center of the films the spherical pore shape is recovered. Third, herein we proof that, in addition to pore size and shape, the chemical composition within the pore can be controlled. In effect, by using amphiphilic block copolymers having different composition we observed either a regular distribution (PS45-b-P(PEGMA300)34) or a radial distribution (PS40-b-P(PEGMA300)48). The latter exhibits a higher amount of block copolymer toward the edge of the film and depletion in the center. Moreover, the chemical composition and the variable pore size porous surfaces have been evaluated to analyze the influence of these variables on the bacterial immobilization. According to our results, the PEGMA block prevents the bacteria from being immobilized. Moreover, in the absence of this block or with a rather low concentration the bacteria immobilization is directed by the size of the pores. Pore sizes similar to the bacteria dimensions appear to be the ideal situation to immobilize bacteria inside of the pores. To the best of our knowledge this is the first example in which by using a simple one-step strategy a multigradient surface can be obtained. Moreover, these initial results can be the base to construct platforms for selective immobilization and isolation of bacteria.

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ASSOCIATED CONTENT

S Supporting Information *

(a) Variation of the pores as a function of the radial distance to the edge for blends containing PS5FS21-b-PS31 and PS and having either 10 or 20 wt % of PS5F21-b-PS31 and 90 or 80 wt % of PS, and (b) variation of the pores as a function of the radial distance to the edge for blends containing PS 40 -b-P(PEGMA300)48 and PS and having either 10 or 20 wt % of PS40-b-P(PEGMA300)48 and 90 or 80 wt % of PS. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare the following competing financial interest(s): The results included in this articles are currently being protected.

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ACKNOWLEDGMENTS This work was financially supported by the MINECO (Projects MAT2010-17016, MAT2010-21088-C03-01, and COST Action MP0904 SIMUFER). A.M.-B. gratefully acknowledges the MINECO for her Juan de la Cierva postdoctoral contract and A.S.d.L. thanks the Ministerio de Educación for his FPU predoctoral fellowship. A.L.C. thanks Marie Curie COFUND “AMAROUT-Europe” Programme and European Commission International Reintegration Grant (IRG-246688) for financial support.

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REFERENCES

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Formation of Multigradient Porous Surfaces for Selective Bacterial

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