Selective Protein Adsorption on Polymer Patterns Formed by Self

Jul 8, 2009 - Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Kraków, Poland, Niewodniczański Institute of Nuclear Ph...
8 downloads 14 Views 4MB Size
Biomacromolecules 2009, 10, 2101–2109

2101

Selective Protein Adsorption on Polymer Patterns Formed by Self-Organization and Soft Lithography Joanna Zemła,† Małgorzata Lekka,*,‡ Joanna Raczkowska,† Andrzej Bernasik,§ Jakub Rysz,† and Andrzej Budkowski*,† Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Krako´w, Poland, Niewodniczan´ski Institute of Nuclear Physics, Polish Academy of Science, Radzikowskiego 152, 31-342 Krako´w, Poland, and Faculty of Physics and Applied Computer Science, University of Science and Technology, Mickiewicza 39, 30-059 Krako´w, Poland Received February 19, 2009

Thin films, with both isotropic and ordered patterns of polymer domains, are used as substrates to study selective adsorption of two proteins (concanavalin A and lentil lectin) and to test reconstruction of polymer patterns by these proteins. Integral geometry approach is used to compare quantitatively fluorescence micrographs of protein patches with AFM images of original isotropic patterns, formed during blend casting of polystyrene/poly(methyl methacrylate) and PS/poly(ethylene oxide). Preferential adsorption of both lectins to PMMA phase domains, enhanced for PS/PMMA interfaces is concluded. In turn, protein binding to PS phase regions of PS/PEO blends is highly selective. Ordered protein grouping is obtained as a result of selective adsorption to alternating stripes of polystyrene (partly brominated to enable identification) and cross-linked PEO, prepared with solvent-assisted micromolding applied to PBrS/PEO bilayers. Biological activity test, performed with concanavalin A, confirms preserved functionality of a complementary protein, carboxypeptidase Y, adsorbed to polymer patterns.

1. Introduction Protein microarrays, that is, arrays of a large number of proteins distributed spatially in a very small space,1 have numerous applications in many fields, such as biology, biomedical devices, biosensor technology, and tissue engineering.2-18 This is because interactions between proteins, antibodies, and other biomolecules play a crucial role in a number of biological processes: cell signaling, immune responses, cell adhesion, or cell cycle. Protein microarrays form a powerful tool allowing for simultaneous characterization of complex analyte solutions with regard to many features.1 This may be extremely useful, for example, for biomedical diagnosis as the levels of different proteins can be correlated not only with detection but also with progression and prognosis of various diseases.15 Protein microarrays may also be used as substrates for inhomogeneous cell seeding, which offers control over cell-cell and cell-extracellular matrix interactions3,19 and yields a fundamental insight into cell growth, differentiation, and adhesion.16 There are a number of techniques leading to protein microarrays formation.17 Proteins can be patterned directly on a given substrate20-27 or adsorbed nonspecifically28 to a prepatterned surface, which in many cases is polymeric: commonly used methods to fabricate protein microarrays on polymer surfaces include conventional photolithography, electron lithography, soft lithography, pin spotting, and microfluidics.8,11-14,20,29-38 However, also nonconventional methods, such as micropatterning of proteins on breath-figures,4 can be adapted. Experiments performed by Morin et al.,39 Sousa et al.,40 as well as Li et * To whom correspondence should be addressed. Phone: +48 12 66 28 271 (M.L.); +48 12 66 35 550 (A.B.). Fax: +48 12 66 28 089 (M.L.); +48 12 63 37 086 (A.B.). E-mail: [email protected] (M.L.); [email protected] (A.B.). † Jagiellonian University. ‡ Polish Academy of Science. § University of Science and Technology.

al.41,42 show an additional possibility of selective protein adsorption to the domains of phase-separated polymer blends. Polymer blend films are attractive as their surface properties (e.g., hydrophobicity, charge) are varied at length scales comparable to both single molecules (tens of nanometers) and whole cells (tens of micrometers).39-46 In the present paper we study the selective protein adsorption to surface patterns of polymer domains leading to pattern reconstruction by the proteins. The polymer patterns are formed by self-organization (spontaneous phase-separation) during blend casting and by a soft lithographic method of solvent-assisted micromolding, SAMIM,47 applied to precast polymer bilayers. Two fluorescently labeled lectins, concanavalin A (Con A) and lentil lectin (LcH), are used to examine the adsorption to the isotropic patterns of spin-cast self-organized films of polystyrene (PS)/poly(methyl methacrylate) (PMMA) and PS/poly(ethylene oxide) (PEO) blends (the latter cross-linked by ultraviolet). Recently, we have demonstrated that the surface organization of adsorbed proteins can be described by an extension of integral geometry approach,48-58 applied to fluorescence micrographs to provide a complete set of morphological measures, reflecting the coverage, lateral shape, and connectivity of (fluorescently labeled) protein patches.59 Here we compare quantitatively the replicated patterns of adsorbed (fluorescent) proteins with the original isotropic patterns of PS/PMMA and PS/PEO blend domains recorded by atomic force microscopy and analyzed within the same analytical approach.54 Because both patterns are formed on large area surfaces, their morphological (Minkowski) measures are determined based on the series of surface images recorded at various spots and with different scan range. The morphological measures are simply compared to collate different surface patterns54 and to examine pattern replication.55 This is in contrast to another approach,39,41,42 where the overlap of both compared patterns is tested at the same spots.

10.1021/bm900598s CCC: $40.75  2009 American Chemical Society Published on Web 07/08/2009

2102

Biomacromolecules, Vol. 10, No. 8, 2009

Motivated by high protein selectivity to PS/PEO blends we introduce a simple method to form well-ordered protein patterns, which is based on protein adsorption to micromolded bilayers of PEO and PS, the latter partly brominated to provide contrast for surface characterization. To show that deposited proteins preserve their biological specificity, we present the results confirming the specific interaction of glycoprotein-lectin pair, that is, carboxypeptidase Y, adsorbed to the polymer pattern, and concanavalin A.

2. Experimental Section 2.1. Preparation of Polymer Films. Polymers used in this study were polystyrene PS (weight-average molecular weight Mw ) 64000, polydispersity Mw/Mn ) 1.06), poly(methyl methacrylate) PMMA (Mw ) 31000, Mw/Mn ) 1.03), poly(ethylene oxide) PEO (Mw ) 42700, Mw/Mn ) 1.25) and partly brominated polystyrene PBrS (Mw ) 185000, Mw/Mn ) 1.04, with 10.7% segments brominated). Partly brominated polystyrene, used to enable polymer domain identification (see section 2.2), has physicochemical properties similar to those of PS.60 Isotropic Polymer Patterns. To prepare thin polymer films with isotropic patterns, the binary blends of PS/PMMA (1:2 mass fractions) in toluene and PS/PEO (1:1 mass fractions) in chloroform were prepared, with total polymer concentration cp ) 40 mg/mL. The solutions containing PEO were admixed with cross-linking agent, pentaerythritoltriacrylate (PETA, cPETA ) 4 mg/mL). Polymer films were prepared from solutions by spin-casting with coater KW-4A (Chemat Technology) onto SiOx wafers. The PS/PMMA blend films were used as cast (i.e., without any postbaking), while the PS/PEO films were additionally cross-linked using the standard procedure,61-63 employing 2 h long exposure to ultraviolet radiation (high pressure Hg lamp, 400 W) to avoid PEO dissolution during subsequent protein deposition. Ordered Polymer Patterns. The ordered polymer patterns were prepared using SAMIM technique.47 The solutions of PBrS in toluene and PEO (admixed with PETA, cPETA ) 4 mg/mL) in chloroform were prepared with polymer concentration cp ) 20 mg/mL. Then thin films of PEO were spin-cast with coater KW-4A onto SiOx wafers and crosslinked using the standard procedure.61-63 Subsequently, thin films of PBrS were spin-cast onto the underneath PEO layer. Finally, elastomer stamps with various micropatterns (linear bas-reliefs with period of 10-35 µm, symmetric and asymmetric with elevations more narrow than grooves), wetted with toluene (selective solvent for PBrS), was put in contact with the PEO/PBrS bilayer for 5 min, resulting in creation of elevated PBrS stripes, separated by depressed PEO regions. 2.2. Surface Characterization. Surface topography and lateral domain arrangement in the studied polymer films were examined by atomic (AFM) and lateral (LFM) force microscopy (The Academia System, Nanonics Imaging Ltd., Israel) working in contact mode.64-66 Polymer phases and their vertical arrangement for films with patterns formed by self-organization were identified after selective PS dissolution (resulting from film immersion for 60s in cyclohexane). For PBrS/PEO bilayer films micromolded with SAMIM technique, surface domain arrangement was confirmed by the composition mapping mode of dynamic secondary ion mass spectrometry dSIMS (VSW apparatus equipped with high resolution ion gun, liquid metal source, produced by Fei company), yielding with submicrometer lateral resolution the distribution maps of carbon (C2- ions, m/z ) 24), bromine (Br- ions, m/z ) 79), and oxygen (O- ions, m/z ) 16).60,67,68 2.3. Protein Adsorption. To test protein adsorption onto patterned polymer surfaces, two lectins (Biokom, Poland) were used: concanavalin A from CanaValia ensiformis (Con A) and lentil lectin from Lens culinaris (LcH). The main difference between used lectins is their different affinity constants to oligosaccharides and their structure at pH 7.0 (concanavalin A is a tetramer and lentil lectin is a dimer). These lectins were used in our earlier paper59 to demonstrate that the integral geometry approach can be used to compare protein adsorption to different polymer surfaces. Moreover, lectins are the proteins that

Zemła et al. recognize the oligosaccharides’ component, covalently attached to membrane proteins. The recognition occurs in a highly specific way comparable with that observed for antigen-antibody interaction. Both lectins were fluorescently labeled, ConA with the fluorescein isothiocyante (FITC), absorbing blue light (λabs ) 490 nm) and emitting green fluorescence (λemit ) 525 nm) and LcH with tetramethylrodamine isothiocyante (TRITC), absorbing green light (λabs ) 557 nm) and emitting red fluorescence (λemit ) 576 nm). The solutions of proteins in phosphate buffer saline (PBS, pH 7.4, Sigma) were prepared, with concentration c ) 125 µg/mL. The drop of each solution was placed on the patterned polymer substrate for incubation time t ) 15 min, then the sample was rinsed carefully with PBS and placed in a PBS buffer. All fluorescence measurements were performed for the samples immersed in PBS buffer, using Olympus BX51 microscope equipped with a 100W mercury lamp. 2.4. Images Analysis. A special care was taken to calibrate length scale for fluorescence images matching exactly that for AFM. First, AFM length scales were confirmed with calibration grids. Next, standard samples with isotropic fluorescent polymer patterns, spin-cast from PS/ poly(3-alkylthiophene) blends,64-66 were imaged with both fluorescence microscopy FM and AFM. Finally, Fourier transforms of these FM and AFM images were compared and the fluorescence length scale was normalized against that of AFM. Integral Analysis Approach. To analyze AFM and fluorescence micrographs quantitatively, an extension of integral geometry approach was applied48-58 using the procedures described in details in our earlier papers.54,59 In short, each micrograph represents an array of pixels set to a various local levels i(x,y), proportional to the local values of height (for AFM images) or fluorescence intensity (for fluorescence micrographs). Each pixel of the image can be reset to either white or black depending on whether its level value i(x,y) is larger or lower than threshold variable q. The procedures to determine the specific threshold value q, necessary to generate the binary images, have been rigorously determined for AFM54 and fluorescence59 images: For bimodal pixel distribution of AFM images, as recorded in this study (see Figure S1a and a′ in the Supporting Information), the threshold value q ) (h1 + h2)/2 is set54 by dominant height levels h1 and h2. In turn, for monomodal pixel distribution of fluorescent micrographs (as observed here, Figure S1b and b′ in the Supporting Information), the specific q value equals q ) f0 + hwmh/2). It is determined59 by the mean of the distribution and its full-width-at-half-maximum. Each binary (black-and-white) image is fully characterized by three morphological (Minkowski) measures, reflecting area fraction (coverage) F, boundary length (lateral shape) U, and the Euler characteristic (connectivity) χE of the white regions.48-53 Characteristic measures (F, U, χE) describe unequivocally topographical images of polymer patterns54 and fluorescence micrographs of protein patches.59 For each image, the Minkowski measures were computed (normalized by the analyzed area) with the software developed in our laboratory,54 using the algorithm of ref 52 (simpler but equivalent to that of ref 48). Their average values (, , ), obtained from the analysis of several (up to 9) images recorded at various spots with various scan ranges (and subject to the same standard background subtraction method), enable morphological comparison of the patterns of polymer domains and adsorbed proteins. 2.5. Biological Activity Test. To verify the biological activity of proteins adsorbed to polymer patterns, the experiment on recognition between a protein carboxypeptidase Y (CaY, Sigma) and a lectin ConA was performed. Carboxypeptidase Y is a protein possessing N-glycan branched oligosaccharides’ moiety composed of N-acetylglucosamine and mannose. This oligosaccharide structure is specifically recognized by two lectins, concanavalin A and lentil lectin, with high affinity comparable to that observed for antigen-antibody interaction. First, thin films of PS/PEO blend were prepared (see section 2.1). Then the sample was immersed in CaY solution in PBS (125 µg/mL) for 15 min and rinsed carefully to remove nonattached molecules.

Protein Adsorption on Polymer Patterns

Biomacromolecules, Vol. 10, No. 8, 2009

2103

Figure 3. AFM (a,b) surface images of self-organized PS/PEO films as spin-cast (a) and after immersion in cyclohexane to remove PS (b).

Figure 1. Polymer patterns formed by self-organization in spin-cast blend films of PS/PMMA and PS/PEO. Films of PS/PEO blend, cast from solution with added pentaerythritoltriacrylate PETA, were crosslinked by ultraviolet to avoid PEO dissolution in water-rich protein solutions.

Figure 2. AFM (a,b) surface images of self-organized PS/PMMA films as spin-cast (a) and after immersion in cyclohexane to remove PS (b).

Subsequently, one-half of the sample was immersed in bovine serum albumine (BSA, Sigma) solution in PBS (4 mg/mL) for 15 min and rinsed again with PBS. Such prepared samples, divided into two regions, one covered with BSA (blocking specific interactions) and the second with CaY (binding specifically to ConA), were used as substrates to study the lectin adsorption (as described in section 2.3).

3. Results and Discussion 3.1. Patterns Formed by Self-Organization. Polymer Films. The procedure to prepare films with self-organized domains of different polymers is presented schematically in Figure 1. Both investigated binary blends of strongly incompatible polymers, PS/PMMA and PS/PEO, were spin-cast from solution (Figure 1a), resulting in thin films with isotropic surface domain patterns (Figure 1b,d) of coexisting polymer phases. The self-organization mechanism involves here phase separation, initiated by solvent extraction, and the arrangement of demixed domains into lateral film structure, reflected by surface topography due to different solidification rates of various polymers.64-66,69 PS/ PEO films (with PETA additives) were additionally cross-linked by ultraviolet (Figure 1c) to avoid PEO dissolution during subsequent protein deposition process. The resulting surface topography and lateral domain arrangement of PS/PMMA films, examined with AFM, is presented in Figure 2. The elevated, island-like structures, visible in the topographical image (Figure 2a), are formed by one polymer phase, whereas the depressed regions are the regions of the second phase. Additional AFM surface examination after

selective dissolution of PS (by immersion in cyclohexane for 60s, Figure 2b) clearly reveals that elevated regions correspond to PMMA phase (similarly to69,70). The results of analogous surface examination of PS/PEO films (with admixed PETA) are presented in Figure 3. AFM images collected prior (Figure 3a) and after (Figure 3c) selective dissolution of PS clearly reveal that elevated, island-like regions, visible in a topographical image (Figure 3a), correspond to PS phase. Protein Adsorption to Polymer Patterns Formed by SelfOrganization. Thin polymer blend films, PS/PMMA and PS/ PEO, with isotropic domain patterns formed by self-organization have been used as the substrates to study the selective adsorption of two fluorescently labeled lectins: ConA and LcH. Representative results obtained for PS/PMMA films are presented in Figure 4. AFM image depicting the original surface pattern of PS/PMMA blend (Figure 4a) is compared with the fluorescence micrographs recorded for ConA (Figure 4b) and LcH (Figure 4c) adsorbed to this substrate. Direct examination reveals an island-like structure of lectin patches resembling that of PMMA phase domains. In addition, singular spots with higher fluorescence intensity indicate protein clusters that can be formed even on surfaces exposed to low concentration solutions.71 Positions of these singular clusters do not match the polymer patches. In addition, a more careful inspection, enabled by line scan analysis of fluorescence intensity (Figure 4b′ and c′), indicates preferential protein adsorption to PMMA phase (elevated plateau in Figure 4a′), which is enhanced at PS/PMMA interfaces. To verify these specific properties of protein adsorption observed locally with a quantitative and nonlocal analysis, the integral geometry approach was applied to the series of several micrographs. Usually, the quality of the pattern replication is based on the comparison of two patterns: the original one and the one mimicking the original. One solution to solve this problem, advocated by Li et al.41,42 is based on the relative coverage of different overlapping elements (and interfaces) of both patterns, measured at the same spots. This approach cannot, however, quantify any of the compared patterns separately, especially in a manner describing its morphology. We propose another approach where the comparison of patterns formed on large area surfaces is performed using integral geometry analysis of the series of surface images (recorded at various spots and with different scan range). Such way of surface characterization enables the statistical sampling over large areas. Thus, the morphological (Minkowski) measures,48 representative for polymer and protein patterns, were determined from micrograph series as average values (with the estimated error bars). The obtained average values of three Minkowski measures (, , ), which characterize the

2104

Biomacromolecules, Vol. 10, No. 8, 2009

Zemła et al.

Figure 4. AFM image (a) of self-organized polymer pattern PS/PMMA and fluorescence micrographs (b,c) of lectins, ConA (b) and LcH (c), adsorbed to the pattern. Topographic cross-section (a′) and intensity scans (b′,c′) correspond to the lines marked in (a,b,c). Representative black-and-white images (d,e,f) are derived from the originals (a,b,c). White regions, corresponding to PMMA phase domains (d) and adsorbed lectins, ConA (e) and LcH (f), are described (see Table 1) by covered area F, boundary length U, and connectivity χE (i.e., difference between number of white domains and black holes), normalized by imaged area. Table 1. Mean Values of the Minkowski Measuresa PS/PMMA



[µm-1]

[µm-2]

PMMA (elevated) ConA LcH

0.46 ( 0.02 0.51 ( 0.03 0.52 ( 0.01

0.64 ( 0.02 0.85 ( 0.04 0.81 ( 0.01

0.019 ( 0.002 -0.024 ( 0.01 -0.002 ( 0.001

a Calculated for topographical images of (elevated) PMMA phase domains in PS/PMMA (upper row) as well as for fluorescence micrographs of ConA (middle row) and LcH (bottom row), adsorbed to this polymer pattern.

morphology of PMMA phase domains and protein (ConA and LcH) patches, are presented in Table 1. To discuss the values in Table 1 we recall that within this analytical approach54,59 all original micrographs (Figure 4a,b,c) are transformed into representative black-and-white images (Figure 4d, e, and f, respectively), described by the area fraction F, perimeter U, and connectivity χE of the white domains, corresponding to elevated (PMMA) phase (Figure 4d) and adsorbed lectins (Figure 4e,f). The Euler characteristic (connectivity) χE is the difference between the number of separate white and black domains. All morphological measures are normalized by imaged area to provide easy comparison between the micrographs with different scan ranges. The area fraction occupied by PMMA phase domains, equal to 46 ( 2%, is only slightly increased for the surface coverage by lectins, with 51 ( 3% and 52 ( 1% for ConA and LcH, respectively (see Table 1). Overall, preferential protein adsorption to PMMA phase domains is concluded. Slight increase in the value (fraction of white domains in Figure 4e and f as compared to d) could be related with singular protein clusters that do not match the polymer pattern. However, if such clusters were dominant in the morphology, they would reduce

the average perimeter of protein patches. In contrast, the boundary length of surface features is increased from 0.64 ( 0.02 µm-1 for PMMA phase to 0.85 ( 0.04 or 0.81 ( 0.01 µm-1 for ConA and LcH, respectively. Extended perimeter of lectin domains is correlated with drastic reduction of the Euler characteristic (0.019 ( 0.002 µm-2 for PMMA phase, but -0.024 ( 0.01 and -0.002 ( 0.001 µm-2 for ConA and LcH). Nonpositive values reflect strong presence of the regions without proteins (black holes) separated within the protein patches with boundaries extended as compared to PMMA domains (cf. white domains in Figure 4e, f, and d). All these changes in the Minkowski parameters (, , ) can be explained by protein adsorption enhanced at PMMA/PS interfaces. In addition, absolute connectivity || value is lower for adsorbed LcH as compared to ConA, indicating more compact nonadsorbing regions inside protein patches (centered on PMMA phase domains). This makes the enhancement of LcH adsorption to PMMA/PS interfaces more pronounced as compared to ConA. Similar conclusions can be drawn from the comparison of locally observed protein binding (cross sections in Figure 4b′ and c′). Enhanced binding to the polymer/polymer interfaces, in fact, PS/PMMA has been observed previously for two other proteins: fibrinogen39 and human serum albumin (adsorbed from the solutions with lower concentrations).41,42 Proteins are highly surface active and their binding to the interfacial regions (between the PS and PMMA phases exposed to protein solution) could result in the lowest free energy of the system.39 Such preferential interfacial adsorption is disrupted for increased both protein concentration and adsorption time.39,41,42 These two parameters, in addition to ionic strength (pH) of protein

Protein Adsorption on Polymer Patterns

Biomacromolecules, Vol. 10, No. 8, 2009

2105

Figure 5. AFM image (a) of self-organized polymer pattern PS/PEO and fluorescence micrographs (b,c) of lectins, ConA (b) and LcH (c), adsorbed to the pattern. Topographic cross-section (a′) and intensity scans (b′,c′) correspond to the lines marked in (a) and the insets to (b,c). Representative black-and-white images (d-f) are derived from the originals (a-c) with integral geometry approach: White regions, corresponding to elevated PS phase domains (d) and adsorbed lectins, ConA (e) and LcH (f), are described (see Table 2) by fractional coverage F, perimeter U, and the Euler characteristic χE, normalized by imaged area. Table 2. Mean Values of the Minkowski Measuresa 42,72,73

solution, control protein distribution at the interface and on polymer domains.39,41,42 As a result, relative protein binding was observed to be lower, similar or higher for PMMA as compared to PS. We note that also polymer film preparation (used as cast from solvent,40,72 postbaked,59 thermally-39,41,42 or solvent-annealed40) could modify protein adsorption results. This is why the observed lectin binding is higher for PMMA than PS phase (in films with no post-treatment), while a reversed relation was observed for postbaked films of pure polymers.59 The major role in protein adsorption to polymer surfaces is played by hydrophobic interactions. This is probably why lectins (i.e., a special class of proteins) increase their binding properties with growing polymer hydrophobicity (i.e., decreasing polar component of polymer solubility parameter δp ) 17.4, 11.0, and 5.7 MPa0.5 for PEO, PMMA, and PS, respectively).9 However, when protein adsorption to two polymers of similar magnitude, as PMMA and PS, occurs, other effects become more important as it has been already reported.39,41,42 For example, proteins might expose hydrophobic and hydrophilic regions, which tend to bind to the domains of polymer patterns with lower (PS phase) and higher (PMMA phase) surface energy. As a result, enhanced adsorption can be observed at PS/PMMA interface. Such effect was observed for fibrinogen and albumin.39,41,42 While the hydrophobic interaction is expected for both PS and PMMA phase, the latter could exhibit also hydrogen bonding (between polymer carbonyls and protein amides) and dipole-dipole interactions. Balance between the interactions responsible for protein binding to the polymer surface can be affected by the solvent present in the spin-cast

PS/PEO



[µm-1]

[µm-2]

PS (elevated) ConA LcH

0.32 ( 0.04 0.25 ( 0.04 0.27 ( 0.03

0.35 ( 0.04 0.34 ( 0.03 0.32 ( 0.03

0.024 ( 0.005 0.023 ( 0.002 0.020 ( 0.001

a Calculated for topographical images of (elevated) PS phase domains in PS/PEO (upper row) as well as for fluorescence micrographs of ConA (middle row) and LcH (bottom row), adsorbed to this polymer pattern.

films. This effect can be enhanced when the solvent prefers one polymer (e.g., toluene is more abundant in PS than PMMA phase).70 This tentatively explains why the lectins preferentially adsorb to PMMA when PS is usually considered to be an excellent substrate for proteins. Representative micrographs recorded for PS/PEO films are shown in Figure 5. Fluorescence images reflect structures formed by ConA (Figure 5b) and LcH (Figure 5c) adsorbed to the polymer pattern. The structure of protein patches resemble that of PS phase domains in the original pattern, depicted by AFM image (Figure 5a). In contrast to the previous situation for PS/ PMMA, lectin adsorption to polymer phase domains is here highly selective. Additional inspection, enabled by line scans of fluorescence intensity (Figure 5b′ and c′), reveals no detectable signs of protein binding to polymer/polymer (PS/ PEO) interfaces. These specific and locally observed properties of protein adsorption are confirmed by the quantitative results of nonlocal analysis performed within the integral geometry approach. Obtained average Minkowski measures (, , ) given

2106

Biomacromolecules, Vol. 10, No. 8, 2009

Figure 6. Polymer patterns PBrS/PEO formed by soft lithography. Bilayers PEO/PBrS (d), prepared by successive spin-casting from selective solvents (a,c) separated by cross-linking PEO (with added PETA, b), were patterned by solvent-assisted micromolding SAMIM (e,f).

in Table 2 characterize the morphology of elevated PS phase domains and protein (ConA and LcH) patches, illustrated by the white domains in the representative black-and-white-images (Figure 5d-f). The perimeter and connectivity of PS phase regions (0.35 ( 0.04 µm-1, 0.024 ( 0.005 µm-2, respectively) are within experimental error bars identical to those computed for ConA (0.34 ( 0.03 µm-1, 0.023 ( 0.002 µm-2) or LcH (0.32 ( 0.03 µm-1, 0.020 ( 0.001 µm-2). In turn, the area fraction covered by lectins (25 ( 4% for Con A and 27 ( 3% for LcH) is slightly lower as compared to the original value for PS phase domains (32 ( 4%). These characteristic features of compared Minkowski measures reflect selective protein binding to PS phase domains but also the absence of protein adsorption to PS/PEO interfaces. For protein patches the latter effect should result in somewhat reduced , much smaller decrease in , and unchanged value. The difference in hydrophobicity between PEO and PS is larger than between PMMA and PS. Therefore, the effects related to the presence of the solvent (chloroform) in the ascast blends cannot affect preferential binding to PS phase domains. In addition, PETA additives, enabling cross-linking, have been reported to enhance lectin adsorption to PS phase.59 3.2. Patterns Formed by Soft Lithography. Polymer Films. Because highly selective lectin adsorption has been concluded for the binary blend PS/PEO, this polymer pair was chosen to prepare well-ordered polymer patterns guiding formation of protein arrays. To provide contrast necessary for the composition mapping mode of dynamic secondary mass spectrometry dSIMS,60,67,68 PS was replaced by its partly brominated counterpart PBrS (with similar physicochemical properties).60 Ordered polymer patterns were prepared with two-step procedure, presented schematically in Figure 6. First, the bilayers of PEO/PBrS were prepared by successive spin-casting (Figure 6a-d), separated by the cross-linking of PEO layer (with added PETA, Figure 6b). Then the bilayers were patterned by solventassisted micromolding technique SAMIM47 (Figure 6e), resulting in formation of highly ordered alternating stripes of depleted PEO regions and elevated PBrS domains (Figure 6f). Regular polymer micropatterns with different size and shape (symmetric, or asymmetric with wider elevated PBrS stripes) are formed in this way over broad sample areas (Figure 7). To verify that ordered surface patterns are formed exclusively by the polymers (PEO and PBrS), the micromolded bilayers

Zemła et al.

Figure 7. Micromolded PEO/PBrS bilayers with regular polymer patterns (period 10-35 µm) imaged with optical microscopy.

Figure 8. AFM (a) and LFM (b) surface images and dSIMS distribution maps (50 × 50 µm) of carbon (traced with C2- ions; (c)), oxygen (Oions; (d)), and bromine (Br- ions; (e)) recorded for the micromolded bilayers, which form (here asymmetric) surface patterns of elevated (wider) PBrS and depleted (more narrow) PEO.

were examined with AFM and LFM (Figure 8a,b) as well as with composition mapping dSIMS mode (Figure 8c-e). Comparison between AFM and LFM surface images (Figure 8a and b, respectively) clearly reveals that depressed and elevated stripes must be composed of different materials (resulting in visible LFM contrast). In turn, homogeneous dSIMS map of carbon concentration, determined with mass-resolved C2- ions (Figure 8c), clearly shows that the whole surface is covered only with polymer materials, PBrS and PEO. Thus, the crosslinked PEO layer (adjacent to the SiOx substrate) is not interrupted by the micromolding SAMIM procedure, and the alternating stripes of PBrS and PEO are formed. For the asymmetric surface patterns, the composition of elevated (wider) pattern domains (Figure 8a) is additionally confirmed by the dSIMS map of PBrS distribution determined with Br- ions (Figure 8e). Protein Adsorption to Polymer Patterns Formed by Soft Lithography. Representative fluorescence micrographs (Figure 9) illustrate proteins adsorbed to polymer bilayers patterned with the SAMIM technique. Ordered grouping is manifested for both lectins, ConA (Figure 9a) and LcH (Figure 9b), which replicate the well-ordered polymer patterns PBrS/PEO. An obvious

Protein Adsorption on Polymer Patterns

Figure 9. Fluorescence images of the lectins ConA (a) and LcH (b) adsorbed selectively on the polymer patterns PBrS/PEO formed by soft lithography. Intensity scans (a′,b′) correspond to the lines marked in (a,b).

hypothesis, suggested by the experiments with PS/PEO blends (section 3.1), is that the proteins adsorb selectively to PBrS (similar to PS) and not PEO regions. This is indeed confirmed by Figure 9a, where the proteins cover the wider stripes of polymer pattern, which for the used elastomer stamps correspond always to PBrS domains. In addition, the cross-sectional analysis of recorded fluorescence micrographs (Figure 9a′,b′) suggests that the selectivity of polymer-protein interactions is higher for the PBrS/PEO patterns than for the structures of PS/PEO blends (cf. Figure 5b′,c′). 3.3. Verification of Biological Activity. The crucial issue, relevant for potential biomedical applications of protein patterns, is their biological activity since immobilization can sometimes block access to specific part of a protein. To examine whether the applied protein grouping procedure (using nonspecific but selective adsorption to prepatterned polymer surfaces) reduces protein activity, the following experiment was performed. First, the PS/PEO surface patterns were exposed to the solution of nonfluorescent carboxypeptidase Y (CaY) to allow its selective adsorption. Carboxypeptidase CaY, used here as a model protein, possesses a carbohydrate moiety that is specifically recognized by lectins such as concanavalin A. Then one-half of each such sample was covered by bovine serum albumin (BSA, region II), which is commonly used to block any specific interactions. Finally, the whole samples were immersed in the solutions of fluorescent lectins ConA. Representative fluorescence micrograph, recorded after this step was completed, is presented in Figure 10. It clearly shows the presence of ConA exclusively in the areas not covered with BSA (region I), where the lectins could specifically bind to CaY and thus reproduce the surface CaY distribution guided by PS domains of PS/PEO. The adsorption of proteins to any surface usually results in randomly oriented protein molecules. To apply such surface for example in biosensors, there is a need to ensure that protein binding sites are exposed to exterior, and proteins are able to interact in a specific way, preserVing their biological activity. Our test of biological activity confirms that presented novel method to form well-ordered protein patterns, based on protein adsorption to micromolded bilayers of PS top and cross-linked PEO bottom lamellae, is suitable for potential biomedical applications: The oligosaccharides’ moieties of adsorbed CaY

Biomacromolecules, Vol. 10, No. 8, 2009

2107

Figure 10. Results of biological activity test for proteins adsorbed to prepatterned polymer films. Micrographs of fluorescent lectins ConA bound specifically to CaY proteins, which adsorbed earlier to the PS domains of PS/PEO patterns (regions I). Note that lectins do not bind to the areas covered by bovine serum albumin BSA blocking specific interactions (region II).

seem to be exposed to the exterior, as they participate in specific glycoprotein-lectin interactions. Affinity of such binding is comparable to that of antigen-antibody pairs.74

4. Summary and Conclusions Selective protein (ConA and LcH) adsorption to polymer patterns formed by self-organization (blend films PS/PMMA and PS/PEO) and soft lithography (micromolded bilayers PBrS/ PEO) is demonstrated in this paper. Protein patches, resulting from preferential adsorption to polymer blend surfaces, are compared quantitatively with the original patterns (isotropic blend structures) to test the quality of pattern replication. Results of nonlocal integral geometry analysis, applied to the series of several (fluorescence, AFM) micrographs, are expressed in terms of average morphological measures describing area fraction F, perimeter U, and connectivity χE of both protein patches and polymer patterns. Two types of pattern replication are described by different relations of two sets of these measures (see Tables 1 and 2): Enhanced adsorption of both proteins (ConA and LcH) to polymer/polymer (PS/PMMA) interfaces, in addition to preferential binding to one (PMMA) of the polymer blend phases, leads to the “holes” in poorly replicated domains with extended boundaries. This is reflected in drastic χE reduction to nonpositive values, as well as in increased U and F (Table 1). In turn, highly selective protein adsorption to polymer (PS) phase domains with no signs of adsorption activity at polymer/ polymer (PS/PEO) interfaces results in nearly prefect pattern replication. The U and χE values are comparable, while F is slightly reduced (Table 2). We believe than the integral geometry approach, which is demonstrated in this paper, could contribute to the protein adsorption studies, as it describes in a quantitative and relatively easy way the morphological organization of proteins with nonuniform surface distribution, reflected in various types of micrographs.4,8,11,17,20,25,29-31,36,38-42,46,75-78 Here this approach is used to select the polymer phase pair (PS/PEO) with different polymer-protein interactions, which can be used to form polymer templates for ordered protein grouping (protein mi-

2108

Biomacromolecules, Vol. 10, No. 8, 2009

croarrays). A novel method is proposed to form such templates with different shapes and sizes over broad areas, using solvent assisted micromolding SAMIM applied to the bilayers of PEO and PS (partly brominated to enable identification). Ordered grouping of two lectins (ConA, LcH) is obtained. Moreover, the biological activity test, performed for carboxypeptidase Y-concanavalin A molecular complex, shows that protein grouping obtained due to nonspecific but selective adsorption to prepatterned polymer surfaces do not alter the functionality of adsorbed proteins, which makes this approach suitable for potential biomedical applications. Acknowledgment. This work was partially supported by the Reserve of the Faculty of Physics, Astronomy and Applied Computer Science of the Jagiellonian University. J.R. is grateful to the Foundation for Polish Science for support. Supporting Information Available. Supplementary figures representing bimodal pixel distribution obtained for AFM images and monomodal pixel distribution of fluorescent micrographs. This material is available free of charge via the Internet at http:// pubs.acs.org.

References and Notes (1) Angedent, P. DDT 2005, 10, 503–511. (2) Schwarz, U. Soft Matter 2007, 3, 263–266. (3) Khetani, S. R.; Bhatia, S. N. Curr. Opin. Biotechnol. 2006, 17, 524– 531. (4) Zhang, Y.; Wang, C. AdV. Mater. 2007, 19, 913–916. (5) C, J.; Lee, C. J.; Blumenkranz, M. S.; Fishman, H. A.; Bent, S. F. Langmuir 2004, 20, 4155–4161. (6) Lin, C. C.; Co, C. C.; Ho, C. C. Biomaterials 2005, 3655–3662. (7) Veiseh, M.; Zareie, M. H.; Zhang, M. Langmuir 2002, 18, 6671– 6678. (8) Yang, Z.; Belu, A. M.; Liebermann-Vinson, A.; Sugg, H.; Chilkoti, A. Langmuir 2000, 16, 7482–7492. (9) Garcı´a, A. J. AdV. Polym. Sci. 2006, 203, 171–190. (10) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60. (11) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408–2413. (12) Chen, C. S.; Jiang, X.; Whitesides, G. M. MRS Bull. 2005, 30, 194– 201. (13) Patrito, N.; McCague, C.; Norton, P. R.; Petersen, N. O. Langmuir 2007, 23, 715–719. (14) Welle, A.; Chiumiento, A.; Barbucci, R. Biomol. Eng. 2007, 24, 87– 91. (15) Christman, K. L.; Maynard, H. D. Langmuir 2005, 21, 8389–8393. (16) Kumar, G.; Ho, C. C.; Co, C. C. AdV. Mater. 2007, 1084–1090. (17) Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Soft Matter 2006, 2, 928–939. (18) Sapsford, K. E.; Bradburne; Delehanty, J. B.; Medintz, I. L. Mater. Today 2008, 11, 38–49. (19) Liu, W. F.; Chen, C. S. Mater. Today 2005, 8, 28–35. (20) Douvas, A. M.; Petrou, P. S.; Kakabakos, S. E.; Misiakos, K.; Argitis, P.; Sarantopoulou, E.; Kollia, Z.; Cefalas, A. C. Anal. Bioanal. Chem. 2005, 381, 1027–1032. (21) Bernard, A.; Delamarche, E.; Schmidt, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. A. Langmuir 1998, 14, 2225–2229. (22) James, C. D.; Davis, R. C.; Kam, L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. Langmuir 1998, 14, 741–744. (23) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971–3975. (24) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. AdV. Mater. 2000, 12, 1067–1070. (25) Tan, J. L.; Tien, J.; Chen, C. S. Langmuir 2002, 18, 519–523. (26) Bernard, A.; Fitzli, D.; Sonderegger, P.; Delamarche, E.; Michel, B.; Bosshard, H. R.; Biebuyck, H. A. Nat. Biotechnol. 2001, 866–869. (27) Renault, J. P.; Bernard, A.; Juncker, D.; Michel, B.; Bosshard, H. R.; Delamarche, E. Angew. Chem., Int. Ed. 2002, 41, 2320–2323. (28) Quinn, A.; Mantz, H.; Jacobs, K.; Bellon, A.; Anten, L. Europhys. Lett. 2008, 81, Art. No 56003 (6pp). (29) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6, 2427–2448.

Zemła et al. (30) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. A. J. Am. Chem. Soc. 1998, 120, 500–508. (31) Suh, K. Y.; Seong, J.; Khademhosseini, A.; Laibinis, P. E.; Langer, R. Biomaterials 2004, 25, 557–563. (32) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363–2376. (33) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. Langmuir 1999, 15, 3845–3851. (34) Schwarz, A.; Rossier, J. S.; Roulet, E.; Mermod, N.; Roberts, M. A.; Girault, H. H. Langmuir 1998, 14, 5526–5531. (35) Wang, C.; Zhang, Y. AdV. Mater. 2005, 17, 150–153. (36) Bre´tagnol, F.; Valsesia, A.; Sasaki, T.; Ceccone, G.; Colpo, P.; Rossi, F. AdV. Mater. 2007, 19, 1947–1950. (37) Ionov, l.; Synytska, A.; Diez, S. AdV. Funct. Mater. 2008, 18, 1501– 1508. (38) Petrou, P. S.; Chatzichristidi, M.; Douvas, A. M.; Argitis, P.; Misiakos, K.; Kakabakos, S. E. Biosens. Bioelectron. 2007, 22, 1994–2002. (39) Morin, C.; Hitchcock, A. P.; Cornelius, R. M.; Brash, J. L.; Urquhart, S. G.; Scholl, A.; Doran, A. J. Electron Spectrosc. Relat. Phenom. 2004, 137-140, 785–794. (40) Sousa, A.; Sengonul, M.; Latour, R.; Kohn, J.; Libera, M. Langmuir 2006, 22, 6286–6292. (41) Li, L.; Hitchcock, A. P.; Robar, N.; Cornelius, R.; Brash, J. L.; Scholl, A.; Doran, A. J. Phys. Chem. B 2006, 110, 16763–16773. (42) Li, L.; Hitchcock, A. P.; Cornelius, R.; Brash, J. L.; Scholl, A.; Doran, A. J. Phys. Chem. B 2008, 112, 2150–2158. (43) Whitesides, G. M. Nat. Biotechnol. 2003, 21, 1161–1165. (44) Dalby, M. J.; Riehle, M. O.; Johnstone, H. J. H.; Affrossman, S.; Curtis, A. S. G. Tissue Eng. 2008, 8, 1099–1108. (45) Gadegaard, N.; Dalby, M. J.; Riehle, M. O.; Curtis, A. S. G.; Affrossman, S. AdV. Mater. 2004, 16, 1857–1860. (46) Minelli, C.; Kikuta, A.; Tsud, N.; Ball, M. D.; Yamamoto, A. J. Nanobiotechnol. 2008, 6, art no 3 (11pp). (47) Kim, E.; Xia, Y.; Zhao, X. M.; Whitesides, G. M. AdV. Mater. 1997, 9, 651. (48) Mecke, K. R. Phys. ReV. E 1996, 53, 4794–4800. (49) Mecke, K. R.; Sofonea, V. Phys. ReV. E 1997, 56, R3761-R3764. (50) Mecke, K. R. Int. J. Modern Phys. B 1998, 9, 861–899. (51) Mecke, K. R. In Statistical Physics and Spatial Statistics; Mecke, K. R., Stoyan, D., Eds.; Springer-Verlag: Heidelberg, 2000; Vol. 554, p 111. (52) Michielsen, K.; de Raedt, H. Phys. Rep. 2001, 347, 461–538. (53) Sofonea, V.; Mecke, K. R. Eur. Phys. J. B 1999, 8, 99–112. (54) Raczkowska, J.; Rysz, J.; Budkowski, A.; Lekki, J.; Lekka, M.; Bernasik, A.; Kowalski, K.; Czuba, P. Macromolecules 2003, 36, 2419–2427. (55) Raczkowska, J.; Bernasik, A.; Budkowski, A.; Rysz, J.; Kowalski, K.; Lekka, M.; Czuba, P.; Lekki, J. Thin Solid Films 2005, 476, 358– 365. (56) Gutmann, J. S.; Mu¨ller-Buschbaum, P.; Stamm, M. Faraday Discuss. 1999, 112, 285–297. (57) Becker, J.; Gru¨n, G.; Seemann, R.; Mantz, H.; Jacobs, K.; Mecke, K. R.; Blossey, R. Nat. Mater. 2003, 2, 59–63. (58) Rehse, S.; Mecke, R. K.; Magerle, R. Phys. ReV. B 2008, 77, 051805. (59) Zemła, J.; Lekka, M.; Wiltowska-Zuber, J.; Budkowski, A.; Rysz, J.; Raczkowska, J. Langmuir 2008, 24, 10253–10258. (60) Raczkowska, J.; Bernasik, A.; Budkowski, A.; Rysz, J.; Gao, B.; Lieberman, M. Macromolecules 2007, 40, 2120–2125, references therein. (61) Doytcheva, M.; Stamenova, R.; Zvetkov, V.; Tsvetanow, C. Polymer 1998, 39, 6715–6721. (62) Doytcheva, M.; Dotcheva, D.; Stamenova, R.; Tsvetanow, C. Macromol. Mater. Eng. 2001, 286, 30–33. (63) Doytcheva, M.; Petrova, E.; Stamenova, R.; Tsvetanow, C.; Riess, G. Macromol. Mater. Eng. 2004, 289, 676–680. (64) Jaczewska, J.; Budkowski, A.; Bernasik, A.; Raptis, I.; Raczkowska, J.; Goustouridis, D.; Rysz, J.; Sanopoulou, M. J. Appl. Polym. Sci. 2007, 105, 67–79. (65) Jaczewska, J.; Budkowski, A.; Bernasik, A.; Moons, E.; Rysz, J. Macromolecules 2008, 41, 4802–4810. (66) Jaczewska, J.; Budkowski, A.; Bernasik, A.; Raptis, I.; Moons, E.; Goustouridis, D.; Haberko, J.; Rysz, J. Soft Matter 2009, 5, 234–241. (67) Bernasik, A.; Rysz, J.; Budkowski, A.; Kowalski, K.; Camra, J.; Jedlin´ski, J. Macromol. Rapid Commun. 2001, 22, 829–834.

Protein Adsorption on Polymer Patterns (68) Bernasik, A.; Rysz, J.; Budkowski, A.; Brenn, R.; Kowalski, K.; Camra, J.; Jedlinski, J. Eur. Phys. J. E 2003, 12, 211–214. (69) Walheim, S.; Bo¨ltau, M.; Mlynek, J.; Krausch, G.; Steiner, U. Macromolecules 1997, 30, 4995–5003. (70) Raczkowska, J.; Budkowski, A.; Rysz, J.; Czuba, P.; Lekka, M.; Bernasik, A. J. Nanostruct. Polym. Nanocomp. 2005, 1, 23–35. (71) Rabe, M.; Verdes, D.; Seeger, S. Soft Matter 2009, 5, 10391047. (72) Mielczarski, J. A.; Dong, J.; Mielczarski, E. J. Phys. Chem. B 2008, 112, 5228–5237. (73) Dong, J.; Mielczarski, J. A.; Mielczarski, E.; Xu, Z. Biotechnol. Prog. 2008, 24, 972–980. (74) Sharon, N.; Lis, H. Science 1989, 246, 227–234.

Biomacromolecules, Vol. 10, No. 8, 2009

2109

(75) Lee, S. W.; Oh, B. K.; Sanedrin, R. G.; Salaita, K.; Fujigaya, T.; Mirkin, C. A. AdV. Mater. 2006, 18, 1133–1136. (76) Mack, N. H.; Dong, R.; Nuzzo, R. G. J. Am. Chem. Soc. 2006, 128, 7871–7881. (77) Renault, J. P.; Bernard, A.; Bietsch, A.; Michel, B.; Bosshard, H. R.; Delamarche, E.; Kreiter, M.; Hecht, B.; Wild, U. P. J. Phys. Chem. B 2003, 107, 703–711. (78) Michel, R.; Reviakine, I.; Sutherland, D.; Fokas, C.; Csucs, G.; Danuser, G.; Spencer, N. D.; Textor, M. Langmuir 2002, 18, 8580–8586.

BM900598S