Article pubs.acs.org/Langmuir

Effects of Polythiophene Surface Structure on Adsorption and Conformation of Bovine Serum Albumin: A Multivariate and Multitechnique Study K. Awsiuk,*,† A. Budkowski,† M. M. Marzec,‡ P. Petrou,§ J. Rysz,† and A. Bernasik∥ †

M. Smoluchowski Institute of Physics, Jagiellonian University, Kraków, Poland Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, Kraków, Poland § Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety NCSR Demokritos, Aghia, Paraskevi, Greece ∥ Faculty of Physics and App. Computer Science, AGH University of Science and Technology, Kraków, Poland ‡

S Supporting Information *

ABSTRACT: Protein interactions with surfaces of promising conducting polymers are critical for development of bioapplications. Surfaces of spin-cast and postbaked poly(3-alkylthiophenes), regiorandom P3BT, and regioregular RP3HT are examined prior to and after adsorption of model protein, bovine serum albumin, with time-of-flight secondary ion mass spectrometry, atomic force microscopy, and X-ray photoelectron spectroscopy. The multivariate method of principal component analysis applied to ToF-SIMS data maximizes information on subtle differences in surface chemistry: PCA reveals alkyl side chains and conjugated backbones, exposed for RP3HT and P3BT, respectively. Phase imaging AFM shows semicrystalline microstructure of RP3HT and amorphous morphology of P3BT films. A cellular-like pattern of proteins adsorbed on RP3HT develops with coverage to more uniform overlayer, observed always on P3BT. The amount of adsorbed protein, determined by XPS as a function of BSA concentration (up to 10 mg/mL), is ∼21% lower for RP3HT than P3BT (up to 1.1 mg/m2). Although PCA differentiates protein from polythiophene, relative protein surface composition evaluated from ToF-SIMS saturates rather than increases with amount of adsorbed BSA from XPS. This reflects ToF-SIMS sensitivity to outermost layer of proteins, enabling multivariate analysis of protein conformation or orientation. PCA distinguishes between amino acids characteristic for external regions of BSA adsorbed to P3BT and RP3HT. These amino acids are identified for P3BT and RP3HT as hydrophilic and hydrophobic, respectively, by relative hydrophobicity of amino acid side chains. Alternative identification with BSA domains fails, pointing to substrate-induced changes in conformation and degree of denaturation rather than orientation of adsorbed protein. (RP3HT), thoroughly applied P3AT semiconductor,7 and regiorandom poly(3-butylthiophene) (P3BT) were chosen for this study. Solution-deposited films of regiorandom P3ATs are amorphous, but their regioregular counterparts are semicrystalline with self-oriented crystallites.8 The lamellae, formed in the crystallites by separated layers of conjugated backbones and insulating alkyl groups,13 can orient themselves parallel or normal to the film substrate depending on P3AT regioregularity (e.g., 96% or 81%, respectively, reported for RP3HT14). Parallel lamellar alignment, with P3AT chains oriented edgeon,15 is desirable as it provides strongly enhanced charge mobility as compared to plane-on textured crystallites with normally oriented lamellae.14 Recently, Scarpa et al. concentrated on widely used RP3HT and overcame its biocompatibility problems by applying, besides plasma treatments, protein-based coatings (poly-Llysine, collagen, fibronectin) to enable growth of living cells.16

1. INTRODUCTION Protein interactions with surfaces of conducting polymers are critical for development of diverse applications, such as biosensors, bioseparations, implantable bioelectrodes, tissue engineering, and regenerative medicine.1 This development is synergistically fostered by electronic properties of conducting polymers on one hand, enabling molecular recognition to signal transduction2,3 and electrical control of material and interfacial properties,4,5 as well as easy solution processing, on the other hand, using solution deposition and patterning techniques of “plastic” electronics,6,7 compatible with large area surfaces and flexible substrates. Poly(3-alkylthiophenes) (P3ATs) are a promising conducting polymer family with high charge mobility, 8 good solubility,9,10 and commercial availability.8 P3AT macromolecules are made of conjugated backbone of thiophene rings and solubility-providing alkyl side chains that are attached to the backbone in a pattern specified by head-to-tail (HT) couplings. HT regioregularity induces crystalline order11 that modifies electronic structure11 and dramatically improves conductivity. 11,12 Regioregular poly(3-hexylthiophene) © 2014 American Chemical Society

Received: July 4, 2014 Revised: September 19, 2014 Published: October 27, 2014 13925

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RP3HT (Figure S1 and Table S1 in Supporting Information). Additionally tested regiorandom P3HT (cast as RP3HT) showed substantial morphological changes upon buffer exposure and for this reason was discarded from these studies. Bovine serum albumin (BSA; Cohn Fraction V, purchased form ACROS Organics) was adsorbed to the surface of thin P3AT films through incubation for 1 h at RT from solutions (with concentration CBSA in the range from 16 μg/mL to 10 mg/mL) in 50 mM phosphate buffer, pH 7.4. After incubation the samples were gently washed with the pure buffer and distilled water and then dried under N2 flow. 2.2. AFM Surface Examination. An Agilent 5500 microscope working in noncontact mode was used to examine the poly(3alkylthiophenes) surfaces in air prior to and after incubation in phosphate buffer, either pure buffer or containing different BSA concentration (followed by washing and drying). AFM cantilevers (probe type PPP-FMR, Nanosensors) with force constant of ∼2 N/m, resonant frequency of ∼75 kHz, and AFM tips with standard beam shape and small radius ( 6600 [at C4H5+ (m/z = 53) and C5− (m/z = 60) peak for positive and negative spectra, respectively] was maintained. 2.5. Multivariate ToF-SIMS Analysis with PCA. Three various types of multivariate surface examination were performed with ToFSIMS signals analyzed with principal component analysis (PCA): Surface exposure of alkyl side chains and polythiophene backbones was examined based on the signals of negative ion fragments of P3AT polymers. Outermost surface-sensitive composition of BSA overlayers on P3AT surfaces was evaluated for the peaks corresponding to negative ion fragments of both P3AT substrate and BSA protein. Surface-sensitive amino acid assays of BSA protein adsorbed to P3AT surfaces were performed based on the signals of positive ion fragments of amino acids present in BSA. The different sets of ToF-SIMS peaks selected for PCA analysis in all three cases are specified below in the corresponding figures. Prior to PCA, the intensities of the selected peaks from each spectrum were normalized to the sum of their intensities and then mean-centered. PCA was performed using PLS Toolbox (Eigenvector Research, Manson, WA) for MAT-LAB (MathWorks, Inc., Natick, MA). 2.6. Water Contact Angle Measurements. Static contact angle experiments were performed by the sessile drop technique using a Kruss EasyDrop (DSA15) instrument. Water drops were positioned on pure polymer films in six nonoverlapping spots.

Direct adsorption of proteins to RP3HT was studied by Albers et al., since it is an optimal functionalization method for biosensors, as covalent binding can disturb surface conformation of thiophene rings.17 Higher protein immobilization capacity but nonincreased specific binding ability was observed when RP3HT was exchanged for its more hydrophilic derivative.17 Here, we examine how the surface structure of P3AT films, different for highly regular RP3HT and regiorandom P3BT, modifies adsorption and conformation of bovine serum albumin (BSA)a model protein used often to block biosensor surfaces.18−20 This work extends to conducting polymers recent studies on BSA behavior regulated by subtle surface modifications. Cho et al. showed that molecular disorder of hydrocarbon chain monolayer induces stronger BSA adhesion.21 Michel et al. reported that decreased surface concentration of polar poly(ethylene glycol) involves conformational changes of adsorbed BSA with surface-exposed hydrophobic amino acids pointing to higher degree of protein denaturation.22 To study BSA adsorption and conformation, we apply X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry with information depth comparable with the dimensions of whole protein23 and its outermost parts exposed away from the surface,24 respectively. The amount of adsorbed protein is determined from XPS, based on the ratio of signals unique for protein and substrate.18,23,25−27 In turn, subtle differences in surface chemistry are resolved28 when the multivariate method of principal component analysis is applied29−34 to large ToF-SIMS data sets representing the multitude of ion fragments from protein amino acids and substrate.29,30,35 Spectroscopic characterization is complemented with atomic force microscopy, providing insight into lateral (micrographs) and vertical structure (height histogram measures, e.g., surface roughness) of adsorbed proteins.25,36 The main section 3 of this paper is organized as follows. We start with multivariate ToF-SIMS and AFM analysis of dissimilar surface structure in the films of RP3HT and P3BT. Then, differentiated BSA adsorption to various P3ATs is examined with AFM and XPS. Finally, double multivariate ToF-SIMS analysis is presented. The first one, confronted with XPS results, confirms the method sensitivity to outermost parts of adsorbed BSA. The second one shows and explains different amino acid assays of BSA adsorbed onto RP3HT and P3BT.

2. EXPERIMENTAL SECTION 2.1. Sample Preparation. Polymers used in this work were poly(3-alkylthiophene)s (P3ATs) purchased from Aldrich Chemical Co. Poly(3-butylthiophene) (P3BT) was reported by the supplier to have the 1:1 ratio of head-to-head:head-to-tail (HT) linkages. In turn, regioregular poly(3-hexylthiophene), RP3HT (molecular weight Mw ∼ 47 600, polydispersity Mw/Mn ∼ 2.1), was specified by HT regioregularity >90.0%. Silicon wafers (Simat, Germany) with native oxidized silicon layers (SiOx) were cleaned by sonication in toluene and dried with a nitrogen stream. Polymer films were spin-cast (coating speed ω = 2.2 krpm) from analytical grade toluene (P3BT, solution concentration CP = 15 mg/mL) or thiophene (RP3HT, CP = 12 mg/mL). Before subsequent protein adsorption, the films were postapply baked for 1 h at 60 °C. To test the impact of buffer, used in protein adsorption, on P3AT thin films, the polymer substrates were immersed in 50 mM phosphate buffer, pH 7.4, for 1 h at RT, followed by washing with distilled water, and dried under N2 flow. AFM examination of the P3AT films prior to and after incubation in pure buffer, quantified with height distribution measures, revealed not altered surface topography for both P3BT and 13926

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Figure 1. (a) PC1 scores plot from PCA of the negative ion ToF-SIMS spectra from RP3HT (open circles) and P3BT films (solid squares) spin-cast from tiophene and toluene, respectively, and postbacked for 1 h at 60 °C. (b) PC1 loadings plot corresponding to the scores plot shown in (a). The PC1 is loaded in positive direction by ion fragments C2nH− (n = 1−3) of alkyl side chains (and S− from thiophene rings) pointing to (c) surface exposure of alkyl chains. In turn, the PC1 is loaded in negative direction by secondary ions C2nHS− (n = 0−5) that are (d) fragments of polythiophene backbones (with alkyl side chains) that are easily accessed by ToF-SIMS (shaded sampling region).

Figure 2. Topographic (a, c) and phase (b, d) AFM images of RP3HT (a, b) and P3BT (c, d) films showing semicrystalline microstructure (fibrils in (b)) and amorphous morphology, respectively. RMS roughness error limit is standard error of the mean (expressed in nm).

3. RESULTS AND DISCUSSION 3.1. Surface Structure of P3AT Films. The films of P3AT polymers, P3BT and RP3HT, were spin-cast at room temperature from good solvents with a low vapor pressure, toluene (Pvp = 2.9 kPa) and tiophene (Pvp ∼ 8.3 kPa), respectively. In addition, to minimize any solvent effects on protein adsorption,38 the cast polymer substrates were post apply baked for 1 h at 60 °C. Such P3AT films were examined with surface-sensitive techniques, ToF-SIMS and AFM. Surface Exposure of Alkyl Side Chains and Polythiophene Backbones from Multivariate ToF-SIMS Analysis. To enhance sensitivity in detecting subtle variations in surface chemistry,

different ToF-SIMS signals are inspected simultaneously within principal component analysis. The data set used for multivariate PCA analysis is formed by the intensities of 22 peaks (specified in Figure 1b) corresponding to negative ion fragments of P3AT polymers (with m/z up to 152.98) and acquired from multiple ToF-SIMS spectra of P3BT and RP3HT films (Figure S2 in Supporting Information). The main direction of uncorrelated major variations, the so-called first principal component (PC1), captures 95.77% of the total variance in the data set. The PC1 scores plot clearly separates P3BT films, having negative PC1 values, from their RP3HT counterparts exhibiting positive PC1 scores (Figure 1a). The negative scores are related with 13927

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Figure 3. Representative topographic AFM images of RP3HT (a−c) and P3BT (d−f) after incubation with (a, d) 16 μg/mL, (b, e) 400 μg/mL, and (c, f) 2 mg/mL BSA solution for 1 h, washing and drying. RMS roughness error limit is standard error of the mean (expressed in nm).

abundant secondary ions C2nHS− (n = 0−5) which are the fragments of polythiophene backbones (with alkyl side chains) and load PC1 negatively (Figure 1b). In turn, the positive PC1 scores are due to the positive PC1 loadings of the secondary ion fragments C2nH− (n = 1−3) of alkyl side chains (and S− from thiophene rings). The results of Figures 1a and 1b can be explained having in mind that the sampling depth of ToF-SIMS (shaded areas in Figures 1c and 1d) is limited to outermost regions of substrate (reported as 1−1.5 nm for amorphous polymers24). The C2nHS− ions show that polythiophene backbones (with attached alkyl side chains) are easily accessed by ToF-SIMS for P3BT. This is not true for RP3HT, where the C2nH− signals point to alkyl groups that must be exposed, protecting deeper located conjugated backbones. Microstructure of P3AT Films from AFM. Recorded AFM data (Figure 2), especially phase images, indicate clearly semicrystalline and amorphous morphology for RP3HT and P3BT, respectively. Phase contrast originates from different mechanical properties of crystalline and amorphous regions with higher signal corresponding to stiffer crystalline domains.39 Fibrillar morphology similar to that of Figure 2b has been reported for RP3HT previously.8,40 In addition, RP3HT nanofibrils have been suggested to be related with edge-on oriented P3AT chains.44 It has been shown in the literature that crystalline order and mesoscale microstructure observed for regioregular P3AT films depend on detailed conditions of solution deposition,8 such as P3AT molecular weight,8,40,41 deposition rate specified by the solvent used,42 casting method,8,41 and coating speed43 as well as additional thermal treatment.40,44 While the high molecular weight of RP3HT polymer can ruin the semicrystalline morphology (as observed for the films cast from chloroform with Pvp ∼ 23.5 kPa),40,41 this effect is most probably counteracted here by the solvent

with a low vapor pressure Pvp. Additionally, relatively smooth spin-cast polymer surfaces (with low roughness RMS values; see Figure 2 and Table S1 in Supporting Information) are secured by Pvp values of the solvent used, lower than the onset value (10 kPa) for film roughness induced by rapidly evaporating solvent (Marangoni instabilities).45 3.2. BSA Adsorption to P3AT Films. Morphology of BSA Coverage Provided by AFM. Representative AFM images of P3AT surfaces with proteins adsorbed from solution with different BSA concentration CBSA are shown in Figure 3. The structures formed as a result of surface coverage with protein molecules are different from the topographies of P3AT substrates, as indicated by the changed surface roughness RMS values (cf. Figure 2). For RP3HT, adsorbed proteins form distinct cellular-like patterns, with interconnected dendrites of patches fusing with increasing CBSA into more uniform coverage with separate uncovered areas of molecular size. The latter morphology resembles that recorded on P3BT for all BSA concentrations. However, the nonpositive surface skewness Sk determined for proteins on RP3HT indicates that holes are dominant structures in surface topography. In contrast, the positive Sk value reflects for P3BT and all the CBSA values dominance of surface asperities in noncontinuous protein coverage. Protein patterns similar to that of Figure 3 have been reported for BSA adsorbed to polystyrene46 and surfaces covered with methyl-terminated monolayers of silanes21 and thiols.47 Figure 3 indicates that the morphology of protein coverage is different for RP3HT and P3BT, pointing to a different impact of BSA aggregation into patches during protein adsorption to both polymer substrates. We will come back to this issue when discussing amount of adsorbed protein determined with XPS as a function of BSA concentration. 13928

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terminated thiol monolayers (0.97 mg/m2)47 and polystyrene (3.15 mg/m2).46 Reference surface density of an ideal, closely packed BSA monolayer (Mw of 66 500) is close to ∼2.0 or ∼2.2 mg/m2 for BSA globular molecules with side-on orientation, described as ellipsoid-like (4 × 4 × 14 nm3)47 or heart-shaped (9.5 × 5.5 × 5.5 nm3),48 respectively. The determined maximal surface density of BSA on P3BT surface is close to the value expected for an inefficiently packed protein monolayer, obtained from random sequential adsorption with saturation coverage of 55%.49 The amount of BSA adsorbed on RP3HT is lower than P3BT (Figure 4). This reduced surface density of BSA could reflect protein deformation. BSA is known to demonstrate altered conformations on hydrophobic surfaces.50,51 Closer inspection of the data in Figure 4 shows, more clearly for P3BT, that an increase at high solution concentration CBSA in relative adsorbed BSA amount does not stop completely (forming a plateau) but exhibits a slow rise. Such adsorption behavior, described as a Freundlich isotherm, reflects not only surface interactions with adsorbates but also interactions between adsorbed molecules.52,53 This is consistent with the observed patch-like morphology of protein coverage on RP3HT at lower solution concentrations CBSA (Figure 3), pointing to aggregation due to BSA−BSA interactions. Similar relation between Freundlich-type isotherm and morphology of adsorbed protein patterns was reported previously.46 Interactions between adsorbed proteins have been recently related with their conformational changes, based on results of BSA adsorption to self-assembled monolayers with different terminal groups (and various water contact angle 22° ≤ Θ ≤ 110°).47 It has been argued47 that protein unfolding or denaturation, expected upon adsorption to hydrophobic surfaces,54 can expose internal domains of BSA molecules providing sites for BSA−BSA interactions, resulting in protein clusters formed by diffusion of adsorbed proteins.55 A similar mechanism can be involved here. Distinct patches of protein coverage on RP3HT suggests conformational changes much stronger than for BSA on P3BT. Substrate-induced modifications in BSA conformation cannot be expected based merely on slightly different wettability of RP3HT and P3BT, with water contact angle 97.4(5)° and 92.7(1.5)°, respectively. They must be evidenced, as presented in the final section 3.3 of this work. 3.3. Amino Acid Assays of Outermost Regions of Adsorbed BSA. ToF-SIMS Sensitivity to External Part of Adsorbed Proteins. Before amino acid composition of outermost regions of adsorbed BSA proteins would be analyzed, we examine first the vertical extent of these outermost regions, accessible with ToF-SIMS, relative to vertical extent of the whole BSA proteins, sampled with XPS. For this purpose the relative amount of BSA adsorbed on P3AT substrates, yielded as the ratio (XN/ZN)/(XS/ZS) from XPS, is confronted with protein surface composition provided by the scores of the first principal component PC1 (Figure 5b). Corresponding PCA analysis examines the ToF-SIMS intensities of 18 negative ion fragments (specified in Figure 5a) of BSA protein and P3AT polymers recorded for BSA adsorbed to both P3BT and RP3HT. PC1 (capturing 93.77% of the variance) is loaded negatively by P3AT ion fragments (mainly S−, C2nHS−, and C2nH−) and positively by secondary ions corresponding to protein (mainly fragments of amide bonds). Such a loading plot (Figure 5a) makes the PC1 a suitable variable to distinguish the scores corresponding to the P3AT surfaces with different outermost composition of BSA coverage.

Amount of Adsorbed Protein Evaluated from XPS. XPS was used to evaluate the variation in the elemental composition of surface prior to and after protein adsorption (see Figure S3 in Supporting Information). Two XPS signals, characteristic for protein (N 1s) and P3AT substrate (S 2p), depend on surface concentration of adsorbed BSA. Adsorbed BSA molecules increase the XPS signal from protein but reduce that from P3AT substrate. Therefore, the ratio of XPS intensities (XN/ ZN)/(XS/ZS) evaluates the relative amount of adsorbed protein. The ratio of XPS intensities XN/XS was measured at takeoff angle Θ = 45o and recalculated with sensitivity factors Si (contained in MultiPak’s ESCA database, ULVAC-PHI) to yield N and S atomic concentrations (see Table S2). They are normalized with respect to molar fractions Zi of the atoms emitting N 1s and S 2p photoelectrons. As values for molar fractions Zi we take the atomic concentrations determined from independent XPS analysis of bulk protein material (ZN = 15%27) and bare P3AT films (ZS = 8.0% and 8.2% for RP3HT and P3BT, respectively). The ratio (XN/ZN)/(XS/ZS) characterizes both homogeneous and nonuniform protein films. For uniform ovelayers (thinner than triple attenuation lengths 3λi of N 1s and S 2p photoelectrons, λN = 2.7 nm and λS = 3.1 nm37), it could be used to determine protein surface density Γ.18,23 The latter is related (using protein specific volume, 0.73 cm3/g, as a scaling factor) with the XPS thickness D, that describes effective attenuation of photoelectrons: XN /Z N 1 − exp( −D/λN cos Θ) = XS / ZS exp( −D/λS cos Θ)

(1)

The ratio of normalized XPS signals (XN/ZN)/(XS/ZS) that evaluates, for both P3BT (solid squares) and RP3HT (open circles) substrates, the relative amount of adsorbed BSA is plotted in Figure 4 versus BSA solution concentration, CBSA. In addition, protein surface density Γ is determined from eq 1 for maximal CBSA values (uniform BSA overlayers). The highest amount of absorbed BSA for RP3HT is approximately 21% lower than that for P3BT (Γ = 1.1 mg/m2). This value can be compared with amount of adsorbed BSA reported for methyl-

Figure 4. Ratio of normalized XPS signals (XN/ZN)/(XS/ZS), unique for protein and P3AT substrate. This ratio evaluates for P3BT (solid squares) and RP3HT (open circles) the relative amount of adsorbed protein, plotted as a function of BSA concentration. 13929

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Figure 5. (a) PC1 loading plot from PCA of the negative ion ToF-SIMS spectra from poly(3-alkylthiophenes) (P3ATs) films with adsorbed BSA protein. Protein and P3AT fragments load the PC1 in the positive and negative direction, respectively. (b) Scores of PC1, defined in (a), plotted for BSA adsorbed to P3BT (solid squares) and RP3HT (open circles) as a function of the normalized XPS signals (XN/ZN)/(XS/ZS). Plots in (b) reflect ToF-SIMS sensitivity to the outermost regions of adsorbed BSA proteins.

Figure 6. (a) PC2 scores plot from PCA of the positive ion ToF-SIMS spectra of BSA protein adsorbed to P3BT (solid squares) and RP3HT (open circles) films. (b) PC2 loadings plot corresponding to the scores plot shown in (a). PCA distinguishes between amino acids abundant in the outer regions of BSA adsorbed to P3BT and RP3HT that load the PC2 in the positive and negative direction, respectively.

presented in Figure 6a, reveal that the second principal component PC2 (that captures 7.65% of total variance) shows interesting direction of the uncorrelated variation in the data set. It turns out that the PC2 scores plot (Figure 6a) clearly separates amino acids of outermost regions of BSA adsorbed on P3BT, having positive PC2 values, from those immobilized on RP3HT, exhibiting negative PC2 scores. These positive and negative PC2 scores (Figure 6a) are induced by positive and negative, respectively, loadings on PC2 presented in Figure 6b and originating in different magnitude of various amino acids (see Table S3). The PC2 loadings plot and its interpretation is a subject of our next analysis (see the next section). In contrast, PC1 is not informative. Interpretation of Multivariate ToF-SIMS Assays. Subtle differences in the outermost surface chemistry of adsorbed proteins, revealed by PCA analysis of ToF-SIMS data, are caused mainly by the substrate-induced changes in orientation or conformation of immobilized proteins.56 Therefore, the PC2 loadings plot (Figure 6b) is examined with two models taking

The scores of PC1 (defined in Figure 5a) are plotted in Figure 5b for BSA adsorbed to P3BT (solid squares) and RP3HT (open circles) as a function of the ratio (XN/ZN)/(XS/ ZS) from XPS. Relative ToF-SIMS surface composition evaluated by PC1 saturates rather than increases linearly with the ratio of normalized XPS signals. This shows that surface protein composition determined with ToF-SIMS, sampling only the outermost top regions of adsorbed proteins, increases slower than analogous parameter evaluated with XPS, sampling the whole proteins. This reflects ToF-SIMS sensitivity to the outmost regions of adsorbed BSA proteins. Amino Acid Assays of Adsorbed BSA with Multivariate ToF-SIMS. To detect differences in amino acid composition of outermost regions of BSA proteins adsorbed to P3BT and RP3HT, PCA analysis is applied on a set of ToF-SIMS signals originating only from amino acids. A list of 28 peaks corresponding to positive ion fragments of 17 amino acids is specified in Figure 6b (and in Table S3, Supporting Information). The results of performed multivariate analysis, 13930

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Figure 7. (a) Loadings on PC2 (shown in Figure 6b) from amino acid ion fragments of outer regions of BSA molecules adsorbed to P3BT (positive PC2 values) and RP3HT (negative PC2 values) plotted for each amino acid as a function of its side-chain hydrophobicity, defined as the difference in RPC retention time ΔtR, relative to Gly peptide, of a peptide analogue differing only by one amino acid residue.58 Amino acids identified for P3BT and RP3HT as more hydrophilic and more hydrophobic than Gly, respectively, are marked schematically by ellipses. (b) Analogous loadings on PC2 from amino acid ion fragments of outer BSA regions plotted for each amino acid as a function of its relative amount in the BSA domain: I (squares), II (circles), and III (triangles).59

against the loadings on PC2 corresponding to ion fragments of this amino acid. No correlation might be indicated, based on Figure 7b, relating the PC2 loadings with substrate-induced orientation changes of adsorbed BSA protein.

into account BSA conformational modifications and, independently, orientation changes. Conformational changes of proteins on modified substrates have been reported previously to be revealed by PCA and ToFSIMS data separating amino acids in two groups, identified simply as hydrophobic and hydrophilic.22,24,28,34,57 Here we propose a more rigid approach, introduced by Monera et al.,58 that uses a relative measure of hydrophobicity of amino acid side chains. It is provided by the results of reversed phase chromatography (RPC) and defined as the difference in retention time, ΔtR, relative to glycine peptide, of a peptide analogue differing only in one amino acid residue (substituted on the hydrophobic face of amphiphatic α-helix).58 For each of 17 amino acids resolved by ToF-SIMS its side chain hydrophobicity is plotted in Figure 7a against the examined loadings on PC2 corresponding to ion fragments of this amino acid. It is evident from this plot that amino acids, identified as abundant in outer regions of BSA molecules on P3BT (positive PC2 loadings) and RP3HT surfaces (negative PC2 values), are more hydrophilic (ΔtR < 0) or more hydrophobic than glycine (ΔtR > 0), respectively, as marked schematically by ellipses. This indicates substrate-induced modifications in BSA conformation. As hydrophobic amino acid residues are commonly located inside the protein in a “native” state, their predominant presence on molecules adsorbed on RP3HT substrates, detected by ToF-SIMS and PCA (Figure 7a), points to a higher degree of protein deformation and denaturation. BSA molecule is a single polypeptide chain with three distinct domains.59 It has been suggested48 that BSA adsorption with preferred side-on orientation, as concluded here (section 3.2), is related with favored molecular orientation of the domains I and II toward substrate and the domain III away from surface. To examine whether the observed substrateinduced changes in adsorbed BSA molecules are related with modified exposure of BSA domains, we perform an analysis similar to that discussed above. For each amino acid resolved by ToF-SIMS its relative content in the domain I, II, and III (marked with different symbols)59 is plotted in Figure 7b

4. CONCLUSIONS Regioregular polythiophene RP3HT films with crystalline order, known to provide strongly enhanced charge mobility, affect both conformation and adsorption of BSA protein relative to amorphous surfaces of regiorandom P3BT. Substrate-induced modifications in BSA conformation are not expected based merely on slightly different wettability of RP3HT and P3BT. Higher degree of protein deformation and denaturation on RP3HT films is evidenced by hydrophobic residues, resolved by PCA and not expected for a “native” protein. Further studies, relevant for bioapplications, focused into P3AT polymer-induced changes in biological activity of adsorbed proteins are necessary. Such experiments should also clarify whether surface behavior of protein could be modified by the variation in alkyl chain length not accompanied by the modification of crystalline order. Multiple surface sensitive techniques (AFM, XPS, ToFSIMS) enhanced by multivariate PCA examination of ToFSIMS data reveal a complementary picture of substratedependent protein conformation and adsorption. For BSA proteins on RP3HT films, the PCA analysis (Figure 7a) reveals more exposed hydrophobic amino acids and advocates more deformed proteins. Enhanced exposure of the hydrophobic residues enabling BSA−BSA interactions,47 consistent with Freundlich type of adsorption isotherms (mimicked by Figure 4, from XPS), leads to protein clusters observed as a pronounced patch-like morphology of protein coverage (Figure 3, from AFM). In turn, stronger protein deformation accords with lower amount of adsorbed BSA (Figure 4). Several novel extensions of surface sensitive analysis methods are presented in this study. First, surface exposure of alkyl side chains and polythiophene backbones is resolved by PCA analysis of ToF-SIMS data. Second, combined analysis of ToF13931

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SIMS and XPS data provides insight into the vertical extent of outermost protein regions with respect to whole adsorbed proteins. Third, a more rigorous approach to examine protein conformational changes from ToF-SIMS data is proposed, relating PCA loadings for different amino acids with their sidechain hydrophobicity instead of the commonly used simplified classification as merely hydrophobic or hydrophilic residues.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on AFM examination of P3AT surfaces prior to and after incubation in pure phosphate buffer as well on XPS (C 1s and N 1s core level spectra) and ToF-SIMS analysis (signals used for PCA analysis) of BSA adsorption onto P3AT surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel +48 12 664 4734; Fax +48 12 664 4905; e-mail kamil. [email protected] (K.A.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Polish National Science Center (NCN) under Grant UMO-2011/03/N/ST5/04764, equipment purchased thanks to the financial support of the European Regional Development Fund (POIG.02.01.00-12023/08).

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REFERENCES

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