PAA Brushes with Switchable Properties Toward

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Design of Mixed PEO/PAA Brushes with Switchable Properties Toward Protein Adsorption M. F. Delcroix,† G. L. Huet,† T. Conard,‡ S. Demoustier-Champagne,† F. E. Du Prez,§ J. Landoulsi,∥ and C. C. Dupont-Gillain*,† †

Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Croix du Sud 1 (L7.04.01), 1348 Louvain-la-Neuve, Belgium ‡ IMEC, Kapeldreef 75, 3001 Leuven, Belgium § Polymer Research Group, Ghent University, Krijgslaan 281 S4-bis, 9000 Ghent, Belgium ∥ Laboratoire de Réactivité de Surface, UMR 7197 CNRS, Université Pierre and Marie Curie - Paris VI, 4 Place Jussieu, Case 178, 75252 Paris, France S Supporting Information *

ABSTRACT: Adsorption of proteins at interfaces is an ubiquitous phenomenon of prime importance. Layers of poly(ethylene oxide) (PEO) are widely used to repel proteins. Conversely, proteins were shown to adsorb deeply into brushes of poly(acrylic acid) (PAA), and their subsequent partial release could be triggered by a change of pH and/or ionic strength (I). Mixed brushes of these polymers are thus promising candidates to tune protein adsorption onto new smart surfaces. In this work, the synthesis of such mixed brushes was performed based on a “grafting to” approach, the two polymers being either grafted sequentially or simultaneously. Detailed characterization of the obtained brushes using static water contact angle measurements, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and polarization−modulation reflection−absorption infrared spectroscopy is presented. While sequential grafting of the two polymers for different reactions times did not give rise to a broad range of composition of mixed brushes, simultaneous grafting of the polymers from solutions with different compositions allows for the synthesis of a range of mixed brushes (mass fraction of PEO in the mixed brushes from 0.35 to 0.65). A key example is then chosen to illustrate the switchable behavior of a selected mixed PEO/PAA brush toward albumin adsorption. The adsorption behavior was monitored with a quartz crystal microbalance. The mixed brush could adsorb high amounts of albumin, but 86% of the adsorbed protein could then be desorbed upon pH and I change. The obtained properties are thus a combination of the ones of PEO and PAA, and a highly switchable behavior is observed toward protein adsorption.

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INTRODUCTION Protein adsorption is a spontaneous and nearly irreversible phenomenon of major importance in biomaterials and biomedical science.1 Biofouling is often uncontrolled and nondesirable. Conversely, the adsorption of cell-adhesive proteins is crucial for cell and tissue colonization of materials surfaces.2,3 Protein adsorption may be controlled using interfaces modified with polymer brushes. Polymer brushes refer to an assembly of polymer chains that are tethered by one end to a surface in such a way that the grafting density is high enough for the attached chains to stretch away from the surface.4 They can be stimuli-responsive, which means that they can answer to an external stimulus (temperature, pH, light, solvent, ionic strength, electric field, etc.) in a desired way (change in color, wettability, protein adsorption, etc.).5−8 A binary mixed polymer brush is a surface layer that consists of two different types of polymer chains that are end-grafted to a substrate.9 Such binary brushes can be switched between © XXXX American Chemical Society

different surface energetic states upon exposure to different surrounding media.10−14 They could therefore expose alternately one of the two components and thus constitute interesting platforms to build stimuli-responsive (or smart) surfaces. Polyelectrolyte brushes have been well investigated for their swelling−deswelling behavior. Among them, weak polyelectrolyte brushes are of particular interest. Indeed, the fraction of charged repeating units depends on local conditions, such as pH, salt concentration, valence of counterions, and grafting density.15,16 Poly(acrylic acid) (PAA) is a weak polyacid that is widely studied for its pH-dependent deprotonation of COOH groups along the chains, as well as for the nonmonotonic dependence of its brush thickness as a function of ionic Received: October 23, 2012 Revised: December 7, 2012

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dx.doi.org/10.1021/bm301637h | Biomacromolecules XXXX, XXX, XXX−XXX

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strength (I).17−25 At very low I, at a pH where the charge density is high, electrostatic repulsion between charged repeating units leads to a certain level of swelling of the brush by water. A rise of I of the solution leads to an increase of counterion condensation inside the brush. Therefore, the polymer chains expand further due to the osmotic pressure of localized counterions (osmotic regime). With further I increase and subsequently decreased Debye screening length in solution, the brush enters the salted regime and collapses.24,26 Proteins are slightly repelled by the shrunk brush while they were shown to deeply enter inside the swollen brush, whatever the isoelectric point of the protein.27−35 One of the most studied protein-repellent polymers is poly(ethylene oxide) (PEO), also known as poly(ethylene glycol) (PEG). The resistance of PEO to protein adsorption is generally attributed to a steric repulsion effect, by which the polymer prevents the protein from reaching the substrate surface to adsorb.36−38 Indeed, in aqueous environments, PEO molecules are highly mobile and strongly hydrated, attaining extremely large exclusion volumes.39,40 The steric repulsion has an osmotic (due to the solvation of the PEO chains) and an elastic (due to the conformational entropy of the PEO chains) component. These repulsive components become effective when the protein reaches the interphase by diffusion and compresses the PEO layer.41 However, it is becoming increasingly clear that the protein-repellent properties of PEO-containing molecules should be ascribed to a combination of phenomena.42,43 Grafting density, length, and conformation of PEO chains have been identified as parameters of prime importance.41,44−47 Mixed brushes made, on the one hand, of a proteinadsorbing and, on the other hand, of a protein-repellent polymer may be favorable for the control of protein adsorption.48−50 Hoy et al. were able to vary the hydrophobic−hydrophilic interactions with a multicomponent mixed polymer brush made of a hydrophilic homopolymer, PEO, and an amphiphilic block copolymer, polystyrene−poly(acrylic acid) (PS-b-PAA). PEO and PS were alternately exposed at the interface through variation of the PAA swelling depending on specified conditions (pH, I, and the presence of Ca2+).50 Uhlmann et al. tuned the interfacial properties of a mixed brush made of two weak polyelectrolytes, namely poly(2-vinylpyridine) (P2VP), a polybase, and PAA, a polyacid. They could change the amount and the mechanism of αchymotrypsin and α-lactalbumin adsorption through the variation of pH and ionic strength of the surrounding solution.49 However, to the best of our knowledge, switchable protein adsorption from very low to high amounts has not yet been reported. Such systems could, for example, be useful for the design of reusable biosensors or for the capture of proteins in a defined time frame of a biotechnological or biomedical process. The surface of inorganic and organic substrates can be coated by polymers using a variety of techniques.51 Two main approaches are used to graft polymer brushes: the “grafting from”51−53 and the “grafting to” methods.41,42,45,46,54−57 The “grafting from” process includes the immobilization of one or two initiators (such as azo-, peroxide-, or photoinitiators) of the polymerization on a substrate surface. Monomers are then brought in solution, and the evolving brush grows away from the interface. With this technique, very dense brushes can be prepared, but the chain length distribution cannot always be accurately controlled. In the “grafting to” technique, end-

functionalized polymer molecules react with complementary functional groups located on the surface to form tethered chains. This technique allows precise selection of the polymer properties (i.e., chain length distribution) and the mixing of chains of different natures, but the availability of adequate endfunctionalized polymers may be an issue.4,6,43,54,58−60 The majority of authors use silane chemistry to graft brushes on substrates via the “grafting to” procedure.49,61−63 Here, the chemisorption of thiols on gold is preferred because of the poor stability of the silane layers in saline solutions.64−67 The objective of this work is the synthesis of a range of mixed brushes of PAA and PEO using the “grafting to” method, with a view to tune protein adsorption on the created interfaces. Thiol-terminated PEO and PAA bearing a disulfide bond in its center are used to synthesize the brushes on a gold substrate. The grafting kinetics of each polymer is first studied to identify useful conditions for the synthesis of mixed brushes. Two types of strategies are then adopted to create mixed brushes: either grafting one of the two polymers and then grafting the second one (sequential grafting, see Scheme 1a,b) Scheme 1. Illustration of the Methods Used to Graft PEO and PAA on Gold to Form Mixed Brushes: (a) Sequential Grafting Starting with PEO; (b) Sequential Grafting Starting with PAA; (c) Simultaneous Grafting

or mixing the two functionalized polymers in the grafting solution (simultaneous grafting, see Scheme 1c). The synthesized brushes are characterized by means of X-ray photoelectron spectroscopy (XPS), contact angle measurements, electrochemical impedance spectroscopy (EIS), quartz crystal microbalance with dissipation monitoring (QCM-D), and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Finally, the properties of such mixed brushes are illustrated through the study of the effect of pH and I on human serum albumin (HSA) adsorption.

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MATERIALS AND METHODS Synthesis of the Polymer Brushes. Thiol-terminated PEO methyl ether with Mn = 1100 g/mol (about 25 repeating units) and polydispersity index of 1.08 was purchased from B


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Figure 1. Chemical structure of PEO (a) and PAA (b).

with a monochromatized Al X-ray source (powered at 10 mA and 15 kV). The samples were fixed on a standard stainless steel multispecimen holder by using a piece of double-sided insulating tape. The pressure in the analysis chamber was about 10−6 Pa. The direction of photoelectrons collection was perpendicular to the sample surface. Analyses were performed in the hybrid lens mode with the slot aperture, and the resulting analyzed area was 700 × 300 μm2. The pass energy was set at 160 eV for the survey scan and 40 eV for narrow scans. In the latter conditions, the full width at half-maximum (fwhm) of the Ag 3d5/2 peak of a standard silver sample was about 0.9 eV. Charge stabilization was achieved by using the Kratos Axis device. The following sequence of spectra was recorded: survey spectrum, C 1s, O 1s, S 2p, Au 4f, and C 1s again to check for charge stability as a function of time and the absence of degradation of the sample during the analyses. The C-(C,H) component of the C 1s peak was fixed to 284.8 eV to set the binding energy scale. Molar fractions (%) were calculated using peak areas measured after linear background subtraction, and normalized on the basis of acquisition parameters, experimental sensitivity factors, and transmission factors provided by the manufacturer. Data treatment was performed with the CasaXPS program (Casa Software Ltd., UK). The procedure used for peak decomposition is explained in details in the Supporting Information section and allowed the relative fractions of repeating units of PEO and PAA in the obtained brush, respectively gPEO and gPAA, to be extracted. The synthesis of polymer brushes and subsequent adsorption of HSA (Sigma, Belgium, A3782) was also monitored in situ using QCM-D. Measurements were performed with a Q-Sense E4 System (Gothenborg, Sweden) at a controlled temperature of 20 ± 0.1 °C. The crystals used were thin AT-cut quartz coated with a 100 nm Au film purchased from Q-Sense. They were cleaned like the gold substrates prior to use (see above). Oscillations of the crystal at the resonant frequency (5 MHz) or at one of its overtones (15, 25, 35, 45, 55, 65 MHz) were obtained when applying AC voltage. The variations of the resonance frequency (Δf) and of dissipation (ΔD) were monitored upon bringing different solutions (solvents, polymers, HSA) in contact with the crystal. Solutions were flowed into the measurement cell using a peristaltic pump (Ismatec IPC-N4) at a flow rate of 50 μL/min except if otherwise stated. The baseline was established with ultrapure water for a minimum of 30 min. Then, the grafting solution containing the polymer was flowed for 30 min, and a rinsing step of 30 min with ultrapure water was performed. For the test of the stimuli-responsive behavior of the brushes, saline solution at pH 5 and I = 10−5 M was subsequently flowed for 30 min, then the protein solution (200 μg/mL, same pH and I) was flowed for 90 min at a flow rate of 25 μL/min. Rinsing was performed by three successive steps of 30 min: first, the same saline solution as previously (pH 5 and I = 10−5 M), then another saline solution (pH 9 and I = 10−1 M) to trigger the desorption, and finally ultrapure water. NaCl

Polymer Source Inc. (Dorval, Canada). PAA with a disulfide bond in its center was synthesized, using a homemade protocol based on atom transfer radical polymerization of ethoxyethyl acrylate from a disulfide-containing initiator.68,69 The Mw is 5800 g/mol (about 2 × 40 repeating units) and the polydispersity index is 1.30. Chemical structures of the two polymers are presented in Figure 1. Grafting solutions were prepared by dissolution of the functionalized polymers at a total concentration of 1 g/L in ultrapure water. Stock solutions of PAA and PEO at 1 g/L were directly used for the synthesis of homobrushes or for sequential grafting. For simultaneous grafting, solutions with PEO/PAA mass ratios of 99/1, 90/10, and 50/50 were prepared by mixing the same stock solutions with the given ratio in volume. Gold substrates were fabricated in clean room environment by evaporation (100 nm) on top of a silicon wafer, with the presence of a titanium interlayer. Cleaning of cut gold samples was performed just before the grafting process by immersion in a piranha solution [2:1 mixture of concentrated sulfuric acid (VWR BDH Prolabo, Leuven, Belgium) and 30% hydrogen peroxide solution (VWR BDH Prolabo, Leuven, Belgium)] for 2 min followed by 10 times rinsing with ultrapure water. (Caution: “piranha” solution reacts violently with many organic materials and should be used with extreme care). After that, they were flushed with ethanol and quickly dried under gaseous nitrogen flow, submitted to UV/ozone cleaning for 15 min, rinsed again with ethanol and quickly dried under gaseous nitrogen flow. Synthesis of the mixed brushes was performed with two different methods (see Scheme 1): (i) sequential grafting by immersion of cleaned gold in a solution of one of the two functionalized polymers, rinsing with ultrapure water and drying under gaseous nitrogen flow, followed by immersion of the same sample in a solution containing the other functionalized polymer, rinsing with ultrapure water, and drying under gaseous nitrogen flow; (ii) simultaneous grafting by immersion of cleaned gold in a solution containing the two functionalized polymers, rinsing with ultrapure water, and drying under gaseous nitrogen flow. The duration of immersion of the samples into the grafting solution will be specified for each experiment in the following section. All characterizations were performed on freshly prepared samples. Surface Characterization Methods. Static water contact angles (θw) were measured at ambient temperature using the sessile drop method and image analysis of the drop profile. The instrument, using a CCD camera and an image analysis processor, was purchased from Electronisch Ontwerpbureau De Boer (The Netherlands). The ultrapure water droplet volume was 0.3 μL, and the contact angle was measured 5 s after the drop deposition on the sample. For each sample, the reported value is the average of the results obtained on at least three droplets. The typical recorded error on the measurements is about 1°. XPS analyses were performed on a Kratos Axis Ultra spectrometer (Kratos Analytical, Manchester, UK) equipped C


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(Sigma-Aldrich), HCl (Merck), and NaOH (Sigma-Aldrich) were used to prepare saline solutions of adjusted pH and I. For a deposited layer whose properties are similar to those of quartz, a negative shift in frequency accounts for an increase in mass on top of the crystal, while the dissipation shift should be negligible. Δf and ΔD values were always measured between the baseline established before polymer grafting or albumin adsorption and the values recorded after rinsing in the same solution as the one used to establish this baseline. All observed ΔD/Δf ratios were lower than 0.4 × 10−6 Hz−1. In this case, the deposited layer is considered rigid. Therefore, the Sauerbrey equation, which relates the deposited mass Δm to the observed frequency shift, can be used to extract the mass per unit area of the layer.65 EIS experiments were carried out in a one compartment Teflon cell at room temperature with a Pt counter electrode (surface area of about 3.6 cm2) and a Ag/AgCl reference electrode, and were controlled using an EG&G Princeton Applied Research 273A potentiostat/galvanostat. The working electrode is the created substrate sealed with the Teflon cell (active area of about 0.2 cm2). All the solutions in contact with the system were degassed at least 10 min with gaseous nitrogen. Grafting of the polymers was performed in situ on a cleaned gold substrate and in contact with a saline solution (NaCl 10−3 M) of the polymers (1 g/L). Data were acquired in the frequency range 10−1 Hz to 105 Hz, and were analyzed by fitting with the most common equivalent circuit model, the simplified Randles cell, schematically represented in Figure 2. It

To estimate the quality of the grafted layer, the R2 parameter, which represents the resistance to charge transfer, will be normalized to the maximum value of the concerned experiment to obtain values between 0 and 1 and will be chosen as the significant parameter of EIS. This resistance indeed increases as the layer is formed, while R1 and CPE1 should remain quite constant. It was already shown that EIS is particularly well-suited to study the progressive evolution from a noncomplete to a complete coating.72,73 Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) spectra were recorded on a commercial NICOLET Nexus spectrometer. The external beam was focused on the sample with a mirror, at an optimal incident angle of 65°. A ZnSe grid polarizer and a ZnSe photoelastic modulator, modulating the incident beam between p- and spolarizations (HINDS Instruments, PEM 90, modulation frequency = 37 kHz), were placed prior to the sample. The light reflected on the sample was then focused onto a nitrogencooled MCT detector. The detector output was sent to a twochannel electronic device that generates the sum and difference interferograms, which were processed and underwent Fourier transformation to produce the PM-IRRAS signal, which is expressed as the differential reflectance spectrum of the adsorbed surface species (ΔR/R0) = (Rp − Rs)/(Rp + Rs), where Rp and Rs are the amplitude of the radiation collected parallel and perpendicular to the polarized radiation, respectively. All reported spectra, recorded at 8 cm −1 resolution, were obtained by coaddition of 128 scans. The use of a modulation of polarization enabled us to perform rapid analyses of the sample without purging the atmosphere or requiring a reference spectrum. Spectra were corrected against the Bessel function using a program integrated in OMNIC software.

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Figure 2. Equivalent circuit of the simplified Randles cell used to treat the data obtained by EIS.

RESULTS AND DISCUSSION This results section is divided into four parts: first, the grafting kinetics of each polymer is studied; second, the results related to the elaboration of mixed brushes by sequential grafting are developed; third, the results obtained using simultaneous grafting are presented; finally, the responsive behavior of the created brushes is tested in the presence of HSA in saline solutions of different pH and I. Grafting Kinetics of Each Polymer. Figure 3 illustrates the formation of polymer homobrushes with time (left: PEO, right: PAA). This Figure shows the R2 parameter (left axis, red

includes an ohmic resistance of the bulk electrolyte solution R1 and a double layer capacitor CPE1 in parallel with a charge transfer resistance R2. This latter actually depends on the thin film associated with the electrode surface.70 A constant phase element CPE1 is used instead of the double-layer capacitance reflecting the space-distributed capacitance of the thin film associated with the electrode surface.71 However, their physical meanings are very close to each other.71

Figure 3. Evolution of R2 (EIS, red line), characteristic C 1s component molar fraction related to the total carbon fraction (XPS, blue squares) and water contact angle (green triangles) as a function of the grafting time of (left) PEO and (right) PAA. Insets give a detailed view of R2 at short times. D


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QCM-D experiment with nonfunctionalized PEO and PAA of comparable molar mass was performed and showed that thiol or disulfide functions are mandatory to get a coating that is resistant to water rinsing (not shown here). Moreover, previous studies have shown that dialkyl disulfide and alkanethiol (for an alkyl chain of the same mass and stoichiometry) present indistinguishable physical and chemical properties on Au surfaces,75,76 while differences in the grafting kinetics behavior are observed. 77,78 However, Paik et al. observed that chemisorption of thiols and disulfides occurs with different electrochemical reactions: thiol molecules are grafted on Au through an anodic reaction, whereas dialkyl disulfides are grafted through a cathodic reaction. They also suggest that an appropriate control of the potential during the reaction gives rise to monolayers of higher integrity in a shorter time interval.67 Jung et al. compared film formation of alkylthiol, dialkyl disulfide, and alkyl sulfide of the same length and found that film formation is the fastest for the thiol.78 Biebuyck et al., however, showed that layers based on alkylthiols and dialkyls disulfides of the same length present indistinguishable rates of formation at short times. However, the rates of displacement of molecules from an already assembled monolayer by thiols were much faster than by disulfides.77 Besides, Vanderah et al. have shown that the organization of layers of PEO functionalized by thiol or disulfide is strongly different: the amount of grafted disulfide-modified PEO is lower, and the layer is less organized but more protein-repellent than the layer formed by thiolfunctionalized PEO.79 Finally, the mixing of disulfides and thiols (see following sections) could give rise to a very complex behavior,76 especially if they are different in mass and in chemical composition. Our results suggest that the composition of a PEO-modified gold surface is changing rapidly from a relatively pure PEO grafted layer to a more contaminated layer as a function of the grafting time. Usually, long grafting times between 12 h and 3 days are used to graft thiol-terminated PEO.79−82 Studies with short PEO grafting times are scarcely reported. Additionally, it was reported that a minimum grafting time of 16 h is required to fully cover the gold substrate with longer PEO chains (Mw = 5000) at 6 g/L in an ethanolic solution.45 Most of the time, the “grafting to” process of alkane thiols on gold is indeed performed from solutions in ethanol.41,45,56,68 By XPS analysis, we have compared the grafting of the polymers used in the present study from ethanol and ultrapure water solutions and noticed no significant difference (results not shown). Since the synthesized surfaces will be further used in physiological media and with biomolecules, we have therefore preferred to always use ultrapure water as solvent hereafter. Optimum grafting time is subject to change by modifying solvent, chain length, or depending on the presence/absence of an alkyl chain, which stabilizes the layer. Here, we show that short and disordered thiol-terminated PEO chains form the most complete layer at short grafting times. Nevertheless, our data evidence that a short time of grafting (i.e., from 15 min to 4 h) results in the most dense and pure PEO layer. This is supported by the study of Unsworth et al.83 Layers formed by PEO of Mw ranging from 600 to 2000 g·mol−1 with grafting times from 10 min to 4 h were studied. The grafting density was increasing with time, but the hydration of the layer and its protein-resistance showed an optimum close to 30 min of grafting.83 For the formation of PAA layers, Van Camp et al. recommended a grafting time of 3 days.68 Our results show

line), which represents the resistance to charge transfer evaluated by EIS, as well as water contact angle values (right axis, green triangles), which are a measure of the hydrophobicity of the surface and, finally, the fraction of the C 1s peak area attributed to the typical component of each polymer, as recorded by XPS (right axis, blue squares, C-O-C for PEO and COOH for PAA; complete XPS data for these samples are available in the Supporting Information, Tables S-1 and S-2 and Figures S-2 and S-3). For PEO (Figure 3, left), the water contact angle is close to 40° and does not vary much with the grafting time, with, however, a minimum value observed between 2 and 4 h of grafting. On the contrary, the R2 parameter shows a maximum for very short grafting times. Indeed, R2 increases slightly from the beginning of the grafting to 15 min, then decreases markedly up to 8 h and finally remains constant at longer times. The resistance to charge transfer is therefore the highest at very low grafting times (∼ 15 min), meaning that the substrate coverage by PEO grafting reaches its highest level in these conditions. Besides, the fraction of the C 1s peak recorded by XPS attributed to the CO-C component is quite high (∼75%) from the beginning of the grafting up to 4 h when a maximum is observed. It undergoes a 10% decrease after 8 h, but then remains constant. This decrease is the result of an increase of the other components of the C 1s peak, attributed to contamination (see Figure S-2). For PAA (Figure 3, right), the water contact angle is close to 45° and is quite constant with the grafting time. This is also the case for the COOH fraction of the C 1s peak, even if a slight drop is recorded from 8 to 24 h. The chemical composition of the outermost layer does not change substantially with the grafting time, but the quality of the layer is constantly improving, as evidenced by the continuous increase of R2 with the grafting time. QCM-D analyses (see Figures S-4 and S-5) show a rapid (∼1 min) frequency drop evolution after injection of PEO or PAA solution in contact with the gold-coated crystal. The frequency shift then stabilizes and does not change markedly upon rinsing with ultrapure water. The corresponding hydrated mass per unit area estimated after a 30 min of grafting followed by rinsing are 620 ± 67 ng/cm2 for PEO and 499 ± 49 ng/cm2 for PAA, where the confidence interval is given at the 95% level and accounts for 16 and 14 repetitions for PEO and PAA, respectively. Regarding the grafting reaction, decomposition of the S 2p peak (less than 3 atomic %) recorded by XPS gives evidence for bond formation between the sulfur atoms and the gold substrate, according to the assignments made by Castner74 (results not shown). Two doublets are well-defined and can be assigned to bound thiolate (S2p3/2 binding energy near 161.7 eV45,74,75) and unbound thiol/disulfide (S2p3/2 binding energy near 163.2 eV) in a roughly 3:2 ratio, except for PAA grafted for very short times (1 h) for which the ratio approaches 2:3. These unbound thiol molecules could be either lying on top of the layer or partially penetrating into the layer.74 Note that the signal from bound sulfur is more attenuated than the one from unbound sulfur coming from the upper lying chains, due to the layer thickness, and bound sulfur is thus underestimated. No oxidized sulfur species (S2p3/2 binding energy above 166 eV) are detected. S 2p peak decomposition of well-formed layers thus shows that thiolated PEO and PAA with disulfide bond are well grafted on gold, even if part of the molecules are also probably physisorbed or embedded in the layer. A control E


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polymers can lead to a wide range of compositions. For example, amine-terminated chains of polystyrene of different lengths and of PEO were grafted on silica and formed mixed brushes of variable composition as a function of the grafting time of each polymer.55 Moreover, other studies of sequential grafting usually showed a larger proportion of the polymer grafted first in the obtained layer.5,84,85 Therefore, in order to broaden the range of composition of the mixed brushes as well as to enhance the mass proportion of PEO in mixed brushes, PEO was grafted first for 1 or 24 h while PAA was grafted in a second step for 30 min, 2 h, 8 h, or 24 h. The wettability and composition of these mixed brushes was analyzed by means of water contact angle, XPS and PM-IRRAS. In Figure 4, typical PM-IRRAS spectra from 2000 to 1000 cm−1 for PAA (top), PEO (down) and a mixed brush (center)

that the obtained layer properties are indeed evolving with grafting time and that a stable PAA layer is formed after long grafting times (i.e., here, up to 24 h). Additionally, XPS measurements of PAA samples prepared with longer grafting times (up to 3 days, see Figure S-3) show no remarkable difference compared to a 24h-grafted sample. In summary, in the case of homobrushes, the optimal grafting time is short for PEO (from 15 min to 4 h), while it is longer for PAA (24 h). Sequential Grafting of the Polymers. The time optima for the grafting of the two polymers are highly different; these times are not easily compatible, which is a priori not favorable for the preparation of mixed brushes in a single adsorption step. Therefore, in order to create mixed brushes, a sequential grafting procedure was first considered (see Scheme 1a,b). In an exploratory phase, a wide range of grafting times was selected: on 3 samples, PAA was grafted first for 1, 8, or 72 h and PEO was then grafted for 24 h; on three other samples, PEO was grafted first for 1, 8, or 72 h and PAA was then grafted for 24 h. The relative fraction of PEO repeating units in the mixed brushes, gPEO, extracted from XPS analyses, is presented for all these mixed brushes in Table 1. Note that since the gold substrate was still largely detected (not shown), it is highly probable that the whole content of the organic layer was probed. Table 1. Relative Fraction of PEO Repeating Units in the Mixed Brushes Created by Sequential Grafting 1st polymer grafted

2nd polymer grafted

gPEO

PAA

PEO

0.55 0.49 0.42 0.45 0.39 0.44

PEO

1h 8h 72 h 1h 8h 72 h

PAA

24 24 24 24 24 24

h h h h h h

Figure 4. Typical PM-IRRAS spectra from 2000 to 1000 cm−1 of PAA (top), mixed brush (center) and PEO (down). Bands attributed to CO stretching (at about 1734 cm−1) and of C−O−C asymmetric stretching (at about 1120 cm−1) are indicated with dashed lines.

are shown as an example. The two pure polymers show typical bands due to the carbonyl function of PAA close to 1725 cm−1 (CO stretching86,87) and due to the ether function of PEO, leading to a broad band close to 1100 cm−1 (C−O−C asymmetric stretching vibration87). Accordingly, these two bands can be used as markers of each polymer to investigate the evolution of the polymer layer in a semiquantitative way. The mixed brush sample shows the typical band of each polymer, again confirming that PAA and PEO are both present in the mixed brushes. The water contact angles of all samples are very close to each other (near 46°). This water contact angle value is closer to the one of PAA homobrushes compared to the one of PEO homobrushes (see Figure 3), indicating a higher proportion of PAA at the outermost surface of the mixed brushes. XPS and PM-IRRAS measurements are reported in Figure 5 through gPEO for XPS (Figure 5a) and through the ratio AνC−O−C/ AνCO of the area of the bands attributed to PEO (AνC−O− C) and to PAA (AνCO) for PM-IRRAS (Figure 5b). From XPS analysis, it can be deduced that PEO and PAA are both present in the mixed brushes that are created by sequential grafting. Indeed, the fraction of PEO repeating units in the mixed brushes is always situated near 0.5, pointing to the fact that PAA and PEO repeating units are present in similar quantities in the mixed brushes. As previously discussed, these fractions reflect a lower mass proportion of PEO in the mixed brush (near 37%) but a higher grafting density of PEO chains

The results show that in all cases, both polymers are well present in the created brushes, with a fraction of PEO repeating units in the range of 40 to 55%. All values for gPEO are thus situated in a quite narrow range. Thus, despite the range of grafting times chosen and the change in the order of grafting of the polymers, we were not able to create sharply different mixed brushes. The results also reveal that PEO and PAA repeating units are present in similar quantities. In term of mass and volume, PEO is therefore always less present in the mixed brushes compared to PAA, whatever the order of grafting and the grafting time, the repeating unit of PEO being lighter than the one of PAA. However, taking into account the molar mass of the polymers, the PEO/PAA molar ratio is ∼2.7 (or ∼1.4 if only the half of the molar mass of PAA is considered, since the disulfide bond is in the center of the polymer chain). Therefore, the grafting density of PEO chains is higher compared to the one of PAA. When PAA is grafted first, its presence in the mixed brush increases with its grafting time since gPEO is decreasing. This agrees with the earlier described kinetics of grafting where the quality of the PAA layer was shown to improve with the grafting time. When PEO is grafted first, however, nearly no difference could be observed between the different grafting times. Our results are not in agreement with previous studies, which showed that sequential tethering of two different functionalized F


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Figure 5. Evolution of (a) gPEO determined from XPS data and of (b) AνC−O−C/AνCO determined from PM-IRRAS data with the grafting time of PAA for mixed brushes created by sequential grafting with PEO grafted first (blue squares: PEO 1 h; red dots: PEO 24 h).

(number of PEO chains/number of PAA chains close to 1.6 if only the half of the molar mass of PAA is considered, since the disulfide bond is in the center of the polymer chain). The fraction of PAA repeating units is increasing with the increase of PAA grafting time, whatever the PEO grafting time (see Figure 5a). This increase is slightly sharper for PEO grafted during 1 h. Except when PAA is grafted for 24 h, the mixed brushes with 1 h of grafting time for the PEO show slightly more PEO repeating units compared to the mixed brushes obtained after 24 h of PEO grafting time. It is again pointing to the fact that a short grafting time is optimal for PEO. PM-IRRAS experiments confirm that all mixed brushes created by sequential grafting contain both PEO and PAA (see Figure 5b). Results show that all brushes display similar ratio between areas of PEO and PAA bands (AνC−O−C/AνCO close to 0.46), accounting for a similar number of CO and C−O−C asymmetric stretching events detected in the mixed brush. Finally, despite several trials of different grafting times for the two polymers, the range of composition of the mixed brushes obtained by sequential grafting is not broad. Therefore, another strategy will be developed in the following section. Indeed, obtaining a wider range of fractions of protein-adsorbing and protein-repelling species in the created layers seems a mandatory condition to reach the objective of the present work, which is to create mixed PEO/PAA brushes to trigger different behaviors toward protein adsorption. Simultaneous Grafting of the Polymers. The second strategy to synthesize mixed brushes of PEO/PAA consisted in performing the grafting process in a unique grafting step in which the polymers are mixed in a single solution (see Scheme 1c). Since PAA tends to dominate the composition (mass fraction) of mixed brushes obtained by sequential grafting (see above), some solutions were strongly enriched in PEO. The formation of three mixed brushes from solutions with PEO/PAA mass ratios of 99/1, 90/10 and 50/50, as well as the one of the two corresponding pure polymer brushes, were monitored in situ by means of EIS. These five brushes were also characterized after 30 min, 2 h, and 24 h of grafting by means of XPS and PM-IRRAS. The normalized R2 parameters from EIS are presented in Figure 6 while XPS and PM-IRRAS measurements are presented through gPEO in Figure 7a and AνC−O−C/AνCO in Figure 7b, respectively.

Figure 6. Normalized R2 parameter of mixed brushes obtained by simultaneous grafting for different PEO/PAA mass ratio in the grafting solution, as a function of the grafting time. Lines are drawn as a guide to the eye.

From EIS data, it can be seen that the grafting time corresponding to the maximum in the R2 parameter is increasing with the proportion of PAA in the grafting solution. For the mixed brush obtained with a PEO/PAA ratio in the grafting solution of 99/1, the optimum grafting time regarding the resistance to charge transfer is as short as the one for pure PEO, i.e., ca. 20 min. For mixed brushes obtained with a PEO/ PAA ratio in the grafting solution of 90/10 and 50/50, a plateau value is reached after 3−5 h while the R2 value is still increasing up to 8 h for PAA. This is in line with the short optimal grafting time for PEO and the longer one for PAA as discussed earlier. XPS results demonstrate that gPEO is situated between 0.5 and 0.75, whatever the grafting time, which at first instance shows that PEO and PAA are both present in all mixed brushes. These gPEO values can be converted in mass fraction of PEO, which is then situated between 0.35 and 0.65: this range is rather broad and centered around 0.5, accounting for similar quantities of PEO and PAA in mixed brushes. Moreover, gPEO of the mixed brushes is increasing with the fraction of PEO repeating units in the grafting solution. Nevertheless, all data points are under the line representing an equal proportion in the grafting solution and in the probed layer, in other words, PEO is always underrepresented in the grafted layer compared to the grafting solution. This effect is particularly pronounced for the mixed brush obtained with a PEO/PAA ratio in the grafting solution of 99/1 since only 1% of PAA repeating units in the grafting solution results in a value of gPAA as high as 0.3. The grafting time has no significant effect. Indeed, grafting G


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Figure 7. Evolution of (a) gPEO determined from XPS data with the fraction of PEO repeating units in the grafting solution and of (b) AνC−O−C/ AνCO determined from PM-IRRAS data with the mass fraction of PEO in the grafting solution for mixed brushes created by simultaneous grafting with different grafting times.

Table 2. Surface Composition Determined by XPS (Molar Fraction in %), Water Contact Angle (± Confidence Interval at 95% Level), Hydrated Mass Per Unit Area Δm Obtained Using Sauerbrey Modeling of Δf Measured by QCM-D (± Confidence Interval at 95% Level) and Average Thickness Measured by XPS (See Supporting Information for Details) of Mixed Brushes Synthesized Using Simultaneous Grafting with a Grafting Time of 30 min S 2p (%) PEO/PAA mass ratio in grafting solution

O 1s (%)

C 1s (%)

unbound

thiolate

total

Au 4f (%)

100/0 99/1 90/10 50/50 0/100

15.3 18.5 19.6 21.3 16.4

39.4 45.1 47.4 51.2 46.4

0.7 0.4 0.4 0.4 0.4

1.0 0.7 0.5 0.3 0.2

1.7 1.1 0.9 0.7 0.6

43.6 35.2 32.2 26.7 36.6

times of 30 min and 2 h always show the same gPEO values, which are slightly higher compared to 24 h of grafting. Therefore, for high grafting times, mixed brushes created by simultaneous grafting tend to be slightly depleted in PEO. This is again in line with the short grafting time that is optimal for PEO. PM-IRRAS results confirm that PEO and PAA are both present in the grafted layer. The areal ratio AνC−O−C/AνC O is always lower than unity, which shows that PAA is predominant in the grafted layer. Although this ratio seems to increase with the PEO mass fraction in the grafting solution, all data points are close to each other for different grafting times, again demonstrating that a change in grafting time does not generate important differences between mixed brushes created by simultaneous grafting. Since 30 min of grafting already provides a wide range of layer composition for mixed brushes created by simultaneous grafting (Figure 7a), this duration has been chosen for further experiments. More detailed characterization of the brushes obtained for this grafting time is presented in Table 2. While XPS analyses were obtained twice, only one set of data is presented here due to the relatively high experiment-toexperiment variability. The second set of data is given in the Supporting Information (Table S-3) and showed similar trends. QCM-D analyses were repeated between 14 and 16 times: the mean value of Δm is given with a confidence interval at 95%. Finally, the thickness was also estimated by means of XPS measurements (experimental and modeling details in Supporting Information). From XPS data, it can be deduced that the coverage of the gold substrate by the polymer layer is better for pure PAA compared to pure PEO. Indeed, the Au mole fractions, coming

θw (°) 35.0 36.0 35.7 33.0 31.8

± ± ± ± ±

4.3 1.2 0.7 0.0 1.4

Δm (ng/cm2)

estimated thickness (nm)

± ± ± ± ±

[2.5−3] [2.5−3] [3.5−4] [4.5−5] [3−3.5]

620 568 742 645 499

67 54 53 82 49

from the substrate, and also, the mole fraction attributed to S, the end group function of the polymers by which they are attached to the gold substrate, are smaller for PAA compared to PEO. This result is linked to higher percentages of O 1s and C 1s, coming from the organic layer. This is in line with the thickness estimation, performed through the modeling of a homogeneous adlayer, which is slightly higher for pure PAA than for pure PEO. For the three mixed brushes, the Au and S percentages decrease with the presence of PAA in the mixture. However, mixed brushes seem to better cover the substrate than the pure polymers do. This better coverage could come from a higher grafting density of the chains, giving a higher thickness of the layer. Thickness estimation is indeed the highest for the mixed brush created with a 50/50 PEO/PAA mass ratio in grafting solution. The S 2p peak is decomposed with two doublets, one near 163.2 eV assigned to unbound thiol/disulfide and another near 161.7 eV assigned to thiolate.74 Interestingly, the ratio between these two doublets varies progressively from PEO to PAA. The fraction of unbound sulfur increases with the presence of PAA in the brush. This points to the fact that the grafting of PEO is favored for short grafting times in comparison to the one of PAA, as well as to a more important physisorption of PAA compared to PEO. However, Δm extracted from QCM-D data seem not to be in agreement with XPS data. In fact, Δm of PEO is higher than the one of PAA, and mixed brushes present the same order of magnitude for Δm as pure PEO. Taking into account the density of PEO (1.13 g/cm3) and PAA (1.074 g/cm3),88 layer thickness deduced from Δm is of 5.5 and 4.7 nm for pure PEO and pure PAA, respectively. Since Δm accounts for a hydrated mass per unit area while XPS is performed on dry samples, the observed differences between QCM-D and XPS data could be H


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are screened by ions from the solution and the brush is shrunk19,53,90 and HSA adsorption on PAA chains under these conditions is decreased.92−94 However, this desorption process is limited since Table 3 shows here that only 30% of adsorbed HSA leaves the PAA brush upon desorption. For the mixed brush, the properties of the two polymers are nicely combined. In fact, at pH 5 and low I, PAA properties are predominant since PAA chains are swollen in solution. Therefore, the brush could adsorb a high quantity of HSA, corresponding to an adsorbed layer thickness of 9.5 nm, higher than a typical albumin monolayer. Interestingly, at high pH and I, PAA chains are shrunk and PEO chains are supposed to be more exposed at the outermost surface. Therefore, HSA is repelled from the surface, with an efficient desorption percentage (86%). These results actually indirectly confirm the presence of both polymers in the interfacial layer. With this example, we clearly demonstrate that HSA adsorption can be tuned by simple modification of the pH and I of the solution in contact with the surface during adsorption. Further studies are now ongoing on the full range of synthesized brushes, including the use of other proteins of different sizes and isoelectric points.

assigned to differences in layer hydration. From this, it can be tentatively concluded that the PEO layer is more hydrated than the PAA layer. Values of contact angle measurements are close to each other and cannot be used to discriminate the different brushes. Moreover, to control the homogeneity of mixed brushes, images were obtained by means of atomic force microscopy (AFM) in tapping mode and time-of-flight secondary ions mass spectroscopy (ToF-SIMS) (see Table S-4). A segregation of the two polymers into domains in the mixed brushes was indeed not desirable. On the contrary, PEO and PAA should be mixed at the molecular scale to finely tune protein adsorption. At the probed scale (∼20 nm for AFM, ∼150 nm for ToF-SIMS), no segregation of the chains could be observed. Additionally, the ToF-SIMS results confirm the presence of both PEO and PAA in the mixed brushes. Switchable Behavior of the Brushes toward Albumin Adsorption. As an illustration, the PEO/PAA mixed brush prepared from a 99/1 grafting solution (see characterization in Table 2) is selected to demonstrate its switchable stimuliresponsive behavior toward albumin (HSA) adsorption. This behavior is compared to the ones of pure PEO and pure PAA brushes. HSA adsorption at pH 5 and I = 10−5 M and possible desorption at pH 9 and I = 10−1 M were monitored in situ by means of QCM-D. Table 3 presents the hydrated mass per unit

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CONCLUSIONS The synthesis and protein adsorption behavior of mixed brushes of PEO and PAA have been presented in this article. First, the grafting kinetics of homobrushes of PEO and PAA was studied with EIS, water contact angle measurements, and XPS. On one hand, the most dense and pure PEO layer was formed after a short grafting time, ranging from 15 min to 4 h. On the other hand, a stable PAA layer was only formed after longer grafting times (up to 24 h). A sequential grafting strategy was then applied with subsequent grafting of PEO and PAA. Unfortunately, despite a wide range of grafting times tested, the composition of the obtained mixed brushes was not varying much (gPEO from 0.4 to 0.6) and reflected a low mass proportion of PEO in the layer (near 40%). Finally, simultaneous grafting of PEO and PAA was used and three mixed brushes with different compositions were created (gPEO between 0.5 and 0.75, i.e., PEO mass fraction from 35 to 65%), and characterized in detail. The mixed brushes seem to better cover the Au substrate compared to homopolymer brushes. Brushes are more hydrated with increasing PEO fraction, while the fraction of physisorbed (compared to chemisorbed) chains increase with the amount of PAA in the brush. AFM and ToFSIMS analyses showed that the mixed brushes were laterally homogeneous at a scale of ∼100 nm. Albumin was adsorbed onto a selected mixed brush designed with a 99/1 PEO/PAA mass ratio in the grafting solution. The amount of adsorbed albumin is larger than the one expected for monolayer coverage, showing the high adsorbing ability of PAA. Interestingly, upon change of pH and I, nearly 90% of the adsorbed protein layer is desorbed, revealing the proteinrepellent properties of PEO. These mixed brushes are therefore promising candidates to tune protein adsorption with an external stimulus (pH and/or I).

Table 3. Hydrated Mass Per Unit Area Computed from QCM-D Data Using Sauerbrey Modeling of Δf after the Adsorption of Albumin (pH 5 and and I = 10−5 M) and after Its Possible Desorption (pH 9 and I = 10−1 M), as Well as Resultant Desorption Percentage PEO/PAA mass ratio in grafting solution

Δmadsorption (ng/cm2)

Δmafter desorption (ng/cm2)

desorption percentage (%)

100/0 99/1 0/100

18 1296 2106

0 180 1476

100 86 30

area found on the crystal after adsorption (Δmadsorption), then after desorption (Δmafter desorption) for the three considered brushes. The desorption percentage is also calculated (desorption percentage = ((Δmadsorption − Δmafter desorption)/ Δmadsorption) × 100). QCM-D graph for the PEO/PAA 99/1 sample is presented in Figure S-7. From these results, it can first be confirmed that PEO inhibits HSA adsorption. Therefore, molar mass and grafting process chosen here are adapted for this purpose. Conversely, PAA brushes are well suited to adsorb a large amount of proteins. Indeed, the thickness of adsorbed HSA layer on PAA-modified gold (calculated considering that albumin density = 1.36 g/ cm3 66) would be 15.4 nm. This is thicker than a monolayer of HSA (HSA has roughly a heart shape with sides of 8 nm and thickness of 3 nm89), confirming the capacity of PAA to adsorb proteins in large amounts. To give a point of comparison, Δmadsorption recorded for HSA adsorption on bare gold at pH 5 and I = 10−5 M was equal to 530 ng/cm2, i.e., 4 times less compared to the value found on a pure PAA brush. In fact, at pH 5 and low I, PAA chains are supposed to be slightly charged and well swollen since nearly no counterions could allow for charge screening.19,90 These chains would therefore allow high amount of HSA entering into the brush.91,92 Upon changing pH and I, PAA brushes could eject a part of adsorbed HSA. In fact, at high pH and high I, the negative charges of PAA chains

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ASSOCIATED CONTENT

S Supporting Information *

This material, including decomposition and exploitation of XPS C 1s peaks, XPS characterization of PEO and PAA homobrushes as well as mixed brushes created by simultaneous grafting, QCM monitoring of PEO and PAA grafting, polymer I


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film thickness estimation by ARXPS measurements, AFM and ToF-SIMS images, and QCM monitoring of the switchable behavior toward albumin adsorption, is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Nesrine Aissaoui (LRS, UPMC), Michel Genet (IMCN, UCL), Marie Henry (IMCN, UCL), Claude Poleunis (IMCN, UCL), Wim Van Camp (PRG, GU) and Sami Yunus (IMCN, UCL) for technical help and fruitful discussions. The work was supported by the Belgian Science Policy through the Interuniversity Attraction Pole Programs (P6/27 and P7/05). The financial support of the Belgian National Foundation for Scientific Research (FNRS) is acknowledged. S.D.-C. and M.F.D. thank the FNRS for their Senior Research Associate and Research Fellow positions, respectively.

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