Biofouling Properties of Nitroxide-Modified Amorphous Carbon Surfaces

Aug 29, 2016 - nafion,24 and their charge transfer properties have been exploited for biomaterials ... overnight at 4 °C with 0.1 mg/mL human serum a...
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Biofouling Properties of Nitroxide-Modified Amorphous Carbon Surfaces Mariantonietta Parracino,† Paola Pellacani,‡ Pascal Colpo,† Giacomo Ceccone,† Andrea Valsesia,† François Rossi,† and Miguel Manso Silvan*,‡ †

Joint Research Center, European Commission, Via Enrico Fermi, 21020 Ispra, Varese, Italy Departamento de Física Aplicada and Instituto Nicolás Cabrera, C/Francisco Tomás y Valiente 7, Universidad Autónoma de Madrid, 28049 Madrid, Spain

‡

ABSTRACT: Amorphous carbon films exhibit attractive optical and surface properties. In this work, modified amorphous carbon films incorporating nitroxide groups (α-CNO) have been obtained by searching for a condensed analogue to classical soft antifouling materials. Thin films deposited by reactive magnetron sputtering in air discharges at varying power conditions were characterized by ellipsometry, atomic force microscopy, and water contact angle. Plasma power was observed to activate the densification and roughness of nanograined films. Most hydrophilic films deposited at 30 W exhibited the lowest refractive index, negligible optical absorption in the visIR, and presented a close to stoichiometric C2NO composition, as derived from X-ray photoelectron spectroscopy. Micropatterns prepared by photolithography validated the transparency−hydrophilicity of the α-CNO, as observed by water condensation contrast imaging. An albumin adsorption experiment evaluated through fluorescence revealed that α-CNO behaves as antifouling with respect to Si. Such thin antifouling films are of interest for the initiation of immobilization cascades in imaging surface plasmon resonance, where they have confirmed their antifouling contrast enhancement role. These results illustrate that the combination of a nanorough surface with nitroxide chemistry induces an antifouling behavior. In association with the optical transparency, the results invite the exploration of the bioengineering dimension of α-CNO films. KEYWORDS: amorphous carbon nitroxide, nanoroughness, hydrophilic, antifouling, surface plasmon resonance

1. INTRODUCTION Micropatterned antifouling surfaces are experiencing a huge demand to control biotechnological processes on solids. In DNA or protein arrays, target recognition sites are surrounded by an antifouling background. This configuration allows inhibiting nonspecific adsorption and maximizes analyzed signal contrast (most often fluorescence). Moreover, antifouling surfaces allow in cellular systems the control of cell localization, polarization, or migration. The required antifouling surfaces have traditionally been produced by using poly ethylenglycol1,2 or carbohydrates such as dextran.3 The steric hindrance effect has been described as the main molecular mechanism to explain the prevention or retardation of protein adsorption.4 However, the antifouling behavior has been more recently attributed also to topographic features and in particular to nanostructured surfaces made of organic,5 inorganic,6 or composite7 materials. Current challenges in the development of antifouling surfaces pass through the production by techniques with increased reliability and productivity. In this sense, compatibility with microelectronics processes is key. Relevantly, micropatterned amorphous carbon (α-C) films deposited by magnetron sputtering have been already proposed for surface plasmon resonance sensing (SPR).8 To increase the surface bioengineering possibilities, C films have been modified with polyelectrolyte multilayers.9 Additionally, denser diamond films © 2016 American Chemical Society

incorporating boron have been described to induce antifouling behavior and exploited as electrodes,10 but the attractive properties of C based films for bioengineering do not restrict to surface fouling and conductivity and span to optical properties.11,12 In this latter frame, the variability of the optical properties is so wide depending on structure and composition that it becomes necessary to optically characterize any specific α-C based film prior to its application. In particular, optical properties, such as refractive index below 2 and extinction below 0.4,13 make of α-C an attractive material to control the behavior of plasmonic metal nanostructures such as those made from Ag,14,15 Cu,16 and Au.17 Particular biomolecular detection platforms have been in fact described for this latter type of Au nanostructures, although the application of α-C films to imaging SPR (i-SPR) devices appears to be restricted.8 This particular configuration of SPR detection is however the most advantageous because it allows a multiplexed array-type detection, it makes possible to perform kinetic affinity studies at each spot of the i-SPR sensor and it is a label free process.18,19 In alternative devices, the SPR can be used to enhance fluorescence, the most common reading signal in conventional microarrays.20,21 Received: July 6, 2016 Accepted: August 29, 2016 Published: August 29, 2016 1976

DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982

Article

ACS Biomaterials Science & Engineering In the present work, we aim at preparing modified α-C films by reactive magnetron sputtering in air based gases and at describing their optical and surface properties. In particular, we focus on the incorporation of nitroxide (NO) species to form α-CNO. We hypothesize that the carbon matrix is able to stabilize the free radical nature of NO, otherwise exploited in biomedical antioxidant applications22 and in polymeric radical batteries.23 First attempts to surface immobilize this radical were performed by wet chemistry on glassy carbon and nafion,24 and their charge transfer properties have been exploited for biomaterials modification through NO radical polymerization.25 Meanwhile, a NO induced modification of the biointerface and optical properties of the C matrix would be welcome for their integration in biomedical devices. In this context, a reduction of optical indexes and increase of band gap can be obtained,26 which shall extend the electric field projections of the surface plasmon.27 As an example a simplified i-SPR device is proposed by using an α-CNO film as antifouling contrast with respect to a gold layer deposited on a prism for an analysis in the Kretschmann28 configuration.

2. EXPERIMENTAL SECTION 2.1. α-CNO Films and Micropatterns. The deposition of α-CNO was performed by reactive magnetron sputtering. Isopropyl alcohol cleaned Si (100) substrates were used as substrates. A high purity carbon target ([C] = 99.995 at%) was used in a mixed Ar−N2−Air discharge (Ar/N2/Air = 5/4/1), optimized by optical emission spectroscopy of relative O and N active species in the plasma and ulterior X-ray photoelectron spectroscopy (XPS) of deposited films. The background pressure was 2.10−5 mbar, the deposition pressure was 2.10−3 mbar adjusted with a guillotine valve, and the operation power varied from 30 to 75 W. Samples were deposited (after 16 min presputtering) during 64 min. Micropatterned samples were fabricated by α-CNO deposition after lift-off photolithography performed using Microposit S 1805 resist, followed by homogeneization in hot plate for 3 min and using MF-321 as developer and acetone as remover. UV irradiation was performed using a Hamamatsu Lightingcure UV-LED system through masks printed on UV transparent foils. The steps for the preparation of the micropatterns on Au coated glass prisms for iSPR are illustrated in Figure 1. An analogue process on Si (100) wafers was carried out for protein adsorption studies. 2.2. Characterization. The screening of the most attractive conditions for deposition of α-CNO was performed by a generalized analysis by ellipsometry, AFM, and water contact angle. Ellipsometry measurements were made in a variable angle spectroscopic ellipsometer (VASE) from J.A. Woollam Co., Inc., model M2000 V. Analyses were performed in the 400−1000 nm range at 55, 65, and 75 incidence angle on samples deposited during 64 min. The results were fitted by using a Cauchy film including roughness corrections onto a Si substrate with native oxide layer. The topography of the different films was studied by atomic force microscopy (AFM) in an NT-MDT with Smena head. Silicon cantilevers with force constant of 5 N/m and first harmonic frequency of 158 kHz were used. Image analysis was performed with NT-MDT software. To trace the wettability of the αCNO surfaces, water contact angles (WCA, DGD Fast/60, GBX technologies) were measured with milli-Q water recording both advancing and receding angles. Vapor selectivity of the micropatterned films was studied by optical microscopy. The microscope objective was inserted in a closed cell with H2O vapor and dry air inlets to simulate atmospheric conditions from nearly 3% to 99% humidity while the substrate remained at 22 °C. XPS measurements were performed in an AXIS ULTRA spectrometer (KRATOS Analytical, UK). The samples were irradiated with monochromatic Al Kα X-rays (hν = 1486.6 eV) using X-ray spot size of 400 × 700 μm2 and normal takeoff angles analysis. Surface charging was compensated by means of a filament inserted in a

Figure 1. Schematic representation of the processing of micropatterned α-CNO films on Au coated glass prisms for i-SPR analysis. magnetic lens system and the C1s hydrocarbon component was set to 284.50 eV binding energy (BE). For the core level spectra, a pass energy of 40 eV was selected, determining a resolution of 0.63 eV as measured both on the Ag 3d5/2 peak and on the Ag Fermi edge. The data were processed using CasaXPS v16R1 (Casa Software, UK). The core level envelopes were fitted with Gaussian−Lorentzian function (G/L = 30) and variable full width half-maximum. 2.3. Protein Assay. Prior to any biomolecular interaction, the patterned surfaces of α-CNO were incubated overnight at 4 °C in 5 mM TRIS-HCl buffer pH 8.5 for physiological stabilization. They were further washed with 5 mM acetate buffer at pH 5 and then incubated overnight at 4 °C with 0.1 mg/mL human serum albumin (HSA) diluted in the same buffer. After incubation, three washing steps were performed with 5 mM buffer acetate containing 0.1% of Triton, each of 10 min in agitation, in order to remove the unbound protein. The presence of surface linked protein was evaluated by fluorescent labeling with 8-anilino-1-naphthalenesulfonic acid (ANS) dye. Then 10 nM ANS in MES buffer were used to trace the HSA bonded onto the micropatterned surface. ANS binds preferentially to hydrophobic cavities and with lysine/arginine residues, giving fluorescence enhancement from the interaction of the charged group of lysine and arginine with the sulfonate group. The binding was carried out at RT for 1 h in agitation, followed by three buffer washing steps. α-CNO micropatterns were imaged after protein interactions with a fluorescence microscope (Epifluorescence NIKON, Inverted Optics) through 10× and 40× dry objectives, and snapshots were recorded with a CCD camera (Hamamatsu). To image ANS, Filter Cube 2 was used (393 CWL, excitation filter bandpass 385−400 nm). 2.4. iSPR. SPR prisms for analysis were purchased from Horiba (SPRi-Biochips) and consisted of 5 nm thick Ti followed by 45 nm of Au bilayers on the glass prisms. The Au surface of the prism was modified with α-CNO square micropatterns. The chip was then rinsed with MQ water, dried with N2, and transferred in the SPR system. The SPR measurements were performed with SPRi-PLEX+ instrument 1977

DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982

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ACS Biomaterials Science & Engineering

Figure 2. Ellipsometry measurements (dotted lines) and fitting models (continuous lines) corresponding to α-CNO films deposited at 30 W (a) with corresponding derived optical properties (b). Idem for α-CNO films deposited at 75 W (c, measurements, and d, optical parameters).

Table 1. Properties of α-CNO Films Deposited by Reactive Magnetron Sputtering at Different Power Conditions (th, Thickness; thR, Optical Roughness; n, Refractive Index at 550 nm; Rrms, Root Mean Square Roughness As Obtained from Scanning Areas of 5 × 5 μm2)

(GenOptics, Paris, France), which is equipped with a 12-bit chargecoupled-device (CCD) camera. The Kretschmann configuration for plasmon resonance was used with a fixed incident beam (810 nm). The different areas of interest were selected using a CCD image. Then the angle of the incident beam with the sample surface was varied (from 53° to 63° with a step of 0.30°) and the SPR curves from each area of interest were obtained. The working angle was then determined as the maximum of the first derivative of the reflectance as a function of the angle of incidence. The working temperature was fixed at 25° and equilibrated for 5 min in 5 mM MES pH 6 buffer, with a flow rate of 100 μL/min. Then two concentrations of HSA in 5 mM MES buffer (1 and 10 μg/ μL) were injected consecutively and the binding monitored. The system allows obtaining differential images to provide the contrast of biomolecular immobilization on the different regions of the interface. The HSA binding kinetics was analyzed using the software Scrubber 7. The difference images were analyzed with ImageJ.

ellipsometry P (W) 30 45 60 75

th (nm) 47 38 41 45

± ± ± ±

5 5 5 5

thR (nm) 13 19 31 29

± ± ± ±

2 2 3 2

AFM n (550 nm) 1.79 1.81 1.96 1.99

± ± ± ±

0.01 0.01 0.01 0.01

Rrms (nm) 2.8 2.8 3.6 4.3

± ± ± ±

0.6 0.5 0.5 0.8

analogue representation of ellipsometry spectra and optical parameters are shown in Figure 2c,d for the sample deposited at 75 W, which shows a moderate increase of ca. 10% of n, with an almost negligible k as in the previous case. Table 1 shows that the increase of n is gradual for increasing deposition power, which can be related to an increasing densification of the deposited α-CNO films. Although the thickness is not significantly affected by the deposition power, the optical roughness is influenced, with a ca. 100% increase from the 30 W to the 75 W deposited sample. To get a clear picture of the sample’s topography, an AFM study was performed on the α-CNO films. Parts a and b of Figure 3 show the 2 × 2 μm2 noncontact topography images of the samples deposited at 30 and 75 W, respectively. It can be observed that both films are composed of nanograined structures, as generally observed in α-C29 and DLC and in contrast to particular α-CN films.30 A study of the roughness of all the α-CNO films was performed by scanning 5 × 5 μm2 images. Values of the mean root square roughness (Rrms) are reported in the last column of Table 1. The results are

3. RESULTS 3.1. Properties of α-CNO Films. An initial screening of αCNO surfaces was performed by determining basic characteristics such as optical properties, morphological features, and wetting behavior. Figure 2 shows the optical characterization by ellipsometry for α-CNO deposited at the lowest and highest magnetron power. Figure 2a shows the experimental and simulated data for an α-CNO film deposited at 30 W at three different measuring angles. The model used fits well the optical behavior of the films in the visible and near-infrared range with slight deviations only close to the UV edge. The optical parameters derived from these simulations are plotted in Figure 2b, showing the typical monotonous decay for n and a constant behavior for k. Most relevant optical properties derived from the model, such as film refractive index at 550 nm, film thickness, and optical roughness, are detailed in Table 1. An 1978

DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982

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ACS Biomaterials Science & Engineering

Figure 3. AFM images from the surfaces of α-CNO films deposited at 30 W (a) and 75 W (b) scanned over 2 × 2 μm2.

remaining α-CNO surfaces, being the only one with final WCA below 60°. In view of the interest of this lower WCA, we further explored the wettability of the α-CNO surfaces deposited at 30 W by performing water condensation studies under controlled atmosphere in micropatterned areas. The inset images in Figure 4 show the surfaces of micropatterned αCNO films deposited at 30 W before (left) and after (right) the aperture of the water vapor saturated air inlet. Arrows indicate the localization of the α-CNO stripes, which are almost invisible in view of their thickness and optical properties. After contact with water vapor saturated air, the α-CNO stripes become clearly visible as a result of a selective condensation. This result confirmed the higher hydrophilicity of α-CNO surfaces deposited at 30 W with respect to the Si background. This behavior can be influenced by both the nanograin structured surface and the composition of the surface. In view of the potential functionality of the most hydrophilic films, the chemical nature of α-CNO surfaces deposited at 30 W was further studied by XPS. The elemental composition of the surfaces was obtained from survey spectra, which demonstrated that the surfaces are composed by C, N, O, and Si (apart from an undetermined amount of H), as reported in Table 2. These data show that the stoichiometry of the films

consistent with roughness values of the previously referenced αC and α-CN films and confirm that an increase of the surface roughness is induced by an increase in the deposition power. However, the AFM estimation is 1 order of magnitude below the optical estimation. This may be due to the limitations of AFM scanning taking into account the tip curvature (ca. 10 nm), which makes it difficult to characterize nanoscale features of high aspect ratio. In that sense, the surface roughness may be higher than measured by AFM. The wetting behavior of the surfaces of α-CNO films deposited at different power was probed by measuring WCA in dynamic mode. Figure 4 shows the evolution of the WCA for

Table 2. Surface Composition of α-CNO Films Deposited at 30 W (at %) As Derived from XPS

Figure 4. Dynamic water contact angle measurements of α-CNO films deposited at 30, 45, 60, and 75 W. Inset: optical microscope image of α-CNO micropatterned films deposited on Si at 30 W prior to (left) and after (right) moisture exposure.

sample compd at %

C

N

O

Si

30 W α-CNO

47.9 ± 0.7

20.3 ± 0.6

25.7 ± 0.5

6.1 ± 0.3

is close to C2NO, with a N/O atomic ratio slightly below 1 (0.8). Additionally, the Si substrate is detected in spite of the nominal thickness of the α-CNO films (ca. 40 nm) being higher than the mean electron scape depth (ca. 10 nm). This result suggests again that the coatings present an irregular topography, and their roughness may be higher than that estimated by AFM. Furthermore, it shows that the slight deviation in the N/O atomic ratio can be corrected due to the partial oxidation of the underlying Si substrate. C1s, N1s, and O1s core level spectra are depicted in parts a, b, and c of Figure 5, respectively. The C1s core level spectrum could be fitted with three contributions, which correspond well

the different α-CNO surfaces from droplet volumes of 1 up to 4 and then down to 2 μL. All the surfaces showed a very similar behavior in the advancing WCA, which was even comparable to that of the Si substrate. However, the behavior was observed to change in the receding WCA, where α-CNO surfaces were observed to be more hydrophilic than the Si substrate. Relevantly, the sample deposited at the lowest power (30 W) was observed to present a lower contact angle than the 1979

DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982

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ACS Biomaterials Science & Engineering

Figure 5. XPS measurements from the surfaces of α-CNO films deposited at 30 W. (a) C1s core level spectrum, (b) N1s core level spectrum, and (c) O1s core level spectrum.

to C−C at BE 284.5 eV, C−O(N) at BE 286.5 eV, and (N)C O, O−C−O(N) at BE 288.2 eV (where “(N)” refers to a potential presence or absence of N to comply with the N/O ratio below 1 of the film).31 Although these attributions are based on polymer structures, these could also fit well on previously prepared α-CNO films in which pure C−N, CN, and CN bonding energies are considered.32 In any case, the degree of integration of O and N in the current work surpasses the levels of the previously referenced work (especially with regards to O) so that the assignment of C1s peaks with no pure CN bonding and in comparison with softer compounds as polymers is justified. The analysis of the N1s core level spectrum confirms what is suggested by the C1s and elemental composition data. A unique wide component centered at 400.0 eV can be observed in the spectrum displayed in Figure 5b. This band does not match any of the previously described assignments in α-CNO, which are treated as different levels of C−N bonding hybridization but fits much better other unintentionally O doped CN films.33 Relevantly, the O1s core level spectrum reveals two different components at BE 531.7 eV and BE 534.0 eV. In view of the slight excess of O with respect to N, these two peaks may be assigned to CO and C−O−N, respectively, in agreement with the assignments of O1s core level peaks to polymers incorporating single- and doublebonded oxygen functionalities.34 Relevantly, the presence of CO polar groups identified in the C1s and O1s spectra may be at the origin of the slightly hydrophilic behavior of these films. 3.2. Biofunctionality of Micropatterned α-CNO. The surface properties of α-CNO in terms of biomolecular interactions have been studied on α-CNO micropatterns that could reveal adsorption/adhesion contrasts with respect to Si. Our first efforts were devoted to determine the natural affinity of the synthesized films for generic serum proteins. Figure 6 shows the fluorescence imaging results of adsorption of HSA onto two different types of α-CNO micropatterns on Si as revealed by ANS staining. Parts a and b of Figure 6 correspond to a spot background micropatterned as observed in the green channel (ANS active) and red channel (ANS blind channel). The green channel image reveals that HSA adsorbs

Figure 6. Grayscale fluorescence microscopy images of α-CNO micropatterned films deposited at 30 W on Si after incubation with HAS and detection of adsorption by ANS. (top) An α-CNO background micropattern showing Si spots (green, a) and red, b) pass band images) and (bottom) α-CNO squares on a Si background (green, c) and red, d) images).

preferentially on the Si spot areas, while the red channel images serves as control, evidencing that detected emission is due to ANS. To confirm this result, inverted micropatterns where fabricated by deposition of α-CNO squares on a Si background. Parts c and d of Figure 6 confirm that lower green fluorescence intensity (image in grayscale) is observed on αCNO areas with respect to the Si background. This result can be considered as a first indication of an antifouling behavior of the α-CNO films. The previous results led to additional tests in a real bioanalytical system. α-CNO background micropatterns were fabricated on Au surfaces to carry an iSPR study. The test comprised two steps of exposure of the surfaces to increasing concentrations of HSA. Figure 7a shows specific adsorption kinetic curves for the adsorption of HSA at 1 and 10 μg/mL onto gold and α-CNO (bottom axis). The related statistical analysis is illustrated by the spot charts comprising data from 10 1980

DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982

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ACS Biomaterials Science & Engineering

Figure 7. (a) Particular adsorption kinetics of HAS solutions (1 μg/mL from 0 min and 10 μg/mL from 875 min, bottom scale) onto micropatterned α-CNO background areas (bottom line) and on Au SPR squares (top line). Average adsorption saturation at the two injected HSA concentrations (top scale) for micropatterned α-CNO background areas (squares) and on Au SPR squares (circles). (b) Pixel readout and differential stripe image after HSA adsorption saturation (10 μg/mL) on micropatterned Au squares with α-CNO background.

increased hydrophilicity) of the low power α-CNO films, the biofunctional studies were concentrated on these films. Such films exhibit a net antifouling behavior as demonstrated by fabricating micropatterned α-CNO films on Si. The adsorption of HSA, determined by an ANS fluorescence, was in fact observed to be depleted on α-CNO areas with respect to the uncoated Si substrate. The relatively low refractive index, almost no optical absorption, and limited thickness associated with the 30 W deposited α-CNO films were considered to be optimal for implementing an iSPR study. We exploited surface antifouling and optical properties in the development of an iSPR system based on α-CNO background micropatterns deposited on Au coated glass prisms. The precise properties (roughness, surface composition) of the α-CNO on Au shall be further studied and compared with those deposited on Si. In any case, the adsorption of serum proteins on the α-CNO films was observed to reduce the adsorption by ca. 40% to the sensing surface. Differential adsorption images evidence the possibility of inducing contrasts by using an α-CNO antifouling complement as a base for iSPR and invites further exploration of the biomedical potential of α-CNO films.

different adsorption kinetic curves (top axis). It is shown that, even at the lowest HSA concentrations, the protein adsorption of HSA onto α-CNO (circle spots) is significantly reduced with respect to the Au surface (square spots). At higher HSA concentrations of 10 μg/mL the α-CNO surface induces a net decrease of ca. 40% adsorption with respect to the Au surface. To illustrate the relevance of such adsorption contrast, a row of Au spots with the α-CNO surrounding background was imaged at the saturation state with 10 μg/mL HSA solutions. The image (difference image with respect to contrast at zero HSA exposure), as well as the pixel reading, are shown in Figure 7b. The image shows a clear contrast of dominant HSA adsorption on the Au squares with a remarkable decrease of intensity in the α-CNO background areas. Both the image and the pixel reading show some heterogeneity of the signal in the Au active sites, which may be attributed to effects of the multistep processing of the Au coated glass prisms. This suggests that the whole α-CNO film deposition process on Au should be compared in terms of surface roughness and composition with what obtained on the Si control samples.

5. CONCLUSIONS Thin α-CNO films have been grown by reactive magnetron sputtering on Si and Au substrates and their biofouling properties have been studied. The modification of the film properties upon variation of deposition power was analyzed, showing that rougher and denser surfaces are produced by increasing the power. The surfaces exhibit a rather hydrophilic behavior, especially at low power deposition regime (30 W), as derived from both dynamic contact angle measurements and water vapor condensation experiments. These particular surfaces present a composition close to a stoichiometric C2NO. The presence of polar groups on the surface, such as CO, could explain the wetting behavior of the films, although an influence of the nanograined surface cannot be excluded. In view of the dual attractive properties (lower optical indexes and

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 914974918. Fax: +34 914973969. E-mail: miguel. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We thank Giovanni Maselli for his technical assistance during materials processing. Marie Curie funding provided by the European Commission through FPVII grants IMPRESS (IAP GA 286262) and THINFACE (ITN GA 607232). 1981

DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982

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ACS Biomaterials Science & Engineering

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DOI: 10.1021/acsbiomaterials.6b00381 ACS Biomater. Sci. Eng. 2016, 2, 1976−1982