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Article pubs.acs.org/JPCC

Depth-Resolved Structural and Compositional Characterization of Ion-Implanted Polystyrene that Enables Direct Covalent Immobilization of Biomolecules Marcela Milena Marie Bilek,*,† Alexey Kondyurin,† Stephen Dekker,† Bradley Clifton Steel,† Richard Arthur Wilhelm,∥,⊥ René Heller,∥ David Robert McKenzie,†,◆ Anthony Steven Weiss,#,▽,○,◆ Michael James,‡,§,◆ and Wolfhard Möller∥,◆ †

Applied and Plasma Physics Laboratory, School of Physics (A28), The University of Sydney, Sydney, New South Wales 2006, Australia ‡ The Bragg Institute, Australian Nuclear Science and Technology Organisation (ANSTO), Locked Bag 2001, Kirrawee DC, New South Wales 2232, Australia § The Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia ∥ Institute of Ion Beam Physics and Materials Research, Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstraße 400, 01328 Dresden, Germany ⊥ Technische Universität Dresden, 01069 Dresden, Germany # School of Molecular Bioscience, University of Sydney, Sydney, New South Wales 2006, Australia ▽ Charles Perkins Centre, University of Sydney, Sydney, New South Wales 2006, Australia ○ Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia S Supporting Information *

ABSTRACT: A polystyrene film spun onto polished silicon substrates was implanted with argon ions using plasma immersion ion implantation (PIII) to activate its surface for single-step immobilization of biological molecules. The film was subsequently investigated by X-ray and neutron reflectometry, ultraviolet (UV)−visible (vis) and Fourier transform infrared (FTIR) ellipsometry, FTIR and Raman spectroscopy, as well as nuclear reaction analysis to determine the structural and compositional transformations associated with the surface activation. The ion irradiation resulted in a significant densification of the carbon structure, which was accompanied by hydrogen loss. The density and hydrogen profiles in the modified surface layers were found to agree with the expected depths of ion implantation as calculated by the Stopping and Range of Ions in Matter (SRIM) software. The data demonstrate that the reduction in film thickness is due to ion-induced densification rather than the removal of material by etching. Characterization by FTIR, atomic force microscopy (AFM), ellipsometry, and X-ray reflectometry shows that polystyrene films modified in this way immobilize dense layers of protein (tropoelastin) directly from solution. A substantial fraction of the immobilized protein layer remains after rigorous washing with sodium dodecyl sulfate solution, indicating that its immobilization is by covalent bonding.

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INTRODUCTION

groups, or oxidation of the surface upon exposure to the atmosphere. Because of the energy loss of the ions along their path, the modifications vary strongly with distance from the surface. Polymers modified using PIII have found applications in aeronautics,5 microelectronics,6 and medicine.7,8 There is mounting evidence that PIII modification also increases the density of protein attachment and the longevity of bioactivity of immobilized proteins on polymeric materials9−11 and provides strong, robust immobilization through covalent bonds.12,13

Ion implantation is a versatile technique for the modification of polymer surfaces.1−3 In plasma immersion ion implantation (PIII), a bias potential is applied to a substrate immersed in a plasma.4 Ions crossing the plasma sheath formed around the substrate are accelerated as they approach the substrate. At sufficiently high voltage, the ions are implanted below the surface of the substrate. The interactions between the target atoms and the incoming ions lead to displacement and excitation of atoms and electrons along the paths of the ions. These interactions may cause chemical and structural changes such as cross-linking between polymer macromolecules, scission of the polymer backbone, degassing of volatile side © 2015 American Chemical Society

Received: May 30, 2015 Published: June 3, 2015 16793

DOI: 10.1021/acs.jpcc.5b05164 J. Phys. Chem. C 2015, 119, 16793−16803

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The Journal of Physical Chemistry C

conditions used. This fluence range corresponds to treatment times between 40 and 1600 s for 20 kV pulses applied at 50 Hz, while 400 s for 2 kV at 1600 Hz achieves a fluence of 1016 ions cm−2. The ion fluences delivered in the PIII system were determined by comparison of changes in optical properties with reference samples implanted in a beamline apparatus, where fluence was directly measured.3 Thin-Film Characterization (Bulk). Visible (M-2000V) and IR variable-angle spectroscopic ellipsometers (VASE) from J. A. Woollam (USA) were used to determine the thickness and optical properties over the visible and infrared wavelengths, respectively, of the polystyrene films before and after PIII treatment and after incubation in protein solution. FTIR transmission spectra were recorded using a BOMEM FTLA 2000 FTIR spectrometer. Micro-Raman spectra (λ = 532.14 nm) were obtained in the backscattering mode using a Horiba Jobin Yvon, HR800 LabRam spectrometer. Atomic force microscopy (AFM) images of the protein-coated samples were collected using a Pico SPM instrument in tapping mode. Thin-Film Characterization (Depth-Resolved). X-ray and neutron reflectometry20−26 were carried out at the Australian Nuclear Science and Technology Organization (ANSTO), using a Panalytical X’Pert Pro reflectometer with Cu Kα radiation (λ = 1.54056 Å) and the Platypus time-offlight neutron reflectometer27,28 with a cold neutron spectrum (2.8 ≤ λ ≤ 18.0 Å) at the OPAL 20 MW research reactor, respectively. X-ray reflectometry is sensitive to the electron density profile normal to the surface, while neutron reflectometry probes the variation in neutron scattering length density normal to the surface and is particularly sensitive to hydrogen content. X-ray reflectometry has been used for studies of protein and cell adsorption on a range of surfaces including surfaces modified by plasma.29−35 The fitting (performed with MOTOFIT software) of both X-ray and neutron reflectivity data achieves its highest precision at low film thickness. Therefore, thinner films (with thickness 15 and 25 nm) and correspondingly low irradiation energy (2 keV) were employed for these studies. Hydrogen depth profiles were obtained from nuclear resonance reaction analysis using the reaction 1H(15N,αγ)12C. The limited depth resolution of the nuclear reaction profiling requires a larger thickness of the modified layer compared with the one that is optimum for X-ray and neutron reflectivity measurement. Therefore, 100 nm films modified by 20 keV ion irradiation were chosen for this analysis. Integrated carbon and oxygen contents of the films were obtained from the nuclear reactions 12C(d,p0)13C and 16O(d,α0)14N, respectively. Protein Immobilization Study. For coating with a protein layer, the samples were incubated in a 10 μg/mL tropoelastin in PBS (phosphate-buffered saline) solution at room temperature. The recombinant human tropoelastin, corresponding to amino acid residues 27−724 of GenBank entry AAC98394 (gi| 182020), was expressed and purified as previously described.36,37 The incubation time (55 min) was that required for saturation of the protein coverage, as measured by visiblelight ellipsometry. After incubation, the samples were rinsed in Milli-Q water and dried in atmosphere at room temperature. To identify protein that is covalently immobilized, we washed the samples in 2% w/v SDS (sodium dodecyl sulfate) in water solution at 80 °C for 55 min and rinsed them in Milli-Q water. SDS is a detergent that is used to unfold proteins and to remove physically adsorbed proteins from surfaces. The SDS cleaning procedure is well-established in the literature38−40 and

These findings suggest promising future applications in proteinbased biosensors,14 microarrays,15 and implantable biomedical devices.12,16 In many applications, the mechanical integrity of the modified layer is important. A previous study by Koval17 of ion-beam-etched poly(methyl methacrylate) (PMMA) thin films on Si using low-energy Ar ions (250 eV) revealed a sequence of strata within the polymer including a graphitized surface layer, a cross-linked layer beneath it, and a lowmolecular-weight layer (of density lower than unmodified PMMA) beneath that. They showed that the presence of the low-molecular-weight layer compromises the structure’s strength and durability, causing the graphitized and crosslinked layers to detach under stress. Furthermore, the depth of the modified layer has been shown to determine the longevity of the covalent coupling capability for biomolecules.12 It is not known whether an underlayer of reduced density such as that which leads to loss of mechanical integrity in PMMA occurs in other polymers, and the extent to which the depth profile of the modified layer in polymers is predictable from widely used simulation strategies such as the Stopping and Range of Ions in Matter (SRIM) software18 is not yet well tested. An understanding of these structural effects of ion beams on polymers requires knowledge of how the structure and composition vary as a function of depth in PIII-modified polymers. The present work combines several complementary diagnostic techniques (X-ray and neutron reflectometry, nuclear reaction analysis, ultraviolet−visible (UV−vis) and Fourier transform infrared (FTIR) ellipsometry, and FTIR and Raman spectroscopy) to gain an understanding of the subsurface structure and composition in PIII-modified polystyrene. We demonstrate also that films modified in this way are capable of single-step covalent immobilization of the extracellular protein tropoelastin, and we investigate the structure of surface-immobilized protein layers on the ionmodified surfaces.

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EXPERIMENTAL METHODS Full experimental details are provided in the Supporting Information (SI). Polystyrene films were spin-coated onto (100) polished silicon wafers to nominal thicknesses of 15, 25, and 100 nm from solutions (4, 6, and 20 g/L) of polystyrene (Austrex 400) in HPLC toluene. The wafers were spun at a rate of 3250 rpm for a total time of 20.0 s. The first 1.0 s consisted of a linear acceleration ramp up to this speed. These parameters gave a uniform (±1%) thickness distribution of the polymer film across the silicon wafer. Ion Implantation of the Polymer Films. The spin-coated wafers were attached to a stainless-steel sample holder, held 45 mm beneath a stainless-steel mesh to which the sample holder was electrically connected. This entire assembly was placed within the diffusion chamber of a PIII system previously described.19 An inductively coupled radio frequency (RF) plasma (100 W forward power at 13.56 MHz) in argon (4.4 × 10−2 Pa) was used to provide a source of ions for implantation. High-voltage pulsed bias was applied to the sample holder and mesh assembly to achieve the implantation of ions from the plasma. The length of the pulses was 20 μs, while the applied bias (2 or 20 kV) and the pulse frequency (1600 or 50 Hz) were adjusted to suit the thickness of the sample being treated. Treatment times were chosen to achieve desired fluences of ions in the range from 5 × 1014 to 2 × 1016 cm−2 for each set of 16794


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The Journal of Physical Chemistry C more recently reviewed12,13 as a test of covalent attachment to surfaces. SDS is an ionic surfactant that unfolds proteins and disrupts the forces responsible for physical adsorption, while leaving the covalent bonds intact.41 Covalent bonding can be inferred when SDS washing under the same conditions completely removes protein from a more hydrophobic control with similar surface roughness as the test sample.40,42 Further discussion and references are given in the SI. The adsorbed tropoelastin layer before and after SDS elution was characterized using X-ray reflectometry, AFM, FTIR, and ellipsometry.

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RESULTS AND DISCUSSION Structure and Composition of the Modified Layer. Raman and FTIR spectroscopy show that the ion-implanted PS is transformed into a disordered graphitic structure that undergoes oxidation when exposed to the atmosphere. After treatment with a fluence of 1016 ions/cm2, a strong broad band, typical of graphite-like amorphous carbon,43 appears in the Raman spectrum (Figure 1), while the polystyrene lines

Figure 2. FTIR transmission spectrum of PS modified by PIII. The arrows indicate the lines of chemical groups introduced by the PIII modification and residual hydrocarbon structures. The spectra are baseline-corrected and have the spectrum of the uncoated silicon wafer subtracted. Polystyrene was spin-coated to a thickness of 16 nm (0.4% solution, 3500 rpm) on silicon and PIII-treated (Ar+, 2 kV pulse bias, 20 μs, 1600 Hz, 400 s).

coefficient after PIII treatment, consistent with a high degree of carbonization and densification, as would be expected with a transition of the film structure to graphite-like amorphous carbon. The ellipsometric model fitting showed that the PS film thicknesses were reduced after the PIII treatment. For example, a film of 16 nm was reduced to 12.4 nm upon treatment with an ion fluence of 1016 ions/cm2. Depth-Resolved Structure and Composition. Figure 3 shows the X-ray scattering length density (SLDx) depth profiles obtained by fitting X-ray reflectivity (XRR) data (shown in Figure S3 in the SI) from (a) an untreated film, and from argon ion PIII-modified films (2 kV, 1600 Hz) after treatment times of (b) 80 and (c) 400 s. For untreated polystyrene, a very good fit to the data was obtained using a single-layer model with film thickness of 26.4 ± 0.1 nm and a constant SLDx of 9.4 ± 0.1 × 10−6 Å−2 (Figure 3a). This refined SLDx equates to a film mass density of 1.03 g/cm3 and is typical of PS thin films prepared by spin coating. The Si/film interfacial roughness and the surface roughness of the film were refined in the model as 0.3 ± 0.1 and 0.4 ± 0.1 nm, respectively. For the argon PIII-modified films (Figures 3b,c), variable composition and density precluded fitting with a single-layer model, and hence the discrete density profile (DDP) method described by Fullagar and coworkers45 was used. In this fitting method, the number of layers used in the model is chosen based on the quality of the data, as expressed by Qmax, the value of Q where the specular reflectivity is no longer discernible above the background (see Figure S3 in the SI). With the exception of the uppermost layer, the thickness and interfacial roughness of the layers were fixed at 2.0 and 0.4 nm, respectively, and only the SLD of each layer was refined to generate a smoothly varying profile. Excellent fits to the XRR data were obtained for the PIII-modified films of thickness 24.2 ± 0.1 (80 s, Figures 3b) and 20.0 ± 0.1 nm (400 s, Figure 3c) using 12 and 10 layers, respectively. The same films were also characterized using neutron reflectometry. Data from the untreated PS film were fitted using a single-layer model, while data from PIII modified films were modeled using the DDP method. The refined neutron SLD profiles are shown in Figure 4. While both X-ray and neutron reflectivity probe the film thickness and internal structure, the

Figure 1. Micro-Raman spectrum of spin-coated (nominally 100 nm) polystyrene on a silicon wafer treated in argon plasma by plasma immersion ion implantation with bias pulses of 20 kV at 50 Hz for 1600 s (1016 ions/cm2). Component peaks are fitted with Gaussian functions.

observed for the untreated film disappear. Similar features have been observed for polypropylene following PIII treatment.44 The new band can be fitted by a G peak at 1558 cm−1 and a D-peak at 1387 cm−1, which are characteristic of disordered carbon structures containing a majority of sp2 bonds.43 The G-peak position (1558 cm−1) and the integrated peak intensity ratio I(D)/I(G) = 1.76 value show that the nanocrystalline graphitic islands have average size of 1.8 nm. (Details of the calculation are given in the SI.) FTIR transmission spectroscopy of the argon-bombarded PS film (Figure 2) shows vibrational lines of residual hydrocarbon groups in the range 2800−3000 cm−1 attributed to CH stretching, ν(CH), and at 1450 cm−1 attributed to bending vibrations δ(C−H), as well as lines from unsaturated carbon− carbon group vibrations, ν(CC), in the 1600−1650 cm−1 region. Absorptions associated with vibrations of oxygencontaining groups at 3500 cm−1, ν(OH), attributed to hydroxyl, peroxide, carboxyl groups, and at 1720 cm−1, ν(CO), attributed to carbonyl, carboxyl, and ester groups, are also observed. Visible and FTIR ellipsometry measurements (presented in the SI) showed increases in refractive index and extinction 16795


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Figure 3. X-ray SLD profiles from (a) (blue curve) untreated spin-coated PS film; (b) (green curve) PIII-treated PS (argon ions, 2 kV pulsed bias, 20 μs, 1600 Hz, 80 s); and (c) (red curve) PIII-treated PS (argon ions, 2 kV pulsed bias, 20 μs, 1600 Hz, 400 s).

Figure 4. Refined neutron SLD profiles from (a) (blue curve) untreated spin-coated PS film; (b) (green curve) PIII-treated PS (argon ions, 2 kV pulsed bias, 20 μs, 1600 Hz, 80s); and (c) (red curve) PIII-treated PS (argon ions, 2 kV pulsed bias, 20 μs, 1600 Hz, 400 s).

former is sensitive to the electron density of the film, and the latter is sensitive to the neutron scattering length density (SLDn). Both the electron density and the neutron scattering length density depend on a combination of the composition and mass density at any level within the film,46 although neutron reflectometry is much more sensitive to hydrogen content than XRR due to the substantial difference in coherent neutron scattering length (b) between C (6.646 fm = 6.646 × 10−15 m) and H (−3.739 fm). The fact that the hydrogen neutron scattering length is negative means that a hydrogen loss will increase the neutron scattering length density. Although the combined fitting of the X-ray and neutron reflectivity data can lead to an understanding of chemical composition as a function of depth and in principle lead to the extraction of hydrogen depth profiles, independent knowledge of the non-hydrogen composition depth profile, which is not available in this case, is required to do so effectively. Selected complementary results on hydrogen concentration from nuclear reaction analysis are presented in Figures 5 and 6a. The fluence dependence of hydrogen loss observed here agrees

very well with that observed in polyimide (PI) and polyethylene terephthalate (PET) due to implantation of 150 and 200 keV argon ions, as measured by elastic recoil detection (ERD).47 Similar depletion levels were observed to a depth of 80 nm for 50 keV nitrogen ion implantation into poly(styreneco-acrylonitrile) (PSA).48 Figure 6b,c shows the integrated amounts of carbon and oxygen in the polystyrene film as a function of ion fluence. The oxygen increases with fluence up to a fluence of 5 × 1015 ions/cm2 and then decreases again to stabilize at a value of ∼1 × 1016 cm−2, which is higher than the oxygen concentration in the polymer prior to ion implantation. This is consistent with trends previously observed after ion implantation of polymers, which do not include significant amounts of oxygen in their preimplantation compositions.49,50 The oxygen in this case originates from reactions that occur with atmospheric species after the sample is removed from the treatment chamber. The structure’s capacity to react with atmospheric oxygen is reduced as it is transformed into a dense amorphous carbon structure at high fluences.49 In the case of polymers, such as polyimide, that contain oxygen as part of 16796


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Comparing the refined X-ray electron density profiles and neutron SLD profiles for untreated and PIII treated films as well as the profiles obtained from nuclear reaction analysis for argon ion-treated polystyrene (Figures 3−6), a decrease in total film thickness is clearly demonstrated both for 2 and 20 keV Ar+ irradiation. On the contrary, the amount of carbon present remains essentially constant, as shown by the constant areal density of carbon in Figure 6c for the 20 keV case. Thus, the thickness reduction with increasing treatment time must be attributed to structural densification, accompanied by hydrogen loss, rather than to ion-induced etching of the carbon backbone, as previously suggested in the case of 5−20 keV PIII50,51 and observed by others for the case of 1 keV ion bombardment.52 Both X-ray and neutron reflectometry measurements indicate the presence of density gradients within the PIII modified films. In all cases, the uppermost layers have scattering density values higher than the unmodified material that can be attributed to the formation of a carbonized structure with a higher mass density, as also inferred from Raman spectra and ellipsometry. This is consistent with the observed loss of hydrogen (see Figures 5 and 6a) and a correspondingly increased degree of interlinking of the carbon network. The layer adjacent to the silicon wafer has a low SLD and mass density similar to the initial polystyrene coating. Comparison between Figures 3 and 4 can be used to infer further information about dehydrogenation and densification profiles in the ion-modified layers. The fact that X-rays are sensitive to variations in electron density, while neutrons are also sensitive to hydrogen content, allows us to comment on the relative changes in composition and mass density within these films as a function of depth. XRR indicates a maximum in electron density ∼5 nm below the surface for films after both 80 and 400 s treatments with 2 keV argon ions and a slightly higher overall scattering density for the latter. In contrast, the neutron SLD has a maximum value for these modified films essentially at the surface, consistent with a loss of hydrogen, contributing to an additional increase in SLD because of the negative scattering length of hydrogen. The peak in neutron SLD for the film after 400 s (4.4 × 10−6 Å−2) is substantially larger than for the film after 80 s (2.9 × 10−6 Å−2). By itself, this result could indicate a lower hydrogen content or higher mass density; however, when coupled to similar X-ray scattering length densities, it points to the former. When compared with the expected neutron SLD for graphite (7.4 × 10−6 Å−2), the refined values suggest a small residual hydrogen content in this part of the film or a lower mass density. The nuclear reaction analysis for 20 keV irradiation (Figure 5) confirms a significant depletion of hydrogen at the surface with increasing Ar+ fluence. In the layer close to the polystyrene/silicon interface, which is not significantly affected by the irradiation, the hydrogen concentration agrees within the error bars with the nominal hydrogen content of polystyrene (C8H8). Toward the surface, increasing hydrogen loss is observed, being greatest close to the surface. A stationary hydrogen concentration is established at the surface at a fluence above ∼1 × 1016 cm−2. For 2 keV irradiation, the combined Xray and neutron diffraction data suggest that some residual hydrogen remains even at the highest fluence. The neutron SLD rises more steeply at the surface than the X-ray SLD, indicating that the top surface layer is dehydrogenated and has low density compared with the deeper material (Figures 3 and 4) and confirms the similarity between the 2 and 20 keV irradiation results. The dramatic increase in the peak neutron

Figure 5. Hydrogen depth profiles as obtained from nuclear reaction analysis, after irradiation of a 100 nm PS film (Ar+, 20 kV pulsed bias, 20 μs, 50 Hz) at two different fluences (1 × 1016 Ar/cm2 corresponds to a treatment time of 800 s). The trailing edges (on the right-hand side) of the profiles indicate the position of the film/substrate interface. The curves have been drawn to demonstrate trends.

Figure 6. Hydrogen surface concentration (a) and integrated areal densities of oxygen (b) and carbon (c) versus irradiation fluence, as obtained from nuclear reaction resonance analysis. The hydrogen surface concentration (a) has been obtained by extrapolation of the depth profile data (see Figure 5) to zero depth. The dotted line in panel c represents a linear fit, and 1 × 1016 Ar/cm2 corresponds to a treatment time of 800 s.

their monomeric units, the result of ion implantation is typically a reduction in the oxygen content of the surface layer,47 as a significant portion of the incorporated oxygen forms volatile species during ion irradiation and diffuses out of the structure. 16797


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Figure 7. TRIM12 collisional simulation results for 2 (a,c,e) and 20 keV (b,d,f) argon bombardment of polystyrene: (a,b) ion range distributions; (c,d) distributions of deposited energy due to nuclear and electronic collisions; and (e,f) distributions of generated vacancies and corresponding final positions of recoil atoms (for carbon recoils only). The data shown correspond to averages taken over simulations of 10 000 ion impacts.

atoms of the polystyrene macromolecules, the latter causing collision cascades in which some carbon and many hydrogen atoms receive enough energy to be ejected from their original sites and to be permanently displaced. Figure 7 shows the distribution of stopping range of the implanted ions together with the profiles of ion energy dissipated in electronic and nuclear collisions and the distributions of carbon vacancies and associated displaced carbon atoms generated by the ion impacts. A comparison of the vacancy and displaced atom profiles indicates a net inward transport of the carbon atoms, which is consistent with the observed densification with ion fluence. For 2 keV ion irradiation, the width of the carbon recoil distribution profile of roughly 10 nm agrees well with the XRR SLD profile shown in Figure 3. The observed hydrogen profile at 20 keV (Figure 5b) with a maximum depletion depth ∼50 nm is consistent with the nuclear energy deposition profile shown in Figure 7d. Thus, bond breaking by nuclear collisions may be the origin of hydrogen depletion in the present range of energies. However, some contribution of electronic collisions cannot be ruled out. It should be noted that a unique assignment is also hampered by the dynamically developing composition and structure profiles during irradiation, whereas the simulation assumes ion impacts into the unirradiated polymer. Hydrogen depletion in polymers at MeV light ion irradiation, where the nuclear energy transfer is negligible, has been attributed to electronic energy deposition.53 More elaborate descriptions of the hydrogen release from polymers include the model of bulk molecular recombination,54,55 which was originally applied to hydrogenated amorphous carbon (aC:H).56 Hydrogen atoms, which have been collisionally released from their bonds, may locally recombine into molecules, which quickly diffuse out through the surface.57,58 Characterization of the Structure of a SurfaceImmobilized Protein Layer. Recent findings12,13 show that

SLD with increasing treatment time from 80 to 400 s compared with the relatively stable value of the peak in X-ray SLD supports a reduction in subsurface hydrogen concentration with increasing fluence, in agreement with the NRA data as shown in Figures 5 and 6. The observed time dependence of the hydrogen profile may be influenced by water uptake during ion treatment or after exposure to air. This is consistent with the oxygen incorporation shown in Figure 6b. The ion irradiation initially results in some chain scission, leaving a damaged but as yet not strongly densified and cross-linked polymer, which is prone to water and oxygen diffusion. (Note that the maximum amount of oxygen observed corresponds to several tens of monolayers.) At sufficiently high fluence ((1 to 2) × 1016 ions/cm2), the oxygen level decreases toward that observed in the unirradiated film. This reduction in oxygen implies the formation of a densely cross-linked amorphous carbon film that reduces oxygen and water infusion from the atmosphere after treatment. The amount of oxygen observed at the highest fluence, if assumed to be due to water ingress, would contribute significantly to the residual hydrogen concentration (∼1.5 × 1016 O/cm2 corresponding to ∼3 × 1016 H/cm2). If we assume that this residual hydrogen is distributed over the mean depth of the depletion profile (∼30 nm), the hydrogen concentration is ∼1022 cm−3, which corresponds to ∼10 at % H in hydrogenated amorphous carbon. Thus, the observed hydrogen profiles may result from ion-induced depletion combined with postirradiation incorporation of water by diffusion. For further interpretation of the observed hydrogen depletion and film compaction profiles, computer simulations based on the binary collision approximation were performed for both 2 and 20 keV argon irradiation using the SRIM18 code. The penetrating ions deposit their energy in “electronic” collisions with the electrons and “nuclear” collisions with the 16798


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Figure 8. (a) Changes in the FTIR spectrum of a PIII-treated PS film after incubation with tropoelastin. (b) Changes in the FTIR spectrum of the PIII-treated PS film after incubation in tropoelastin and subsequent SDS washing. In both cases, the spectrum of the PIII-modified PS (Figure 2a) was subtracted to reveal the lines due to the protein vibrations (amide A, I, and II (positions indicated by dashed lines)) as well as lines that appear due to reactions of surface radicals with SDS (indicated by arrows). The spectra are baseline-corrected and have the spectrum of the uncoated silicon wafer subtracted. The polystyrene was spin-coated to a thickness of 16 nm (0.4% solution, 3500 rpm) on silicon and PIII-treated (Ar+, 2 kV pulsed bias, 20 μs, 1600 Hz, 400 s).

Figure 9. AFM topography image of tropoelastin attached to untreated PS (a,b) and to PIII treated (Ar+, 20 kV pulsed bias, 20 μs, 50 Hz, 1600 s) PS at a fluence of 1016 ions/cm2 (c,d). Image size is 328.8 nm × 332.7 nm (a), where z = 12.7 nm; and 1000 nm × 1000 nm (b) and 328.8 nm ×332.7 nm (c), where z = 6.4 nm; and 60 nm × 60 nm (d). Distribution of pixel events in area of 5 μm × 5 μm (not shown) for height in AFM image of tropoelastin on untreated (e) and PIII-treated PS (f).

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The Journal of Physical Chemistry C polymer films modified in the ways described above are able to immobilize bioactive protein molecules directly from a contacting solution. Here we show that the polystyrene films of this study have this capability using the extracellular matrix protein tropoelastin as an example, and we describe the characteristics of the surface-immobilized protein layer. Figure 8a shows the FTIR spectrum of a layer of tropoelastin adsorbed on the PIII-treated surface upon contact with a solution containing the protein, and Figure 8b shows the FTIR spectrum after washing of the sample in SDS detergent. The spectra of the modified PS layer with attached tropoelastin show new lines that correspond to the amide A, I, and II lines at 3300, 1650, and 1540 cm−1, characteristic of protein. After washing the sample in SDS, the protein lines remained in the spectra, albeit with reduced intensity consistent with the removal of all except for a surface-contacting layer of protein. In the case of untreated polystyrene, no protein lines remained after SDS washing, indicating that all of the tropoelastin had been removed by the detergent. Because the untreated surface is known to be more hydrophobic than the treated surface59 and therefore would be expected to retain physically adsorbed protein more strongly,60 our observation of protein retention on the PIII-treated surface indicates that a significant fraction of the protein on the ion treated surface was covalently immobilized.13 Additional lines at 1720, 1580, and 1450 cm−1, indicated by the arrows on the Figure, that appear after the SDS washing correspond to groups produced by chemical reaction with SDS. These are discussed in more detail in the SI. IR ellipsometric data were also acquired after incubation in protein solution and drying. Fitting an appropriate ellipsometric model to the data, as described in the SI, revealed absorption features corresponding to the amide I and amide II vibrations of the protein backbone (Figure S5 in the SI) and gave a thickness of 4.9 nm for the protein layer. The same fitting procedure was carried out for ellipsometric data obtained after the sample was washed in SDS. The fitted optical constants of the protein layer were similar to those found prior to the SDS wash, while the thickness of the layer decreased to ∼3.0 nm, indicating that a substantial fraction of the protein layer remained. Figure 9a,b shows AFM measurements of the tropoelastin layers on untreated polystyrene, indicating that the tropoelastin formed an incomplete monolayer. The AFM topography images confirm that the dry tropoelastin layer had large pits. Differences in tip interactions with the surface suggest that the troughs were PS while the elevated regions were protein. The roughness histogram shows the distribution of surface pixel events and displayed two peaks (Figure 9e,f). The 6.4 nm distance between the peaks corresponds to the average distance between the PS surface and the top surface of the tropoelastin layer; this value is consistent with the dimensions of tropoelastin monolayers and the thickness of the tropoelastin molecule.61,62 To obtain an estimate for the protein coverage, we analyzed the AFM image to obtain the fractional coverage of pits >6.4 nm in depth, and the result indicates that tropoelastin covered ∼72% of the surface. The average pit size was in the range of 150−170 nm. Similar protein islands have also been previously observed for the enzyme horseradish peroxidase (HRP) on untreated polystyrene.3,10 The tropoelastin coverage on PIII-modified PS does not have deep pits. AFM phase images (data not shown) indicate that the material in low and high regions is similar. The distribution of surface pixel events (Figure 9f) in this case has only one

peak, and the few small pits in the surface that were observed have a depth of ∼2.7 nm and narrow bases (Figure 9c,d). The AFM image was analyzed to obtain the fractional coverage of pits >2.6 nm in depth, and the result indicates that tropoelastin covered 95% of the surface. XRR was used to analyze the attached tropoelastin on the untreated and PIII-treated polystyrene. First, the measurement was done for a ∼14 nm as-prepared PS coating, before and after incubation in tropoelastin solution. In each case, attempts were made to fit XRR data using a single-layer model. Such a model proved to be appropriate only in the case of untreated PS films before the incubation, with the thickness determined to be 14.2 ± 0.1 nm with a constant SLD of (9.5 ± 0.1) × 10−6 Å−2 and a surface roughness of 0.3 ± 0.1 nm. A single-layer model did not lead to a satisfactory fit to the data of the protein incubated film (χ2 = 0.0091), but a two-layer model gave a substantially better fit to these data (χ2 = 0.0045), leading to a base layer of polystyrene (of 14.1 ± 0.1 nm thickness and SLD (9.0 ± 0.1) × 10−6 Å−2) and a 2.0 ± 0.1 nm thick layer of increased SLD ((9.4 ± 0.1) × 10−6 Å−2) at the surface of the film. On the basis of an average molecular formula for the protein of (C2747H4405N759O743S2), the mass density of the adsorbed layer is estimated to be 1.05 g/cm3. The density of protein is in a range of 1.33 to 1.42 g/cm3.46 The low density we observe must be due to incomplete coverage of the PS surface. The protein coverage calculated from the X-ray fitted data using an average value for the density of protein of 1.35 g/cm3 is 76% and agrees with the AFM measurement of 72%. A study by Le Brun et al.63 examined the structure and surface coverage of tropoelastin adsorbed on hydrophobic and hydrophilic surfaces, both in the hydrated state using neutron reflectometry and as dried films using X-ray reflectometry. These results indicated adsorbed tropoelastin monolayers of extended conformations (∼15−16 nm thick) were present in the hydrated state, while collapsed protein conformations (∼4− 6 nm thick) were present when these surfaces were dried. Such results are consistent with our above observations for an adsorbed tropoelastin monolayer on untreated polystyrene: relatively low surface coverage due to poor adhesion, meaning that when the largely unstructured protein collapses in the dry state it forms a thin adsorbed layer with low molecular density. A protein layer adsorbed on the PS film modified by PIII treatment with 2 kV argon ions for 400 s was also studied by Xray reflectometry. A very good fit of the XRR data from modified PS film prior to the protein attachment was generated with the DDP method (χ2 = 0.005), using 11 layers, giving a total film thickness of 9.9 ± 0.1 nm and a surface roughness of 0.4 ± 0.1 nm. The attachment of protein changed the observed XRR, leading to more closely spaced Kiessig fringes, indicative of an increase in total thickness. Fitting using the DDP method gave a very good fit (χ2 = 0.005) and required 13 layers of total thickness 12.3 ± 0.1 nm, suggesting a thickness of the protein layer of ∼2.4 nm. The 2 to 3 nm surface layer corresponds to a monolayer of adsorbed tropoelastin protein, having a slightly lower refined SLD of ∼12 × 10−6 Å−2 than the graphitized bulk of the PIII film. Although of similar thickness consistent with a dry and therefore collapsed configuration, the tropoelastin layer on this modified surface (1.32 g/cm3) has a significantly higher density than the layer adsorbed on the untreated polystyrene film. Using a value for the average density of protein of 1.35 g/ cm346 gives a coverage of 98%, again in good agreement with the AFM measurement of 95%. 16800



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CONCLUSIONS A combination of Raman, FTIR transmission spectroscopy, FTIR ellipsometry, X-ray reflectometry, neutron reflectometry, and nuclear reaction analysis have been used to establish a model for the depth-resolved structure of polystyrene after PIII modification. In this model, the ion implantation creates a graded structure in which the near-surface layers are highly dehydrogenated with a porous carbon structure that allows diffusion of and reaction with oxygen containing species from atmosphere, with a highly carbonized material with higher density underneath. These modified layers become more dehydrogenated, dense, and cross-linked as the ion fluence increases, and as a result, reactions with atmospheric oxygen in the surface layer are reduced after longer ion treatments. The unmodified bulk below retains the hydrocarbon structure of polystyrene. No evidence of a layer with reduced density compared with untreated polystyrene that would lead to a loss of mechanical integrity was observed. The observed increases in refractive index and SLD for Xrays and neutrons indicate a densification of the material upon ion implantation with increasing fluence. The degree of densification and the stable value of the areal density of carbon, as measured by NRA, show that etching, or the removal of material from the surface by ion impact, contributes far less to the reduction in thickness than compaction of the material due to structural modifications. After incubation of the modified polystyrene in tropoelastin solution, our analyses establish that a protein layer binds to the surface and that this layer is much denser than that simply adsorbed on untreated polystyrene. Detergent washing cannot remove all of this layer because a molecular coating of the protein is covalently bound to the modified surface.

This substantial increase in surface coverage is consistent with the results of Gan et al.,10 who observed full coverage of PIII-treated polystyrene after incubation in a solution of HRP. This was a substantial increase over the HRP coverage (only 27%)10 observed on the more hydrophobic untreated PS surface. It is also in agreement with the findings of Le Brun et al.,63 where the more hydrophilic and negatively charged (in pH 7 buffer) surface of silica adsorbed 1.75 times as much tropoelastin (7 compared with 4 mg/m2 as measured by quartz crystal microbalance with dissipation (QCM-D) monitoring) as a hydrophobic octadecyl trichlorosilane (OTS) surface. The increase in coverage was attributed to the negatively charged nature of silica in pH 7 solution, while tropoelastin is positively charged. A similar mechanism is expected on PIII-treated surfaces because they also acquire a negative charge64 in solution due to the appearance of oxygenated groups such as carboxyl on the surface after PIII treatment.49 In addition, to this charge effect the PIII treatment also endows the surface with covalent coupling capability,12 increasing the irreversibility of the adsorption. This is consistent with the observations of Yin et al.,65 who found 14% more tropoelastin adsorbed (also by QCM-D) on an ion-activated polymeric surface than Le Brun et al.63 This 14% increase is likely to underestimate the effect substantially because the flow rate Yin et al.65 used was 3000 times higher and the adsorption time was shorter, with rinsing carried out prior to saturation of the adsorbed layer’s mass. In summary, a surface-attached protein layer is clearly detected after incubation in tropoelastin solution by FTIR transmission spectroscopy, FTIR ellipsometry, and X-ray reflectometry. The presence of a polypeptide backbone in this layer is revealed by characteristic amide group vibrations in the FTIR transmission and FTIR ellipsometry spectra. Our FTIR ellipsometry (4.9 nm) and AFM (6.4 nm) thickness measurements agree with recent ellipsometry, X-ray reflectometry, and AFM measurements reported by Yin et al.,65 Le Brun et al.,63 Holst et al.,62 and Baldock et al.,66 while the values we obtained by XRR (2.4 ± 0.1 nm) indicated a thinner protein layer. The SLD obtained from XRR corresponds to the expected value for a protein layer.46 The SLD profiles are consistent with a model where the protein molecules sit on top of the coating without penetrating it, and AFM measurements support this interpretation. These two techniques also paint a consistent picture when comparing tropoelastin coverage for untreated and PIII -modified polystyrene films. Analysis of AFM data show an increase in surface coverage from 72% on the hydrophobic untreated surface to 95% on the hydrophilic and negatively charged PIII-treated surface, while X-ray reflectometry indicates an increase in density from 1.03 to 1.32 g/cm3 corresponding to incomplete protein coatings of 76 and 98% coverage, respectively. After washing with SDS detergent, FTIR-ellipsometry and XRR data indicate that the amount of protein on the treated surfaces decreases but that a substantial amount remains on the surface. This supports previous results that propose that the binding of up to a monolayer of protein to polymer surfaces modified by energetic ions can be covalent9−11 due to direct covalent immobilization by reaction with surface-embedded radicals.12

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ASSOCIATED CONTENT

S Supporting Information *

Detailed descriptions of materials and methods including materials purity data and sources, ion implantation system and protocols, and determination of fluence. Details of procedures used for thin-film characterization including variable angle spectroscopic ellipsometry (VASE), FTIR spectroscopic ellipsometry (IR-VASE), FTIR transmission spectroscopy, Raman spectroscopy, AFM, X-ray and neutron reflectometry, and nuclear reaction analysis. Details of protocols and procedures used for protein immobilization and testing of covalency. Supporting results show further data including the optical constants measured by spectroscopic ellipsometry across visible and IR wavelengths. Analysis of Raman data. Raw data obtained in X-ray and neutron reflectometry measurements together with details of fitting models used. IR ellipsometry analysis of the immobilized protein layer and further interpretation of FTIR results shown in the main manuscript. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jpcc.5b05164.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ◆

D.R.M., A.S.W., M.J., and W.M. are equal senior authors.

Notes

The authors declare no competing financial interest. 16801


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ACKNOWLEDGMENTS We acknowledge the Australian Research Council and the Australian Institute of Nuclear Science and Engineering for supporting this project.

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