In Situ Electrochemical–AFM and Cluster-Ion ... - ACS Publications

Feb 13, 2017 - relevant properties are now known. XPS and, more ... In the work now presented, we .... analyzed at the end of the electrosynthesis; th...
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In Situ Electrochemical−AFM and Cluster-Ion-Profiled XPS Characterization of an Insulating Polymeric Membrane as a Substrate for Immobilizing Biomolecules Maria E. E. Carbone,*,† James E. Castle,*,‡ Rosanna Ciriello,† Anna M. Salvi,† Jon Treacy,§ and Peter Zhdan‡ †

Science Department, University of Basilicata, Viale dell’Ateneo Lucano, 10-Potenza, Italy Department of Mechanical Engineering Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, U.K. § Thermo Fisher Scientific, The Birches Industrial Estate, Imberhorne Lane, East Grinstead, West Sussex RH19 1UB, U.K. ‡

S Supporting Information *

ABSTRACT: The electrochemical oxidation of ortho-aminophenol (oAP) by cyclic voltammetry (CV), on platinum substrates in neutral solution, produces a polymeric film (PoAP) that grows to a limiting thickness of about 10 nm. The insulating film has potential use as a bioimmobilizing substrate, with its specificity depending on the orientation of its molecular chains. Prior investigations suggest that the film consists of alternating quinoneimine and oAP units, progressively filling all the platinum sites during the electrosynthesis. This work concerns the evaluation of the growth orientation of PoAP chains, which until now was deduced only from indirect evidence. Atomic force microscopy (AFM) has been used in situ with an electrochemical cell so that PoAP deposition on a specific area can be observed, thus avoiding any surface reorganization during ex situ transport. In parallel with microscopy, XPS experiments have been performed using cluster ion beams to profile this film, which is exceptionally thin, without damage while retaining molecular information.

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INTRODUCTION This work concerns the characterization of a membrane formed on platinum and intended as an active substrate for the immobilization of large organic molecules, such as peptides and enzymes. The membrane is produced, by the electrochemical oxidation of ortho-aminophenol (oAP) on the platinum substrate in neutral media, as an adherent polymer (PoAP) of self-limited thickness.1−3 The PoAP is electrosynthesized by cyclic voltammetry (CV), and the platinum is completely passivated by 20 cycles. The average thickness of the passivating film is on the order of 7 nm as measured by X-ray photoelectron spectroscopy (XPS) using the attenuation of the Pt signal.4 That this film is suitable for immobilizing biomolecules was demonstrated by its employment as an © XXXX American Chemical Society

enzyme-entrapping layer for the production of a glucose biosensor.5 Further bioapplications as a starting substrate could be based on the presence of external functionalities able to anchor interesting biomolecules. Particularly appropriate would be the recognition of peptides prone to aggregate, the kinetics of which are based on the alignment of chains and on their mutual interactions. Interchain H-bonds between peptide groups combined with van der Waals/weak polar forces and hydrophobic and local ionic forces ensure the stability of the supramolecular assembly, which is crucial for the onset of Received: December 2, 2016 Revised: February 4, 2017 Published: February 13, 2017 A

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present work, the monatomic argon gas cluster ion source (MAGCIS) from Thermo Scientific17 has been employed, giving excellent depth resolution within the thin film thickness.

diseases of various kinds associated with irreversible protein misfolding and thus with protein malfunction.6 Amyloid peptides, for example, are those tending to assemble in the form of extended β-sheets, well-ordered supramolecular structures, whose permanency in vivo as indissoluble deposits is responsible for a human disease named amyloidosis.7 Hence, the carbonyl (and imino) terminal groups of PoAP chains can be expected to act in concert as a template surface for specific peptide sequences inducing their adsorption via multiple C O···H−N bonds, reinforced by hydrophobic interactions between PoAP aromatic units and peptide lateral aliphatic chains. Thus, the specific interactions of such films with adsorbates depend on the orientation and packing of the polymeric molecules from which they are composed, and some relevant properties are now known. XPS and, more recently, time-of-flight secondary ion mass spectrometry (ToF-SIMS) investigations have led to the hypothesis that the film composition consisted of short chains, on average 7 nm, made of alternating quinoneimine and oAP units and containing water molecules.4,8 XPS results from smaller numbers of cycles showed that the PoAP composition is the same at any number of cycles.9 Thus, the progressive coverage of the platinum sites with each cycle was postulated and confirmed by use of the electrochemical oxidation of potassium ferrocyanide to measure the remaining exposed surface as a function of the number of cycles. The most recent work8 by ToF-SIMS analysis of the PoAP/Pt interface in the early stage of polymer formation showed that polymer chains tend to interact with the electrode surface preferentially through the nitrogen atom of the oxidized monomers. ToFSIMS as a function of the number of cycles through to full passivity showed that the nitrogen-containing fragment attached to Pt decreases with increasing surface coverage, as it becomes masked by the clustering of polymeric molecules progressively adsorbed on neighboring platinum sites. As this basal fragment decreases in intensity, with coverage, fragments associated with the outer terminal groups increase in intensity.8 Some questions left unanswered concern the growth orientation of polymer chains. Because of its very small thickness, the vertical alignment of polymer chains was deduced only from indirect evidence. In the work now presented, we have attempted to obtain direct evidence for the probable alignment. Atomic force microscopy (AFM) has been used in situ with an electrochemical cell so that PoAP deposition on a specific area can be observed, thus avoiding any surface reorganization during ex situ transport. In parallel with microscopy, XPS depth profiles of the polymeric film were acquired using a cluster ion beam. Monoatomic ion guns mounted on X-ray photoelectron spectrometers are frequently used for depth profiling to determine the distribution of various chemical compounds. However, when a standard Ar+ ion beam was used on the PoAP film, the chemical composition was significantly altered at the earliest stage of etching, and a depth profile analysis did not give reliable information. Cluster ion beams have been shown to overcome such problems, having the ability to profile organic materials while retaining molecular information.10 Several published studies10−16 have already highlighted the ability of these beam sources to provide a depth profile of organic materials with minimum damage. Cumpson et al.14 demonstrated how the use of argon cluster sources is crucial for the characterization of important biomaterials, emphasizing the opportunity to probe interfaces between organic deposits and inorganic substrates. In the

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EXPERIMENTAL SECTION

Chemicals. A monomer of o-aminophenol (oAP) was obtained from Sigma-Aldrich (Germany) and purified by recrystallization in ethyl acetate.18 All other chemicals were of analytical grade and were used without further purification. Monomer solutions were prepared in a supporting electrolyte just before their use. Pure water supplied by a Milli-Q RG unit from Millipore (Bedford, MA, USA) was used throughout. Electrochemical Apparatus and PoAP Deposition for Ex Situ Characterization. Electrochemical experiments were performed using an Autolab II potentiostat/galvanostat (Utrecht, Netherlands) equipped with GPES version 4.9 software (EcoChemie B.V.) for data acquisition and potentiostat control. The electrochemical cell was a standard three-electrode system with a Pt counter electrode, a saturated KCl, Ag/AgCl reference electrode, and a working electrode consisted of a platinum foil (10 × 15 × 0.250 mm3). Pt working electrodes were cleaned by following the optimized procedure described by the authors.19 A PoAP film was electrosynthesized at room temperature by CV using a 5 mM oAP solution in phosphate buffer (KH2PO4/NaOH, I = 0.1 M, pH 7). No action was taken to remove oxygen from solutions. The potential was scanned between −0.1 and +0.9 V (vs Ag/AgCl, KCl saturated) at a scan rate of 50 mV/ s for 20 cycles. The electrodeposition ended at the reduction potential. The sample was then washed with double-distilled water and dried at room temperature in a nitrogen atmosphere before its ex situ analysis. XPS Depth Profile Measurements. The XPS depth profile analysis of the electrochemically synthesized PoAP film was carried out using a Thermo Scientific K-Alpha instrument with microfocused monochromatized Al Kα radiation (hν = 1486.68 eV), a 180° doublefocusing hemispherical analyzer, and a 128-channel detector. The Xray source operated at 12 kV with an emission current of 6 mA (72 W) and a spot size of 400 μm on the sample. A dual-beam flood gun was used to compensate for surface charging during the analysis: an electron beam current of 150 μA and Ar+ ions at 0 eV minimized the possibility of sample degradation. No charge correction was applied, and the energy scale was calibrated using metallic Au, Ag, and Cu. A pass energy of 50 eV and fixed analyzer transmission (FAT) operation mode with channel widths of 0.1 eV were employed. Profiling of the thin insulating PoAP was performed by MAGCIS using 4 keV argon cluster ions with an average cluster size of 2000 atoms. A raster size of 1 mm was used, and the beam current was calibrated to 7 nA. The sample was attached to a standard K-Alpha sample holder using a copper clip to ensure good electrical contact between the plate and the sample surface. Alternating stages of sputtering and XPS analysis enabled an accurate in-depth composition of our polymer. The duration of the etch cycles were 10 s for the first 25 levels, 30 s for 9 levels, and finally 60 s per cycle for the last 3 levels. The estimated sputter rate varies from 0.093 nm/s at the beginning of etching and then decreases to 0.001 nm/s once the Pt substrate is exposed (vide infra). The acquired spectra were elaborated using Shirley background subtraction and peak area normalization with proper sensitivity factors, as provided by Thermo Scientific Avantage software. Electrochemical−Atomic Force Microscopy (EC−AFM) Analysis. The EC−AFM experiments were performed with a Nanoscope III atomic force microscope (Digital Instruments, Santa Barbara, CA, USA) using a J scanner with a maximum scan size of 100 μm interfaced to a VMP3 potentiostat/galvanostat (BioLogic Science Instruments, Claix, France) controlled by EC-Lab software (version 10.2x). Triangular silicon nitride cantilevers covered on one side with a Au mirror (NT-MDT, tip curvature radius of about 10 nm) with a spring constant of 1.45−15.1 N/m (manufacturer’s data) and rectangular Si cantilevers (Nanosensors, Wetzlar-Blankenfeld, Germany) with a nominal resonance frequency and force constant of 10− 17 kHz and 0.07−0.29 N/m, respectively, were used for imaging in contact mode in a 5 mM oAP solution. In the electrochemical cell B

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Figure 1. Static EC-AFM experiment: topography of PoAP/Pt electrode obtained for 1 (A), 5 (B), and 20 (C) voltammetric scan cycles in the presence of a 5 mM monomer solution in phosphate buffer. Scan size 20 μm × 20 μm. The corresponding section analysis taken along the white lines in topographic images is reported, respectively, in panels A′−C′. employed: the average roughness, Ra, and the root-mean-square roughness, RRMS.24

provided with the AFM instrument, there is a provision for placing counter and pseudoreference electrodes (Pt wires), whereas a conventional Pt disk was used as the working electrode. Platinum substrates were polished to a submicrometer diamond finish. Electrolyte insertion was done from a separate aperture in the cell. Electrosynthesis of the insulating PoAP was carried out by CV as already described. Because the polymer morphology is related to the voltammetric scan rate, 50 mV/s was adopted in order to allow a direct comparison with our previous ex situ results.9,19 AFM images were acquired and analyzed using the commercial Nanoscope software (version 4.0, Digital Instruments, USA) implemented by the auxiliary Gwyddion program20 for more profound processing of the AFM data. Comparing the recorded images, it is also possible to note a slight movement in the scanned region. This is a consequence of the thermal drift, inherent in microscope piezoelectric scanners, which was impossible to avoid during the long time of experiments in the electrochemical AFM cell.21 However, easily recognizable features could be located throughout a complete set of images, allowing for detailed analysis of selected areas. Cai et al.22 have reported that during in situ EC-AFM experiments a scanning or oscillating AFM tip can influence the morphology and the kinetics of synthesis over the scanning area. This effect is more evident at high interacting forces between the tip and the sample and is enhanced by inducing an oscillation in the AFM tip. During the course of our contact AFM experiments, using the approach proposed by Suarez et al.,23 we did not observe a significant difference in the PoAP morphology between the scanned area and an adjacent region analyzed at the end of the electrosynthesis; therefore, we are confident in asserting that the tip does not affect the polymer deposition or damage the obtained film even though contact mode was adopted. The surfaces of bare Pt and of polymeric deposits were characterized by 512 × 512 pixel images with usual areas of 20 μm × 20 μm. Two different methods for roughness evaluation were

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RESULTS AND DISCUSSION In Situ EC−AFM Growth of Insulating PoAP. The majority of the research on the PoAP structure and morphology has been based on ex situ experiments after washing, drying, or treatment under vacuum on the polymeric films.1,2,4,8,9,19 Here, an in situ EC−AFM investigation of the stages of its electrochemical deposition on platinum electrodes was attempted because it affords fundamental insight into the simultaneous study of the surface morphology and growth mechanism.25 PoAP was electrochemically synthesized on Pt as described in the Experimental Section, and two different approaches for the morphological image registration were adopted. With the expression “static mode”, we refer to the AFM images recorded in the electrolyte solution shortly after each voltammetric scan cycle. Thus, an alternation of electrochemical growth and morphological characterization steps was repeated, obtaining a representation of the sample after each CV scan in the same spatial region of the electrode surface, except for a small shift due to the drift of the piezoelectric scanner. In this way, subsequent images well account for the details of the PoAP growth. AFM images of the bare electrode surface were also recorded. These results were compared with those obtained in dynamic mode in which the recording of images and the electrosynthesis of the insulating PoAP occur simultaneously. In dynamic mode, the AFM tip scans during the polymerization allowing a morphological image to be obtained that is C

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the scanned area and then study the variation of the morphology exactly on the same section line, as shown in Figure 2. Here, deposited polymer chains do not grow in height proceeding with CV cycles, and only smoother and slightly broadened profiles are seen in the 5th cycle and further refined at the 20th cycle, demonstrating that, along the selected line, “nearly” all platinum sites were filled by PoAP chains after the first scan. A comparative examination of the section analysis before starting the electrosynthesis (Figure S1B) and during the polymerization scans (Figures 1 and 2) definitely proves that small differences in the length measurements of PoAP chains are expected because of the intrinsic roughness of the starting Pt electrode, as evaluated and reported in Table 11. Furthermore, because the images are composed of arrays containing the xyz values for each pixel, it is possible to create an image in which the xy values are aligned and the z values, i.e., the height of each point of the image, are obtained by the subtraction of successive images. In Figure 3A, the difference image of 10 and 5 voltammetric scans along the same apportioned area is reported. From this image, given the reduced influence of the platinum surface structure and of other deposit irregularities, it is possible to appreciate the progress of film deposition noting some light areas ascribable to new PoAP chains bonded to the electrode substrate. The subtracted image was further studied with section analysis (Figure 3 B) by focusing our attention both on a dark zone (profile 1), where there has been only a very small change between the 5th and 10th cycles, and on light zones (profiles 2 and 3), which are typical locations where there has been substantial growth. Measuring the height of these light features, we obtained values comparable with the film thickness (in each case, about 10 nm in height), again in agreement with our suggestion on the mechanism of polymer deposition. These observations have been confirmed by measurements of the volume of the deposit, taking the entire 400 μm2 area into account. Figure 4 shows the volume lying above base planes set at three threshold levels above the minimum value of height in the micrographs. The plot clearly shows that the increase in the deposit takes place between the 5th and 10th cycles and that the volume measured increases as the threshold is lowered to the level of the substrate. The threshold setting at 5 nm captures the bulk of the volume of material deposited during the cycles; raising the threshold levels, it is evident that much of the deposit exceeds 7 nm in height, but there is a little deposition lying above a threshold of 10 nm. The volume decreases a little after 10 cycles, and there is also a small decrease in the first cycle. Such reductions may be apparent rather than real: the infilling of valleys and small features in the Pt surface by adsorbed material raises the reference depth for the threshold plane, and thus the observed reduction corresponds to a general flattening of the underlying substrate. However, the volume decrease between 10 and 20 cycles may also be a reflection of polymer refinement, as revealed by Figure 2C, due to a sort of compacting of the polymer chains occurring in the final stage of cycling, most likely as a consequence of the complete detachment of short segmented oligomers deposited in previous scans. Thus, the gradual deposition of polymer chains, of about 10 nm in length, on the underlying bare platinum electrode occurs until all available sites are occupied and the steric hindrance of neighboring chains prevents further deposition.

dependent not only on the spatial coordinates but also on the elapsed time and consequently on the applied potential. Static Mode. Each experiment started with the acquisition of an image of bare platinum assembled in the electrochemical cell containing an electrolytic solution of oAP (5 mM in phosphate buffer): a starting image of the platinum electrode surface in the electrolyte solution with the corresponding section analysis is reported in the Supporting Information (Figure S1) and shows slight scratches and defects that can be considered to be typical of bare Pt. The general 2D view of the progressively modified PoAP/Pt electrode, obtained by increasing the voltammetric scan cycles, illustrated the gradual covering of the platinum surface during polymerization. Among all of those collected, some selected images are presented in Figure 1 to give the best account of the evolution of the surface profile with the deposit and to allow comparison with the images acquired ex situ.9 Interestingly, the in situ comparison has further unveiled characteristic aspects of PoAP deposition that were already noticeable in ex situ experiments. All AFM images are affected by the topography of the underlying platinum electrode because of the thinness of the film4,8,9 and show a general smoothing and broadening of features with an increase in the number of cyclic voltammetric scans. Moreover, in situ recording of the same substrate areas also confirms the sporadic presence of simultaneous precipitates being the result of the kinetically induced deposition of suspended oligomers, favored by repeat cycles, that would eventually desorb with time after a thermodynamically driven refinement of PoAP chains. These precipitates were likely trapped on the substrate surfaces after the sudden interruption of potential scans and were generally recognized as patches in ex situ images.9 All occurring phenomena contribute to any quantitative measurement performed over the full area of the micrograph as a function of the number of CV cycles. A confirmation is given by the roughness data reported in Table 1, which moderately increase with cycles, particularly between the 5th and 10th, accounting for the limited thickness variation of the deposit. Table 1. Static EC-AFM Mode: Ra and RRMS Roughness of the PoAP/Pt Electrode after Various CV Cycles CV cycles

Ra (nm)

RRMS (nm)

0 1 5 10 20

2.3 2.2 2.4 2.9 2.9

2.9 2.9 3.0 3.8 3.7

On the basis of these premises, the morphology of PoAP after the 1st (Figure 1A), 5th (Figure 1B), and 20th (Figure 1C) CV scans is shown, with their relevant section analysis, for selected lines of the scanned areas that are differently located but similarly visually unaffected by unwanted patches. Remarkably, from the height profiles provided it is possible to note that, from the first cycle, there is a deposit that is about 10 nm tall that does not increase in height during the electrosynthesis. On increasing the number of CV scans, we observed only a progressive filling of platinum sites by the polymer chains with about the same height at any cycle, suggesting their vertical alignment. Using the same criteria, notwithstanding the xy drifts in the AFM images, it was possible to recognize the same features on D

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Figure 2. Static EC-AFM experiment: topography and section analysis on exactly the same line of the PoAP/Pt electrode obtained upon 1 (A), 5 (B), and 20 (C) voltammetric scan cycles in the presence of a 5 mM monomer solution in phosphate buffer. Scan size 20 μm × 20 μm. The corresponding section analysis taken along the white lines in topographic images is reported, respectively, in panels A′−C′.

Figure 3. Static EC-AFM experiment: difference image of PoAP obtained by subtracting the image relevant to 5 voltammetric scan cylces of growth from that of 10 cycles after a proper correction for xy drift (A) and the relative section analysis (B).

This result is completely different from the evidence reported in the literature23,26 on the mechanism of the unrestrained growth of conducting polymers, such as polybithiophene and polypyrrole. For these, in situ AFM measurements showed that the polymerization proceeds through the preferential growth of initially formed active nuclei. In an early stage, the polymeric clusters fuse into one another covering the whole electrode surface. This three-dimensional growth mechanism prevails in the first stages of polymer deposition, whereas after a compact polymer layer has formed on the Pt substrate, bidimensional growth is preferred.26 In this connection, Tucceri27 has recently asserted that, during the synthesis of PoAP film in the conductive mode, two or more stages of the polymerization process are also distinguishable: in the first, islands of the polymer are formed at the substrate surface, and then a continuous film, which is compact and nonporous, is formed by

the fusion of these islands. Further growth takes place above this compact layer, giving an external porous part of the film. The reason for this difference in behavior between conducting and insulating polymerization modes is ascribable to the electrical insulating properties of the latter, which prevent any further growth. As reported in previous work, different polymerization mechanisms were derived for insulating and conductive PoAP, depending on the pH of electrosynthesis.4,28 In neutral media, the polymerization route starts with oAP oxidation to quinoneimine with subsequent monomer addition. The derived structure, made of alternating quinoneimine and monomer units, does not provide a conjugation able to promote electron hopping, thus justifying the insulating behavior of the polymer, revealed by the full abatement of its CV profile4 and proved by permeability tests carried out with redox probes.5 In acidic E

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Figure 4. Static EC-AFM experiment: the estimated volumes of polymer chains for different threshold heights (5, 7, and 10 nm) as a function of the voltammetric scan cycles.

Figure 5. Dynamic EC−AFM experiment: morphological images registered for a platinum electrode before (A) and during the first CV scan (B) of PoAP electrosynthesis. The relative section analysis area is reported respectively in panels A′ and B′. It should be noted that the y-axis scales are different for the two images because of the polymer deposition. The AFM scan time needed for each image was 40 s.

as the thickness of the finite film. Therefore, it is suggested that, soon after the oAP oxidation, a fast chemical process takes place to form chains of about 10 nm of length. This kind of ECAFM approach further corroborates the hypothesis of their vertical alignment. It should be considered that, after the first few cycles, a progressive loss of resolution was found. This could be rationalized in terms of the high AFM scan rate employed, which is necessary to achieve an image representative of each CV cycle, but also because, during electrosynthesis, troubles typical of EC-AFM analysis may occur. It is well known that an experimental problem is the stability of the laser signal that could consequently influence the AFM image quality. Probably the movement of the suspended oligomers over the cantilever induced scatter in the AFM laser beam, compromising its reflection on the photodiode detector.23 Ar Cluster Ion Depth Profiling of Insulating PoAP. The main aim of this investigation was to determine if any interpretable oscillation of the carbon/nitrogen/oxygen ratio could be observed in the etch profile. As will be shown later, the possibility of structure in the profile that would point to a vertical alignment could be envisaged. The spectral superimposition of PoAP/Pt at different level of etching is shown in Supporting Information Figure S2. The comparison of these XPS spectra revealed a progressive attenuation of C 1s, O 1s, and N 1s intensities and an increase in the platinum signal during the etching. It is worth noting

media, monomer oxidizes to radical cations. Their coupling gives linear dimers that fill all suitable Pt sites and undergo cyclization.28 Polymerization proceeds by the addition of oxidized monomers to the initial covering layer. The conjugated phenazine-like closed structure gives reason for the conducting behavior of the film, characterized by reversible oxidation/reduction processes assisted by deprotonation/ protonation steps.29 Dynamic Mode. EC−AFM was also employed for the simultaneous film deposition and AFM characterization in a dynamic experiment, which allowed us to better understand the interaction of the polymer with Pt, focusing our attention especially on the early stages of polymerization. Indeed, our previous investigation5 performed by electrochemical quartz crystal microbalance (EQCM) has shown a net mass increase on the platinum electrode during the first voltammetric scan; therefore, the evolution of platinum morphology was sought to be better investigated by EC−AFM at the beginning of polymer deposition. In Figure 5, the acquired images and the relative section analyses exactly on the same position of the platinum electrode before (A) and during the first CV scan (B) of PoAP electrosynthesis are reported. Considering also the measured height profile, it is clearly evident that the deposition of the film starts in the first half of the image, i.e., during the first anodic scan, after the initial monomer oxidation. PoAP chains, bonded to the underlying platinum in this stage, have the same length F

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Figure 6. XPS curve-fitted detailed C 1s, O 1s, and N 1s regions acquired for PoAP after 50 s of etching.

components, a new one, O3 at 534.0 eV, was clearly identified and associated only with H2O content. Thus, of the total content of water, one part is still incorporated under peak O2 and the remaining part is represented by peak O3 at a slightly higher BE. Importantly, the interactions between PoAP chains and water, previously advanced by curve fitting the achromatic spectra,4,9 are better resolved here using the monochromatic source. Moreover, the confirmation of aminic (N1) and iminic (N2) components of equal intensity in the N 1s region, used as the reference region for the overall semiquantitative analysis, further supports the proposed PoAP composition. To compare the PoAP composition at different levels of etching, the spectral quantification was based on the total area under each region, evaluated using Avantage software, and the polymer and substrate composition so obtained are plotted in Figure 7. The curves show the smooth increase in the Pt

that, consequently, there is also a change in the shape of the background associated with individual photoelectron peaks. However, the spectral features ascribable to the polymer are still evident after long sputtering times. To determine if argon cluster ion sputtering can remove material without causing significant subsurface damage, the spectra after the fifth etching cycle (i.e., 50 s) were curve fitted (Figure 6), and the obtained values are reported in Table 2. These may be compared to Table 2. Curve-Fitting Results of PoAP after 50 s of Etching BEs (eV)

FWHMs (eV)

corrected areas (a. u.)

At %

assignments

C 1s

284.8 285.5 286.4 287.4 288.6

1.22 1.22 1.22 1.22 1.22

O 1s

531.4 533.0 534.1

1.74 1.46 1.46

N 1s

399.1 400.0

1.72 1.72

Pt 4f

71.4 74.7

1.11 1.11

159 318.80 63 727.60 111 523.20 31 863.76 7965.96 374 399.32 29 543.94 38 436.97 26 551.82 94 532.73 28 966.03 31 572.97 60 539.00 29 150.91 21 108.82 128 232.05

43 17 30 8 2 100 31 41 28 100 48 52 100 23 77 100

Carom C−N−C C−O−C/CN CO CO···H2O CTOT CO C−O−C/H2O H2O OTOT CN C−N NTOT Pt 4f7/2 Pt 4f5/2 PtTOT

those obtained by XPS of unetched surfaces.4,9 It is worth noting that overall and cross-checked mass balances gave the same chemical composition as proposed in the previous works,4,9 confirming the weak damaging effects of the argon cluster ion gun. Furthermore, because of the monochromatic source, some spectral details could be better resolved and new small features could be noticed. In the C 1s region, for example, a small peak at 288.6 eV due to the interaction of carbonyl terminal groups with water molecules through H-bonds is present. This additional signal was not clearly observed in the previous studies4,9 probably because of its low intensity, which made it indistinguishable from the spectral background. Another difference could be envisaged in the O 1s region, where two components were previously identified, with the first one, O1, assigned to carbonyl groups and the second peak, O2, which was broader, assigned to a combination of an ether-like group and water oxygen.4,9 Here, in addition to O1 and O2

Figure 7. Evolution of elemental composition during etching. The left scale of the y axis represents the elemental atomic percentage of polymer. The right scale of the y axis represents the substrate percentage, fixed to 100% Pt intensity at the end of etching.

substrate signal as etching progresses. Carbon, nitrogen, and oxygen intensities decrease progressively with the Pt increase. Using the proper inelastic mean free path value,30 it was possible to calculate the sputtering rates from the change in Pt 4f intensities: it is 0.093 nm/s between 50 and 100 s, i.e., at the beginning of etching, and a progressive decrease was observed because a value of 0.001 nm/s was estimated at around 300− 600 s. The final elemental ratio corresponds to the formula composition of the monomer, C/O/N = 6:1:1. At early stages G

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Figure 8. Deduced kinetics of etching of PoAP/Pt estimated as the evolution of O 1s/N 1s (A), O1/(O2 + O3) (B), C 1s/O 1s (C), and C 1s/N 1s (D) ratios over etching time.

1.25. Finally, the O1/(O2 + O3) ratio reaches a value of 1.0 when all of the water is removed from the polymer film (O/N ≈ 1.0). As a confirmation of this finding, the curve fitting of two additional spectra, at 130 and 210 s, representative of the second and third ranges, respectively, is reported in Figure S4 and the relevant data are reported in Table S3. It is clearly evident that the progressive loss of water causes a decrease in O2 intensity and the abatement of the O3 signal. In the last stage of etching, the remaining oxygen peaks, O1 and O2, have the same area, as expected from the proposed structure of the PoAP repetition unit. In addition, carbon peak CO···H2O at the highest binding energy (288.6 eV) is absent. Similar behavior of the C 1s/O 1s ratio is shown in Figure 8C. The theoretical ratio of 4 (6/1.5) is observed after a few instants of etching time as a result of the removal of carbon contamination. The red line interpolating the experimental ratio shows that this value remains constant for a certain time but after 200−250 s it increases to about (6.5), in agreement with the value expected from the polymer structure without the excess water. In Figure 8D, the C 1s/N 1s area ratio is reported as a function of the etching time. In fact, at the beginning of the experiment, the measured carbon is slightly higher than the expected value of 6 because of contamination, probably located on top of the polymer. On the basis of standard methods,30,31 the excess carbon corresponded to a contribution to the total thickness of 0.3 nm. This is negligible, and in any case, the

of the etch, it is clear from the plots that the concentrations of N and O are not equal but that oxygen is present in excess. The trend in the O 1s/N 1s ratio with the etching time is seen clearly in Figure 8A. Theoretically, the presence of water inside the film increases the O 1s/N 1s ratio from 1 (for the monomer) up to 1.5. This oxygen excess, already justified in terms of water entrapped in polymer chains,4 is removed in two consecutive stages. Indeed, the average O 1s/N 1s ratio (red lines in Figure 8A) goes from an initial value of about 1.5 to about 1.25 in the first 120−130 s and then to the theoretical value of 1 after 200 s. Thus, it seems that two different kinds of interactions between PoAP and H2O molecules coexist in this thin film. The significance of the water is its role as a binder between adjacent polymer strands, able to hold them in place and giving an opportunity for more permanent molecule-tomolecule hydrogen bonds to form, essentially giving a zipperlike mechanism. The two stages suggest that as the density of polymer increases at the base, water is progressively expelled but remains in place at the lower-density surface. To evaluate changes in the oxygen components, the ratio O1/(O2 + O3) over etching time was examined, as shown in Figure 8B. Water contributes to the oxygen in both the O2 and O3 peaks (Figure 6 and Table 2), and its presence determines the value of the denominator in this ratio. The ratio varies from an initial value of about 0.5, representative of the usual content of water trapped between PoAP chains, to about 0.7 in the second range of etching, i.e., corresponding to the O/N decrease from 1.5 to H

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Langmuir

electrode, at which point steric hindrance between neighborhood chains prevents further PoAP deposition. The result of this work, that chains are oriented perpendicularly to the surface, implies that the reactive end groups are available for biomolecule capture. In perspective, the employment of PoAP can be envisaged either as a biosensor for peptide aggregates or by preadsorbing or imprinting the given peptide sequence to catalyze the aggregation of similar sequences by self-recognition assembly. In the next phase of work, this will be investigated using combined surface techniques such as AFM and XPS.

average C 1s/N 1s ratio (red line in Figure 8D) remains very close to the expected value of 6 as etching proceeds. Really, the final average ratio is slightly higher than the theoretical one because of oscillations resulting from inconsistencies in the automatic measurements of the peak area of the carbon envelope. This is possibly because the overall width of the C 1s components is greater than that of the other elements, which might have given a slight error in measuring the full intensity. Nevertheless, the achievement of a stoichiometric ratio approximately equal to that of the monomer (C/O/N = 6:1:1) at this point in the profile confirms that a monolayer consisting of the monomer structure is present on the electrode surface. The Pt etch profile Figure 7 shows that by the time the monomer ratio is achieved so also the Pt signal approaches its maximum value. We assume that when the sputtering is prolonged almost all of the polymer has been removed and etching is no longer effective and probably just the first monolayer, at the inner PoAP/Pt interface, remains strongly bound to the electrode surface. It has a monomer-like ratio in agreement with our previous ToF-SIMS results.8 In conclusion, the distribution of water molecules in the structure is good evidence of the alignment of PoAP chains perpendicular to the substrate. The observation made feasible by the use of cluster ion etching provides a useful example of the etching efficiency of an argon gas cluster ion source: the preservation of chemical PoAP features through the profile is a typical indicator of the minimal damage effects from the MAGCIS source.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b04335. Static EC-AFM experiment, stacked plots relevant to C1s, O 1s, N 1s, and Pt 4f spectra, curve-fitting results of PoAP after etching, and XPS curve-fitted detailed C 1s, O 1s, and N1s regions acquired for PoAP after etching (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

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James E. Castle: 0000-0002-2949-6076

CONCLUSIONS Chain orientation and the alignment of electrosynthesized PoAP on Pt electrodes was evaluated by in situ EC-AFM and argon cluster XPS depth profiling, showing that both methods are valuable for the study of thin insulating polymer films, especially during the early stages of their growth. In situ AFM was successful, giving direct evidence of PoAP deposition and registering images in liquid, thus avoiding surface reorganization that could occur during ex situ transport. It revealed that from the first cycle of PoAP electrosynthesis there is a deposit of about 10 nm in height that does not increase during the following voltammetric scan cycles, showing that the formation of polymer chains occurs soon after the initial monomer oxidation. Increases in the surface roughness and volume of polymer chains, particularly between the 5th and the 10th cycles of electrosynthesis, were revealed, probably ascribable to the elapsed time necessary for PoAP refinement. The subtraction of AFM images within this range of cycles, by reducing the influence of initial features on the Pt surface and of deposit irregularities, has confirmed the estimation made in prior work on the PoAP thickness and its morphology. Cluster ion profiling enabled XPS spectra to be obtained throughout the whole thickness of the film. These confirmed the strong attachment of the polymer to the Pt surface, where the residual signal was the same as that of the monomer. They showed the presence of tightly bound water molecules within the structure and added new information, of a greater fraction of these molecules in the outer region of the polymer. These new results add information, suggesting that the short PoAP chains are vertically aligned and progressively fill all platinum sites until a homogeneous compact layer is formed on the

Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.langmuir.6b04335 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.6b04335 Langmuir XXXX, XXX, XXX−XXX