Iridium Oxohydroxide, a Significant Member in the Family of Iridium

Article pubs.acs.org/JPCC

Iridium Oxohydroxide, a Significant Member in the Family of Iridium Oxides. Stoichiometry, Characterization, and Implications in Bioelectrodes A. M. Cruz,† Ll. Abad,† N. M. Carretero,† J. Moral-Vico,† J. Fraxedas,‡ P. Lozano,§ G. Subías,∥ V. Padial,⊥ M. Carballo,⊥ J. E. Collazos-Castro,⊥ and N. Casañ-Pastor*,† †

Institut de Ciència de Materials de Barcelona, CSIC, Campus de la Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain ‡ Centre d’ Investigació en Nanociència i Nanotecnologia, CIN2-CSIC-ICN, Campus de la Universidad Autónoma de Barcelona, E-08193 Barcelona, Spain § Instituto Universitario de Investigación en Nanociencia de Aragón (INA) Edificio Interfacultativo II, Universidad de Zaragoza, C/Pedro Cerbuna 12, E-50009 Zaragoza, Spain ∥ Instituto de Ciencia de Materiales de Aragón, CSIC-Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain ⊥ Hospital Nacional de Parapléjicos, SESCAM, Finca La Peraleda s/n, 45071 Toledo, Spain S Supporting Information *

ABSTRACT: Iridium oxohydroxide thin coatings have been prepared by a dynamic oxidation electrodeposition method from complex oxalate solutions that induce template effects in the final coating at the nanoscale. The preparation method induces the formation of a oxohydroxide with reproducible stoichiometry and sponge-like quasiamorphous open structure, high ionic mobility, and significant behavior as compared with other reported iridium oxides as derived from X-ray diffraction, XPS, and TGA. Reproducible mixed valence states are also observed and a local rutile structure that allows ion exchange and facile redox changes. Rather significant is the large affinity for organic compounds observed and the behavior as substrate for cell culture, the best observed to date. Optimal cell response seems to be related to such open structure, which suggests this coating as ideal for devices implanted in the nervous system.

1. INTRODUCTION Electrostimulation electrodes implanted in biological systems are subject to voltage and charge capacity limitations due to the secondary reactions that occur at their interface, mainly radical formation. Also, impedance effects yield a heating response that damages biological tissue.1−10 Usual materials are inert noble metal electrodes and their alloys,11,12 whose charge is stored in a capacitive way, but it has been clear that other materials like iridium oxide allow an increase in that charge capacity. Electroactive intercalation materials undergo charge transfer reactions themselves, and thus, phases like iridium oxide behave in a faradic way, increasing the charge capacity that can be delivered by 1 order of magnitude, without damage to © 2012 American Chemical Society

biological tissues. In recent years an attempt has been done on studying the effects of electric field application in possible regeneration of tissues in the nervous system. Significantly, guiding axon and dendrite growth has been proven using external electrodes, a significant fact in possible future tissue repair.13,14 However, if that is to be applied in a living organism, electrodes need to be implanted and will suffer the same compatibility criteria than functional electrostimulation electrodes. They must be safe for the cellular media in which they are Received: December 20, 2011 Revised: January 30, 2012 Published: February 1, 2012 5155

dx.doi.org/10.1021/jp212275q | J. Phys. Chem. C 2012, 116, 5155−5168

The Journal of Physical Chemistry C

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70 mm pieces) coated previously with a Ti (5 nm) adhesion film. IrOx thin films were obtained by constant current methods, as reported by Petit et al.,27 and also by a new dynamic potential sweep method. In both cases, the solution was prepared from 0.2 mmol of IrCl3.H2O (Aldrich 99.9%), 1 mmol of oxalic acid, H2C2O4.2H2O (Aldrich 99%), and 5 mmol of K2CO3 (Aldrich 99%) dissolved in water in that sequence. The final 50 mL solution has a pH of 10. The solution was kept at 37 °C for 4 days and stored at 4 °C prior to use. The aging period changes notably the color of the solution and the UV− vis spectra, showing a change in the iridium ion coordination sphere. A VMP potentiostat (Biologic) is used for electrodeposition control. A three-electrode cell system is used: a platinum counter electrode and a working electrode with the same dimensions and a Pt quasi-reference electrode with a potential of 0.0 V vs Ag/AgCl. Positive and negative electrodes are placed in a parallel arrangement by two Teflon pieces that keeps a reproducible distance of 1 cm between electrodes assuring reproducibility in the applied electric field and, therefore, in the resulting coating. Thin films were obtained by two different methods: the previously reported method based on galvanostatic control27 at (35 μA/cm2) and by a new potentiodynamic protocol involving 50 or 100 potential sweeps with speeds of 2 or 10 mV/s between open circuit potential (near 0.0 V) and 0.55 V vs Pt. The thickness of the coating was kept below the transparency limit for further use in cell culture.17 Some samples were also reduced in saline sodium phosphate buffer 0.1 M, pH 7.35, (2.5 g/L NaCl added), in K2CO3 0.1M, and in presence of L-PLL solution (4 mg/mL), using several potentials or currents related to CV observations. The resulting films were studied by X-ray grazing angle diffraction (GIXRD) carried out at 0.3°, 0.35°, and 0.5° incidence angles on a Siemens D500 Diffractometer equipped with parallel beam attachment in the diffracted beam and using 0.05° step in data acquisition. The same diffractometer permitted X-ray reflectometry and thin films thickness measurement. Thermal treatment of samples was performed to evaluate structural changes up to 600 °C. For that purpose some samples were prepared on quartz coated slides of the same dimensions and with the same metal bilayers, and treated with heating ramps of 10 °C/h up to 600 °C in air. TGA analysis of the deposited materials were carried out on a PerkinElmer TGA7 in air (25 to 300 °C) and in argon/H2 (5%) (25° to 600 °C) at speeds of 2 °C/min with ambient temperature and final temperature plateaus to evaluate water weight loss and possible reduction to metallic iridium. Scanning electron microscopy (SEM) was carried out on QUANTA FEI 200 FEG-ESEM at 5 kV and a pressure of 50 Pa, to contrast the thickness evaluation made by X-ray interferometry, without metalizing, with coherent results. X-ray photoelectron spectroscopy (XPS) measurements were performed at room temperature using an AXIS Ultra from Kratos hemispherical analyzer with monochromatic Al KCuα radiation. XAS measurements at Ir L3 edge were performed on BM29 beamline at the ESRF (Grenoble, France). X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectra were recorded at room temperature in fluorescence mode using a 13-element Ge solid-state detector. Minimizing Pt Lα fluorescence line from the substrate, partially overlapping with Ir Lα fluorescence line, incident X-rays are at a glancing angle of 0.15° (above the critical angle) with respect to the sample surface were used. The Ir L3-edge energy, i.e., 11.220 eV, was calibrated using

implanted and create no secondary effects like radical formation or heating due to impedance effects. In that sense, in the search for possible nerve regeneration methods, neural stimulation using gold-based cuff electrodes have been tested, using different coatings.15 It is significant that new electroactive materials like iridium oxide is also being tested in a first stage as substrate for neural growth,16,17 along with other oxides like TiO2.18 Carbon nanotubes and also organic conducting materials such as polypyrrole and poly(3,4-ethylenedioxythiophene) (PEDOT) have been studied as possible candidates, although the cell culture observations are rather contradictory depending on the counterion they carry.19−23 It is significant, though, that in IrO2 each preparation method may yield a substantially different material. Several procedures such as sputtering,24 chemical anodization of the metal Ir,25,26 or electrodeposition from Ir(III) or Ir(IV) salts27,28 have been described, and cell studies done only in sputtering materials. Although all of the resulting materials are being considered the same in literature, some features show that this is not the case,11,16,28−32 including the information reported here. Mostly they seem to differ in purity (Cl residues are very common),33 homogeneity, oxygen content and structure.34 The final redox state and therefore electrochemical properties, charge capacity and impedance at the interface, are highly dependent on the preparation method and the resulting structure. High temperature physical-based synthesis like sputtering yield a crystalline structure and hydrolysis and electrodeposition seem to yield other phase. On one side,35 hydrolysis from Ir(III) chloride, in presence of lithium ions that could be removed easily, has been reported. The final phase is quasiamorphous with 5 broad peaks in X-ray diffraction related to the rutile phase, and contains chloride. On the other hand, galvanostatic electrodeposition as proposed by Petit et al.,27 from Ir(III) oxalate aged solutions, yields a product that turns out to be different from products in absence of oxalate. The final phase has very low density and is shown here to be quasi-amorphous. The number of electrons removed during electrochemical oxidation does not correspond with any expected valence change in iridium and we observed a significant improvement in cell growth in an attempt to define the best iridium oxide coatings for biomedical applications, and its special unique features. Within this context, we have grown an iridium oxide by oxidation electrodeposition methods with an optimized dynamic procedure that allows us to obtain thinner transparent electrodes for further bio applications. The final oxygen stoichiometry (IrOx) is also analyzed in terms of various components. Although all materials share essential features, this paper shows that the electrodeposited IrOx material seems to be significantly different from other materials, and that is formed by a template effect that yields actually an amorphous oxohydroxide, whose open structure allows ionic transport and exchange, and furthermore, the best results in cell growth observed to date. We propose that cell response is related to the more open structure that results from the method itself, and that is characterized here. IrOx has also found applications in other fields like catalysis,36 electrochromic devices and ferroelectric memories,37 properties that derive from the same electronic structure that confers the material mixed valence and conductive properties. For that reason, the study shown here may also be significant in other areas.

2. MATERIALS AND METHODS IrOx was electrodeposited on substrates prepared by evaporating thermally Pt (12 nm) on soda lime glass (24 × 5156



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The cells were seeded at high density (25000 cells/cm2) for quantification of survival at 4 days in vitro (DIV) and also at low density (500 cells/cm2) for measurement of axon and dendrite lengths. The cells were cultured in Neurobasal supplemented with L-glutamine, B27 supplement, penicillinstreptomycin and gentamicin (all from Invitrogen/Gibco), after being plated in DMEM medium containing 10% fetal calf serum (Invitrogen/Gibco, DMEM: Dubelccós modified Eagles medium) that was replaced 2 h after plating to remove cell debris and subsequently at 4 DIV. Visual inspection showed adhesion in all cases. Cultures were then kept at 37 °C in a humid atmosphere (5% CO2) and after 4 DIV they were fixed in Neurobasal containing 2% paraformaldehyde at room temperature for 12 min. Indirect double-immunofluorescent labeling combined with Hoechst 33342 (Molecular Probes, 2 μL/ml in PBS applied for 50 min after fixing the cells) nuclear staining was used for definition of cell phenotype and identification of condensed and fragmented nuclei. Briefly, fixed cultures were incubated for 30 min in 0.1 M saline phosphate buffer (PBS) pH 7.4 containing 0.2% Triton and 5% normal goat serum, rinsed with PBS and then incubated overnight at 4 °C with combinations of the following primary antibodies: rabbit policlonal anti-TAU (Sigma T-6402, 1:100), mouse monoclonal anti-MAP-2 (Sigma M-4403, 1:500), mouse anti-GFAP (Dako Z-0334, 1:500), rabbit anti-NF (Affinity NA1297, 1:750), or mouse antivimentin (Neomarkers, MS-129, clone V9, 1:1000). Alexa-488 antirabbit (Molecular Probes, 1:500) and Alexa-594 antimouse (Molecular Probes, 1:1000) were used as secondary antibodies. At 4 DIV more than 50% of the neurons were still in developmental stages 2−3 (morphological classification38), limiting the use of MAP-2 and Tau staining for classifying neural processes because of the lack of specificity. Therefore, processes were distinguished by morphological criteria and neurons were classified from stages 1 to 5 according to their morphology.38 In stages 1 and 2, neurites were defined as cell processes arising from the soma with lengths over 10 μm. In more advanced stages, axons were defined as neurites with a length of >50 μm and more than twice the length of other neurites, which if longer than 10 μm were regarded as dendrites. For quantification of cell survival, pictures at high resolution (2776 × 2074 pixels) were taken using a digital microscope system (Olympus DP50) and the fluorescent images of both Hoechst and antibody staining were combined. Sixteen radial fields (563 × 401 μm each) were systematically photographed in each sample and cell counts were extrapolated to the total surface area. Axon and dendrites were counted and measured four and ten days after culturing the cells at low density on the different substrates. Averages from at least 100 neurons in stages 1−2 and 3−4 were obtained for each sample type. One-way analysis of variance (ANOVA) and Holm-Sidak postest (0.050 of alpha level) were used to compare the data utilizing commercial software (SigmaStat 3.11, Systat). Electric field on cell cultures was applied with IrOx being the cathode, through 10 min dc current, at −0.30 V, and through 10 s pulses, during a period of one hour, using a counterelectrode and a reference electrode of platinum 99.99% Goodfellow, cut in 2 mm strips. The IrOx coating was used as substrate-cathode. Survival evaluation was performed by simple inspection under microscope of inhibition and detachment.

metallic Ir recorded simultaneously in transmission. IrO2 commercial oxide was used as reference (Sigma Aldrich 99.9%). Atomic force microscopy (AFM) used was an Agilent Technologies model 5400 SPM, in tapping and current sensing mode, for topography and electrical conductivity. Image processing and analysis was done using Mountains Map Premium software (Digital Surf Co.). Electrochemical behavior of the coatings, was studied by cyclic voltammetry (CV) with the same potentiostat described above, using various electrolytes as sodium phosphate buffer (pH 7.2) or in Neurobasal TM cell culture media containing L-glutamine and B27 supplements (all from Invitrogen/Gibco, Carlsbad, CA).38 The anodic synthesis process was studied by simultaneous cyclic voltammetry measurements (CV) and electrochemical quartz microbalance (ECQM) that show mass changes during electrochemical deposition. For that, a SEIKO 922 quartz electrochemical microbalance was coupled to the potentiostat. These experiments were performed on platinum coated quartz crystals (AT cut), 7 mm in diameter, and with fundamental frequency near 9 MHz. Intercalation processes occurring during reduction were also evaluated with the same set up on coatings deposited directly on the Pt coated quartz (surface 0.35 cm2). Contact angle measurements were performed with a goniometer (Pocket, model PG2) using 1 μL droplets of distilled Milli-Q water or Neurobasal culture medium supplemented with L-glutamine and B27 (all from Invitrogen/Gibco, Carlsbad, CA), immediately after placing the drop on either polylysine (L-PLL) coated or uncoated surfaces. Cleaning and L-PLL deposition was performed as for the cell cultures. (B27-supplemented Neurobasal is a serum-free cell culture medium optimized for long-term growth of central nervous system neurons.38 Neurobasal contains inorganic salts, aminoacids, vitamins, glucose, phenol red, buffers, and sodium pyruvate, whereas the B27 supplement is composed of several vitamins, hormones and proteins (albumin, catalase, insulin, superoxide dismutase, and transferrin)). L-PLL was deposited from 40 mg/mL aqueous solutions and incubated at 37 °C during 30 min. The final nominal concentration in the surface was 4 μg/cm2. The presence of the polypeptide adhered was confirmed by confocal fluorescence microscopy (Leica TCS SP2 AOBS, 0,2 μm resolution) using fluorescein isothiocyanate (FITC). Coatings were immersed in ionic or biological media to evaluate possible debris formation upon time, with negative results for up to a 3 month period, as chemical analyses showed (ICP, Inductive Couple Plasma UB services). Cell culture studies were performed in the following way. Prior to cell plating, both IrOx films and Borosilicate H cover glasses were cleaned by sonication in sterile distilled water, one hour-immersion in distilled water at 70−80 °C and finally covered with a solution of poly-L-lysine (PLL, 45 μg/mL, Sigma-Aldrich) for 30 min at 37 °C. PLL was used in the study of IrOx-neuron interactions because the simple chain of lysine residues makes the development of cells on PLL-coated substrates less likely to be altered by the orientation of the polypeptide in the coating layer. Moreover, we have successfully used PLL to promote neuron adhesion and differentiation on TiO2 rutile and anatase surfaces,18 and those studies provide a baseline for comparison of the present results. Neurons were obtained from the cerebral cortex of E18 Wistar rat embryos. The isolated cortices were dissociated for 30 min at 37 °C in HBSS supplemented with pyruvate, albumin, trypsin and DNAase (all from Sigma-Aldrich), followed by trituration with fire polished Pasteur pipettes.

3. RESULTS The original iridium solution changes in color during aging, indicating a change in the iridium coordination and the 5157



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intense peaks (110) and (220) in similar positions to the previous broad features.39,40 Thus, as-prepared films may contain the local structure of the final rutile iridium oxide. Further fitting of EXAFS data shown below supports that idea. The resultant thickness, as measured by X-ray Interferometry was in the 170 nm range for the 50 CV-cycles electrodeposited films and 330 nm for the 100 cycles as prepared samples. SEM analysis shows the same thickness (140 nm for 50 cycles) (Figure 2). Powder extracted from the films was analyzed by TGA in air, showing low temperature weight losses typical of hydration water, in several nonresolved steps. There is a significant weight loss between 30 to 300 °C, evidencing the possible existence of zeolitic water and also of covalently bonded H2O and OH−. Although is not possible to resolve the peaks due to the existing overlap, it can be said that the solid retains water in its structure with different binding energies. The total amount of water cannot be calculated exactly from this experiment, although it can be estimated as 2% weight in the commercial iridium oxide and about 16% in the electrodeposited material. 3.2. Surface Characterization of IrOx Thin Films. According to AFM measurements, IrOx films present cauliflower-like morphology at the nanoscale (Figure 3). Constant current electrochemical synthesis yields films with loose particles that interact with the AFM tip showing a nonreal surface (Figure 3A). A significant number of microscopic cracks are observed in SEM for these samples (also described by Petit and others24,27). However, cycled depositions yield adherent coatings with no loose particles or cracks. The grain size increases with film thickness as shown by AFM (Figure 3A,C and 3A,D). The surface morphology is completely different from the materials grown by physical methods like sputtering, PLD or ion beam assisted deposition where the typical morphology described is granular and cracks usually appear in the surface.16,28−32,41,42 PLL adhesion as evaluated through fluorescence images evidence (Figure 2, Supporting Information) is significant on IrOx as compared with blanks, in fact up to 1.5 times larger than in tested PEDOT polymers. Hydrophilicity observed through contact angle measurements is similar for all asprepared samples, with very different values for water or cell culture media (Figure 4). On the other hand, when the material is reduced, hydrophylicity decreases (Figure 4) in water and, even more, in cell culture media. When L-PLL is coating the samples, differences in hydrophilicity for several oxidation states are much smaller, so that the final surface exposed is not different in this aspect. This is a significant aspect in view of the possible applications of these coatings in electrodes for biological systems. It is rather significant the larger affinity of the IrOx electrodeposited material for the cell culture solution than for water or for purely ionic media such as sodium chloride solutions. That is probably related to a great affinity of IrOx for proteins and other organic molecules in the media. Such affinity decreases upon reduction, (Figure 4B) and also once the material is coated with L-PLL, as expected if it derives from a strong interaction of the oxide with amine or carboxylate groups from biomolecules. 3.3. Oxidation Process Mechanism. The oxidation process that involves deposition has been evaluated by EQCM experiments, evidencing significant data with respect to the final coating. Charge to mass ratios, Q/m, were calculated on the basis of the integrated current and the mass changes measured simultaneously during electrodeposition. For

formation of an oxalate complex of unknown composition that stabilizes iridium ions in solution at pH 10. Studies shown below suggest that such complex is Ir(C2O4)3x−. A certain change in the oxidation state of iridium is also possible, since the final blue color in thin films may also indicate mixed valence properties as the observed UV−vis spectra shows charge transfer bands in the near IR region. Irreproducible deposition is observed without that aging or without oxalate. Thick films show black color. Anodic electrodeposition at constant current yields coatings with low adherence (as seen by AFM) and macroscopic cracks. For that reason, a dynamic potentiostatic procedure was devised, which showed a much better adherence and quality of the coating. Since CV for the solution shows a wave at 0.55 V vs Pt, and constant current experiments stabilize their potential at that value during deposition, the window for this dynamic method was chosen from 0 to 0.55 V vs Pt (Figure 5). Cycling speed was optimized at 10 mV/s. Thickness control was performed by changing the number of cycles: 2, 50, or 100. The coatings obtained at the anode are blue in all cases suggesting the formation of a mixed valence iridium oxide. Golden coatings are also obtained at the cathode and they will be object of a different study. 3.1. Structural Features of the Coatings. GIXRD of the coatings, (Figure 1A), evidence peaks corresponding to the

Figure 1. GIXRD of an IrOx electrodeposited sample (0.55 V, 10 mV/ s, 50 cycles). (A) As prepared coating on Pt coated glass and (B) coatings on platinum coated quartz treated at different temperatures after preparation.

underlying Pt, and two broad features centered at 28° 2θ and 58° 2θ that may correspond to the underlying glass and to an amorphous phase. Using Pt coated quartz substrates the coatings may be heated up to 700 °C and as seen in Figure 1B gradual crystallization of a rutile structure occurs with the most 5158



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Figure 2. (A) SEM images of a transverse section of the IrOx electrodeposited coating and (B) macroscopic images of coatings.

XPS data). Such final phase shows reproducibly the same characterization parameters. Gas formation, electrode surface changes due to CO2 evolution and subsequent hydrolysis may also explain the somewhat cyclic nature of the Q/m ratio observed. On the other hand, the envisaged mechanism suggests that the final low density open structure may come from the presence of large oxalate species acting as template, during the hydrolysis process, as well as ion incorporation in the structure. 3.4. Electrochemical Behavior of As-Prepared Films. Once formed, the film has a reproducible electrochemical response in the oxidation and reduction parts. Two main reduction waves are observed at −0.23 and −0.53 V vs Pt. (Figure 8) when CV is performed on the same electrode geometry as prepared. Both evidence intercalation occurring simultaneously, as observed by mass increase in ECQM experiments and therefore electronic changes in Ir states. The Q/m ratio upon cathodic intercalation reactions is positive, as expected from intercalation of positive ions during cathodic processes. The mass increase can be related to an effective molecular weight of such species, using Q/m ratio, since the number of moles needs to be equivalent to the number of iridium atoms reduced for the same charge. Such calculation yields an average molecular weight of 13 g/mol, which does not correspond to Na+ ions, H+, neither hydrated protons, and it may be a combination of OH− release and H(H2O)+ or Na+ intercalation, since OH− seems to be rather mobile in this structure (T loss near 100 °C). Charge capacities were estimated in constant potential reductions at −0.6 V vs Pt (20 s), with integrated currents resulting in 40 mC/cm2, if the geometrical area is considered. Such value is significantly large for a thin film. Since the material is truly being porous and electroactive through the bulk, the charge capacity may also be given better as 130C/g, using the mass derived from microbalance experiments. Such values are larger than those reported for noble metals like Pt (in the range of μC/cm2), and even larger than those found in bulk IrO2 (1 mC range).1 Safe working potentials, without radical formation can be estimated between +0.4 V and −0.9 V vs Pt A third very small peak, of reproducible intensity, is observed at much lower potentials in CV, −0.82 V, a potential where CO32‑/C2O42‑ reduce as seen in blank carbonate solutions. It is possible that an open channel structure may retain either oxalate or carbonate. According to AFM studies, reduction induces a decrease in roughness that can be interpreted as swelling (Figure 3B). TGA analysis in air does not show weight stabilization. However, in Ar/H2(5%), there is a significant plateau at 300°, corresponding to the loss of 2.7 O/Ir. The

50 cycle preparations, reproducible mass and charge were 13.2 μg and 22 mC. That involves an electrochemical oxidation that forms the IrOx layer with a total Q/m ratio of 3.9 ± 0.3 electrons/iridium atom (including molecular formula corrections described below). An oxidation process related to a valence change for iridium, would yield a smaller Q/m, and therefore there is a complex mechanism for the reaction. When the data is analyzed cycle by cycle, no mass increase is observed up to the fourth cycle, and even a mass decrease is evident at the start of the process (Figure 6). The Q/m mass changes for subsequent cycles follow a significant evolution from 6 to 2 electrons per iridium per cycle. Since the final product is a hydrated iridium oxide as shown below (XPS), a possible interpretation that would account for Q/m values and original mass decrease would be the release of a gas on the surface of the quartz crystal. It has been tested that only oxalate, apart from iridium ions, can be oxidized at that potential and that the final products derived from oxalate would be either carbonate ions or CO2. Thus, we can identify a possible product, CO2 gas. Once oxidized, free Ir (III, IV) aqueous ions would be released and undergo hydrolysis at that pH. The original mass decrease, and subsequent increase, can be considered a proof that this is the acting mechanism in the reaction. Also numerically, Q/m evolution (Figure 7) for each cycle indicates that the mechanism suggested is consistent. Oxalate oxidation involves 2 electrons per oxalate if it yields CO2. Considering that Ir(III) or Ir (IV) are usually six-coordinated, very possibly there are 3 oxalate bidentate ligands coordinated to it, Ir(C2O4)3x−, oxalate oxidation would involve 6 electrons per iridium. Iridium may also oxidize partially and may contribute to Q in smaller values. In fact mixed valence states, as deduced from the blue color and presence of near IR charge transfer bands, suggest so. After oxalate starts being consumed, the number of oxalates in the iridium complex may vary until final hydrolysis (3 to 2, 1, or 0 oxalates), and simultaneously the number of electrons per iridium would decrease. A complex hydrolysis seems to be the main process occurring at the electrode after electron transfer. Initial quantities evaluated by ECQM, are intrinsically affected by a larger error due to the formation of CO2 gas, with subsequent changes in electrode surface in contact with the solution, and as the amount of gas decreases, the error decreases. The solubility of the various species of iridium possible in solution during oxalate oxidation may induce precipitation of mixed species, oxides, hydroxides, oxohydroxides and also hydroxo-oxalates, with counterions such as K+. Thus, the final phase obtained may be clearly different from physical methods preparations, and with final different properties, as further characterization below indicates (see 5159



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Figure 3. A. AFM images of IrOx electrodeposited samples in tapping mode. (A) Galvanostatic synthesis, (B) dynamic mode synthesis, 0 to 0.55 V, 2 mV/s, 17 cycles, (C) dynamic mode synthesis, 0 to 0.55 V, 10 mV/s, 50 cycles, and (D) dynamic mode synthesis, 0 to 0.55 V, 10 mV/s, 100 cycles. B. AFM profiles of IrOx electrodeposited samples and their changes upon reduction in two extents (number of electrons per iridium). rms represents “root mean square” surface roughness.

mass loss continues until about 600 °C without stabilizing, probably the reduction to metallic iridium in Ar/H2. 3.5. Surface Composition. XPS studies (Figure 9) of the resulting coating show the expected elements: Ir, O, and also K. Significantly, Cl atoms are totally absent, while commercial oxide, and many reported IrOx, show Cl−.43,44 Quantification based on XPS surface data (Figure 9A) yields reproducibly a molar ratio O/Ir of 4.1 ± 0.4, which does not correspond to a pure oxide IrO2. The high resolution O 1s spectra (Figure 9B) show three different contributions: O2‑, OH− and H2O. By

deconvolution of the O 1s peak we can conclude the possible unit formula: IrO1.1(OH)2.7·0.4H2O. This stochiometry seems to correspond to an oxidation state of Ir4.9+. The same deconvolution for the commercial oxide, yields O/Ir = 2.9 and would also involve hydration or hydroxyl ions. Sputtered samples have been reported to yield different O/Ir values 2−3. It is therefore evident that the electrodeposited material is different from others, and is better described as a hydrated oxohydroxide. Comparison with the materials prepared by other methods suggest that we are dealing with a non5160



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Figure 6. ECQM measurements of an IrOx sample electrodeposition by dynamic methods (0.55 V, 10 mV/s, 50 cycles) showing the mass change for each cycle independently.

Figure 4. Contact angle measurements (water and culture medium drops) for electrodeposited as prepared IrOx and for further reduced samples, with (●) and without (○) L-polylysine coating. Measurements on Pt (*) and Borosilicate H (■) are shown for comparison. Error bars are smaller than circles.

Figure 7. Q/M evolution as number of electrons per iridium, as a function of cycle number (with number of cycles for anodic IrOx electrodeposition). Figure 5. Electrochemical synthesis of an IrOx sample on platinum coated quartz resonator cyclic voltammetry (0.55 V, 10 mV/s, 50 cycles).

stoichiometric material that retains reproducibly a specific local structure for iridium, shows larger hydration and a more amorphous structure and, as shown below, supports better neural growth than other iridium oxides. All types of phases yield the crystalline IrO2 rutile phase upon heating. Also significant is that XPS of these electrodeposited samples shows reproducibly K, with a K/Ir ratio of 1.7, but that this ion is easily removed by soaking in pure distilled water. No evidence of color change, rest potential, or voltammetry are observed after soaking, so a K+ - H+ exchange with no redox changes in iridium, is the more plausible explanation. C 1s signal in energies near carbonate (oxalate) region is also observed. A C/Ir ratio 3.6, is somewhat larger than expected from ambient contamination, and larger than in the commercial

Figure 8. Cyclic voltammetries (2 mV/s) of an IrOx sample electrodeposited by dynamic potential methods (0.55 V, 10 mV/s, 50 cycles) in phosphate pH 7.1 and cell culture medium. (Note that CV starts near 0.0 V vs Pt.) 5161



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between metallic and cationic iridium. In the high resolution Ir 4f spectra (Figure 9C) the Ir 4f7/2 Ir and 4f5/2 at 62.8 and 65.8 eV respectively, are observed, consistent with other literature values for the IrO2.30,45 On the basis of all the above, we propose that this “wet” IrOx is a hydrated oxohydroxide phase clearly different from other IrOx possible structures present in literature.45 A direct hydrolysis reaction from iridium trichloride appears to be also an oxohydroxide with different stochiometry,35 but apparently not the phases prepared by physical methods: PLD or sputtering41 or by anodization42 of the metal. It is possible that hydration could be easier in some non crystalline phases and yield an oxohydroxide of variable stochiometry, while it would disappear upon crystallization of the rutile phase. This variability in the O/OH stoichiometry is related to the opensponge like structure and needs to be related to Ir oxidation state. It is therefore essential in the understanding of the mixed valence, electronic properties of the material, and its structural flexibility. Hydration plays an essential role in the mixed valence properties of the material, through the acceptance of a variable H+ or M+ in its open structure as suggested also for RuOx phases.15,31 3.6. Bulk XAS Studies. Figure 10 shows normalized XANES spectra at Ir L3 edge of the as-prepared sample

Figure 9. (a) XPS survey of an IrOx sample electrodeposited by dynamic potential methods (0.55 V, 10 mV/s, 50 cycles) as prepared and soaked in water 7 days and (b) O1s and Ir 4f XPS signals for the same samples.

oxide, 1.8. However, the value observed is small with respect to the expected if oxalate remained coordinated. Thus, part of this C is aliphatic (78% of the total), as if the material had great affinity for this type of carbon (as described before for L-PLL adhesion), and the rest, 22% corresponds to a carbon bound to oxygen, involving about 0.5 C−O per Iridium. Iridium has been reported to absorb carbon dioxide as well as nitrogen oxides in fact.44 However, in this case the synthesis method starting from oxalate may favor, through a template effect, a small oxalate/ carbonate retention in the oxide final structure. Taking all those factors into consideration, the XPS data suggest an empirical formula K1.7(CO3)0.2IrO1.1(OH)2.7·0.4H2O, with an oxidation state of Ir3.6+, or between Ir3.2+ and Ir3.6+ since the C−O can be as CO32‑, HCO3−, or C2O42‑. Additional analysis show that when K+ is removed by soaking in water the stochiometry changes yields the formula IrO0.3(OH)30.4H2O that implies also the same oxidation state, Ir3.6+, and no redox changes. Significantly the amount of OH from XPS quantification 2.7 O/ Ir, coincides with the observed weight loss at 300 °C in TGA in Ar/H2(5%). XPS Ir 4f signal is not sensitive to oxidation state changes between Ir3+ and Ir4+, although it differentiates

Figure 10. (A) XANES of the Ir oxides: rutile IrO2 and as prepared, soaked and reduced IrOx samples. (B) Plots of kχ(k) vs k (upper panel) and corresponding Fourier transforms (lower panel) for commercial IrO2 and selected IrOx samples.

compared to the commercial IrO2 oxide and how it changes upon inmersion in water or reduction. A shift of the position of the white line peak to lower energies is observed for all the IrOx samples as compared with reference IrO2. This shift is accompanied by a decrease of the white line intensity, 5162



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Figure 11. Representative microphotographs and neural survival of cerebral cortex neurons seeded at 25 000 cells/cm2 and cultured 4 days on electrodeposited IrOx samples (A) and (C) Tau immunocytochemical staining and (B) and (D) Hoescht staining of the nuclei corresponding to the cells in (A) and (B). And E neurons survival for each type of sample as compared to borosilicate-H control.

coatings. From the structural data of trivalent iridium oxalate, a compound with six oxygen atoms in an octahedral coordination, Hüppauff and Lengeler34 have calculated that the Ir−O distance of a Ir3+ must be close to 2.02 Å. Using a linear regression for the Ir−O bond length variation versus Ir oxidation state, Ir atoms in the films are determined to be Ir3.4+ in very good agreement with observations based on XPS O 1s peak deconvolution. Furthermore, the short-range order (IrO6) in the as prepared film is concluded to be of the rutile type. EXAFS confirms the crystallization to the rutile type structure upon heating at 450 °C (inset of figure 10b). However, the reduced films show an increased structural disorder marked by an apparent reduction in coordination number, implying that reduction increases disorder in the IrO6 octahedra. Both, a particle size decrease and a true coordination change could account for such local disorder. In conclusion, XAS data evidence mixed valence around Ir3.5+ in the electrodeposited IrOx films extending the XPS surface observations also to the bulk material. The local structure of the as prepared material, that results optimal for neural growth,17 can be described as a disordered IrO2 rutile-like structure, where IrO6 octahedra are similar to those found in the crystal structure. The lack of intense shells in the Fourier Transforms of the EXAFS signal beyond the nearest oxygen neighbors indicates that the films do not display long-range three-dimensional order. It is likely that the presence of structural water disrupts the three-dimensional rutile structure, providing transport pathways for both electrons and protons. This interplay between ionic and electronic transport, which in turn, is controlled by the degree of hydration and local order was already suggested for related RuOx(OH)y materials15,31 and now for IrOx. 3.7. Transport Properties. The homogeneity of transport properties across the Iridium oxide surface have been measured by AFM working in current sensing mode and using a silicon AFM tip coated with a boron-doped diamond conducting film. Transport measurements across oxide surfaces have been

indicating that the oxidation state of Ir in the IrOx samples is reduced from Ir4+ in the reference IrO2 compound. No differences are observed during inmersion and K−H exchange but the intensity of the white line strongly decreases as the films are reduced. The lack of a good reference for Ir3+ prevents us to determine the iridium valence, but we can conclude that electrodeposited samples contain Ir lower than 4+. Due to the quasi-linear correspondence between the interatomic Ir−O distances and the iridium valence, we have analyzed EXAFS spectra to determine changes in the bond lengths that can be related to changes in oxidation state. Figure 10b shows the Fourier transforms of the EXAFS signals, kχ(k) extracted using the Athena (version 0.8.058) code,46 without phase correction calculated between 3 and 12 Å−1 using a sine window. The model used for fitting the data is the rutile or hollandite crystal structure based on the X-ray observations made before. Closed circles represent experimental data and the solid lines best fits obtained from a statistical analysis of the EXAFS data based on a rutile structure (up to 4 Å), that is, including the first oxygen shell and the second two Ir shells. Best fits to the data were performed using the Artemis program (versión 0.8.013)47,48 in the R-space fitting mode. All of the samples show similar nearest-neighbor oxygen octahedral environments (IrO6) but the amplitude of the Ir−Ir peaks beyond 2 Å was considerably damped for the IrOx samples, indicating that these oxyhydroxides are highly disordered.49 We note here that even the commercial powder is not a well-crystallized phase. However, the outer two iridium shells up to 4 Å are detected and EXAFS spectrum resembles that of a well-crystallized IrO2 powder.34 EXAFS analysis of the commercial IrO2 yields an average Ir−O distance of 1.97 ± 0.01 Å, which corresponds to the rutile structure in agreement with previous XAS works.34,49−51 On the other hand, an increase of the Ir−O distance from 1.97 to 2.00 Å has been found for all the studied electrochemically prepared IrOx samples, in line with a formal oxidation state for Ir in lower than 4+ in the 5163



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reported, but it is known that they are highly dependent on the effective contact area, geometry of the tip, applied surface force or surface roughness. For this reason this method is an estimate of how good conductor is the film. AFM contact images of the IrOx surface and corresponding current−voltage curves at different points of the image are given in the Supporting Information. No clear differences in resistance were observed from top to valley parts of IrOx as-prepared samples, with values in the order of 60 Ω for positive or negative voltages. Therefore the surfaces are homogeneous and with high conductivity for such small thickness. (Figure 1, Supporting Information) 3.8. Neural Cell Behavior on IrOx Surfaces. The specific cells used to evaluate the material as substrate for the adhesion and growth of primary, mammalian cerebral cortex neurons, are taken as a first step in assessing the suitability of this material as electrode for brain-implantable neuroprostheses. Among possible choices for such evaluation, embryonic primary neurons do not divide after cell differentiation, thus precluding the assessment of the effects of the material surface on cell proliferation. However, compared to dividing cell lines, embryonic cortical neurons resemble more closely the properties of normal, adult brain neurons targeted by implantable electrodes. Neurons seeded at 25 000 cells/cm2, on IrOx films had good appearance and numerous cell processes both at 4 DIV (Figure 11) and 7 DIV even better than those in the borosilicate-H glass used as control. At 7 days, coatings tend to lose adhesion to the glass/Pt substrate in presence of cells and not in aqueous media, possibly through the effect of cell metabolites on it. However, no change is observed in the material, neither debris is found in the supernatant solutions, and therefore no interaction of cells with micro or nanoparticles is occurring. The results shown below for iridium oxohydroxide are highly reproducible, as compared with other materials tested. Immunocytochemical studies in combination with Hoechst nuclear staining revealed that at 4 DIV the cultures contained almost exclusively neurons, other cells representing less than 0.1% of the total. In the absence of L-PLL, neurons did barely attach to either control or IrOx surfaces. In contrast, the neurons seeded at 25 000 cells/cm2 on as-prepared, PLL-coated IrOx films appeared healthy and had numerous and long neurites. The quantification of healthy cells (i.e., those having cell nuclei exhibiting no condensation or fragmentation with Hoechst staining) on IrOx electrodeposited at 25, 50, and 100 cycles showed that all films worked very well as cell substrate (Figures 11−13), without significant differences between them. We also performed low-density cell cultures (500 cells/cm2) to quantify neuronal differentiation and growth on IrOx. In low density cultures, cells develop on IrOx, at 4 DIV in a similar way to control and emit less neuritis (Figure 13 and Figure 3 in the Supporting Information), and eventually less dendrites, in a similar fashion to the observations made on TiO2.18 At 4 DIV, approximately 50% of the neurons were in stages 1−2 and the other 50% were in stages 3−4 in both, IrOx films and borosilicate glass. However, less neurons advanced from stage 3 to 4 on IrOx compared to borosilicate glass (Figure 13 and Figure 3 in the Supporting Information), implying that the cells normally differentiated axons on both surfaces but failed to differentiate dendrites on IrOx. This was confirmed by counting and measuring the cell processes, finding that the number of neurites (for stages 1−2) and dendrites (for stages 3−4) decreased about 40% on IrOx compared to borosilicate

Figure 12. Neuron survival in high-density (25 000 cells/cm2) cultures after 4 DIV on IrOx samples electrodeposited by cyclic voltammetry (0.55 V, 10 mV/s) as prepared and reduced in K2CO3 0.01 M pH 11 and Phosphate 0.1 M pH 7 at 0, 18, and 120 s. The data represent the mean ± statistical significant difference at p < 0.05 compared to controls. It seems to be that the reduction medium is a key factor, more significant than the potential, in terms of survival, achieving values of survival statistically equivalents to the values obtained with the as prepared layers.

Figure 13. Neuron survival in low-density (500 cells/cm2) cultures after 4 DIV in the different stages of development of hippocampal neurons in culture (stage 1: lamellipodia/0.25 days in culture, stage 2: minor/0.5 days in culture, stage 3 axonal/1.5 days in culture, stage 4: dendritic/4 days in culture, and stage 5: maduration/>7 days in culture [ref 38]).

glass. On the contrary, neurons in stages 1−2 appeared to have longer neurites on IrOx films, and the length of dendrites and axons of neurons in stages 3 and 4 was equivalent to the control values indicating that PLL-coated IrOx allowed appropriate elongation of cell processes. The effect of reduction prior to culture was also studied, and a significant variation was observed in terms of cell survival depending on the medium used for such reduction (Figure 12). For long 120 s reductions, if K2CO3 media was used as electrolyte cells show much worst behavior a low overpotentials (−0.23 V vs Pt) than if phosphate buffer had been used. More negative potentials yield down to 20% cell survival during culture. Significantly reductions at −0.23 V vs Pt in phosphate buffer, show cell cultures with little variation with respect to as prepared samples. The apparent toxic effect of reduction in carbonate is significantly canceled in low time reductions (18 s). The significance of this data refers to the ability of the material to undergo redox processes during dc or ac electric field application. The study shown in simple 5164



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IrOx on metals and subsequently activating the oxide by electrochemical procedures to reduce their electrical impedance, thus achieving the extracellular recording of action potentials with good signal-to-noise ratio32 or effective electrostimulation of neurons in culture.31 Sputtered IrOx has also been recently analyzed as substrate for insect and chicken brainstem neurons.16 In this case, coatings of different morphology and oxidation state were obtained varying the O2 partial pressure during the sputtering, and applying poly-Dlysine to the oxide surface allowed the adhesion and growth of chicken neurons. Our study extends those results to the case of mammalian cerebral cortex neurons on electrodeposited, amorphous IrOx, demonstrating that it allows neuronal adhesion and survival with elongation of axons and dendrites. These results sharply contrast with previous studies in which very poor adhesion and growth of rat cerebral cortex neurons occurred on sputtered, amorphous IrOx films.11,30 In that study, the best cellular outcomes were obtained on poly-Dlysine coated, crystalline IrOx, although the cell counts were still only 30% of those found on control surfaces. Some neuronal survival was also reported on films of sputtered Ir with subsequent anodization52 although, at 4 DIV the cells appeared clustered and showed signs of retraction from the oxide surface. In terms of cell material interactions and their effects, a significant discussion is worth to make since the interaction initially occurs at the cell membrane and hence, the specific proteins, carbohydrates and phospholipids expressed at the membrane of different cell types will influence the manner in which cells respond to the material. That has been deeply studied in the case of nanoparticles, a case quite different from this since no nanoparticles are released from the coating and therefore no fagocytosis is expected here. The term “cell vision” has been proposed for the varied capabilities that cells with different phenotypes have to interact with nanoparticles.53 This phenomenon probably underlies the differential cytotoxicity that iron oxide nanoparticles show depending on the cell line. For instance, they are much more toxic to neuroblastoma (BE2-C) and glioblastoma (A172) cell lines compared to kidney 293T and human cardiac miocyte (HCM) cell lines.53,54 In this regard, the question arises if other cell types will show the same behavior that we report here for primary neurons on IrOx surfaces. The electrochemical properties of IrOx nanoparticles in solution differ from those in electrofflocculated films,55 and the aim of the present work was not to study the interaction of IrOx nanoparticles with mammalian cells. Instead, we had the specific objective of evaluating electrodeposited IrOx as substrate for the adhesion and growth of primary, mammalian cerebral cortex neurons, as a first step in assessing the suitability of this material as electrode for brain-implantable neuroprostheses. Embryonic primary neurons do not divide after cell differentiation, thus precluding the assessment of the effects of the material surface on cell proliferation. However, compared to dividing cell lines, embryonic cortical neurons more closely resemble the properties of normal, adult brain neurons targeted by implantable electrodes. There is no obvious reason to expect a toxic or growth-inhibitory response of other mammalian cells to IrOx, provided that the oxide surface is appropriately functionalized for cell adhesion and does not dissolve or delaminate from the subjacent metallic electrode. In fact, our results add to other studies that showed good cell growth of chicken and insect brainstem neurons on poly lysine-coated IrOx,16 despite the different phenotype of those neurons compared to rat cerebral cortex neurons. On the other hand,

ionic media evidence that pulses lower than 18 s would not affect cell survival. As a proof of concept, an electric field was applied in situ during cell culture, using the IrOx as cathode and substrate, in the same configuration than standard cell cultures, with a counterlectrode inserted in the cell media. The use of dc currents (−0.2 V vs Pt, resulting in 2 mA max current), induced cell death after 12 h. In such time, the charge delivered constitutes about 90% of the available for the material. However, the use of 1 s pulses with 5 s rest among them, during 10 times and 3 h interval, did not affect the final cell survival. This study may be extended in the future to a different set of conditions and applied electric fields, but in itself, it demonstrates the feasibility of using IrOx coatings for electroestimulation electrodes, and a range in which no cell death occurs.

4. DISCUSSION The results shown above evidence that anodic electrodeposited IrOx material is a hydrated oxohydroxide material with an open channel structure able to exchange ions, within the structure, with or without simultaneous redox changes, and with a large affinity for carbon. Electric fields can be applied through it, while the material absorbs the charge through intercalation reactions, instead of radical formation, with rather large charge capacities.Very small changes in structure, hydrophilicity and topography are observed during redox changes. The specific amorphous structure obtained by electrodeposition may be derived from the method itself, where oxalate ions may be acting as template for the formation of an open structure, that allows ion exchange with and without redox changes, with a reproducible stoichiometry and behavior clearly differentiated from the material obtained by other methods. Ionic affinities would also explain pH sensor capabilities, while possible cationic exchange may be at the root of the significant cell growth on these surfaces reported before. A possible structural path from channel type disordered structure hollandite- type yielding rutile crystalline phase can be envisaged. The observed molecular weight of the intercalated species upon reduction also implies the presence of large vacancies, and high ion mobility. That same channel structure may be at the origin of the nice behavior in electrostimulation and cell culture since redox changes occur on an open structure, and therefore are faster. Comparison with phases obtained from physical methods evidence that each preparation method yields a significant different phase with common local features. The present results also demonstrate that electrodeposited, amorphous IrOx is a noncytotoxic substrate that efficiently adsorbs polycations such as PLL and allows adhesion and growth of mammalian cerebral cortex neurons. This opens the possibility of exploiting the versatility of electrodeposition for creating coatings of IrOx on a variety of electronic neuroprostheses, thus enhancing the electric charge delivery capacity while promoting neural cell growth on the electrode surface. Electrodeposition is a inexpensive technique, as compared with sputtering methods, that may produce IrOx coatings on electrodes of varied shapes. Multiple, small electrodes are necessary to achieve high spatial resolution of electrostimulation and signal recording with neuroprosthetic devices. However, the large electrical impedance of the microelectrode/ tissue interface limits charge transfer in those applications and hence, coating the microelectrodes with IrOx is a potential strategy to increase their electric performance. Planar multielectrode arrays (MEAs) have been fabricated by sputtering 5165



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the biomolecules used for coating the nanoparticle determine to a great extent the biophysicochemical interactions at the cellnanomaterial interface, 56,57 in the same manner that biomolecules on artificial substrates specify the type of cell that adheres and the rate of cell growth.22 Given its polycationic nature, PLL electrostatically interacts with both negatively charged cell membranes and inorganic oxides,18 thus allowing excellent cell adhesion to those materials. However, PLL is unspecific or, in other words, it promotes the adhesion of numerous cell types independently of its embryonic origin; whereas neuroprosthetic applications will need of mechanisms to enable the adhesion of selected cell populations to the electroactive surface. This can be effectively achieved binding specific biomolecules to the material surface,58 for which several methods are currently available. In fact, the chemical strategies used for covalent attachment of adhesive peptides to Ti oxide59 may be adapted for IrOx or, alternatively, composites containing IrOx60 may be developed to facilitate biomolecule immobilization. Summarizing, the highly hydrated amorphous oxohydroxide phase created by this electrodeposition method accounts for the large differences between cultures reported here and those of previous studies that tested mammalian cerebral cortex neurons on IrOx with much lower survival and development rate.11,16,30,52 The comparison shows that hydrated, amorphous IrOx films formed by electrodeposition provide a much more favorable surface for PLL adsorption and neuronal growth on the electrode, suggest that electrodeposition is probably the best technique for producing IrOx coatings for neuroprosthetic applications. Although less neurites/dendrites developed from neurons on IrOx, their elongation did not appear to be inhibited, neither axonal growth. The impairment of dendrite development was very similar to that occurring for neurons on PLL-coated TiO2 surfaces18 although the underlying mechanisms remain unknown. Nevertheless, the use of molecules other than PLL as biofunctionalization of the oxide film will likely overcome this problem.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +34935801853 Ext. 275. Fax: +34935805729. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Spanish Ministry of Science and Education (MEC) (MAT2005-07683, MAT2008-06643, and MAT2011-24363 and FIS08-03951) and the European Commission FP6 NEST-STREP Program (Contract 028473). A.M.C. and J.M.V. thank the Spanish Ministry of Education for a predoctoral fellowship, and N.M.C. thanks CSIC for a predoctoral JAE. The authors thank also J. Bassas for technical assistance and Prof. C. Suñol for helpful discussions in cell culture evaluation.

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5. CONCLUSIONS This work shows that electrodeposited IrOx thin films, obtained from oxalate solutions are truly a hydrated oxohydroxide with reproducible stoichiometry. This phase constitutes a significant substrate for neural cell growth, with much larger survival rates than other phases obtained by different methods, with non cytotoxic effects when used as electrodes and with a large charge capacity and good affinity with neural media. Its structure is more open and hydrated than other IrO2, exchanges easily ions through chemical exchange or intercalation redox procedures, with a very stable redox chemistry and shows good conductivity and electrochemical response. All aspects observed suggest that the procedure yields a good adhesion coating that can be used in devices implanted in the nervous system.

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ASSOCIATED CONTENT

S Supporting Information *

Figures dipecting the current sensing AFM response (Figure 1), PLL adhesion as imaged by confocal fluorescent imaging (Figure 2), and neuron survival in low-density (500 cells/cm2) cultures after 4 DIV (Figure 3). This material is available free of charge via the Internet at http://pubs.acs.org. 5166



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