Multilayered Polypyrrole-Coated Carbon Nanotubes To Improve

Mar 8, 2011 - (8) Cui, X. Y.; Lee, V. A.; Raphael, Y.; Wiler, J. A.; Hetke, J. F.;. Anderson, D. J.; Martin, D. C. J. Biomed. Mater. Res. 2001, 56 (2)...
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Multilayered Polypyrrole-Coated Carbon Nanotubes To Improve Functional Stability and Electrical Properties of Neural Electrodes Hailan Chen, Longhua Guo, Abdul R. Ferhan, and Dong-Hwan Kim* School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457, Singapore ABSTRACT: The surface of neural electrodes was modified with multilayered, polypyrrole (PPy)-coated, multiwalled carbon nanotubes (MWCNTs) via the layer-by-layer fabrication method. The modified electrode with PPy-coated MWCNTs exhibited a significant improvement over conventional Au electrodes and PPy-coated electrodes with respect to the electrical properties based on charge storage capacity (660 times larger), impedance (5 times lower), and electrochemical stability (3 times more stable). Positive results from the biocompatibility tests conducted on PC12 cells and RAW 264.7 further suggested that our multilayered, PPy-coated MWCNT was an excellent candidate for the surface modification of neural electrodes.

1. INTRODUCTION Recently, there has been an increasing trend in the utilization of implantable electrical devices capable of communicating with biological tissues for the diagnosis and treatment of neurological disorders.1-7 Although the operational capability of such devices has been established,8,9 the long-term performance remains speculative because of the time-dependent deterioration of the stimulation and recording efficiencies, which partly results from material instability and foreign body response.10-12 Much effort has been made to reduce or eliminate problems associated with long-term use of implantable electrical devices, including neural electrodes through chemical surface modification (e.g., polypyrrole (PPy),8 poly(3,4-ethylenedioxythiophene) (PEDOT),13,14 hydrogel15 and carbon nanotubes16), new electrode designs (protruding microelectrodes17) and incorporation of bioactive molecules (nerve growth factor (NGF)6), cell adhesion peptides (CDPGYIGSR8), and R-melanocyte-stimulating ormone (MSH)).18 Conducting polymers, including PPy, PEDOT, and their derivatives, have been widely used to modify the surface of neural eletctrodes.8,15,19 Among the conducting polymers, PPy has been extensively studied for the surface modification of neural electrodes because of its aqueous solubility, low oxidation potential, high conductivity, and biocompatibility.20,21 Despite the great applicability of PPy, the degradation of PPy in the course of time makes it difficult to use for long-term applications.22 One study reported that 95% of the initial activity of PPy films could be lost within 16 h under continual stimulation at 0.4 V.23 Therefore, there is a demand to improve the long-term functional stability of PPy for application in neural electrodes. Carbon nanotubes (CNTs) are particularly attractive candidates to interface electrodes with tissues because of biocompatibility and excellent mechanical and electrical properties.24-26 CNTs have been reported to support neuronal growth,27 boost r 2011 American Chemical Society

neural signal transmission,28 and improve device recording capabilities.29 Improvements in interfacial electrostability and conductivity have been observed when electrodes were modified with composites of CNTs and either conducting polymers30 or polyelectrolytes.31 Few studies, however, have been performed on the multilayered structures of CNT and PPy composites. In the present study, we investigated the functional stability and biocompatibility of a multilayered composite of CNT and PPy fabricated via the layer-by-layer method. We prepared three different types of materials (i.e., multilayered PPy, PPy-coated multiwalled carbon nanotubes (MWCNTs) and copolymerized MWCNTs and PPy) using the process schematized in Figure 1. For simplification, the materials were named ‘mPPY’, ‘LBL’, and ‘CO-POLY’, respectively. With dramatically lower production cost and ease of handling, MWCNTs would be more attractive for commercialized products than single-walled CNT; hence, MWCNTs were adopted in this study. After the modification of Au electrodes with the aforementioned materials, the structural properties, electrical properties, electrochemical stability, and biocompatibility of these materials in RAW 264.7 (mouse leukemic monocyte macrophage) and PC12 (pheochromocytoma) cells were investigated. Our results show that the LBL exhibited a significantly higher performance based on the charge storage capacity and impedance compared with the bare electrodes, mPPY and CO-POLY. We also observed a dramatic increase in the electrochemical stability of LBL films. In addition, we have shown that the LBL films support the growth, adhesion, and neurite extension of PC12 cells. Received: December 2, 2010 Revised: February 17, 2011 Published: March 08, 2011 5492

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Figure 1. Schematic of the fabrication process of multilayered mPPY, LBL, and CO-POLY.

2. EXPERIMENTAL DETAILS 2.1. Materials Preparation. MWCNTs of 95% purity, with a diameter of approximately 20-30 nm and a length of approximately 50 μm, were purchased from Chengdu Organic Chemicals Co. Ltd. The MWCNTs were initially refluxed in dilute hydrochloric acid for 8 h to remove residue from the metallic catalyst particles. They were then functionalized with carboxyl and hydroxyl groups by treatment with concentrated sulfuric acid and nitric acid.32 Then 5 mg of the resulting functionalized MWCNTs was dispersed in 10 mL of deionized water under sonication (Ultrasonic Cell Disruptor, SCZJY92-IIDN, Zil Sci, Singapore) to obtain a 0.5 mg/mL MWCNT solution. Large bundles were removed by centrifugation at 10 000 g for 30 min. Pyrrole (Sigma) was distilled before use. All other chemicals were research grade and used as received. The fabrication of materials was performed on an Au-disk electrode (4 mm in diameter) for the evaluation of electrical properties and on an Au-coated Si wafer (5 mm  5 mm) for the in vitro biocompatibility test. Before utilization, the Au-disk electrodes were polished with a series of 1-, 0.3-, and 0.05-μm aluminum powder, sonicated in distilled water for 5 min, and swept between -0.4 and 1.2 V in 0.5 M sulfuric acid (H2SO4, Fisher) with a scan rate of 0.5 V/s until the graph was stable. Three different types of materials were fabricated in the present study (mPPY, LBL, and CO-POLY): mPPY. mPPY was prepared by electrochemical polymerization from a solution containing 0.1 M pyrrole monomer and 0.05 M poly(potassium and sodium styrene sulfonate) (PSS, Sigma) using a CHI electrochemical workstation (CHI 660, CH Instruments, Austin, TX) at a constant current of 1 mA. The solution containing the pyrrole monomer was purged with pure N2 for approximately 10 min before use to prevent oxidation of the monomers prior to electrochemical polymerization. A platinum foil (1 cm 1 cm, CH Instruments) and a saturated calomel electrode (SCE, CH Instruments) were used as the counter and reference electrodes, respectively. The PPy film was dried at room temperature, and an additional layer of PPy was fabricated under the same experimental conditions as the previous PPy polymerization. The multilayered PPy films were fabricated by repeating the electrochemical polymerization (60 s) and drying steps (Figure 1A). LBL. A 20 μL aliquot of 0.5 mg/mL MWCNT dispersion was cast on the electrode surface and dried under ambient conditions. Then, PPy (0.1 M) was electrochemically polymerized on the

MWCNT films for 5 s, 10 s, 40 s, 60 s, and 120 s under the same experimental conditions as the PPy polymerization. After characterizing the morphology of PPy-coated MWCNTs, the 60-s PPy polymerization time was chosen to fabricate each layer of LBL. This process was repeated to fabricate the multilayered LBL (Figure 1B). CO-POLY. A layer of the CO-POLY was fabricated by copolymerization of the MWCNTs and PPy from a solution containing 0.5 mg/mL MWCNT, 0.05 M PSS, and 0.1 M pyrrole monomer under the same experimental condition as the PPy polymerization (Figure 1C). A polymerization time of 10 s for one layer of CO-POLY was chosen to optimize the best electrical properties of CO-POLY, and the polymerization process was repeated to produce the multilayer of CO-POLY. 2.2. Surface Characterization. The surface morphologies and cross-sections of the multilayered films of the mPPY, LBL, and CO-POLY were characterized by scanning electron microscopy (SEM, JSM-6700F, Japan). 2.3. Analysis of Electrical Properties. The analysis of electrical properties of multilayered films was performed on a CHI electrochemical workstation using a standard three-electrode system with 0.1 M phosphate-buffered saline (PBS) (pH = 7.0) as the electrolyte. A platinum wire and a SCE were used as the counter and reference electrodes, respectively. Cyclic voltammetry (CV) was performed between -0.6 to 0.6 V vs SCE at a scan rate of 100 mV/s to determine the charge storage capacity of the electrodes. Five hundred continuous cycles were performed on the multilayered mPPY, LBL, and CO-POLY to determine their electrostabilities. The charge storage capacity was calculated using the formula shown below. The capacitance of the first stable curve was set as 100% to determine the original charge storage capacity, and the following cycles were normalized as percentage of the first stable cycle. area inside CV curve 2  scan rate Using the same setup as the CV measurement, alternating current electrochemical impedance spectroscopy (AC-EIS) was performed over a range of frequencies (1-100 000 Hz) with an AC sinusoidal signal of 5 mV in amplitude. A specific comparison of the impedance at 1 kHz, which is near the bioactive frequency, was performed between different materials. 2.4. In Vitro Biocompatibility Test. The PC12 and RAW 264.7 cells purchased from American Type Culture Collection (ATCC) were grown on the multilayered mPPY, LBL, and charge storage capacitance ¼

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Figure 2. SEM images of (a) MWCNT, and (b-f) PPy-coated MWCNTs on Au-coated Si-wafer with polymerization times of 5, 10, 40, 60, or 120 s.

CO-POLY to evaluate their biocompatibility. PC12 cells were grown in a complete culture medium consisting of RPMI-1640 supplemented with 2 mM L-glutamine (Hyclone), 100 IU/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 10% heatinactivated fetal bovine serum (FBS, PAA), and 5% horse serum (HS, Gibco). The cells were maintained at 37 °C in a saturatedhumidity atmosphere of 95% air and 5% CO2. The RAW 264.7 were used for the cytotoxicity test of the mPPY, LBL, and CO-POLY. The cells were grown in a complete culture medium consisting of Dulbecco’s Modified Eagle Medium supplemented with 2 mM L-glutamine (DMEM, PAA), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10% heatinactivated FBS. The cells were maintained at 37 °C in a saturated-humidity atmosphere of 95% air and 5% CO2. MTT Assay. The multilayered mPPY, LBL, and CO-POLY were soaked in distilled water for 24 h to remove free MWCNTs and pyrrole monomers before being sterilized in 75% ethanol for 2 h. The sterilized films were then washed three times with PBS, placed in a 24-well plate, and seeded with 1 mL of 5  104 PC12 cells or RAW 264.7. The cells grown in the well without any sample were used as the negative control. Response to PC12 Cells. The PC12 cells were grown on poly-Llysine-coated mPPY, LBL, and CO-POLY for two days prior to the addition of 20 ng/mL of NGF (2.5S, Sigma) to the growth medium. Three days after NGF was applied to the PC12 cells, the length and number of neurites per cell were quantified under SEM observation. Prior to the SEM observation, the cells were rinsed with PBS and fixed with 2.5% glutaraldehyde (Sigma) overnight. They were then postfixed with 1% osmium tetroxide (Sigma) for 1 h and rinsed three times with PBS. Next, the samples were dehydrated through an aqueous replacement process by soaking in a series of graded alcohol (50%, 60%, 70%, 80%, 90%, and 100% three times) for 10 min each. The critical drying point was performed by immersing the samples in 50%, 70% of 1,1,1,3,3,3-hexamethyldisilazane (HMDS, Sigma) in ethanol, and 100% of HMDS for 20 min each. The samples

were then dried at room temperature and coated with platinum for SEM. 2.5. Statistical Analysis. The statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by a multisample comparison test. P-values less than 0.05 were considered statistically significant.

3. RESULTS 3.1. Sample Fabrication and Optimization. Figure 2 presents the SEM images of PPy-coated MWCNTs with different polymerization times. Neither particles of amorphous carbon nor other impurities were observed after purification of the carboxyl-functionalized MWCNTs (Figure 2a). After 5 and 10 s of polymerization of PPy on the surface of the MWCNTs, the diameter of MWCNTs increased from ∼20 nm to ∼40 nm (5 s) and ∼80 nm (10 s, which indicates that the PPy polymerized on the surface and coated over the fibrous structure of the MWCNTs. After 120 s of PPy polymerization, the fibrous structure of the MWCNTs eventually disappeared, and a rough surface similar to the morphology of PPy appeared. Because a 60 s interval of PPy polymerization on the MWCNTs produced a porous film structure and encapsulated the MWCNTs with a sufficient amount of PPy (Figure 2e), 60 s of polymerization was selected to create each sublayer of the LBL film. There was no significant difference in microscopic surface morphology of the CO-POLY at any of the examined polymerization times between 10 and 60 s (data not shown). Hence, the polymerization time for CO-POLY was optimized based on electrochemical properties, electrochemical stability and a study on delamination of the films during the polymerization processes and continuous CV scanning. A polymerization time of 10 s was selected to create each sublayer of the CO-POLY film. The surface morphologies of the first and the fifth layer of mPPY, LBL, and CO-POLY are shown in Figure 3. Although the microscopic morphologies of LBL are different from mPPY and 5494

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Figure 3. SEM image of the surface structure of the 1st (1st row) and 5th (2nd row) layer, and the cross-sectional view (3rd row) of multilayered mPPY, LBL, and CO-POLY (from left to right).

Figure 4. Electrical properties of multilayered mPPY, LBL, and CO-POLY. (a and b) CV and impedance of one-layered and five-layered films. (c and d) Comparison of charge storage capacity and impedance at 1 kHz between mPPY, LBL, and CO-POLY. Error bars represent the standard deviation.

CO-POLY, there was no obvious morphologic difference between mPPY and CO-POLY. The mPPY and CO-POLY films were highly dense with some hill-like structures on the surface, whereas LBL exhibited a porous structure with marked roughness. The SEM images of the cross-section of five-layered mPPY, LBL, and CO-POLY are shown in Figure 3g-i. Each layer of mPPY and LBL appeared to be discrete, but the CO-POLY appeared as a single layer with no distinguishable boundaries between neighboring layers. As expected, the thickness of the

CO-POLY increased as the polymerization time increased (the maximum observed time was 60 s). The thickness of the COPOLY (10 s of polymerization time for each layer) was significantly thinner than that of mPPY (5.06 ( 0.21 μm, 60 s of polymerization time for each layer), whereas the thickness of the CO-POLY obtained from 60 s of polymerization was 4.04 ( 0.16 μm, which was comparable with the mPPY. The LBL was much thicker (7.53 ( 0.21 μm) than the mPPY and CO-POLY, which was partly due to the additional layers of MWCNTs. 5495

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Figure 5. (a-c) CV of bare electrode, the 1st, 100th, 300th, and 500th cycle of multilayered mPPY, LBL, and CO-POLY. (d) The charge storage capacity of five-layered mPPY, LBL, and CO-POLY over 500 continuous CV cycles between -0.6 and 0.6 V with a scan rate of 100 mV/s in 0.1 M PBS (pH = 7.0) (n g 3, normalized as a percentage of the original charge storage capacity of each coating). (e) Optical images of the typical delamination of films from the electrode surface during CV scanning. Error bars represent the standard deviation.

3.2. Electrical Properties. The electrical properties of mPPY,

LBL, and CO-POLY were characterized with CV and electrochemical impedance spectroscopy (EIS) (Figure 4). Cyclic Voltammetry. Although the capacitance of the singlelayered mPPY and LBL were relatively similar, the capacitance of the five-layered films was considerably different (Figure 4a). The capacitance of the five-layered LBL was significantly larger than that of the mPPY and CO-POLY. As the number of layers increased, the charge storage capacity of the multilayered coatings increased (Figure 4c). For mPPY, the charge storage capacity initially increased but reached a plateau at the fourth to fifth layers. The charge storage capacity of the LBL and COPOLY increased almost linearly throughout all the layers. The capacitance of the LBL, however, was significantly larger than that of the CO-POLY. Moreover, the increment for LBL after each layer was relatively larger than that of mPPY and CO-POLY. At the fifth layer, the charge storage capacity of LBL was almost twice that of mPPY and CO-POLY and 660-times larger than that of the bare electrode. Interestingly, the enhancement for mPPY and CO-POLY was less than 400-times larger than the bare electrode. Impedance. Compared with the bare electrode, the impedance of all three types of coatings decreased from 2 orders of magnitude at low frequency to several ohms at high frequency (Figure 4b), which was consistent with previous reports.15 Comparing the impedance of the three types of films at 1 kHz, the frequency of a neuronal action potential,33 the impedance of mPPY and CO-POLY slightly increased with the number of layers after the first layer of coating (possibly the lowest obtainable impedance) (Figure 4d). Conversely, LBL showed a continuous drop in the impedance as the number of layers increased. This may have resulted from the increase of interfacial area and the enhanced adhesion between the LBL coating and the electrode surface as opposed to poor adhesion properties of

pure PPy.34 In our study, more than 50% of the mPPY coatings were delaminated during the fabrication process and CV scanning. Interestingly, the same results were observed with the COPOLY modification (Figure 5e). The impedance values of the five-layered mPPY, LBL, and CO-POLY at 1 kHz were 22.8, 14.7, and 18.1 Ω, respectively. The impedance of LBL is approximately five-times lower than bare electrode. 3.3. Electrochemical Stability. Five hundred cycles of CV scans were performed on the multilayered mPPY, LBL, and COPOLY to evaluate their electrostabilities (Figure 5). The capacitance of all the five-layered films gradually decreased with an increasing number of cycles due to the intrinsic nature of mPPy.23 The percentage of the charge storage capacity lost for LBL, however, was significantly less than that of mPPY and COPOLY. After 500 cycles, the loss of charge storage capacity for mPPY, LBL, and CO-POLY was approximately 70%, 20%, and 50%, respectively (Figure 5). The incorporation of MWCNTs into mPPy in a layered manner (i.e., LBL) increased the electrostability from 30% to 80%, which was calculated from the remaining charge storage capacity. The incorporation of MWCNTs via the copolymerization method (i.e., CO-POLY) hardly increased electrostability, even with a much smaller thickness. Interestingly, during the 500 cycles of CV scans, almost half of the mPPY and 10% of the CO-POLY films were delaminated from the electrode surface, but no delamination was observed for the LBL-modified electrodes. The enhanced electrical properties and electrostability of LBL compared with mPPY and CO-POLY can be partially explained by the tight adhesion of the LBL coating to the electrode surface by the MWCNT grafting. 3.4. In Vitro Biocompatibility Test. An in vitro biocompatibility test was performed on the mPPY, LBL, and CO-POLY using PC12 and RAW 264.7 cells. Compared with controls, PC12 and RAW 264.7 cells grown on the multilayered mPPY, 5496

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Figure 6. Results of biocompatibility tests on PC12 cells. (a) Viability of PC12 cells grown on the negative control, mPPY, LBL, and CO-POLY. (b and c) Average neurite length and number of neurites per cell in PC12 cells treated with NGF for three days (n g 3). (d-f) SEM images of PC12 cells treated with NGF for three days. Error bars represent the standard deviation.

LBL, and CO-POLY for three days did not show any obvious difference in cell viability (Figure 6a), which was consistent with previous reports on the biocompatibility of PPy and MWCNTs30,35-37 (RAW 264.7 data not shown). To further study the cellular morphology and dendrite extension, PC12 cells grown on the mPPY, LBL, and CO-POLY were imaged via SEM. After three days of NGF treatment, as expected, neurite extensions of PC12 cellswere observed regardless of the tested samples (Figure 6d-f). Cells grown on the mPPY and CO-POLY tended to cluster together, whereas those grown on the LBL were more likely to grow individually. The average neurite length of PC12 cells grown on the LBL was significantly larger than the neurites from the cells grown on CO-POLY and slightly larger than those on mPPY (Figure 6b). Moreover, each PC12 cell grown on LBL had a larger number of neurites compared with the cells on mPPY and CO-POLY. Although all three materials supported PC12 cell growth and adhesion, the microscopic morphologies and quantitative analysis of the neurites suggested that the biocompatibility of the LBL films was superior to that of mPPY and COPOLY. Thus, LBL offers the potential for surface modification of neural electrodes.

4. DISCUSSION In the present study, the functional stability and biocompatibility of mPPY, LBL, and CO-POLY were investigated. Measurements of charge storage capacity, impedance, and electrochemical stability demonstrated that layering conducting polymers with MWCNTs (i.e., LBL) resulted in a porous structure, which exhibited superior performance compared with mPPY and CO-POLY. In the initial stage of polymerization, the pyrrole monomers likely attached to the surface of the MWCNTs where they were subsequently oxidized to PPy. Other pyrrole monomers in the vicinity were subsequently attracted to the PPy-coated MWCNTs, which resulted in a thickening of the PPy coating and increased the diameter of the porous structures. If the polymerization of PPy continued, all the pores in the multilayered films would eventually disappear. In the case of the

CO-POLY, MWCNTs were copolymerized with PPy and PSS. PSS tends to serve as a dominant dopant in the case of coexistence of two dopants of MWCNTs and PSS, which may have led to the limited amount of MWCNTs incorporated in the CO-POLY. An optimal neural electrode should maximize the charge storage capacity arising from the movement of charged ions in the electrolyte toward or away from the electrode and minimize the site impedance.38 The geometric area of the recording site and the site impedance, however, are directly related to the specificity and sensitivity of the electrodes. Indeed, a small geometric area is needed to isolate the electrical signal from individual neuron, and the impedance is proportional to both the thermal noise and the signal loss through shunt pathways.39-42 A decrease in the geometric area usually results in an increase in the impedance.39-41 The porous structure of the LBL coating significantly enhances the surface area over a given geometric space and promotes an effective ion exchange between the recording site and the surrounding tissue. This results in a dramatic increase in the capacitance, which has been shown to correspond to a decrease in impedance.9 It was observed that the impedance of mPPY and CO-POLY increases as the number of layers whereas that of LBL decreases. This can be explained with difference in the effective interfacial surface area of the samples. Electrochemically deposited PPy on electrode surface substantially reduces the impedance at 1 kHz and excessive deposition of PPy tends to exhibit slight increase in the impedance compared with the optimal condition of PPy deposition. The small increase in the impedance is attributed to the fact that excessive deposition of PPy beyond the optimal condition makes the PPy film fully dense and thickened, thus providing relatively small interfacial surface area for charge transport compared to the optimal PPy coatings.6,43 Similarly, the increases in the impedance at 1 kHz for mPPY and CO-POLY could be explained with the theory of the fully dense and thickened film. However, unlike mPPY and CO-POLY, the additional layers of MWCNTs in LBL provides porous scaffold for PPy growth attributing to the 5497

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The Journal of Physical Chemistry C increase of overall interfacial surface area for charge transport. As a result, the impedance of LBL gradually decreases as the number of layers increases. Surface modification of neural electrodes with CNT/conductive polymer composites can dramatically increase the charge storage capacity. Keefer and co-workers have reported that a CNT/PPy composite shows an increased charge storage capacity of 755 mF/cm2 compared with unmodified electrodes.29 In addition, charge storage capacity values of 1244 mC/cm2 and 45 mC/cm2 were reported for a PPy and single-walled carbon nanotubes composite on a Pt electrode (100 μm in diameter)22 and a MWCNT- polyelectrolyte composite on a Pt-Ir electrode (75 μm in diameter),31 respectively. Compared with pure PPymodified electrodes, SWNT/PPy-modified electrodes revealed a 1.76-fold increase in charge storage capacity,22 and the layer-bylayer addition of MWCNTs to PPy (i.e., LBL) showed a charge storage capacity of 244.4 mC/cm2, resulting in a 1.86-fold enhancement. In addition to the enhancement in the charge storage capacity and impedance, the electrostability of the multilayered LBL was dramatically enhanced compared with the mPPY, in which no MWCNTs were incorporated. In general, the poor electrochemical stability of PPy originates from the irreversible reaction of PPy backbone.23 Doping PPy with CNTs could enhance electron flow and ion transfer in the PPy films, reduce the electrode polarization, and retain electrochemical reversibility of PPy by providing a conductive path that is not subject to oxidative degradaton.22,44 On the other hand, highly porous structures of PPy generated by the CNTs make PPy films more resistive to internal strain that often causes delamination of the coatings. Furthermore, due to the anion intercalation during the oxidation of polymer, the CNTs could act as charge-balancing anionic dopants to minimize the volume changes of PPy films.45,46 On the basis of these factors, we believe the MWCNTs incorporated in the LBL could enhance the electrochemical and mechanical properties of the LBL by preventing the irreversible PPy reactions and minimizing internal strains and volume changes of PPy films. Among the approaches to enhance the electrochemical stability of PPy, multilayered MWCNTs combined with polyelectrolytes showed a 5% loss of charge capacity in 300 continuous cycles,31 which was comparable to the data obtained with LBL in our study. We note that delamination of the films during the fabrication process and CV scanning was observed on the mPPY and the CO-POLY, but not on the LBL.

5. CONCLUSION Alternative layers of PPy and MWCNTs were successfully fabricated over electrodes through a layer-by-layer method. Compared with PPy-modified electrodes, electrodes modified with multilayered LBL showed higher charge storage capacity, lower impedance, and dramatically improved electrostability. Biocompatibility tests proved that these multilayered LBL coatings support PC12 cell growth and neurite extension with NGF treatment. The large surface area, high charge storage capacity, and exceptional electrostability of the multilayered LBL coating make it an excellent candidate for neural electrodes. ’ AUTHOR INFORMATION Corresponding Author

*Tel: (65) 6790 4111. Fax: (65) 6791 6905. E-mail: [email protected].

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