Graphene oxide doped conducting polymer nanocomposite film for

Biomaterials 35 (2014) 2120e2129

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Graphene oxide doped conducting polymer nanocomposite film for electrode-tissue interface Hong-Chang Tian a, Jing-Quan Liu a, *, Dai-Xu Wei b, Xiao-Yang Kang a, Chuan Zhang a, Jing-Cheng Du a, Bin Yang a, Xiang Chen a, Hong-Ying Zhu a, Yan-Na NuLi c, Chun-Sheng Yang a a b c

National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Shanghai Jiao Tong University, Shanghai, PR China National Engineering Research Center for Nanotechnology, Shanghai, PR China Department of Chemical Engineering, Shanghai Jiao Tong University, Shanghai, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 October 2013 Accepted 21 November 2013 Available online 12 December 2013

One of the most significant components for implantable bioelectronic devices is the interface between the microelectrodes and the tissue or cells for disease diagnosis or treatment. To make the devices work efficiently and safely in vivo, the electrode-tissue interface should not only be confined in micro scale, but also possesses excellent electrochemical characteristic, stability and biocompatibility. Considering the enhancement of many composite materials by combining graphene oxide (GO) for its multiple advantages, we dope graphene oxide into poly(3,4-ethylenedioxythiophene) (PEDOT) forming a composite film by electrochemical deposition for electrode site modification. As a consequence, not only the enlargement of efficient surface area, but also the development of impedance, charge storage capacity and charge injection limit contribute to the excellent electrochemical performance. Furthermore, the stability and biocompatibility are confirmed by numerously repeated usage test and cell proliferation and attachment examination, respectively. As electrode-tissue interface, this biomaterial opens a new gate for tissue engineering and implantable electrophysiological devices. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Graphene oxide Conducting polymer Electrode-tissue interface Microelectrode Electrochemical deposition Tissue engineering

1. Introduction With the rapid development of micro fabrication technology, biomedical devices can be manufactured considerably tiny and structurally diverse, which minimize the damage during and after implantation for both short term and long term requirement [1]. The most important factors to decide the performance of implanted micro device are the efficiency and reliability of microelectrodes, which can be used to diagnose and treat many diseases by electrophysiological recording and functional electrical stimulation respectively [2]. With the help of microelectrodes, researchers have already achieved clinical effectiveness, such as relieving Parkinson’s disease symptoms by deep brain stimulation [3], restoring movement of paralyzed limb through electrical stimulation based on neural recording [4], recovering visual or auditory capability by visual prosthesis and cochlear implants, respectively [5,6]. Nowadays, some dense electrode arrays and tenuous electrodes are developed to undertake complex and precise electrophysiological

* Corresponding author. Tel./fax: þ86 21 34207209. E-mail addresses: [email protected], [email protected] (J.-Q. Liu). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.11.058

research with providing excellent spatial selectivity and low power consumption [7e9].The problem is that smaller size, certainly lowers the damage to the tissue, would inevitably damage the performance and safety of the electrodes [10]. Because decrease in the size of electrode will lead to an increase in impedance and a drop in charge storage capacity (CSC), which, as a result, means poor recording signal quality and high stimulating current that may damage tissue. Considering this fact, the interface material plays a significant role to improve the electrode performance. Many efforts are made to develop more ideal electrode-tissue interface materials with properties including: electrical property containing low impedance, high CSC and high charge injection limit; stability for long term work or implantation without significant property variation; biocompatibility ensuring direct contact with tissue without inducing severe tissue response, toxicity or even necrosis. The most widely applied electrode-tissue interface material is noble metals including platinum, gold, iridium, titanium and their alloys due to long-term stability during implantation process without serious chemical corrosion [11]. Nevertheless, the stimulation and recording performance of bioelectronic devices is restricted mostly by the low charge injection limit and CSC of bare metallic bio-electrodes [12]. Some porous structure and peculiar

H.-C. Tian et al. / Biomaterials 35 (2014) 2120e2129

profile is fabricated to form a rough surface of the material, which would consequently improve the electrical properties and biocompatibility by providing a larger effective surface area of electrode sites [13]. Iridium oxide (IrOx) is another electrode coating material regularly reported which has relatively low impedance and high charge transfer efficiency [14]. However, its oxidation-reduction reaction and poor bonding effect to substrate was observed during electrical stimulation. These defects in some degree restrict IrOx from becoming an ideal electrode-tissue interface as the possibility of causing tissue damage [15]. Widely explored conducting polymers, especially poly (3,4ethylenedioxythiophene) (PEDOT), satisfy these requirements of electrode-tissue interface [16]. Besides, the structure and property of conducting polymers can be changed by doping counterions like polymers, biomolecules and carbon nano material, which provides enormous possibilities for the use of conducting polymer in implantable prostheses [17e19]. However, fragmentation and exfoliation of conducting polymer on the substrate may be induced as expansion and contraction of volumes would take place during electrochemical oxidation-reduction procedures [20,21]. This, as a result, arouses the problem of stability and safety for common conducting polymer modified electrode. As single atom layer structural graphene has excellent electrical, mechanical, thermal and optical characteristics [22,23], it has been applied in various areas, especially in biomedical research, such as acting as the cellular interface of stem cell differential [24], sensing biomolecules [25], promoting cell growth [26], and applying in electrical stimulation [27]. It is reported that mixing graphene, especially water soluble graphene oxide (GO), as counterions into polymers forming new composite materials will improve their performance further [28,29]. For example, multifunctional composite hydrogels consisting of GO and DNA possess high mechanical strength, environmental stability, and dyeloading capacity [30]. Also, hybrid papers fabricated by depositing polyaniline on GO exhibit excellent electrochemical performance and biocompatibility [31]. For these reasons mentioned above, GO was doped into PEDOT to enhance the stability of the composite for its excellent mechanical property, and to promote electrochemical performance by increasing effective surface area for its stacking induced by pep bonds interaction. In this paper, we deposited GO as counterion with PEDOT onto surface of microelectrodes. The original GO doped morphology and structure of electrochemically deposited film were observed and analyzed by scanning electron microscope (SEM), transmission electron microscope (TEM) and atom force microscope (AFM). Electrical characterizations containing electrochemical impedance spectroscopy (EIS), cyclic voltammogram (CV) and charge injection limit were performed to investigate electrical property of PEDOT/GO nanocomposite film. Moreover, the stability of PEDOT/GO was evaluated by enduring repetitive CV scanning. In addition, in order to test biocompatibility and cell adherence of PEDOT/GO, we cultured highly differentiated rat pheochromocytoma PC-12 cells (hdPC-12 cells) and mouse embryonic fibroblasts NIH/3T3 (NIH/3T3 cells) for cell viability, proliferation and attachment experiments. 2. Materials and methods 2.1. Electrochemical deposition Electrolyte for PEDOT/GO polymerization consisted of 2 mg/ml graphene oxide aqueous solution (XFNano, China). EDOT (0.01 M) (SigmaeAldrich) was added into GO solutions, followed by stirred for 2 h to dissolve EDOT. The solution was subsequently purged with nitrogen gas for 10 min to eliminate the oxygen in the electrolyte. PEDOT/GO film was deposited on glass slices sputtered with 150 nm gold and gold wire electrode (diameter of 100 mm) by applying constant current at density of 0.2 mA/cm2 through the electrolyte by CHI 660c (CH Instrument) electrochemistry work station.

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2.2. Material morphology and characterization Morphology of PEDOT/GO film deposited on gold wire electrodes and gold sputtered slices was observed by high vacuum scanning electron microscopy (ULTRA 55, Zeiss, Germany). The inner structure of PEDOT/GO fragment was investigated by transmission electron microscopy (JEM-2100, JEOL Ltd., Japan). Detailed morphology and roughness of PEDOT/GO film were obtained with Multimode Nanoscope V Scanning Probe Microscopy System (Bruker, USA). Morphology of cells cultured on PEDOT/GO film was observed by low vacuum scanning electron microscopy (S-4800, Hitachi, Japan). X-ray photoelectron spectroscopy (XPS) analysis was performed by X-ray photoelectron spectrometer (AXIS ULTRA DLD, Kratos) with an excitation source of Al Ka radiation (l ¼ 1486.6 eV). Fourier transform infrared spectroscopy (FTIR) of GO and PEDOT/GO was obtained by Fourier transform infrared spectrometer (Nicolet iN10 MX, ThermoFisher).

2.3. Electrochemical characterization To investigate the electrochemical property, the PEDOT/GO film was deposited on gold wire electrodes with a site area of approximately 0.03 mm2. Both electrochemical impedance spectrum (EIS) and cyclic voltammogram (CV) were measured by CHI 660c in phosphate buffered saline (PBS, pH 7.4) versus saturated calomel electrode (SCE, CH Instrument). CV was scanned in potentials between 0.6 V and 0.8 V at a scan rate of 50 mV/s. EIS was measured over frequency range from 0.1 Hz to 100,000 Hz.

2.4. Stimulation To study the electrical performance during stimulation, bare gold wire electrode and PEDOT/GO coated electrode were immersed in phosphate buffered saline (PBS, pH 7.4) and connected with anode of electrical stimulator separately. A platinum foil connected with cathode of electrical stimulator and saturated calomel electrode were also immersed in PBS as counter electrode and reference electrode, respectively. A series of amplitude of 1 mA charge balanced, cathodic first, biphasic pulse current at 50 Hz was generated by an electrical stimulator (Master 8, A. M. P. I., Israel). An oscilloscope (TDS-2000, Tektronix, USA) was used for voltage excursions recording.

2.5. Stability test Solid plane electrodes based on silicon substrate with site diameter of 100 mm were fabricated for stability assessment. The PEDOT/GO film deposited on the plane electrode at deposition charge density of 0.36 C/cm2 was sustained 1000 cycles CV scanning ranging from 0.6 V to 0.8 V at rate of 100 mV/s in phosphate buffered saline (PBS, pH 7.4). The electrochemical property of CV and EIS was measured before and after repeated CV scanning for comparison. The mechanical stability was examined by comparison of SEM morphology observed before and after scanning.

2.6. Cell culture To investigate the cell viability of PEDOT/GO film, highly differentiated rat pheochromocytoma PC-12 cells (hdPC-12 cells), and mouse embryonic fibroblasts NIH/3T3 (NIH/3T3 cells) were employed in this study, purchased from Chinese Academy of Sciences. HdPC-12 cells and NIH/3T3 cells cultivated with DMEM (Gibco, USA) supplied with 5% CO2 at 37 C. 2.7. Cell proliferation The hdPC-12 cells and NIH/3T3 cells of 5 103 were seeded onto samples of glass slices sputtered with gold and electrochemically deposited PEDOT/GO film on sputtered gold slices in 48-well flat-bottomed cell plates, respectively, for incubating with DMEM (Gibco, USA) supplied with 5% CO2 at 37 C for 1 day, 4 days and 7 days, respectively. For estimating the cell viability, medium was removed and cells on samples were treated with 150 ml fresh DMEM with CCK-8 for 3 h. Then, OD value (optical density) was measured at 450 nm by microplate reader (Multiskan MK3, Thermo Labsystems, Finland). Six parallel replicates were read for each sample.

2.8. Immunofluorescent staining for cell proliferation For observation of cell proliferation, cells were labeled after 1 day, 4 days and 7 days of cultivation. After the removal of the medium, the cells on samples were washed with PBS, and then fixed for 5 min in 3.5% formaldehyde in PBS. Then they were immersed in 0.1% Triton X-100 for 5 min, followed by washing in PBS for 3 times. After aforesaid processing, the cells on samples were stained with phalloidinTRITC (Sigma, USA) for 1 h at room temperature without illumination. Subsequently, the nucleuses of these samples were quickly stained by 5 mg/ml DAPI (4,6diamidino-2-phenylindole dihydrochloride, Sigma, USA) for 5 min at room temperature. Finally, the cells on samples were washed in vast PBS for removing residual stain, and viewed by a laser scanning confocal microscope (Leica TCS SP5, Leica, Germany).

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2.9. Cell attachment The two kinds of cells of 5 104 were seeded onto samples of glass slices sputtered with gold and electrochemically deposited PEDOT/GO film on sputtered gold slices in 48-well flat-bottomed cell plates, for incubating with DMEM (Gibco, USA) supplied with 5% CO2 at 37 C for 1 h, 6 h, 12 h and 24 h, respectively. Viability of cell attachment on samples was researched with CCK-8 as described in cell proliferation. 2.10. Immunofluorescent staining for cell attachment In 24 h of adhesion, the amount and distribution of cells was observed by DIO (Fanbo, China) dissolved in fresh DMEM at a concentration of 1 mg/ml. The samples were incubated for 10 min, then washed with PBS and fixed in 3.5% formaldehyde in PBS for 20 min. Finally, the cells on samples were washed in vast PBS for removing residual stain, and viewed by a laser scanning confocal microscope.

structure for tissue engineering scaffolds, such as directional extracellular matrices. Moreover, the PEDOT/GO was broken into fragment for inner structure analysis by TEM. It is the composition of homogeneous dispersion of PEDOT among GO layers that confirms the GO enhanced rebar concrete structure mentioned before (Fig. 2b). In addition, the morphology details and surface roughness of 41.2 8.6 nm (n ¼ 4) was further investigated and measured by AFM (Fig. 2c). It can be inferred from the morphology observation results of PEDOT/GO composite film that the enlargement of effective surface area of electrode site acts as a significant benefit to improve electrochemical property through reducing electrodeelectrolyte interfacial impedance.

3. Results and discussion

3.2. Chemical structure

GO, standing as counterion with PEDOT, was deposited onto surface of microelectrodes in galvanostatic mode. As illustrated in Fig. 1, the monomer ethylenedioxythiophene (EDOT) was electrochemically oxidized to form conducting polymer chains firstly. Then positively charged PEDOT chains was combined, by ionic bonds, with negative groups by GO. Thus a PEDOT/GO composite film was formed and subsequently accumulated on the surface of depositing microelectrode. In this composite film, GO disorderly distributed as the structural material to form three dimensional crossover networks, while PEDOT served as stable charge transfer medium was interspersing among the interspaces of graphene nets. Like rebar in concrete, GO doping enhanced the mechanical property of conducting polymer film. Meanwhile, the conducting polymer encapsulation prevents GO from dispersing to the tissue during recording or stimulation process, which greatly abates the possibility of cytotoxicity induced by carbon nano material diffusion while contacting with tissue directly.

The chemical structure of the PEDOT/GO composite film was further confirmed by XPS and FTIR. The XPS spectrum of P/G-1800 film shows a primary peak of C 1s at 285 eV, a later peak of O 1s at 532 eV and a following weak peak of S 2p at 164 eV (Fig. 3aec). From detailed analysis, a strong sp2 hybridized graphitic carbon peak at 284.6 eV indicates that large quantity of GO exists on the surface of PEDOT/GO film. And the peaks at 285.4 eV and 288.4 eV are attributed to CeO and C]O configurations, respectively [32,33]. Besides, the presence of PEDOT is proved by a weak peak at 164.0 eV which corresponds to eSe of thiophene [34]. Moreover, the FTIR spectra (Fig. 3d) of GO and PEDOT/GO is analyzed to confirm the composition of electrochemical deposited PEDOT/GO. For spectrum of GO, the stretching vibration peaks at 1214.9 cm1 and 1722.1 cm1 correspond to CeO and C]O bonds, respectively. And peaks at 3436.5 cm1 corresponds to OeH group [35]. In addition, the spectrum of PEDOT/GO shows chemical structure obviously different from GO, such as C]C and CeC of thiophene at stretching vibration peaks of 1520.5 cm1 and 1333.4 cm1, respectively, the CeOeC peaks at 1202.8 cm1, 1088.1 cm1 and 1057.6 cm1, the CeS peaks at 980.2 cm1, 932.7 cm1 and 838.4 cm1, and CeOeCH2eCH2eOeC vibration peak at 1143.8 cm1 [36,37].

3.1. Morphology Due to the process mentioned above, a rough folding morphology was observed by SEM, contributing to an increase in the effective film surface area. As shown in Fig. 2a, the PEDOT/GO coated surface becomes rougher with the continuously growing amount of GO dopants when increasing the deposition charge density. Interestingly, different from the random distribution of GO dopants deposited on plane substrates, almost all graphene on the film surface aligned along the axis direction of the gold wire because of the cylindrical wire surface and micro dimension. This unique micrometer-size morphology could be applied as functional

3.3. Electrochemical impedance spectroscopy The electrochemical impedance performance of bioelectrodes is a determining factor influencing the quality of electrophysiological signal recording and the effects of bioelectrical stimulation. Especially, the impedance at 1 kHz is an important parameter, relating to the frequency of neuronal recording and power consumption

Fig. 1. Schematic illustration of chemical structure of PEDOT/GO composite film. Dashed line connected the upper molecule chain (PEDOT) and lower molecule net (graphene oxide) refers to ionic bond.


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Fig. 2. (a) Scanning electron microscopy (SEM) of PEDOT/GO coatings on plane gold sputtered glass slices (upper) and cylindrical gold wires (under) with different deposition charge density of 0.12 C/cm2, 0.24 C/cm2, 0.36 C/cm2 and 0.48 C/cm2, respectively. (b) Transmission electron microscopy (TEM) of PEDOT/GO fragment morphology in (left) low and (right) high magnification. (c) Atom force microscopy (AFM) of PEDOT/GO film deposited on plane substrate in 3D view (left) and in front view (right).

during electrical stimulation [38]. As shown in Fig. 4a, the impedance at 1 kHz decreases sharply from 20,590 256 U (n ¼ 4) of uncoated gold electrodes to 997 52 U (n ¼ 4) of coated gold electrodes. And the impedance of electrodes deposited with PEDOT/GO for 600 s, 1200 s, 1800 s and 2400 s at frequency below

S 2p

O 1s

Intensity (a. u.)

b

0

C 1s Origin Sum C=C C-O C=O

Intensity (a. u.)

Survey

C 1s

a

10 Hz continuously decreases along with the increase of deposition time. The phase plots show that the frequency angles are approximately 0 at high frequencies and 90 at low frequencies, which demonstrates that the PEDOT/GO acts as resistive material and capacitive material, respectively. In Nyquist plots (Fig. 4b), small

200 400 600 800 1000 1200 Binding Energy (eV)

c

282

d 160 Origin -S-

158 160 162 164 166 168 170 Binding Energy (eV)

% Transmittance

Intensity (a. u.)

S 2p

284 286 288 290 Binding Energy (eV)

292

GO PEDOT/GO

120 80 40 4000

3000 2000 1000 Wavenumbers (cm-1)

Fig. 3. XPS spectra for the (a) survey scan, (b) C 1s region and (c) S 2p region of the PEDOT/GO film. (d) FTIR spectra of GO (red line) and PEDOT/GO (black line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Electrochemical impedance spectroscopy (a) impedance curves (upper) and phase curves (under), and (b) Nyquist curves of PEDOT/GO film for different deposition time. P/ G-600 indicates PEDOT/GO film with deposition time of 600 s, and so do others. Circuit model fitting analysis of EIS of PEDOT/GO coated electrodes: impedance and phase curves (c) and Nyquist curves (d). Msd and Cal indicate experimentally measured data and fitting calculated data, respectively. In Nyquist curves, the insertions exhibit the high frequency parts. (e) Cyclic voltammogram curves of PEDOT/GO coated electrodes for different deposition time. (f) Linear fit of the variation of charge storage capacity (green line) and impedance at 1 kHz (orange line) during the increment of deposition charge density, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

arcs at high frequencies and straight lines at low frequencies indicate the charge transfer procedure is controlled by electrochemical reaction and ion diffusion, respectively. In addition, the steeper gradient of PEDOT/GO film shows faster ion diffusion rate and more approximation to the ideal capacitor, which is desired to perform electrical stimulation safely [33]. Here we introduce an equivalent circuit model suggested by Danielsson et al. to further analyze the EIS of PEDOT/GO composite film [39,40]. As shown in the insertion of Fig. 4d, the circuit model for analysis of conducting polymer coated metal electrode consists of a solution resistance RS, a double layer capacitance Cdl in parallel with a charge transfer resistance Rct, and a bounded Warburg element ZD in series with bulk capacitance Cd. The bounded

Warburg element ZD representing diffusion process in thin layer of electrolyte is defined as follows:

ZD ¼

pffiffiffiffiffiffiffiffiffiffi jus pffiffiffiffiffiffiffiffiffiffi D jusD

sD coth CD

where sD and CD represent diffusion time constant and diffusion pseudocapacitance, respectively, and diffusion resistance RD is defined as sD =CD . As displayed in Fig. 4c and d, the equivalent circuit model provides fairly good fitting to measured data as the fitting degree generally smaller than 0.0015. The numerical fitting results of each components of PEDOT/GO coated gold electrode are shown

H.-C. Tian et al. / Biomaterials 35 (2014) 2120e2129 Table 1 The numerical fitting results of each equivalent circuit component of PEDOT/GO coated gold electrodes with increasing deposition time. Deposition time (s)

Cdl (F)

Rct (ohm)

ZD (S$sec^0.5)

Cd (F)

c2

600 1200 1800 2400

1.528e-9 6.834e-10 5.868e-10 5.693e-10

615.4 864.5 897.3 942.7

1.58e-4 5.734e-5 4.382e-5 3.398e-5

1.16e-6 2.041e-6 2.7e-6 3.192e-6

4.738e-4 1.005e-3 9.644e-4 1.404e-3

in Table 1. The charge transfer resistance Rct increases along with the deposition time, because the thickness increase of PEDOT/GO film would induce slow charge transfer rate, which subsequently leads to diminishment of a double layer capacitance Cdl and

bounded Warburg element ZD. The bulk capacitance Cd, largely related to electroactive surface area, increases along with the effective surface area, because of accumulative fold GO doping during deposition process [19]. 3.4. Cyclic voltammogram Cyclic voltammogram (CV, shown in Fig. 4e) was measured to evaluate the redox characteristics and the charge storage capacity of the electrodes. The CV curves approach to parallelograms without obvious redox peak indicates that the PEDOT/GO film acts as electric double-layer capacitor during the charge transfer procedure of CV scanning, which is in accordance with the EIS results. Moreover, the CSC increases continuously from 8.27 1.32 mC/cm2

b 1 ms

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-3

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-3 -3 0.00 2.50x10 5.00x10 Time (s)

6 5

Bare gold PEDOT/GO Linear fit of PEDOT/GO

4 3

y =1.32+3.49x

2

R 2=0.9824

1 0 0.0

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Pulse Width (ms)

Fig. 5. (a) Voltage excursions of the PEDOT/GO coated (blue) and bare gold (red) electrodes under 0.1 mA charge balanced current pulse stimulation (black). (b) Linear fit of charge injection limit of PEDOT/GO coated (blue line) and bare gold (red line) electrodes for increasing pulse width. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. (a) The SEM morphology of PEDOT/GO film before (left) and after 1000 cycles CV scanning (right). The middle figure exhibits the 1000 cycles CV scanning procedure applied on PEDOT/GO film. CV curves (b) and impedance and phase plots of EIS (c) of PEDOT/GO film before (blue) and after (red) 1000 cycles CV scanning. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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a

1 day

4 days

7 days

4 days

7 days

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Da

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ys

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Au

Ce

PE

ll p

DO

pa

d

lat

T/

e

GO

d 2.0

e at 450 nm

1 day

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b

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6

0.5 0.0

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ys

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PE

Ce

DO

ll p

pa

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T/

lat

e

GO

Fig. 7. CLSM image of hdPC-12 cells (a) and NIH/3T3 cells (b) proliferation on PEDOT/GO film for 1, 4 and 7 days, respectively. Cell viability data diagram of hdPC-12 cells (c) and NIH/3T3 cells (d) proliferation on bare gold (black), cell plate (red), and PEDOT/GO (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(n ¼ 4) of uncoated gold electrodes to 86.75 7.44 mC/cm2 (n ¼ 4) of coated gold electrodes with the increasing of deposition charge density. These enhancements of electrochemical characters are resulted from good electrochemical property of PEDOT/GO film and the increment of film thickness and effective electrode-tissue interface area. While the deposition charge density increasing, the CSC and impedance at 1 kHz exhibit approximately linear increase (R2 ¼ 0.9896) and sharply decrease, separately, as shown in Fig. 4f.

3.5. Stimulation We also investigated the voltage response and measured charge injection limit of bare gold electrode and that coated with PEDOT/GO to evaluate the electrical stimulating performance. The safe charge injection limit is defined as the maximum charge quantity that the electrode-tissue interface is polarized to water electrolysis potential (when Emc reaches 0.6 V shown in Fig. 5a) [41,42]. Therefore, it is preferred that electrode-tissue interface injects large quantity of charge without inducing high voltage

amplitude while performing current pulse stimulation, which is safe for tissue stimulated. As exhibited in Fig. 5a, the voltage excursion and the waveform distortion for PEDOT/GO coated electrode is much lower than that of uncoated gold electrode when applying same current pulse. Furthermore, the charge injection limit of PEDOT/GO coated electrode for different applied current pulse width is much higher than that of uncoated gold electrode, and exhibits a nearly linear increase (R2 ¼ 0.9824) relating to the pulse width (shown in Fig. 5b). Particularly, it increases from 0.16 0.05 mC/cm2 (n ¼ 4) of uncoated gold electrode to 4.71 0.18 mC/cm2 (n ¼ 4) of PEDOT/GO for pulse width of 1 ms. As a consequence, the PEDOT/GO electrode-tissue interface provides efficient electrical stimulation of high charge injection with safely low voltage amplitude.

3.6. Stability Stability is another crucial factor that influences the long term performance of electrode-tissue interface. Since electrochemical oxidation-reduction procedures could induce fragmentation or


a

1h

2127

6h

12 h

24 h

6h

12 h

24 h

250 µm

1h

c Absorbance at 450 nm

2.0

1.5

*

Au pad Cell plate PEDOT/GO

d

2.0

Absorbance at 450 nm

b

1.0

0.5

0.0

1.5

Au pad Cell plate PEDOT/GO

** **

1.0

0.5

0.0 1

12 6 Time (hour)

24

1

6 12 Time (hour)

24

Fig. 8. The amount and distribution of hdPC-12 cells (a) and NIH/3T3 (b) cells attached on PEDOT/GO substrate for 1 h, 6 h, 12 h and 24 h observed by LSCM, respectively. Data diagram of hdPC-12 cells (c) and NIH/3T3 (d) cells adhesion on PEDOT/GO (blue). Cells cultured on gold (black), and cell plate (red) were test for control. Significantly different in comparison with the gold control (*p < 0.05, **p < 0.01, n ¼ 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

exfoliation of conducting polymer, it is strict to investigate the electrochemical stability of PEDOT/GO film by applying large number of repetitive CV scanning [20,21]. As observed in Fig. 6a, though the volume shrinks after CV scanning, the entire and detail morphology varies slightly before and after scanning. Also, there is little variation of CV curve pattern after the scanning. This indicates the electrochemical nature of PEDOT/GO film barely changes during repetitive usage. Moreover, the CSC of PEDOT/GO decreases from 260.0 35.8 mC/cm2 (n ¼ 4) before scanning to 165.1 22.4 mC/cm2 (n ¼ 4) after scanning (Fig. 6b), which mainly attributes to the decrease of the nanocomposite film volume. As shown in Fig. 6c, the impedance and phase plots after repeated CV scanning merely change comparing with that before scanning, which also confirm the electrochemical stability of PEDOT/GO film. Since the mechanical property of conducting polymer mainly depends on the characteristic of negatively charged counterions, the stability of PEDOT/GO nanocomposite film is enhanced by the GO doping in all probability.

3.7. Biocompatibility The possibility that materials with nanometer size yield cytotoxicity by entering cell body worries researchers about the reliability of carbon nano material like graphene and carbon nanotubes. In order to evaluate biocompatibility of PEDOT/GO for cell in vitro, highly differentiated rat pheochromocytoma PC-12 cells (hdPC-12 cells) and mouse embryonic fibroblasts NIH/3T3 (NIH/3T3 cells) were cultured on PEDOT/GO film which deposited on sputtered gold glass slices. The two kinds of cells were chosen for PEDOT/GO compatibility analysis because PC-12 cells frequently serve as neural cells for neural tissue engineering studies, and NIH/ 3T3 cells are mammalian cells generally used for cell deformation, attachment, migration and proliferation researches [43,44]. Both hdPC-12 cells and NIH/3T3 cells were cultured for 1, 4 and 7 days to investigate cell proliferation on PEDOT/GO, sputtered gold and cell culture plate substrates. The morphologies of cell growth for 1, 4 and 7 days were observed by confocal laser scanning

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Fig. 9. SEM images of cells. (a) and (b) are SEM morphology of hdPC-12 cells and NIH/3T3 cells cultures on PEDOT/GO for 1 day, respectively. (c) and (d) are partial enlarged SEM morphology of (a) and (b), respectively. Details of pseudopodia are shown in the inserted images. The scale bars of inserted images are 5 mm.

microscope (CLSM), as shown in Fig. 7a (hdPC-12 cells) and Fig. 7b (NIH/3T3 cells). With the increase of the cell number during the proliferation process, hdPC-12 cells grow long neuritis used to build interconnection with others. Meanwhile, NIH/3T3 cells grow healthily on the sample material as explicitly fusiform shape. In the later period of proliferation, excellent growth vigor in quantity was observed for both kinds of the cells grew closely to each other. The morphology of nerve cells and fibroblasts demonstrates good growth and proliferation on PEDOT/GO coating material. As shown in Fig. 7c and d, proliferation quantities of two species of cells are identical on three kinds of substrates. Compared with the other two materials, PEDOT/GO neither promotes nor inhibits cell proliferation, which indicates that it has excellent biocompatibility on longterm cell response as gold and cell plate. Cells adhesion to PEDOT/GO substrate was also investigated as an important component of biocompatibility. The morphology and viability of both kinds of cells incubated on PEDOT/GO and control substrates for 1, 6, 12 and 24 h to study the initial cell attachment [45]. As shown in Fig. 8a, the profile of hdPC-12 cells is deformed into nerve cell shape in 6 h after cell plantation, and continuously increases in next 18 h. As regards NIH/3T3 cells (Fig. 8b), cell quantity increases remarkably in initial 6 h, and the cell morphology begins to change into fibrous form in the following 12 h. Furthermore, from the quantitative viability test results of cell adhered on three different substrates, the PEDOT/GO substrate significantly develops hdPC-12 cells (*p < 0.05) and NIH/3T3 cells (**p < 0.01) attachment compared with gold substrate for 24 h (shown in Fig. 8c and d). The SEM observation results show details of both kinds of cells interfacing to the PEDOT/GO substrate (Fig. 9a and b), indicating that both hdPC-12 and NIH/3T3 cells adhere tightly to PEDOT/GO coating material. As exhibited in Fig. 9c and d in detail, pseudopodia extend out from both kinds of cells as pointed by white arrows, while their extending ends and PEDOT/ GO substrate almost fuse together. All the results proves PEDOT/GO coating material possessed a good capability of cell adhesion. 4. Conclusions In summary, we develop a GO enhanced conducting polymer composite coating for electrode-tissue interface by electrochemical

deposition. The unique graphene doped molecular structure not only enhances the mechanical property, but also enlarges the effective area of electrode site. Compared with that of bare gold electrode, the impedance of electrode modified with PEDOT/GO film decreases nearly two orders of magnitude at 1 kHz, and the charge storage capacity increases sharply as well. Moreover, PEDOT/GO coated microelectrodes have outstanding ability to perform electrical stimulation for the high charge injection limit. Also, the stability of PEDOT/GO composite film is proved by numerous repetitive usages. In addition, as confirmed by cell viability, attachment and proliferation tests, nontoxic PEDOT/GO film induces little cell response as gold and promoted cell adhesion. For its unique composite structure, excellent electrochemical performance, stability and biocompatibility, electrochemically deposited PEDOT/GO film is a potential biomaterial as electrode-tissue interface for tissue engineering and further implantable electrophysiological devices. Acknowledgments The authors thank to partly financial support from the National Natural Science Foundation of China (No. 51035005, 61176104), 973 Program (2013CB329401), Shanghai Municipal Science and Technology Commission (No. 11JC1405700, 13511500200), NDPR Foundation of China (No. 9140A26060313JW3385). The authors are also grateful to the colleagues for their essential contribution to this work. References [1] Receveur RAM, Lindemans FW, de Rooij NF. Microsystem technologies for implantable applications. J Micromech Microeng 2007;17:50e80. [2] Cogan SF. Neural stimulation and recording electrodes. Annu Rev Biomed Eng 2008;10:275e309. [3] Perlmutter JS, Mink JW. Deep brain stimulation. Annu Rev Neurosci 2006;29: 229e57. [4] Ethier C, Oby ER, Bauman MJ, Miller LE. Restoration of grasp following paralysis through brain-controlled stimulation of muscles. Nature 2012;485: 368e71. [5] Weiland JD, Humayun MS. Visual prosthesis. Proc IEEE 2008;96:1076e84. [6] Waltzman SB. Cochlear implants: current status. Expert Rev Med Devices 2006;3:647e55.

H.-C. Tian et al. / Biomaterials 35 (2014) 2120e2129 [7] Kim S, Bhandari R, Klein M, Negi S, Rieth L, Tathireddy P, et al. Integrated wireless neural interface based on the Utah electrode array. Biomed Microdevices 2009;11:453e66. [8] Sodagar AM, Wise KD, Najafi K. A wireless implantable microsystem for multichannel neural recording. IEEE Trans Microw Theory Tech 2009;57: 2565e73. [9] Kozai TDY, Langhals NB, Patel PR, Deng X, Zhang H, Smith KL, et al. Ultrasmall implantable composite microelectrodes with bioactive surfaces for chronic neural interfaces. Nat Mater 2012;11:1065e73. [10] Grill WM, Norman SE, Bellamkonda RV. Implanted neural interfaces: biochallenges and engineered solutions. Annu Rev Biomed Eng 2009;11:1e24. [11] Merrill DR, Bikson M, Jefferys JGR. Electrical stimulation of excitable tissue: design of efficacious and safe protocols. J Neurosci Methods 2005;141:171e98. [12] Rose TL, Robblee LS. Electrical stimulation with Pt electrodes. VIII. electrochemically safe charge injection limits with 0.2 ms pulses. IEEE Trans Biomed Eng 1990;37:1118e20. [13] Rui YF, Liu JQ, Yang B, Li KY, Yang CS. Parylene-based implantable platinumblack coated wire microelectrode for orbicularis oculi muscle electrical stimulation. Biomed Microdevices 2012;14:367e73. [14] Lu Y, Wang T, Cai Z, Cao Y, Yang H, Duan YY. Anodically electrodeposited iridium oxide films microelectrodes for neural microstimulation and recording. Sens Actuat B 2009;137:334e9. [15] Cogan SF, Guzelian AA, Agnew WF, Yuen TG, McCreery DB. Over-pulsing degrades activated iridium oxide films used for intracortical neural stimulation. J Neurosci Methods 2004;137:141e50. [16] Green RA, Lovell NH, Wallace GG, Poole-Warren LA. Conducting polymers for neural interfaces: challenges in developing an effective long-term implant. Biomaterials 2008;29:3393e9. [17] Berggren M, Richter-Dahlfors A. Organic bioelectronics. Adv Mater 2007;19: 3201e13. [18] Gilmore KJ, Kita M, Han Y, Gelmi A, Higgins MJ, Moulton SE, et al. Skeletal muscle cell proliferation and differentiation on polypyrrole substrates doped with extracellular matrix components. Biomaterials 2009;30:5292e304. [19] Luo X, Weaver CL, Zhou DD, Greenberg R, Cui XT. Highly stable carbon nanotube doped poly(3,4-ethylenedioxythiophene) for chronic neural stimulation. Biomaterials 2011;33:5551e7. [20] Abidian MR, Kim DH, Martin DC. Conducting-polymer nanotubes for controlled drug release. Adv Mater 2006;18:405e9. [21] Abidian MR, Corey JM, Kipke DR, Martin DC. Conducting-polymer nanotubes improve electrical properties, mechanical adhesion, neural attachment, and neurite outgrowth of neural electrodes. Small 2010;6:421e9. [22] Geim AK, Novoselov KS. The rise of graphene. Nat Mater 2007;6:183e91. [23] Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, et al. Graphene and graphene oxide: synthesis, properties, and applications. Adv Mater 2010;22:3906e24. [24] Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, et al. Enhanced differentiation of human neural stem cells into neurons on graphene. Adv Mater 2011;23:263e7. [25] Liu Y, Yu D, Zeng C, Miao Z, Dai L. Biocompatible graphene oxide-based glucose biosensors. Langmuir 2010;26:6158e60. [26] Ruiz ON, Fernando KAS, Wang B, Brown NA, Luo PG, McNamara ND, et al. Graphene oxide: a nonspecific enhancer of cellular growth. ACS Nano 2011;5: 8100e7.

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[27] Heo C, Yoo J, Lee S, Jo A, Jung S, Yoo H, et al. The control of neural cell-to-cell interactions through non-contact electrical field stimulation using graphene electrodes. Biomaterials 2011;32:19e27. [28] Li D, Muller MB, Gilje S, Kaner RB, Wallace GG. Processable aqueous dispersions of graphene nanosheets. Nat Nanotech 2008;3:101e5. [29] Stankovich S, Dikin DA, Dommett GHB, Kohlhaas KM, Zimney EJ, Stach EA, et al. Graphene-based composite materials. Nature 2006;442:282e6. [30] Xu Y, Wu Q, Sun Y, Bai H, Shi G. Three-dimensional self-assembly of graphene oxide and DNA into multifunctional hydrogels. ACS Nano 2010;4: 7358e62. [31] Yan X, Chen J, Yang J, Xue Q, Miele P. Fabrication of free-standing, electrochemically active, and biocompatible graphene oxide-polyaniline and graphene-polyaniline hybrid papers. ACS Appl Mater Inter 2010;2:2521e9. [32] Chen P, Xiao T, Qian Y, Li S, Yu S. A nitrogen-doped graphene/carbon nanotube nanocomposite with synergistically enhanced electrochemical activity. Adv Mater 2013;25:3192e6. [33] You B, Wang L, Yao L, Yang J. Three dimensional N-doped grapheneeCNT networks for supercapacitor. Chem Commun 2013;49:5016e8. [34] Yan H, Okuzaki H. Effect of solvent on PEDOT/PSS nanometer-scaled thin films: XPS and STEM/AFM studies. Synth Met 2009;159:2225e8. [35] Zhang J, Yang H, Shen G, Cheng P, Zhang J, Guo S. Reduction of graphene oxide via L-ascorbic acid. Chem Commun 2010;46:1112e4. [36] Xu Y, Wang Y, Liang J, Huang Y, Ma Y, Wan X, et al. A hybrid material of graphene and poly (3,4-ethyldioxythiophene) with high conductivity, flexibility, and transparency. Nano Res 2009;2:343e8. [37] Kvarnström C, Neugebauer H, Blomquist S, Ahonen HJ, Kankare J, Ivaska A. In situ spectroelectrochemical characterization of poly(3,4-ethylenedioxythiophene). Electrochim Acta 1999;44:2739e50. [38] Lu Y, Li T, Zhao X, Li M, Cao Y, Yang H, et al. Electrodeposited polypyrrole/ carbon canotubes composite films electrodes for neural interfaces. Biomaterials 2010;22:3906e24. [39] Danielsson P, Bobacka J, Ivaska A. Electrochemical synthesis and characterization of poly(3,4-ethylenedioxythiophene) in ionic liquids with bulky organic anions. J Solid State Electrochem 2004;8:809e17. [40] Asplund M, von Holst H, Inganäs O. Composite biomolecule/PEDOT materials for neural electrodes. Biointerphases 2008;3:83e93. [41] Green RA, Hassarati RT, Bouchinet L, Lee CS, Cheong GLM, Yu JF, et al. Substrate dependent stability of conducting polymer coatings on medical electrodes. Biomaterials 2012;33:5875e86. [42] Venkatraman S, Hendricks J, King ZA, Sereno AJ, Richardson-Burns S, Martin D, et al. In vitro and in vivo evaluation of PEDOT microelectrodes for neural stimulation and recording. IEEE Trans Neural Syst Rehabil Eng 2011;19:307e16. [43] Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair and regeneration. Annu Rev Biomed Eng 2003;5:293e347. [44] Ryoo SR, Kim YK, Kim MH, Min DH. Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. ACS Nano 2010;4:6587e98. [45] Dong Y, Li P, Chen C, Wang Z, Maa P, Chen GQ. The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD. Biomaterials 2010;31:8921e30.