Poly(3,4-ethylenedioxythiophene) Nanohybrid as an

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Biological and Medical Applications of Materials and Interfaces

Bionanotube/poly(3,4-ethylenedioxythiophene) nanohybrid as an electrode for neural interface and dopamine sensor Sathish Reddy, Qiao Xiao, Haiqian Liu, Chuping Li, Shengfeng Chen, Cong Wang, Kin Chiu, Nuan Chen, Yujie Tu, Seeram Ramakrishna, and Liumin He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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ACS Applied Materials & Interfaces

Bionanotube/poly(3,4-ethylenedioxythiophene) nanohybrid as an electrode for neural interface and dopamine sensor Sathish Reddy1, Qiao Xiao2, Haiqian Liu1, Chuping Li1, Shengfeng Chen1, Cong Wang3, Kin Chiu4, Nuan Chen3, Yujie Tu2, Seeram Ramakrishna1, 3, Liumin He1,2* 1

Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), MOE Joint International Research Laboratory of CNS Regeneration, Jinan University, Guangzhou, Guangdong, 510632, China.

2

College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, 510632, China. 3

Department of Traditional Therapy, the Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, 510120, China.

4

State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong SAR, 000000, P.R. China.

5

Center for Nanofibers and Nanotechnology, Department of Mechanical Engineering, Faculty of Engineering, National University of Singapore, 117576, Singapore.

Corresponding author: Liumin He ([email protected]).

1

ACS Paragon Plus Environment

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Abstract Poly(3,4-ethylene dioxythiophene) (PEDOT) is a promising conductive material widely used for interfacing with tissues in biomedical fields because of its unique properties. However, obtaining high-charge injection capability and high stability remains challenging. In this study, pristine carbon nanotubes (CNTs) modified by dopamine (DA) self-polymerization on the surface (PDA@CNTs) were utilized as dopants of PEDOT to prepare hybrid films through electrochemical deposition on the ITO electrode. The PDA@CNTs-PEDOT film of the nanotube network topography exhibited excellent stability and strong adhesion to the ITO substrate compared with PEDOT and PEDOT-pTS. The PDA@CNTs-PEDOT-coated ITO electrodes demonstrated lower impedance and enhanced charge storage capacity than the bare ITO. When applying exogenous electrical stimulation (ES), robust long neurites sprouted from the dorsal root-ganglion (DRG) neurons cultured on the PDA@CNTs-PEDOT film. Moreover, ES promoted Schwann cell migration out from the DRG spheres and enhanced myelination. The PDA@CNTs-PEDOT film exhibited excellent electrochemical sensor for detection of DA in the presence of biomolecule interferences. Results would shed light into the advancement of conducting nanohybrids for applications in multifunctional bioelectrode in neuroscience. Key words: PDA@CNTs-PEDOT, Bioelectrode, Electrical stimulation, Neurites outgrowth, Electrochemical sensor.

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1. Introduction In recent years, advances in artificial intelligence associated with ever-increasing interests in healthcare monitoring and therapy have rapidly accelerated the development of smart materials for biomedical applications. Among various smart materials, electroactive biomaterials are a burgeoning generation that allows direct delivery of electrical, electrochemical, and electromechanical signals for specific stimuli-responsive properties. Electroactive biomaterials comprise a large family, including conductive polymers, piezoelectrics, photovoltaic materials, and electrets, which show wide application to drug delivery systems, cardiac pacemakers, bioactuators, biosensors, neural recording, and tissue engineering. As a novel type of electroactive biomaterial, conducting polymers show great advantages because of their mechanical compatibility with cells and tissues. Moreover, the physicochemical and electrical properties of these polymers can be easily but effectively tailored for targeting functions.1 Thus, such materials have gained increasing attention in biomedical fields. Poly(3,4-ethylene dioxythiophene) (PEDOT), which has a high electrical conductivity of up to 3000 S/cm and a long-term electrochemical stability, is one of the most studied conducting polymer in the last decade.2 PEDOT possesses better thermal, electrical, chemical, and environmental stability than polypyrrole (Ppy), another commonly utilized conducting polymer.3 To date, PEDOT is one of the most promising candidates for constructing biosensors because it can effectively improve the electrochemical performance of the deposited electrodes.4 As documented, PEDOT modification enhances the substantial reactivity and detection sensitivity for dopamine (DA),5 ascorbic acid (AA),5 and pesticides.6 PEDOT has also been widely used in biological and biomedical areas, wherein PEDOT directly interacts with cells or tissues. Whether used alone or combined into composites, the conductive nature of versatile PEDOT allows cells or tissue to be stimulated.7 For example, PEDOT-based substrates cannot only promote the adhesion and proliferation of neural stem cells but 3



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also enhance their differentiation into neurons, indicating their potential applications in neural regeneration.8 Moreover, the ability to promote the adhesion and neurite outgrowth of adult neurons and to form stable interface with delicate neural tissue make PEDOT attractive in the application of neural implants.8 PEDOT needs a counterion for charge balancing to render its conductive properties, called dopant. One commonly used dopant is poly(styrene sulfonate) due to its high electrical conductivity and easy availability on a large scale.9 Additionally, various biological molecules have been utilized as dopants to improve the biocompatibility, including hyaluronic acid,10 chitosan,11 fibrinogen,12 and collagen.13 The dopants, however, were also documented to have influence on the own physical properties and the consequent performance of conducting polymers.14 For example, the incorporated biomolecules may change the morphology of the conducting polymer and consequently decrease the electrical properties.15 Negatively charged carbon nanotubes (CNTs) have been widely utilized as counterions doped into conducting polymers.16 CNTs are well-known for extraordinary mechanical, electrical, and optical properties and high stability and low biofouling in biological environments; CNTs are also considered one of the most promising nanomaterials for applications in nanobioelectronics over the past few decades.17 CNTs have a high specific surface area due to their unique one-dimensional geometry, enhancing the electroactivity of CNTs-based microelectrodes. Luo et al. demonstrated that PEDOT/MWCNT coating decreased the impedance of microelectrodes.16 CNTs are excellent candidate dopants of PEDOT for electrode coating.18 Zhou et al. coated platinum microelectrode with carbon nanotube (MWCNT)-doped multiwall PEDOT composite films by electrochemical

polymerization.

PEDOT/MWCNT

films

showed

enhanced

electrochemical performance and improved stability of the coatings compared with the coatings prepared by polystyrene sulfonate doping and potentiostatic polymerization.18 The long-term stability of PEDOT-based conductive materials on the solid substrates ensures the reliable performance of the resultant device, which, however, is 4


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a serious concern and remains a great challenge nowadays.19 The coated PEDOT films would be broken into small pieces and cracks and would delaminate from the substrates during processing and sterilization, even after implantation,20 causing undesirable risks. Given that PEDOT films are mostly prepared via electrochemical deposition, lack of strong chemical interactions or bonding between PEDOT and substrate surface is responsible for such failure. The coating film strength can be improved by subsequent treatment after deposition, e.g., cross linking.21 The effect, however, is usually inadequate. To solve this problem, specific chemical interactions are expected between the conductive polymer and the substrate. As previously reported, creation of the thiol group on the electrode surface by using self-assembled monolayer helped the adhesion of PEDOT on platinum or gold electrode surface.22,23 Ouyang et al. demonstrated a covalently bonded and strongly adherent PEDOT film on substrates by electrografting an amine-functionalized 3,4-ethylenedioxythiophene (EDOT) derivative (EDOT-NH2) onto conducting substrates.19 Despite considerable efforts devoted to high electrochemical performance, long-term stability, and good biocompatibility in the application of bioelectronic electrodes, a major hurdle still remains in conductive coatings. Here, we used a PDA@CNTs as a bionanotube for strong adhesive to the ITO electrode and simultaneous doping with PEDOT films. The PDA@CNTs was prepared from DA π–π stacking and spontaneous self-polymerization on CNTs. Such a strategy showed remarkable advantages over acid treatment in effectively modifying CNTs while simultaneously reserving their intrinsic properties because no chemical reactions occurred in CNTs.24 PDA exhibited a negative zeta potential in neutral aqueous solution because of the deprotonation of the phenolic groups.24 Therefore, negatively charged PDA@CNTs would act as dopants to balance the positive charge in the backbone of the PEDOT during the PEDOT electropolymerization. Reportedly, PDA helped highly adherent coating on organic, inorganic, and conducting polymer substrate.25,26 Moreover, amine moieties were previously documented to form covalent bonds on variety of conducting substrates by electrochemical deposition 5



method.19,

27-29

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The amino moieties of PDA on the surface of CNTs would help to

form covalent bonds with the conducting electrode by electrochemical deposition method. Therefore, in this work, we prepared a stable PDA@CNTs-PEDOT nanocomposite film on ITO electrode via electrochemical deposition method (details see in Sheme.1). The PDA@CNTs-PEDOT films were characterized by Raman spectroscopy, cyclic voltammetry (CV), and X-ray photoelectron spectroscopy (XPS) to confirm the successful modification of PEDOT with PDA@CNTs. Electrochemical characterization was performed to determine the effect of PDA@CNTs combined with PEDOT films on the electrochemical impedance, the charge storage capacity (CSC), and electrochemical stability of the films. As the neural electrode, the interactions between neurons and conductive coating materials are of great importance. Therefore, in the current study, dorsal root ganglion (DRG) neurons were used to study the neuronal development of the resultant PDA@CNTs-PEDOT-coated ITO. Particularly, electrical stimulation (ES) was performed on the cultured DRG by applying a pulsed direct current electric field. ES has been widely proposed as a selective nondrug approach for neurological and psychiatric disorders and neural regeneration in both preclinical and clinical studies.30, 31

Neurite outgrowth, Schwann cell (SC) migration, and myelination of the axons

were thoroughly examined. DA detection and selectivity for DA detection in presence of uric acid (UA), ascorbic acid (AA), and tryptophan (Tryp) were also investigated to examine

the

application

of

PDA@CNTs-PEDOT

nanocomposite

as

an

electrochemical sensor. Our study would shed light on the advancement of conducting nanohybrid for application in multifunctional bioelectrodes. 2. MATERIALS AND METHODS 2.1.

Preparation

of

bionanotube/PEDOT

(polydopamine@carbon

nanotube

[PDA@CNTs]-PEDOT) 2.1.1. Materials DA hydrochloride and AA were purchased from J&K Chemical (Shanghai, China). Pristine MWCNTs (>95% purity, OD: 10–20 nm, length: 0.5–2 mm) were 6


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purchased from MK Impex Corp. (Toronto, Canada). Silicon oil was purchased from Hang Ping Chemicals Industries (Fujian Sheng, China). EDOT and sodium p-toulene sulfonate (pTS) were purchased from Sigma-Aldrich. Phosphate buffered saline (PBS, pH 7.4, 10 mM sodium phosphate, and 0.9% NaCl) was purchased from Sigma-Aldrich. All other chemicals were of analytical grade, and Milli-Q water from a Millipore Q water purification system was used throughout all experiments. The morphology of the bionanotube/PEDOT (PDA@CNTs-PEDOT) film on ITO was observed via scanning electron microscopy (SEM, JSM-TE300, JEOL, Japan) at an accelerating voltage of 3 kV after coating with gold by using a sputter coater (JEOL JFC-1200

Fine

Coater,

JEOL,

Tokyo,

Japan).

The

roughness

of

the

PDA@CNTs-PEDOT surface was measured by atomic force microscopy (AFM) (Bioscope Catalyst Nano-scope-V, Veeco Instruments, NY, USA) with a Digital Instruments Dimension 3000 using Si3Ni4 tap-ping mode cantilevers. The thickness was measured by the step height between the PDA@CNTs-PEDOT surface and uncoated ITO substrate. XPS with a focused monochromatic Al Ka source (1486.7 eV) for excitation was conducted using an ESCALAB 250 X-ray photoelectron spectrometer (Thermo Fisher Scientific, Waltham, MA). Raman spectra were monitored using a Raman’s spectrometer (a Horiba HR800 Raman’s system. A 100× objective lens was used to focus a laser beam on a sample and to collect Raman signal. 2.1.2. Preparation of PDA@CNTs and electrodeposition of bionanotube/PEDOT (PDA@CNTs-PEDOT) PDA@CNTs as bionanotubes were synthesized according to the method reported by our group previously.24 The preparation method is as follows. First, pristine CNTs (0.5 mg/mL) in the Tris-HCl buffer (10 mM, pH 8.5) solution was ultrasonicated for 30 min and then DA (0.1 mg/mL) was added to the solution. This suspension was ultrasonicated for another 2 h, followed by further stirring for 48 h at 25 °C. Then the bionanotube (PDA@CNTs) black solution was kept for 24 h until it settled. The residue solution was filtered first on a Whatman filter paper. Then, the 7



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supernatant solution was filtered on it. The solid black bionanotube powder was collected by repeated washing with Tris-HCl solution (10 mM, pH 8.5) and dried at 50 °C. Third, the bionanotube powder (10 mg/mL) was re-dispersed in Tris-HCl (10 mM, pH 8.5) solution by ultrasonication for approximately 20 min and kept overnight until it settled. The upper solution was collected and added EDOT monomer solution (0.02 M). The final solution contained 0.02 M EDOT, and the bionanotube was used for electrochemical deposition on the ITO electrode by applying constant current (50 A/cm-2) by using chronopotentiometry technique. The film thickness was controlled by adjusting the electrochemical deposition time (300, 600, 900 and 1200s). 2.1.3. Experimental section All

electrochemical

measurements

were

conducted

using

CHI

660E

electrochemical workstation from Chen Hua Instruments Co. (Shanghai, China). A conventional three-electrode electrochemical system was used for all the electrochemical experiments, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode. ITO glass (Zhuhai Kaivo Optoelectronic Technology Co., Ltd.) and PDA@CNTs-PEDOT/ITO were used as working electrodes (Ø 10.0 mm). All electrochemical potentials in the study were reported versus SCE. The adhesion strength of PDA@CNTs-PEDOT, PEDOT, and PEDOT-pTS films coated on ITO substrate was examined via an ultrasonication test (mechanical test). This test was performed by immersion of PDA@CNTs-PEDOT, PEDOT, and PEDOT-pTS-coated ITO glass in glass vial filled with deionized water. Then, these glass vials were placed in a KQ-700QE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., China) with a power of 300 W at 25 °C for 10 s, 10 min, and 60 min. 2.2. DRG cell culture 2.2.1. Experimental section Materials and Reagents: Sprague-Dawley (SD) rats were supplied by the Laboratory Animal Center of Southern Medical University, China.

L-glutamine,

neurobasal-A medium, B27, penicillin-streptomycin solution, L-15 medium, 24 well 8


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dishes, and nine well dishes were purchased from the Nest Biotechnology Co., Ltd. Goat serum was supplied by Abcam. Anti-NF200 antibody produced in mouse was purchased from Sigma-Aldrich. PSD-95 antibody produced from mouse was purchased from Sigma-Aldrich. MBP antibody and S100 antibody produced from rabbit were purchased from Abcam. Both donkey anti-mouse IgG 488 (dilution 1:1000) and donkey anti-mouse IgG 55 (dilution 1:1000) were procured from Millipore Company. 2.2.2. DRG ex vivo preparation This procedure was performed by modifying a reported procedure.32 Whole DRGs with redundant roots were dissected from postnatal 1 to 3 days (P1) SD rats and collected in a cold neurobasal medium. The redundant nerve roots were detached under a stereomicroscope, and ~25 DRGs were counted and plated in each PDL precoated on a PDA@CNTs-PEDOT-ITO glass, cultured with neurobasal medium containing 0.3% L-glutamine, 2% B27, and 100 ng mL−1 NGF in a 37 °C incubator with 5% CO2 and 92% humidity. The medium was changed every other day. Myelination study: This procedure was conducted by modifying a reported procedure.32 Whole DRGs with redundant roots were dissected from postnatal 1 to 3 days (P1) SD rats and collected in a cold neurobasal medium. The redundant nerve roots were detached under a stereomicroscope, and ~25 DRGs were counted and plated in each PDL precoated on a PDA@CNTs-PEDOT/ITO glass, cultured with neurobasal medium containing 0.3% L-glutamine, 2% B27, and 100 ng mL-1 NGF in a 37 °C incubator with 5% CO2 and 92% humidity. At 4-7 days after plating, 50 μg mL-1 of AA was added to the medium of the DRGs to trigger myelination by the endogenous SCs. Cells receiving AA were fed with the same media thrice per week. Electrical stimulation: The procedure was performed according to a reported study with slight modification.33 ES of DRG culture on PDA@CNTs-PEDOT/ITO glass was conducted by applying voltage of 3-4 V, time of 30 min/days, frequency of 10 Hz, current of 1 mA, duty cycle of 2%, and dead time of 1%.

9



2.2.4. DRG neuron neurite extension assay The length of DRG neurites grown on PDA@CNTs-PEDOT-coated ITO glass was quantified manually under confocal microscopy after 7 days of incubation. DRG with extended neurites were calculated. A small circle was drawn to indicate the DRG boundary, and a concentric large circle was drawn to reach the longest neurites. The longest distance of neurite extension was calculated by the radius of the large circle minus the radius of the small circle. The number of SCs per each circle was also calculated. The maximum length measured for each neurite was the distance between the end of neurite growth and DRG body. Statistical analysis of all data was performed via t-test by using Prism software (www.graphpad.com). 2.2.5. Immunofluorescence staining After 14 days, triggering for myelination, the co-cultured cells were fixed by 4% paraformaldehyde in PBS for 2 h, rinsed, permeabilized, and blocked with 0.1% Triton X-100 and 10% normal goat serum in PBS for 30 min. Cells were incubated with primary antibodies against NF-200 (dilution 1:300) and MBP (dilution 1:300) for 30 min at room temperature, followed by secondary antibodies conjugated to Fluor 488 (dilution 1:500) or Fluor 555 (dilution 1:500) incubation for 45 min. Similarly, after 7 days, co-cultured cells were incubated with primary antibodies against NF200 (dilution 1:300) and S100 (dilution 1:300) for 30 min at room temperature, followed by secondary antibodies conjugated to Fluor 488 (dilution 1:500) or Fluor 555 (dilution 1:500) incubation for 45 min. Cells were then rinsed for 2×10 min in PBS before DAPI staining and mounting. The fluorescence was observed and imaged under a confocal microscope. 2.2.6. RNA extraction and real-time polymerase chain reaction (RT-PCR) Both these procedures were conducted by modifying a reported procedure.34 Total RNA was extracted from DRG cultured at 14 days duration after ES by using Trizol (Ambion, North America, USA) method. RNA concentration was analyzed spectrophotometrically using a Nanodrop 2000C instrument (Thermo Scientific, 10


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Pittsburgh, PA, USA). In addition, the purity was measured using the A 260/280 and A 260/230 ratios in the Nano Drop software (Thermo Scientific, Pittsburgh, PA, USA). For quantitative RT-PCR analysis, first strand cDNA was prepared from 500 ng total RNA by using a Prime Script Kit (TakaRa, Dalian, China), following the manufacturer’s procedure. RT-PCR was carried out using SYBR Premix EX Taq II (TakaRa, Dalian, China), and investigation was conducted on a 20 L mixture containing 10 L SYBR Premix Ex Taq II, 2 L diluted cDNA, and 0.8 L forward primers and reverse primers. The amplification was carried out as follows: 95 °C for 1 min, followed by 45 cycles at 95 °C for 10 s, 52 °C for 30 s, and 72 °C for 30 s. 2.3. Dopamine sensor 2.3. Material and experiment 2.3.1 Materials DA, UA, AA, and Tryp were purchased from J&K Chemical (Shanghai, China). MWCNT-COOH was purchased from M.K Nano Co., Ltd. (Mississauga, Canada). Silicon oil was purchased from Hang Ping Chemicals Industries (Fujian Sheng, China). 2.3.2. Bare paste electrode CNTs paste electrode was prepared by taking different silicon oils (20%) and MWCNT-COOH powders (80%). This mixture was thoroughly mixed in an agate mortar for approximately 30 min and packed into a homemade Teflon cavity current collector and polished using a soft paper. 2.3.3. Preparation of PDA@CNTs-PEDOT electrode The solution containing 0.02 M EDOT and PDA@CNTs was used for electrochemical deposition on CNTs paste electrode by applying constant current (50 A/cm-2) via chronopotentiometry technique. Sensor optimization was determined via the electrodeposition time (15, 20, 25, 40, 50 and 100s). 2.3.4. Experimental section All electrochemical measurements were conducted on CHI 660E electrochemical workstation from Chen Hua Instruments Co. (Shanghai, China). A conventional three-electrode electrochemical system was used for all the electrochemical 11



experiments, an SCE as the reference electrode, and a platinum wire as the counter electrode. Bare paste electrode and PDA@CNTs-PEDOT electrode were used as the working electrodes (Ø3.0 mm). All potentials in the study were reported versus SCE. 3.0. Result and Discussions 3.1. Morphology The properties of the modified electrode strongly depend on the surface conducting materials after deposition, including mechanical, electrical, and biological properties.35,36 The surface morphology of PDA@CNTs-PEDOT films on ITO electrode was observed by a scanning electron microscope, as shown in Fig. 1. PDA@CNTs-PEDOT was uniformly distributed on an ITO substrate in a random form, appearing as porous and rough network. Meanwhile, many small granules were observed, which can be ascribed to the PEDOT aggregation. Moreover, we can find that the density of PDA@CNTs-PEDOT network increased with increasing electrochemical deposition time. These observations confirmed our hypothesis that negatively charged PDA@CNTs could act as dopants with positively charged PEDOT during the electrodeposition. Notably, the nanotubes were separately distributed on the substrate, quite different from multilayered CNTs composites in our previous studies, showing nanotube bundles of a certain amount.24,37,38 These results indicated that electrodeposition might have advantages over the method of layer-by-layer assembly in preparing CNTs-based nanocomposites. Atomic force microscope (AFM) was further employed to study the morphology of PDA@CNTs-PEDOT films electrochemically deposited for different time (300, 600, 900 and 1200 seconds), which showed rough surface as shown in Fig. 1E-1H. The root-mean-square roughness of the PDA@CNTs-PEDOT films was measured, which showed roughness of PDA@CNTs-PEDOT surface increased with increasing electrochemical deposition time (Fig. 1I). The thickness was evaluated by measuring the mean step height between the PDA@CNTs-PEDOT films and the uncoated ITO substrate. PDA@CNTs-PEDOT films showed a constant increased thickness with increasing electrochemical deposition time (Fig. 1J), in a good agreement with the 12


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density differences as observed by SEM. 3.2. Adhesion stability Whether acting as biosensors or surface modification of tissue engineering scaffolds and microelectronic devices, the mechanical stability of the coating film plays an important role, which would greatly affect its lifetime and performance.39 The adhesion strength of PDA@CNTs-PEDOT on the ITO substrate was examined by

an

ultrasonic

test

at

25

°C.

PEDOT

and

PEDOT-pTS

films

electrochemical-deposited on ITO substrate were used as comparison. Before ultrasonic test, PEDOT and PEDOT-pTS were both uniformly deposited on the ITO surface (Fig. 2). After ultrasonic test for 10 s, PEDOT film was quickly dissipated into small fragmented pieces. Despite the advantages and promising outlook of PEDOT, relatively poor mechanical performance hinders its use as a coating material for electrodes.20 Substitution of multi/monovalent counter-anion or small dopants

40

can improve the mechanical stability. Electrochemical deposition with pTS showed good stability on the substrate. However, most of PEDOT-pTS film was found to flake away from the substrate after 10 min ultrasonic test. Our observation was in a good agreement with a previous report by Ouyang et al.19 By contrast, PDA@CNTs-PEDOT film was strongly coated on the ITO electrode surface with minimal difference in the film after 10 min ultrasonic test. No significant cracks in the PDA@CNTs-PEDOT film were observed even after 60 min ultrasonic test. Such observation indicated that PDA@CNTs possibly formed strong interaction with PEDOT, which might help the adhesion on the ITO electrode. The bio-inspired PDA is well-known to strongly support adherent coating on a variety of organic or inorganic materials.25 Kim et al. thus used PDA to increase Ppy adhesion on the electrode surface.26 Therefore, PDA contributed to the stability of the resultant PDA@CNTs-PEDOT nanocomposite film. In addition, long-term electrochemical activity of the PDA@CNTs-PEDOT films could be improved by up to ~ 40 to 50%, whereas PEDOT and PEDOT-pTS showed a loss ~ 97% of electroactivity over the course of 30 min ultrasonication test as shown 13



in Fig. 2D. Our observation was in a good agreement with results published by Yamato et al,41 who reported 95% activity losses when PPy/PSS films subjected to polarized for 16 h. CNTs incorporation into PPy/PSS improved 50% electrochemical activity was reported.42 This further indicated that CNTs prevented the loss of the electroactivity of conducting polymers. We thus provided a simple yet effective method to create highly adherent coatings of PEDOT-based nanocomposite on the substrates via electrochemical deposition method, which could be highly useful for long-term performance of biomedical devices. 3.3. Raman spectroscopy and cyclic voltammograms Raman spectroscopy, an effective nondestructive analysis tool to characterize carbon materials, was used to gain further structural properties of the nanocomposite films. Fig.3 shows the Raman spectrum of PDA@CNTs, PEDOT, and PDA@CNTs -PEDOT. D and G modes appearing at 1320 and 1570 cm−1 for PDA@CNTs shifted to 1326 and 1567 cm−1 after being doped in PEDOT film (Fig. 3A). According to previous studies,43,44 the red shift indicated the increasing of charge transfer efficiency, whereas the blue shift indicated enhanced phonon excitation energy. Both these phenomena verified that the strong interactions were formed between the CNTs and the conjugated thiophene chain of PEDOT.43 Characteristic peaks of PEDOT appeared in the Raman spectrum of PDA@CNTs-PEDOT, whereas a decrease of Raman peak at 1125 cm−1 exhibited the planar structure of the PDA@CNTs-PEDOT (Fig. 3B). This result was in close agreement with SEM images.45 In addition, Raman peaks at 1266 cm-1 and 1370 cm-1 decreased (arrows in Fig.3B), which indicated that PDA@CNTs successfully interconnected with a conjugated thiophene chain of PEDOT.45 This result was in a good agreement with a recently published paper by Yuting Wu et al.45 Moreover, we could see in Fig. 3B all Raman peak signals decreased in PDA@CNTs-PEDOT as compared with those in PEDOT, which indicated PDA@CNTs doped with PEDOT film.46 Cyclic voltammetry is one of the highly useful electro-analytical techniques for studying electrochemical oxidation and reduction of organic and inorganic 14


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compounds. The cyclic voltammograms of 0.2 M EDOT, PDA@CNTs, mixed PDA@CNTs, and 0.2 EDOT on ITO electrodes were performed in 1 mM Tris–HCl (pH 8.5) solution. We used 1 mM Tris–HCl (pH 8.5) solution as an electrolyte to avoid PDA@CNTs particle aggregations. Fig. 3C displayed that EDOT started electropolymerization at an oxidation potential value of +0.85 V, peaking at +1.18 V. PDA@CNTs exhibited a broad anodic oxidation peak at +0.58 V and a large peak current from the first CV cycle, which decreased and disappeared with the increasing number of CV cycles (Fig. 3D). This phenomenon indicated that PDA@CNTs were electrochemically deposited on the ITO electrode. Our observation was in good agreement with a previously reported investigation by Kim et al.26 The probable mechanism was that free radicals were created by the deprotonation of amine moieties of PDA@CNTs in the first CV cycle, which were then electrodeposited on the electrode surface in the following CV cycles.28 PDA@CNTs and 0.2 M EDOT mixed solution exhibited multiple peaks (Fig. 3E). Several oxidation peaks appeared at +0.32, +0.57, and +0.85 V, and a reduction peak occurred at −0.143 V with a large redox peak current in the 1st CV cycle. The peak potential and current increased with the increasing number of CV cycles. The increases in peak current might be due to the interaction between the positively charged PEDOT with negatively charged PDA@CNTs,47 whereas the increases in peak potential might be due to the electrochemical deposition of PDA@CNTs-PEDOT on the ITO electrode surface.48 Our results were in a good agreement with a previous study on PDA/Ppy electropolymerization26 and PDA-incorporated graphene oxide/PEDOT hybrid thin films by electropolymerization.49 3.4. X-ray photoelectron spectroscopy (XPS) XPS measurement was performed to determine the surface chemical composition and elemental characteristics of PEDOT, PDA@CNTs, PDA@CNTs-PEDOT, and ITO samples. As shown in the survey scan (Figs. 4A and 4B), the peaks for indium (In) and tin (Sn) signals in ITO completely disappeared after PDA@CNTs-PEDOT electrochemical deposition, whereas strong peaks corresponding to oxygen (O), 15



carbon (C), the main elemental components of the PEDOT and PDA@CNTs, occurred. In addition, peaks corresponding to nitrogen (N 1s at 400.5 eV) and sulfur (S 2p at 163.81 eV), which were abundantly present in PDA and PEDOT, respectively, were both observed in the spectrum of the PDA@CNTs-PEDOT-coated ITO. These findings verified that PDA@CNTs-PEDOT hybrid films completely covered the ITO substrate as expected. Notably, binding energy peaks of O 1s, S 2p, and N 1s of PDA@CNTs-PEDOT shifted as compared with those of PEDOT and PDA@CNTs. Given that the chemical state of element was sensitive to the local chemical environment,50 we presumed that certain interactions occurred between PEDOT and PDA@CNTs during electrochemical deposition. High-resolution spectra in the region of the C 1s, N 1s, O 1s, and S 2p envelopes for PEDOT, PDA@CNTs, and PDA@CNTs-PEDOT were further analyzed. Fig. 4C displays the core level spectrum of PEDOT C 1s comprised two main peaks located at 284.8 eV (C-C and C=C) and 285.4 eV (C-S and C-O), whereas the C 1s core level scan spectra of PDA@CNTs can be deconvoluted into three clear peaks located at 284.8 and 285.0 eV (C-C and C=C) and 286.0 eV (C-N and C-O). In addition, a vague and indistinct peak was found to be tentatively located at the binding energy of 289.3 eV that can be attributed to C=O.51 PDA@CNTs-PEDOT displayed C 1s peaks at 284.4 eV (C-C), 285.1 eV (C=C, C-S), 286.3 eV (C-N, C-O), and 288.3 eV (C=O). As compared with PEDOT and PDA@CNTs, peak signals at the binding energies of 286.3 and 288.3 eV showed a significant increase, indicating a good charge distribution and a possible chemical change due to electrostatic interactions between the negatively charged PDA@CNTs and the positively charged PEDOT after electrochemical deposition of [email protected], 52 Fig. 4D displays the XPS O1s core-level spectrum of PEDOT, PDA@CNTs, and PDA@CNTs-PEDOT. Deconvolution of PEDOT O 1S resulted in a large peak corresponding to C=O at 531.4 eV and a small contribution in the spectra at 532.6 eV ascribed to C-O-C.51 PDA@CNTs O 1s deconvoluted into two peaks at 531.7 eV (C=O) and 533.3 eV (C-OH). Hydroxyl functional moieties of PDA@CNTs can help 16


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the interface passivation by acting as a diffusion barrier to minimize the surface oxidation of films,52 resulting in a significant shift to low binding energy and decrease in peak signal after integration of PDA@CNTs and PEDOT. This finding supported again the assumption of chemical changes due to ion pairing and electrostatic interactions between negatively charged PDA@CNTs and positively charged PEDOT. Fig.4E shows the S 2p core-line spectra obtained for PEDOT and PDA@CNTs-PEDOT. The PEDOT peaks at 163.6 and 164.8 eV were attributed to the spin-split doublets of sulfur atom (S).51,53 The signals of peaks for sulfon groups at 167.5 and 169 eV of PEDOT both significantly decreased after integration with PDA@CNTs. This finding suggested that the negatively charged PDA@CNTs act as dopants to balance positive charge in the PEDOT. Fig.4F exhibits the N 1s spectrum of the PDA@CNTs at 400.5 eV, suggesting the presence of secondary amine (R-NH-R) associated with polydopamine or oxidized intermediates.54 The N 1s peak of PDA@CNTs-PEDOT occurred at 399.3 eV, which was assigned to the tertiary/aromatic (=N-R) amine functional groups ascribed to the tautomers of 5,6-dihydroxyindole and 5,6-indolequinone.54-56 Therefore, our data confirmed the study of Zangmeister et al. that starting (DA) and intermediate (C=N-containing tautomers of quinone and indole) species were present in the polydopamine thin films at all deposition times.54 This finding would benefit the formation of strong adhesion of PDA@CNTs-PEDOT composite on the substrate during

electrochemical

deposition

according

to

a

mechanism

of

DA

self-polymerization. Furthermore, electrochemical oxidation of amines was documented to favor the immobilization of nitrogen-containing species onto ITO substrates.57 Consequently, electrografting mechanism of amines might also be responsible for the mechanical stability of PDA@CNTs-PEDOT nanocomposite film on ITO. 3.5. Electrochemical properties When serving as a neural interface, the electrochemical properties of electrode play an important role in transferring and recording signals from neuron cells. The 17



electrode should have low impedance and high CSC. Low impedance at the neural interface allows to works at a low applied voltage, which can avoid cells damage and other harmful effects. Large CSC of a neural interface would delivery more charges, which could be helpful for recording signals and promoting stimulation of neurons cells.58 3.5.1 Charge storage capacity and electrochemical Impedance The redox peak characteristic of PDA@CNTs-PEDOT-coated ITO electrode was investigated using CV curves in 0.01 M PBS (pH 7.4) at a scan rate of 0.05 V s-1. The CV curves exhibited reversible redox peak as indicated by the oxidation and reduction peaks on the CV curves. The integrated area of the CV curve is proportional to the CSC. Thus, CSC can be calculated using CV curves. As shown in Figs.5A and B, the CSC value of PDA@CNTs-PEDOT film increased with the increase in electrodeposition charge. These results exhibited accordance with our expectation because the increase in electrodeposition charge lead to increase several electroactive species and enhanced electro-active surface area. The relationship between the applied electrodeposition charge for PDA@CNTs-PEDOT films on ITO electrode and the calculated CSC exhibited a nearly linear. The linearity was very good even at a very high applied electrodeposition charge for PDA@CNTs-PEDOT films on ITO electrode. The obtained results exhibited difference as compared with previously reported PEDOT films, For the PDA@CNTs-PEDOT film, the CSC can be linearly in which case the calculated CSC falls off for thicker films of PEDOT because of lower doping and more defects.20 increased to more than 11 mC/cm2. While, previously reported PEDOT films stopped linearity at a CSC value of 7 mC/cm2.20 The electrochemical impedance of the PDA@CNTs-PEDOT-coated ITO electrodes was further characterized with electrochemical impedance spectroscopy in 0.01 M PBS (pH 7.4) with a frequency range between 1 and 100,000 Hz, as shown in Fig. 5C. The impedance decreased sharply with increasing constantly coating of PDA@CNTs-PEDOT film on ITO electrode, especially at low frequency regions. For example, the ITO impedance was 1.07×104 Ω at the frequency of 1 kHz, which 18


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decreased to 130 Ω after electrochemical deposition of PDA@CNTs-PEDOT film at an applied electrochemical deposition charge of 130 mC/cm2. This phenomenon could be possibly due to the electroactive surface area enhanced by increasing the density of PDA@CNTs-PEDOT film on ITO electrode, thus leading to the decrease of impedance of PDA@CNTs-PEDOT/ITO electrode. CSC and impedance of the conducting polymer depend on the moving charge in and out of the film. And, the charge movement depends on doping level of the conducting polymer or interfaces between the conducting polymer surfaces and the electrolytes in the solution.59 Therefore, the doping level of the conducting polymer or interfaces between the conducting polymer surfaces and the electrolytes in the solution was of great importance.59 However, the presence of biological dopants on the conducting polymer reduced CSC and increased impedance reported in previous studies.59 But this is not observed in our experiment. PDA@CNTs-PEDOT film showed nanotube-network topography of rougher surfaces, which was beneficial to the migration of small ions in and out of the PDA@CNTs-PEDOT film. Moreover, as reported, rough surface resulted in large effective surface for the mobility of charge ions between the electrode and electrolyte interfaces.35,36 Therefore, we provided an efficient strategy of improving the electrochemical properties of ITO electrode by electrochemical deposition of PDA@CNTs-PEDOT film. 3.5.2. Electrochemical stability A good electrochemical stability of the electroactive electrode can contribute to efficient and safe charge delivery under ES. Here, the electrochemical stability of PDA@CNTs-PEDOT film on the ITO electrode was evaluated using CV by cycling between −0.6 and +0.7 V in 0.01 M PBS (pH 7.4) at a scan rate of 0.1 V s−1. The enclosed CV curve became slightly small at each CV cycling. A certain decrease in the integrated area was only found after the first 100 cycles, which was slightly changed after 200 and 300 cycles (Fig. 6A). Accompanying the decrease in CSC was a corresponding increase in impedance (Fig. 6B). PDA@CNTs-PEDOT film deposition decreased the initial ITO impedance from 1365 Ω to ~56 Ω when applying 19



Page 20 of 46

an electrochemical deposition charge of 75 mC/cm2 at 10 kHz. The impedance increased to ~390 Ω after 100 cycles of CV. Impedance changed slightly to ~460 and ~510 Ω after 200 and 300 cycles of CV, respectively. Similar to previous study by Ouyang et al.19 some loss of conducting material from the electrode surface might be responsible for the changes in the electrochemical properties. In spite of this phenomenon, PDA@CNTs-PEDOT film showed a good performance in long-term electrochemical stability as an electrode. 3.6. Neurite outgrowth and myelination Given that electrical signals are delivered to neural tissue through electrodes, tailored cell interactions on the interface under ES are critical for neuroscience applications.

Therefore,

we

investigated

DRG

neuron

growth

on

PDA@CNTs-PEDOT-coated ITO electrode with ES. DRG neurons cultured on PDA@CNTs-PEDOT-coated ITO electrode without ES were investigated as control. As shown in Figs.7A and B, DRG neurons were well attached on the PDA@CNTs-PEDOT substrates with long neurites and SC migrations away from the seeded DRG sphere after 7 days. The maximum distance of neurite extension was measured to be 1391±410m on a PDA@CNTs-PEDOT scaffold with ES, significantly longer than the longest neurites without ES, which were 864±484 m (n=4, p

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