Electroceutical Residue-Free Graphene Device for Dopamine

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Interfaces, Optics, and Electronics

Electroceutical Residue-Free Graphene Device for Dopamine Monitoring and Neural Stimulation Ho Sang Jung, Hyun Ho Kim, Myeong Hwan Shin, Seongjong Kim, Ki Su Kim, Kilwon Cho, and Sei Kwang Hahn ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01488 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 15, 2019

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ACS Biomaterials Science & Engineering

Electroceutical Residue-Free Graphene Device for Dopamine Monitoring and Neural Stimulation

Ho Sang Jung1,2,⊥, Hyun Ho Kim3,⊥, Myeong Hwan Shin1,⊥, Seongjong Kim1, Ki Su Kim1,4, Kilwon Cho3,*, Sei Kwang Hahn1,*

1

Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), 77-Cheongam-ro, Nam-gu, Pohang, Kyungbuk, 790-784, Korea

2

Advanced Nano-Surface Department, Korea Institute of Materials Science (KIMS), Changwon, Gyeongnam 641-831, Korea

3

Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77-Cheongam-ro, Nam-gu, Pohang, Kyungbuk, 790-784, Korea

4 Department

of Organic Materials Science and Engineering, Pusan National University, 2

Busandaehak-ro 63 beon-gil, Geumjeong-gu, Busan 46241, Korea

⊥

These authors contributed equally to this work.

Correspondence and requests for materials should be addressed to S.K.H. (email: [email protected]) or to K.C. (email: [email protected]). 1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Graphene interface for simultaneous neural signal monitoring and stimulation can allow accurate neurotransmitter regulation for patients in various degrees of neural degeneration disorders. Here, we developed a residue-free graphene device as an effective electrical neural interface for dopamine sensing and secretion. We demonstrated the ultra-sensitive dopamine sensing of residue-free graphene devices cultured with PC12 cells and the on-demand functional electrical stimulation for electroceutical applications. The doping effect of graphene by the released dopamine from living cells was confirmed from the electrical current change. The dopamine release could be also quantitatively analyzed by ELISA. Then, Ca2+ iondependent dopamine release was optically observed by fluorescence microscopy during the stimulation. Taken together, this study confirms the feasibility of graphene surface as a neural interface for electroceutical applications to various central nerve system disorders. Keywords Graphene, Neuro sensing, Neuro stimulation, Electroceutical

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INTRODUCTION Neural interface devices between nerve systems and external environment enable neural disease monitoring and multiple therapeutic applications for central nerve system (CNS) disorders such as Parkinson’s disease (PD),1-3 spinal cord injury,4,5 and visual loss.6 It has been reported that PD, schizophrenia, and attention deficit hyperactivity disorder (ADHD) / attention deficit disorder (ADD) are strongly related with neurotransmitter dysregulations in basal ganglia.1-3,7,8 Taking medicines to control the neurotransmitter release, especially dopamine, is a conventional method for the treatment and alleviation of the symptoms. Recently, neural stimulation of CNS through external devices such as deep brain stimulation (DBS) has emerged as an effective way for therapeutic activation and control of degenerated nerve systems and neural disorders.9-11 Through the electrical neural stimulation, dopamine release can be modulated and utilized to treat diseases related to neurotransmitter dysregulation.12-14 However, since the response and the amount of released dopamine via electrical stimulation depend on the patient’s nerve system and the degree of degeneration, it is important to monitor the amount of dopamine before and after the stimulation of neural cells for further applications to bioelectronics or electroceutical devices. Electroceutical devices provide temporal and spatial control of bioelectronic signals and small charged molecules.15 The bioelectrically triggered drug delivery has been evolved into the concept of electroceuticals. Especially, the neuromodulation of nerves has been considered as an important field of electroceuticals.16,17 Neuro-interfacing materials were usually composed of electrically conductive materials that have less corrosive characteristics under physiological conditions.18 Although the metal interface effectively transfers external electrical stimulation, it is hard to detect target molecules without surface functionalization of receptors. For the achievement of neural stimulation and sensing via one device, the interfacing materials should be highly electroconductive and electroresponsive for target molecules. There are 3 ACS Paragon Plus Environment


several reports on the neurotransmitter detection with conducting materials, including subnanomolar detection of dopamine using surface-modified electrodes.12,19 Among them, CNT and graphene have been recently harnessed for dopamine sensing as a label-free method.20,21 Zhang et al. reported dopamine sensing with the detection limit down to 1 nM using a solution gated transistor with CVD-grown graphene.21 Dopamine solution or released dopamine from living cells could be stacked on CNT or graphene surface via π-π stacking and induced electropotential change of the sensor surface.20,22 Especially, graphene is a promising neural interface material due to its large 2D surface area, superior electrical conductivity, high mechanical strength, optical transparency, and biocompatibility.23-25 Moreover, owing to the low density of state near Dirac point of graphene, it is definetly suitable for high-perfomance sensing devices. Nevertheless, the applications of graphene materials to neural sensing are in the early stage, mostly for in vitro neurotransmitter detection. In this work, a neural interface graphene system for simultaneous neural monitoring and stimulation was developed as schematically shown in Figure 1. For the effective electrical signal transfer and sensitive detection of dopamine on graphene surface, we devised a facile transfer method for residue-free graphene. The graphene exhibited high sensitivity detecting a sub-nanomolar concentration of dopamine in physiological conditions. Then, real-time in vitro monitoring of dopamine and the electrical stimulation of dopaminogenic neuronal PC12 cells via graphene surface for dopamine release were studied at various stimulation conditions. These fundamental studies on graphene-based simultaneous neural monitoring, stimulation and sensing systems are discussed for further treating neural disease patients.


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MATERIALS AND METHODS Materials. Dopamine hydrochloride, BSA, and potassium chloride were purchased from Sigma Aldrich (St. Louis, MO). Fluo-4 calcium assay kit was obtained from Thermo Fisher Scientific (Waltham, MA). Polydimethylsiloxane (PDMS, Sylgard-184) was purchased from Dow Corning (Midland, MI). Biopsy punch was purchased from Miltex Inc, (Bethpage, NY). PC12 cells were obtained from Korean Cell Line Bank (Seoul, Korea). RPMI 1640, fetal bovine serum (FBS), and horse serum were purchased from Invitrogen Co. (Carlsbad, CA). Dopamine ELISA kit was obtained from Abnova (Taipei, Taiwan). Preparation of Residue Free CVD Graphene. Monolayer graphene was grown on a copper (Cu) foil (Alfa Aesar, product number: 13382) using chemical vapor deposition.26 Poly(methylmethacrylate) (PMMA, Aldrich, MW = 996 kg/Mol) dissolved in chlorobenzene (5 wt%) was spin-coated on graphene/Cu surface as a supporting insulating layer, followed by solvent drying at 120℃ for 30 min. Graphene/Cu/graphene/PMMA structure was transferred onto target substrates (e.g. Si or polymer substrates) via hot pressing process at 150℃ (> Tg of PMMA) and then immersed into 0.1 M ammonium persulfate [(NH4)2S2O8] to dissolve Cu. A reactive ion etcher with oxygen plasma (100 sccm of O2, 250 W for 1 min) was used for the uniform etching of Cu. Graphene/PMMA/substrate samples were put to DI water for rinsing the remaining etchant on graphene surface. The transferred graphene/PMMA films on target substrates were characterized by optical microscopy (OM, Axioplan, Zeiss), atomic force microscopy (AFM, Veeco, NanoScope 8), and Raman spectroscopy (Alpha 300R, WITec, λ = 532 nm). Fabrication of Graphene FET Sensor and Cell Culture. Graphene/PMMA/Si wafer was sterilized by immersing in 70% ethanol solution for 1 min and dried in a clean bench. Cell culture chamber was prepared using a PDMS mold. PDMS solution at a ratio of 10:1 (base: curing agent) was poured onto a 100 Φ petri-dish and placed in vacuum chamber for 2 h. Then 5 ACS Paragon Plus Environment


bubble free PDMS solution was cured on a hot plate at 70℃ for 1 h. PDMS cell culture chamber was cut into a square shape with a dimension of 6 mm × 6 mm and punched with a diameter of 5 mm using a biopsy dermal punch. The prepared PDMS chamber was sterilized in 70% ethanol solution for 1 min and dried in a clean bench at least for 1 h. A PDMS cell culture chamber was placed on the center of the graphene channel and then silver paste was pasted on both ends of the graphene channel as a probe contact electrode. PC12 cells cultured in RPMI 1640 media with 10% FBS, 5% horse serum and 1% antibiotics were prepared prior to seeding on the graphene sensor. PC12 cells at a density of 5,000 cells per graphene cell chamber was seeded and incubated in a 5% CO2 cell culture incubator for 12 h. Stimulation and sensing experiments were carried out after 2 h of replacing cell culture media with fresh media. Characterization of Dopamine Sensing. The dopamine sensing capability of graphene sensor was tested without cells in the chamber. Cell culture medium was filled in the sensor chamber and incubated in a 5% CO2 cell culture incubator for 24 h. PMMA/SiO2 layer and n+-Si Wafer were used as the dielectric layer and the gate electrode, respectively. Drain current and Dirac voltage of graphene were monitored using a semiconductor parameter analyzer (4200-SCS, Keithley) at a drain voltage of 0.01 V for every dopamine hydrochloride solution (1 nM ~ 10 nM) dropping in the sensor chamber using a micropipette. The doping effect of dopamine released from cells on graphene was also characterized by X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Scientific) and Raman spectroscopy (Alpha300R, WITec, λ = 532 nm). Real-Time Stimulation & Monitoring of Dopamine Released from Living Cells. To characterize the electrical stimulation induced dopamine release from living cells, Dirac voltage shift was monitored, while cells were stimulated at a constant drain voltage of 0.1 V. In addition, for the effective stimulation of the cells, we devised a pulse-type interval stimulation method. At every sample, the base drain voltage was fixed at 1 mV for 30 s to apply the pulse-type stimulation. It was noted that the cells on graphene were not activated at 1 mV drain voltage. 6 ACS Paragon Plus Environment

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The pulsed type stimulation conditions were determined as follows. On-time was fixed as 0.1 s at 2 V and off-time was varied from 0.01 to 5 s to observe the current change for the different stimulation frequency. The cells were stimulated more frequently with the shorter off-time. Cell Viability Test and Dopamine ELISA after Electrical Stimulation on Cells. The cell viability of electrically stimulated living cells at 2 V was compared with that of nonstimulated cells via the standard MTT assay. Twenty microliter of MTT solution was added in the graphene sensor chamber containing cells and incubated in a 5% CO2 incubator for 2 h. Then, the supernatant was exchanged with dimethyl sulfoxide (DMSO) and mixed for complete formazan dissolution. The formazan solution was transferred to a 96-well cell culture plate and read at 450 nm using a microplate reader. Cell viability was calculated by using non-stimulated cells as a control (n = 4). Dopamine ELISA was conducted using the supernatant of cell media after electrical cell stimulation (n = 4) according to the manufacturer’s protocol. Frequency-Dependent Current Change and Quantitative Analysis of Released Dopamine. To investigate the effect of pulse-type stimulation frequency on sensor drain current change and its consequent quantitative dopamine release, each sample was stimulated for 10 s at 2 V with the different frequency range from 1 Hz to 10 kHz (on time/off time = 1). The pulsetype frequency was controlled with a function generator (33500B Series, Agilent) precisely adjusted by oscilloscope. The drain current changes were averaged and the experiment was repeated thrice. Then, released dopamine was quantitatively analyzed by dopamine ELISA using the cell supernatant after various electrical stimulations. Ca2+ Imaging of Electrically Activated Cells on Graphene Sensor. For the visualization of electrical stimulation induced cell activation, Ca2+ ion-dependent dopamine release was optically observed after staining PC12 cells cultured on the graphene sensor with Fluo-4 according to the manufacturer’s protocol. The intracellular Ca level increase was measured by monitoring fluorescence intensity change using fluorescence microscopy before and after the



stimulation. The intensity change ratio was calculated by successive subtracting the previous frame fluorescence intensity to exclude the bleaching effect during the measurement.

RESULTS AND DISCUSSION The CVD-grown graphene transfer from catalytic Cu to a target substrate is usually conducted using a supporting layer such as poly(methylmethacrylate) (PMMA) to prevent crack generation.27 However, even after removal of PMMA,28 undesirable residues are left on the graphene surface, causing detrimental effects including the mobility decrease in graphene FET due to the charged impurity scattering29 and the decreased molecular sensitivity of graphenebased sensor.30 For the fabrication of residue-free surface of large-area monolayer graphene, we developed a novel inverse transfer method using hot pressing of graphene/PMMA onto the target Si wafer, as schematically illustrated in Figure 2a. Cu/graphene/PMMA was inversely hot-pressed on SiO2/Si (or plastic substrates) at the temperature above Tg of PMMA and then graphene/PMMA/SiO2/Si structure was fabricated after etching of Cu foil in the aqueous solution of ammonium persulfate. During the hot pressing, PMMA layers became adhesive above its glass transition temperature ( ~105 ℃) and the graphene/PMMA layer could be successfully transferred on the target substrate. For the cell stimulation and measurement experiments, a PDMS chamber was fixed on the graphene surface and the silver paste was covered on the both ends of the device. PC12 cells were then cultured in the PDMS chamber for 24 h before measurement. In-situ sequential optical microscopic images (Figure 2b) showed that Cu foil was removed without generation of cracks and wrinkles on graphene surface. The residue free surface was characterized by AFM after 10 nm pentacene (C22H14) deposition. It showed an epitaxial lying-down pentacene structure grown on graphene surface due to the absence of residues (Figure 2c).28,31 In addition, the sensitivity of residue free graphene was compared with that of PMMA residue remaining graphene surface after dropping 1 nM 8 ACS Paragon Plus Environment

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dopamine solution. The residue-free graphene surface showed remarkable Dirac shift compared to the residue remaining graphene surface reflecting enhanced sensitivity for dopamine (Figure 2d). In addition, it was noted that PC12 cells on graphene could survive even after applying 2 V continuous electrical stimulation for 5 min, which was confirmed by cell viability tests, as shown in Figure 2e. It was reported that the treatment of 2 V DC potential for 24 h enhanced the cell proliferation and elongation forming a dense nucleus.32 Optical microscopy (Figures S1a and S1b) and Raman 2D mapping (Figure S1c) confirmed the aqueous stability of transferred graphene after ultra-sonication in DI water for 5 min. The quality of CVD-grown graphene was also confirmed by SEM (Figure S1d), gate voltage-dependent current measurement (Figure S1e) and Raman spectroscopy (Figure S1f). The SEM image of graphene initially grown on copper foil shows that most of the area is covered by monolayer graphene except a few layer islands and wrinkles caused by the difference in thermal expansion. We could observe the clear Dirac curve under the vaccum condition. In addition, Raman spectroscopy revealed the high quality of CVD-grown graphene with a negligible D peak and a 2D/G intensity ratio higher than 2 reflecting no significant defect generation. The dopamine sensing capabilities of graphene sensor were characterized by measuring Dirac shift using 100 pM - 100 nM dopamine solution. The sensor chamber was filled with cell culture media to make an identical condition to the living cell cultured sensor environment. The drain volatge condition during Dirac measurement was selected as 0.1 V in the case of experiments without cell culture, while 1 mV was selected in the case of cell cultured device. We confirmend that 1 mV showed neglegible stimulation effect on cells, resulting in no significant current change (Figures S2a and S2b). After dropping the dopamine solution, it was observed that the Dirac voltage of graphene field effect transistor (FET) was shifted toward a positive voltage reflecting the p-type doping of graphene surface (Figure 3a). It was previously reported that dopamine could be stacked on graphene surface by π-π interaction and withdraw electrons from the graphene channel, which 9 ACS Paragon Plus Environment


induced the p-type doping of the sensor.20 Furthermore, gate-voltage-dependent drain current was measured for PC12-cell-cultured graphene devices. Interestingly, it showed that the Dirac voltage was positively shifted after electrical stimulation, which might be induced by the doping effect of released dopamine from PC12 cells after the electrical stimulation (Figure 3b). Meanwhile, it was reported that dopamine release from PC12 cells could be activated at high potassium condition due to the opening of Ca2+ ion channels on the cell surface.12,20 In a control experiment of cell stimulation using 50 mM potassium chloride (KCl) solution, it showed similar p-doping effect at every KCl solution dropping, while the amount of Dirac shift was relatively smaller than that of electrical cell stimulation (Figure 3c). Considering no significant Dirac voltage change and drain current change after electrical stimulation on graphene sensor filled with only cell culture media (Figure 3d), we could confirm that Dirac voltage change of PC12 cells after electrical stimulation was induced by the released dopamine from living cells. The current change depending on the dopamine concentration was measured as shown in Figure 4a. Upon every dopamine solution dropping, drain current was gradually increased in real-time for the two-terminal current measurement without applying gate voltage. This result indicates that dopamine can be monitored in real-time. Furthermore, current change induced by the doping of released dopamine on graphene surface was compared in two conditions: continuous and pulse-type stimulation. The applied voltage scheme is shown in Figure S3a. The continuous stimulation was set as applying 2 V potential without interruption and pulse-type stimulation was set as 2 V application with a pulse period of 0.2 s (on : off = 0.1 s : 0.1 s). As shown in Figure 4b, continuous electrical stimulation showed relatively slow current change, whereas the pulse-type stimulation exhibited rapid current increase followed by saturation. Stepwise current change was observed after each repetitive continuous and pulse-type electrical stimulation on PC12 cells. It showed that the pulse-type stimulation induced relatively large current change especially at the first stimulation compared to the continuous stimulation (Figure S3b). The results reflected that the continous stimulation triggered cells one time, whereas the 10 ACS Paragon Plus Environment

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pulse-type stimulation affected cells at each pulse generation. Furthermore, real-time monitoring of current change at various stimulation turn-off times (5 s – 0.01 s) was carried out to observe the cell response at different time intervals. The base current was adopted at 1 mV which showed no significant current increase and the width of stimulation turn-on time was fixed at 0.1 s. The total number of stimulation was fixed at 10 times. The cells were more frequently stimulated with the shorter off-time. As shown in Figure 4c, the shortened turn-off time in one stimulation period induced the relatively large doping effect. The current increase was reduced with increasing stimulation turn-off time. Remarkably, most of the current change occurred at the first stimulation period, which might be related with the initial bursting of dopamine release from living cells and the large doping area remaining on graphene surface. Nevertheless, stepwise increase of drain current could be observed consecutively at every electrical stimulation period. These results are encouraging, because they are the first data suggesting real-time electrical cell stimulation and monitoring of released dopamine from the living cells using one device possibly for electroceutical applications. Other catecholamines including epinephrine and norepiephrine share catechol groups in their chemical structures and have electron withdrawing effect on the sensor surface by π-π interaction.20 It was reported that dopamine, epinephrine, norepinephrine showed similar current effect on FET sensor devices.12 The doping properties of graphene channel after electrical stimulation of cells were characterized by Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). In Raman spectra, G band of graphene channel without cell stimulation was observed at 1582 cm-1, whereas that of electrically stimulated cells cultured on graphene was observed at shifted wavelength of 1593 cm-1 (Figure 5a). The results show the consistency of p-doping effect, since p-doping causes the stiffening and the shift of G peak in graphene due to the Kohn anomaly from the G band and Fermi energy shift, respectively.33 Then, XPS spectroscopic surface analysis was carried out using PC12 cells-cultured graphene before and after the electrical stimulation. Even after the cell fixation process, π-π stacked dopamine with graphene could be 11 ACS Paragon Plus Environment


detected on the graphene channel surface. Compared to PC12 cells on the graphene without electrical stimulation, stimulated samples showed relatively high C-OH peak (286 eV) and CN peak (287.9 eV) in C1s XPS spectra, which might be possibly due to the presence of catechol and amine group in dopamine molecules, respectively (Figure 5b). In addition, increased C-N peak in stimulated cells was also shown in N1s XPS spectra (Figure 5c). Dopamine ELISA was performed for the quantitative confirmation of released dopamine induced by electrical stimulation. Dopamine release at various applied voltages at fixed stimulation time of 30 s showed the proportional increase of released dopamine. The initial dopamine concentration was 28.7 nM, which was increased to 62.9 nM, 87.1 nM and 107.1 nM at 1 V, 2 V, and 5 V electrical stimulation for 10 s, respectively (Figure 5d). We also compared the amount of released dopamine at various stimulation pulse periods at the fixed stimulation time of 30 s. As shown in Figure 5e, the amount of released dopamine increased with decreasing stimulation pulse width, reflecting that cells were more frequently stimulated at the shorter pulse width. The stimulation-induced dopamine sensing from living cells was investigated at varying stimulation conditions. The frequencies were varied from 1 to 10,000 Hz with an identical on/off time ratio at a fixed total stimulation time. The base drain voltage was adjusted at 1 mV and electrical stimulation at various frequencies was induced with a function generator precisely adjusted by the oscilloscope. Because most of dopamine was released at the first stimulation period, initial current change (I/I0) was compared for each frequency group. Remarkably, the current change was increased as the frequency was increased from 1 Hz to 100 Hz. However, it was inversely decreased at relatively high frequency of 100 Hz - 10 kHz (Figure 6a). Then, the released dopamine at various frequencies was quantitatively analyzed by dopamine ELISA (Figure 6b). The released dopamine amount and the current change measured by the graphene substrate were proportional in the range of conventional neuro-stimulating electrical frequencies (1 Hz - 10 kHz). The current change at extremely high electrical frequencies showed no significant correlation with released dopamine amount. Using the in vitro neuro12 ACS Paragon Plus Environment

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stimulation and sensing data, we might be able to control the conditions for further in vivo neuro-stimulation and sensing applications. All these experiments were conducted using randomly distributed samples (n = 5). Finally, to study the dopamine release mechanism from living cells cultured on graphene devices, intracellular Ca2+ imaging was carried out by fluorescence microscopy. It was reported that voltage-dependent Ca2+ channel opening increased the intracellular Ca level and facilitated the extracellular dopamine release.12,20 The Ca2+ labeling dye Fluo-4 was stained on cells prior to the imaging for the real-time monitoring of fluorescence intensity change during the electrical stimulation of cells. The electrical stimulation was applied via the function generator and Fluo-4 signal was recorded in real-time for 2 min. Figure 7a shows the fluorescence after electrical cell stimulation. In addition, the fluorescence change values were calculated by subtracting the fluorescence intensity after and before the stimulation (Figure 7b). From the resutls, we could confirm that dopamine was released by the mechanism of voltage dependent calcium channel opening by the electrical stimulation.



CONCLUSION We developed a graphene neuro-interface device as a new platform for simultaneous neurotransmitter sensing and neuro-stimulation for therapy. Becasue the responses to electrical stimulations are significantly different for patients with various degrees of neural degeneration, the simultaneous electrical sensing and stimulation might be very important to provide electrical responsive levels of neural tissues in patients. Thus, after the analysis of induced dopamine levels using the graphene device, we might be able to control the individual specific neurostimulation conditions for neural disorder patients requiring the deep brain stimulation treatments. Taken together, this graphene device with PC12 cells might be successfully applied to various bioelectronic and electroceutical applications.

ASSOCIATED CONTENT Supporting Information. Optical microscope images, electrical characterization with cell media, and stepwise current measurement.

ACKNOWLEDGEMETNS We would like to thank Prof. Tae-il Kim and Sung-hyuk Sunwoo. This research was financially supported by the Center for Advanced Soft-Electronics (Global Frontier Project, CASE2015M3A6A5072945), the Nano·Material Technology Development Program (Grant No. 2017M3A7B8065278), Global PhD. Fellowship Program (2015H1A2A1034046) and the Basic Science Research Program (Grant No. 2017R1E1A1A03070458) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, Korea.


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Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURES

Figure 1. Schematic illustration for a real-time neuro-sensing, stimulation and monitoring system for electroceutical applications.



a

O2 Plasma, Cu Etching

Hot Pressing above Tg

PMMA-Coated Substrate

Clean Graphene Surface

b

d Pentacene

e

30

Free G Free G + D Residue G Residue G + D

25 20 15

0.5 μm 10

Relative Cell Viability (%)

c

ID (A)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PC12 Cell Culture

120 100 80 60 40 20 0

0

20

40

60

VG (V)

80

Control

2V Stimulation

Figure 2. (a) Schematic illustration for the residue-free transfer of CVD-grown graphene for the fabrication of a simultaneous stimulation and monitoring device. (b) In-situ optical visualization for the Cu foil etching process. (c) AFM image for the 10 nm-thick pentacene deposition on the residue-free graphene surface. (d) Electrical characterization for the dopamine sensitivity between residue-free graphene and graphene with PMMA residues. (e) The relative cell viability before and after electrical stimulation at 2 V for 1 min.


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30 25

ID (μA)

b

Pristine 100 pM 1 nM 10 nM 100 nM

20 15

70 60 50

VD = 0.1 V

10 0

c

90 80

ID (μA)

a

20

VD = 0.1 V

40

40

VG (V)

60

0

80

20

40

60

80

60

80

VG (V)

0.7

d

45 40

ID (μA)

0.6

ID (μA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5 0.4

35 30 25

VD = 1 mV

0.3 0

20

VD = 0.1 V

20

40

60

0

80

VG (V)

20

40

VG (V)

Figure 3. The Dirac voltage shift induced by (a) dopamine solution (100 pM - 100 nM) without PC12 cells, (b) electrical, (c) chemical (KCl) stimulation of living PC12 cells on graphene surface, and (d) electrical stimulation without PC12 cells for four times (0 ~ 4 times).



a

0.92

Current (𝝁A)

Real-time Average 0.91

0.90

0.89 0

100

200

300

Time (s)

Current Change (%)

b

1.5

1.0

0.5

Control Continuous Pulse-type

0.0 0

10

20

30

40

50

200

250

Time (s)

c

1.4 5s 1s 0.1 s 0.01 s

1.3

I/I0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.2 1.1 1.0 0

50

100

150

Time (s)

Figure 4. (a) The stepwise time-dependent current measurement after sequential dropping of 1 nM dopamine. (b) The time-dependent current measurement of graphene with and without culturing of PC12 cells. The cells were stimulated by continous or pulse-type bias. (c) The stepwise current change with increasing time at 1 mV as a function of turn off time in pulsetype stimulation. All stimulations were assessed on 0.1 s and off time was changed from 0.01 s to 5 s. Black arrows indicate each stimulation period (10 cycles per period).


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b

a

c

e

d 140

120

Released Dopamine Dopamine (nM) Released (nM)

Released Dopamine (nM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


120 100 80 60 40 20 0

100 80 60 40 20 0

NC

0.5 V 1 V 2 V Stimulation Voltage

NC

5V

1s 0.1 s Pulse Width Pulse Width

0.02 s

Figure 5. (a) Raman spectra at G peak of graphene. (b) C1s and (c) N1s XPS spectra of PC12adhered graphene film before (black line: control) and after electrical stimulation at 2V (red line: stimulation). Quantitative analysis of released dopamine from living cells on graphene using ELISA as a function of (d) applied voltage and (e) pulse width during the stimulation. The total stimulation time and on/off time ratio in pulse-type stimulation were set to be 30 s and 1, respectively (NC: negative control).



a

b 140

1.5

Released Dopamine (nM)

Normalized Current Change

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.4

1.3

1.2

1.1

z 1H

10

Hz

10

z 0H

0 10

z 0H 1

0 00

120 100 80 60 40 20 0

z 0H

z 1H

Hz 10

z z Hz 0H 0H 00 00 10 10 0 1

Figure 6. (a) The current change (I/I0) as a function of frequency in pulse-type stimulation (on/off time ratio = 1) and (b) the consequent released amount of dopamine by ELISA. The total stimulation time was set to be 10 s for each sample.


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a

b Fluorescence Intensity Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Time (s)

Figure 7. (a) Fluoresence microscopic image of PC12 cells stained with Fluo-4 after the electrical stimulation (scale bar = 25 ㎛). (b) Fluoresence intensity change with increasing time after stimulation at 10 s.



The Table of Contents Entry

Electroceutical Residue-Free Graphene Device for Dopamine Monitoring and Neural Stimulation

Ho Sang Jung1,2,⊥, Hyun Ho Kim3,⊥, Myeonghwan Shin1,⊥, Seongjong Kim1, Ki Su Kim1, Kilwon Cho3,*, Sei Kwang Hahn1,*

ToC Figure


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Electroceutical Residue-Free Graphene Device for Dopamine

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