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Graphene encapsulated copper microwires as highly MRI compatible neural electrodes Siyuan Zhao, Xiaojun Liu, Zheng Xu, Huaying REN, Bing Deng, Miao Tang, Linlin Lu, Xuefeng Fu, Hailin Peng, Zhongfan Liu, and Xiaojie Duan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b03829 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 5, 2016

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Graphene encapsulated copper microwires as highly MRI compatible neural electrodes Siyuan Zhao1,2,3, Xiaojun Liu1,3, Zheng Xu1,3, Huaying Ren2,3, Bing Deng3,4, Miao Tang3,4, Linlin Lu1,3, Xuefeng Fu1,3, Hailin Peng3,4, Zhongfan Liu3,4 & Xiaojie Duan1,2,3* 1 Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China. 2 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. 3 Center for Nanochemistry, Beijing Science and Engineering Center for Nanocarbons, Peking University, Beijing 100871, China. 4 College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

*Corresponding authors. E-mail: [email protected]

Keywords: Neural electrodes, magnetic resonance imaging, graphene biosensing, anti-corrosion, biocompatibility

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ABSTRACT

Magnetic resonance imaging (MRI) compatible neural electrodes are important for combining high-resolution electrophysiological measurements with more global MRI mapping of brain activity, which is critical for fundamental neuroscience studies, as well as clinical evaluation and monitoring. Copper is a favorable material to use in MRI because it has magnetic susceptibility close to water and tissues. However, the cytotoxicity of copper precludes its direct implantation for neural recording. Here, we overcome this limitation by developing a graphene encapsulated copper (G-Cu) microelectrode. The toxicity of copper is largely eliminated, as evidenced by the in-vitro cell tests and in-vivo histology studies. Local field potentials and single-unit spikes were recorded from rodent brains with the G-Cu microelectrodes. Notably, the G-Cu microelectrodes show no image artifacts in a 7.0 T MRI scanner, indicating minimal magnetic field distortion in their vicinity. This high MRI compatibility of our G-Cu probes would open up new opportunities for fundamental brain activity studies and clinical applications requiring continuous MRI and electrophysiological recordings.

Neural interfacing with electrodes constitutes the basis of electrophysiological research and many clinical applications, such as brain-controlled prosthetic devices,1 deep brain stimulation (DBS),2 and preoperative localization of epileptic foci.3 Magnetic resonance imaging (MRI) compatible neural electrodes are important for combining high-resolution electrophysiological measurements with more global MRI mapping of brain activity, which is critical for fundamental neuroscience studies.4 MRI compatible electrodes can also be beneficial in clinical applications, including verification of placement and stability of implanted DBS electrodes5 and long-term 2 ACS Paragon Plus Environment

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epilepsy

monitoring,

in

which

repeated

electrophysiological

measurements

and

anatomical/functional neuroimaging are required.6 Commonly used materials for implantable neural electrodes, including platinum-iridium (Pt-Ir) alloy, tungsten, gold, nichrome (Ni-Cr), stainless steel, etc., usually possess good stability and interfacial electrochemical characteristics. However, these materials, even when nonferromagnetic, may induce severe field distortions due to the mismatch of magnetic susceptibility between the metals and water/tissues, thus producing image artifacts or blind spots around the electrodes in MRI7 that cause inconvenience or interference for anatomical and functional MRI studies.5,

8

Copper (Cu) has magnetic

susceptibility very close to water and should yield negligible image artifacts under MRI. However, Cu cannot be directly used as implantable neural electrodes because of its known toxicity to brain tissues.9 In this study, we demonstrate that this challenge can be overcome by encapsulating Cu microwires with graphene from low-pressure chemical vapor deposition (CVD) (Figure 1a). The graphene encapsulated Cu (G-Cu) microwires show significantly eliminated toxicity to brain tissues, as evidenced by the in-vitro cell tests, in-vivo histology, and MRI studies. Distinct from Pt microwires, the G-Cu microwires exhibit negligible image artifacts under 7.0 T MRI. Local field potential (LFP) and single-unit action potentials of high signal-to-noise ratio were recorded from rat brain using the G-Cu microelectrodes. These results indicate that the G-Cu can be used as highly MRI compatible implantable neural electrodes that would be beneficial in various aspects, from neurophysiological studies of brain activity to clinical diagnoses and monitoring. Furthermore, the concept of using high-quality graphene as a protective or modification layer for biomaterials could be extended to other biomedical applications, which could play an important


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role in improving biological-material interactions for enhanced biosafety and biocompatibility, as well as the development and design of new medically acceptable materials. The graphene encapsulation layer was deposited directly on the Cu microwires surface by lowpressure CVD.10, 11 Compared to transferring the graphene layer on Cu microwires, the CVD approach could provide seamless, full coverage coating of graphene to achieve an impervious barrier for Cu corrosion. A typical scanning electron microscopy (SEM) micrograph of the asgrown G-Cu microwires is presented in Figure 1b, with a magnified image of the dashed box shown in the inset. The narrow, dark lines in the SEM image (red arrow heads) are graphene wrinkles, which is a signature feature of the CVD grown graphene layers on Cu.

10, 12

The

wrinkles crossing the Cu steps and boundaries (green arrow heads) imply the continuity of the graphene film. The flakes with darker colors (yellow arrow heads) indicate graphene with multiple layers, with decreasing brightness attributed to monolayer, bilayer, and trilayer graphene films, respectively. With a PDMS stamp, the graphene layer on a Cu microwire was printing transferred to a 300 nm SiO2/Si substrate,13 as shown in the optical image in the inset of Figure 1c. The clear image of the graphene strip proved the successful growth of graphene on Cu microwires. Raman spectrum of the transferred graphene strip shows a G peak (at ~1582 cm−1) and a 2D peak (at ~2700 cm−1), which are characteristic of graphene. In addition, the small ratio of the integrated peak area between the D (at ~1350 cm−1) and G bands indicates a low defect level in the graphene strip.14 The electrochemical characterization confirmed the effectiveness of the graphene encapsulation layer as corrosion-inhibiting coating for Cu microwires. For bare Cu microwires, the cyclic voltammetry (CV) measurement (blue curve, Figure 1d) in Na2SO4 solution exhibits characteristic anodic and cathodic peaks that can be attributed to the electrodissolution of Cu and 4 ACS Paragon Plus Environment

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the electroreduction of Cu ions. In contrast, G-Cu microwires with the same exposure area in Na2SO4 electrolyte do not feature any peaks, and exhibit dramatically lower current at positive potentials (red curve and inset, Figure 1d). SEM images after the CV scan reveal that there was clear damage to the bare Cu microwire surface with increased roughness, while the G-Cu microwire surface remained undamaged with Cu steps and boundaries clearly shown after the CV measurement (green arrow heads) (Supporting Information Figure S1). Tafel analysis was utilized to quantitatively determine the corrosion rates of the bare Cu and G-Cu microwires. As seen from Figure 1e, for the G-Cu microwire sample, the open circuit potentials (OCPs) where the rates of the anodic and cathodic processes are balanced had a negative shift to approximately -580 mV, compared to -250 mV for the bare Cu microwire sample, concomitant with a sharp decrease in the cathodic reaction rate. We then calculated the corrosion rate for the bare Cu and G-Cu microwires as 6.14×10-12 m/s and 2.45×10-13 m/s, respectively (see the Experimental Section in Supporting Information for details).15-17 These results indicate that the graphene encapsulation layer serves as a barrier between the solution and the Cu surface that effectively prevents the corrosion of Cu in aqueous media.


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Figure 1. Graphene encapsulated copper (G-Cu) microwires. (a) Schematic drawing of the G-Cu implanted neural electrodes. (b) A typical SEM micrograph of the as-grown G-Cu. Inset, magnified image of the dashed box. Scale bar, 50 µm; inset, 10 µm. Red arrowheads refer to graphene wrinkles. The green arrowhead indicates the wrinkle crossing the Cu steps and boundaries. Yellow arrowheads refer to the flakes of graphene with multiple layers. (c) Raman spectrum and optical image (inset) of the graphene printing transferred to 300 nm Si/SiO2 substrate from Cu microwire using PDMS stamp. The arrow in inset marks the graphene strip. Scale bar, 100 µm. (d) CV measurements for bare Cu (blue curve) and G-Cu (red curve) microwire samples. Inset, magnified CV curve of the G-Cu microwire sample. (e) Tafel plots of the bare Cu (blue curve) and G-Cu (red curve) microwire samples. The graphene encapsulation layer significantly slowed down Cu corrosion.


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The cytotoxicity of the G-Cu microwires was accessed in-vitro using PC 12 cell line with cell counting Kit-8 (CCK-8) and live/dead cell staining assay. Briefly, G-Cu and bare Cu microwires of the same diameter and length were added separately to PC 12 cell cultures of the same seeding density and same amount of culture medium. After 12, 24, 36 and 48 h incubation, CCK-8 solution was applied, and the resultant supernatants were assayed by a spectrophotometer. Identical assays without adding any microwires were conducted as a positive control, to which the cell viability was normalized. The results from the CCK-8 assay are shown in Figure 2a. It can be seen that, in contrast to bare Cu microwires which show strong cytotoxic effects, the viability of PC 12 cells cultured together with the G-Cu microwires for different periods of time remains as high as over 93%, indicative of the absence of soluble cytotoxic factors from the GCu microwires. For the live/dead cell assay, G-Cu and bare Cu microwires were put into direct contact with PC 12 cells and incubated under normal culture conditions for various periods of time. Subsequent double staining with the live-marker calcein AM (green) and the dead-marker ethidium homodimer-1 (red) were performed. The fluorescent images of the live/dead-stained PC 12 cells (Figure 2b) illustrate that, for all tested incubation times, the G-Cu microwires (positions marked by the white dashed lines) are surrounded predominantly by dense living cells. Only a few sparsely distributed dead cells are visible. The cell viability and distribution are comparable to the control samples in which no microwires were added (Supporting Information Figure S2). This result indicates that the G-Cu microwires show no adverse effect on cell viability. However, for the bare Cu microwire samples, a large number of dead cells can be found in the area surrounding the wire, which is indicative of high cytotoxicity from the Cu. This toxic effect becomes more prominent with the increase of incubation time, as shown by the increasing 7 ACS Paragon Plus Environment

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number of dead cells. For the incubation time of 48 h, a large empty area forms surrounding the bare Cu microwires, where neither living nor dead cells can be found. The SEM images of the bare Cu microwires after 1 day and 7 days incubation with the culture media under normal culture conditions show obviously increased roughness and cracks on the surface, which indicates the profound corrosion of Cu under the culture conditions, while the G-Cu surface remains intact, indicative of negligible corrosion (Supporting Information Figure S3). All of these results suggest that the G-Cu microwires, with significantly suppressed corrosion in aqueous media, have little effect on cell viability. The non-/low cytotoxicity and high biocompatibility of the G-Cu microwires makes them a promising candidate as implantable neural electrodes that would be beneficial in some special cases.


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Figure 2. In-vitro cytotoxicity test. (a) Normalized viability of PC 12 cells cultured with G-Cu and bare Cu microwire samples for various times measured from a CCK-8 assay. Error bars show s.e.m.; ∗∗∗p < 0.001, n=5, t-test analysis. (b) Representative fluorescence images of the live/dead-stained PC 12 cells cultured with the G-Cu and bare Cu microwire samples for various times. The white dashed lines mark the position of the microwires. Scale bar, 300 µm.

The G-Cu microwires were assembled into implantable neural electrodes after being insulated with a ~10 µm thick layer of Parylene-C through a vacuum vapor deposition process, with the tip area exposed as an electrically active site (Figure 3a). Electrochemical impedance spectroscopy (EIS) measurement gives an average impedance value of ~150 kΩ and phase lag of -71° at 1 kHz for the G-Cu microelectrodes with a diameter of 100 µm (Figure 3b). In-vivo neural recording capabilities of the G-Cu microelectrodes were verified in the CA1 region of the hippocampus of an anaesthetized rat, as schematically shown in Figure 3c. Representative acute electrophysiological recording in the high-frequency range (0.3-6 kHz) is shown in Figure 3d. We were able to detect and isolate two single-unit spikes with peak-to-peak amplitude of 36 and 69 µV and signal-to-noise ratio of ~4 and 7, respectively, as shown by the pile plot (Figure 3e), the mean waveform (Figure 3f), and principle component analysis (Figure 3g). The high signalto-noise ratio of the single-unit recording suggests a close proximity between the firing neurons and the G-Cu microelectrodes. Simultaneously recorded local field potential (LFP) signal and its power spectral density plot are shown in Figure 3h and i, respectively, with the latter data exhibiting a broad maximum at ca. 3 Hz. This broad feature differs from the sharper peaks typically observed in rat hippocampus18 and we believe future studies will be needed to further understand this feature with our new probes. Importantly, our single-unit recordings demonstrate 9 ACS Paragon Plus Environment

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that the G-Cu microelectrodes create effective interfaces capable of recording high quality neuronal activities. Thus, it is found that G-Cu microwires perform as well as conventional metal microwires as neural recording electrodes, but with the advantage of being much more MRI compatible, as discussed below.

Figure 3. In-vivo electrophysiological recording. (a) A four-channel G-Cu microelectrode array assembly placed next to a penny. Two long stainless-steel wires were soldered to the two sides of the assembly for grounding purposes. Inset, SEM image of a G-Cu electrode tip, showing the exposed G-Cu (bright core) as the active recording site and the Parylene insulation layer (dark shell). Scale bar, 100 µm. (b) Magnitude and phase of electrode impedance recorded in 1x PBS (pH 7.4). (c) Schematic of a G-Cu microelectrode implanted into the CA1 region of the 10 ACS Paragon Plus Environment

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hippocampus of a rat brain. (d) Representative acute recording of high-frequency (0.3-6 kHz) electrophysiological signal using a G-Cu microelectrode. (e) Piled single-unit neural recordings over 60 s. Colors serve to distinguish the two distinct single units. (f) Mean waveform for each unit spike. (g) Results from PCA showing two distinct clusters. (h) Raw LFP recorded acutely from the G-Cu microelectrode. (i) Power density spectra across the LFP range.

To assess the in-vivo biocompatibility of the G-Cu microelectrodes, we evaluated the brain tissue reaction to chronically implanted G-Cu microwires through histology studies. Briefly, rat brain tissue was fixed first, and then sectioned using standard procedures following the retrieval of the implanted microwires (see the Experimental Section in Supporting Information for details). Tissue sections were stained using immunohistochemistry for markers chosen to visualize the presence of neuronal nuclei (NeuN), astrocytes (GFAP), microglia (Iba1), and cell nuclei (DAPI). Representative confocal microscopy images of stained brain slices sectioned horizontally at cortical depth 5 weeks post-implantation can be found in Figure 4a. Normalized fluorescence intensity profiles as a function of distance from the insertion site (center of the microwire implants) are plotted in Figure 4b. For G-Cu microwire implants, an increase in the number of GFAP+ and Iba1+ cells compared to the background tissue at the vicinity of the G-Cu implant was observed, concurrent with a moderate decrease in the number of NeuN+ cells. This accumulation of activated microglia and astrocytes (i.e., gliosis), together with the neuronal loss around the neural electrode/tissue interface, is characteristic of brain tissue inflammatory response to implanted microelectrodes, which also occurred for other non-toxic metal or silicon implants.19,

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is comparable to that for the Pt microwire implants of the same diameter. A small difference is that the astrocytes and microglia accumulated right at the Pt microwire-tissue interface, forming a tighter sheath encapsulating the implants. In contrast, for the G-Cu microwires, microglia and astrocytes tend to diffuse and distribute in a larger area away from the implants (Figure 4a and b). We speculate that the anti-fouling surface of graphene accounts for this difference.21 Since the dense cellular sheath that encapsulates the neural electrode and isolates it from the surrounding brain tissue is one of the main causes of reduced electrode performance, 20, 22, 23 the “diffuse” behavior of astrocytes and microglia around the G-Cu microwire implants could be beneficial for maintaining the long-term stability of neural recording. The G-Cu and Pt microwires also showed a similar impact on the surrounding neuronal population, as revealed by the immunofluorescence images and intensity profiles of the NeuN+ cells. Quantitative comparison of the size of the neuron “kill zone”, which is defined as the area around the implants with significantly lowered neuronal density, shows no statistically significant difference between the G-Cu and Pt implants for both acute and chronic time points (1 day and 5 weeks, respectively) (Figure 4c). The fact that the degree of neuron loss was around the same level as the Pt microwire implants suggests that the neuron degeneration was not due to the toxic effects of Cu, but rather resulted from the chronic inflammatory response of the host brain to implantable microelectrodes, which also occurs for other non-toxic metal or silicon implantable electrodes. These results indicate that the G-Cu microwires show negligible toxicity to brain tissues and have biocompatibility and biosafety that are comparable to Pt microwires, at least over early chronic timescales. Conversely, severe necrosis was observed in the vicinity of the bare Cu microwire implant, which is evidence of cytotoxic effects from Cu. Representative immunofluorescence images and 12 ACS Paragon Plus Environment

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intensity profiles 1 day post-implantation show several differences compared to the G-Cu and Pt microwire implants (Supporting Information Figure S4): 1) Enlarged neuron “kill zone” for bare Cu implants (median size ~850 µm) compared to the G-Cu and Pt microwire implants (median size ~150 µm) was observed (Figure 4c). While neuronal loss at this acute time point is mainly due to the displacement of tissue during implantation for the G-Cu and Pt implants,24-26 the extended neuron “kill zone” for the bare Cu implant indicates neuronal degeneration caused by the cytotoxic effects of Cu; 2) There was a slight upregulation of astrocytes around the G-Cu and Pt implants at this acute time point, but the bare Cu implant exhibited a clear astrocyte loss in their close proximity. It is known that astrocytes are intrinsically less capable of withstanding toxic products than giant cells and meningeal elements.9 This result suggests that the cytotoxic signals from the bare Cu are fatal to astrocytes, as well; and 3) A pronounced elevated expression of Iba1 indicates a higher activation of microglia around the bare Cu microwire implant, although the activated microglia is located further away from the bare Cu microwire implants than the G-Cu and Pt microwire implants. Microglia is known to form a front line of defense during acute and chronic inflammatory responses. This higher immunoreactivity of microglia was consistent with the much higher toxicity of bare Cu than the G-Cu and Pt microwires. After 5 weeks implantation, the neuron “kill zone” extended to ~2.2 mm size for the bare Cu microwire implant, which is approximately 10 times larger than that of the G-Cu and Pt microwire implants, suggesting progressive neuronal degeneration due to continuous exposure to noxious agents released from bare Cu (Figure 4c). Meanwhile, the astrocytes loss zone also progressed substantially at this chronic time point, leading to a large hole around the bare Cu implant, in contrast to its accumulation around the G-Cu and Pt microwire implants (Figure 4a and b). Similar effects have been reported in earlier studies of toxic metal implants where 13 ACS Paragon Plus Environment

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necrotic tissues and edema were found to be dominant at the implant-tissue interface.9 Microglia, on the other hand, survived the cytotoxic effects from Cu better, although the majority of the microglia in close proximity to the Cu microwire implants exhibit different cell morphology compared to the background tissue and those at the G-Cu and Pt microwires interface (Supporting Information Figure S5). Figure 4d presents the immunofluorescence image of a brain slice cut coronally at the implantation site 7 days after implanting a G-Cu microelectrode to the dentate gyrus region of the hippocampus. The microelectrode was insulated with ~10 µm thick Parylene, except for the tip area. An elevated expression of Iba1 and GFAP around the implant indicated the formation of gliosis. The neurons in close proximity to the implant maintained high viability and density along the G-Cu microelectrodes, including both the Parylene-C insulated area and the uninsulated tip area at this acute time point (inset of Figure 4d). This result indicates that, after microelectrode fabrication and assembly, graphene at the uninsulated tip area which serves as an active recording site remains intact and effective as a Cu corrosion barrier, ensuring high biocompatibility of the G-Cu microelectrodes. It should be pointed out that the insulation layer Parylene-C can also act as a barrier for Cu corrosion. But it can’t be applied at the exposed recording site where the toxicity of Cu will be more fatal for the recording because it causes the degeneration of neurons right around the recording site. Graphene, on the other hand, can be an effective anti-corrosion layer for Cu microwires without compromising the recording capability of the electrode.


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Figure 4. Histological studies of tissue response to chronically implanted G-Cu microelectrodes. (a) Immunofluorescence images of tissue responses following a 5-weeks implantation of a G-Cu, Pt, and bare Cu microwires. All microwires were 100 µm in diameter. The Pt and G-Cu microwires were implanted contralaterally in the same rat, and the bare Cu microwire was implanted separately. Tissue was labeled for astrocytes (purple), microglia (red), neurons (green), and nuclei (blue). Scale bar, 300 µm. (b) Normalized fluorescence intensity profile as a function of distance from the center of the microwire tract. Error bars show s.e.m. (n=5). (c) Neuron “kill zone” sizes for G-Cu, Pt, and bare Cu microwire implants over1 day and 5 weeks implantation. The G-Cu and Pt microwire implants had a significantly decreased “kill zone” size compared to the bare Cu implant for both 1 day and 5 weeks post-implantation (***p < 0.001, n=5, one-way ANOVA). There was no significant difference in neuron “kill zone” size between the G-Cu and Pt microwire implants for both time points. (d) Fluorescence images of an immuno-stained brain slice sectioned coronally at the implantation site after seven days of implantation with a G-Cu microelectrode. Tissue was labeled for astrocytes (purple), microglia (red), neurons (green), and nuclei (blue). Scale bar, 1 mm. The inset is a magnified image of the white dashed box that shows the tissue response at the tip area where the G-Cu was exposed to act as the recording site. Scale bar, 300 µm.

MRI image artifacts of the G-Cu microwires were studied in comparison to Pt microwires of the same diameter implanted contralaterally in rat brain using a 7.0-T MR scanner (Bruker BioSpin MRI, Germany), as schematically shown in Figure 5a. A significant difference in artifact size between the G-Cu and Pt microwire implants was observed in both the coronal and horizontal sections of the T2*-weighted images, as shown in Figure 5b and c. The artifact size of the Pt 16 ACS Paragon Plus Environment

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microwire was ~2.3 mm in comparison to its real diameter of 100 µm. Remarkably, the G-Cu microwire showed a size of ~150 µm (between one to two pixels in size), indicating a negligible artifact. In T1- and T2-weighted images, the artifact size of the Pt microwire reduced to ~1.6 mm and 1.2 mm, respectively. In addition, the G-Cu microwire, with its presence confirmed by the microcomputed tomography (micro CT) scans, was barely visible under T1- and T2- weighted sequences (Supporting Information Figure S6). The remarkable difference in artifact size between the G-Cu and Pt microwires reflects the higher MRI compatibility of the G-Cu microwires. Artifact-free MRI enables anatomical and functional neuroimaging of brain tissue surrounding implantable electrodes. Visualization of brain structures where electrophysiological signals are recorded or neural stimulation is applied could be beneficial to many clinical applications and fundamental studies, such as the localization of epileptic foci,2 efficacy and safety evaluation of DBS,5 implanted electrode placement and stability verification,5, 6 and the neurophysiological study of fMRI signals.3 Metallic implants in the body would become magnetized under MRI and perturb the nearby static magnetic fields that lead to artifacts. The variation of magnetic susceptibility between the implants and surrounding tissues is usually the dominant cause for the artifacts. The magnetic susceptibility χ of human tissues is usually very close to that of water, χ = −9.05 ppm. Cu has a magnetic susceptibility of -9.63 ppm, almost closest to that of water among all the available conducting materials (see Table S1 in Supporting Information for magnetic susceptibility values of common electrode materials).7 Significantly, our MRI results show that the G-Cu microwires do have negligible artifacts during the various imaging sequences used in routine MRI studies under high field strength MRI (7.0 T), compared to ~20× artifact of the Pt microwires (χ = 279 ppm). It is noted that the microwire implants usually have larger artifacts in 17 ACS Paragon Plus Environment

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T2*-weighted images than T1- and T2-weighted images. T2* relaxation refers to the decay of transverse magnetization caused by a combination of spin-spin relaxation and magnetic field inhomogeneity.27 So, it is more sensitive to the magnetic susceptibility mismatch between the implants and tissues. Since T2* relaxation forms the basis for many magnetic resonance applications, including fMRI,27 the use of G-Cu, with its capability of minimizing image distortion, could play a unique role in functional neuroimaging for both fundamental brain studies and clinical applications. In addition to magnetic susceptibility mismatch, artifacts can also arise due to eddy currents induced in conductive implants by gradient switching and the RF field.28 However, due to the relatively small geometrical size and area receiving the magnetic flux, the induced eddy currents in G-Cu microwires should be quite small, ensuring a negligible artifact under MRI.29 A pairwise comparison between the MRI images of the G-Cu and bare Cu microwire implants 24-h post-implantation (Figure 5d-f, and Supporting Information Figure S7) reveals several interesting characteristics. Firstly, the G-Cu and bare Cu microwire implants showed comparable artifact size, which suggests that the graphene encapsulation layer did not make a noticeable change to the magnetic susceptibility, and no additional field distortion was induced. Secondly, the high signal surrounding the bare Cu microwire implant indicates a circular edematous region, approximately 1.2 mm in diameter in the T2*-weighted image (1.4 mm and 1.0 mm diameters in T1- and T2-weighted images, respectively, Supporting Information Figure S7). The absence of brain edema around the G-Cu microwire indicates that it resulted from the cytotoxic effects of the bare Cu. This result constitutes additional evidence that graphene encapsulation can significantly eliminate the toxicity of Cu to brain tissue.


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Figure 5. MRI artifact properties study. (a) Schematic diagram of a rat implanted contralaterally with a G-Cu and Pt microwire used for MRI. (b, c) Coronal and horizontal sections of the T2*weighted images of a rat implanted contralaterally with a G-Cu and Pt microwire. Blue and red arrows points to the Pt and G-Cu implant, respectively. (d) Schematic diagram of a rat implanted contralaterally with a bare Cu and G-Cu microwire used for MRI. (e, f) Coronal and horizontal sections of the T2*-weighted images of a rat 24 h after contralateral implantation of a bare Cu and G-Cu microwire. Green and red arrows points to the bare Cu and G-Cu implant respectively. All microwire implants were the same diameter of ~100 µm. Graphene has several advantages as anti-corrosion layer for Cu microwires for the purpose of making highly MRI compatible neural electrodes. Firstly, graphene is conductive and will not compromise the recording capability of the electrodes. Secondly, because of the one-atom layer 19 ACS Paragon Plus Environment

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thickness and the close magnetic susceptibility between carbon and water/tissue, the graphene coating doesn't make noticeable change on magnetic susceptibility of the electrodes which is important to preserve the high MRI compatibility of Cu. As indicated by SEM and Raman spectrum, the graphene we used here is mainly single layer with some small bilayer and trilayer islands. Our in vitro cell cytotoxicity test and in vivo histology studies show that single layer graphene is enough to effectively prevent the corrosion of Cu, although we believe that increasing the layer number of graphene, and encapsulation of Cu microwires with large single crystal graphene30,

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could further increase the biosafety and biocompatibility of G-Cu

microelectrodes to even longer timescales. In summary, we achieved a new type of neural microelectrode from graphene encapsulated copper that is artifact-free under high field strength (7.0 T) MRI, and has high biosafety and biocompatibility. The unique MRI compatibility of this electrode would be beneficial to a wide range of applications, from neurophysiological studies of brain activities,32 to patient monitoring which requires continuous electrophysiological recording and anatomical/functional MRI studies.

Supporting Information. Experimental details, supporting figures and table. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *E-mail: [email protected] 20 ACS Paragon Plus Environment

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Author Contributions S. Z. and X. D. designed the experiments. S. Z., H. R., B. D., and M. T. performed the graphene growth and characterization. S. Z. and X. L. conducted the in-vitro cytotoxicity test. S. Z. and Z. X. did the in-vivo electrophysiology recording. S. Z. performed the histology and MRI studies with the assistance of L. L. and X. F. S. Z., Z. X., X. L., and X. D. analyzed the data and wrote the manuscript. X. D. supervised the project. All of the authors discussed the results and commented on the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank Dr. Shaowu Li and Dr. Song Yang from the Functional Neuroimage Department, Beijing Neurosurgical Institute, for help in magnetic resonance imaging; the Core Facilities at the School of Life Sciences, Peking University, for assistance with confocal microscopy; and Dr. Yunlong Huo for help with micro-CT imaging. X. D. acknowledges support from the National Natural Science Foundation of China (No. 21422301, 21373013), the National Basic Research Program of China (No. 2014CB932500, 2016YFA0200103), and China's 1000 Young Talent Award program.


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