Graphene Encapsulated Copper Microwires as Highly MRICompatible Neural ElectrodesSiyuan Zhao,†,‡,§ Xiaojun Liu,†,§ Zheng Xu,†,§ Huaying Ren,‡,§ Bing Deng,§,∥ Miao Tang,§,∥ Linlin Lu,†,§

Xuefeng Fu,†,§ Hailin Peng,§,∥ Zhongfan Liu,§,∥ and Xiaojie Duan*,†,‡,§

†Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China‡Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China§Center for Nanochemistry, Beijing Science and Engineering Center for Nanocarbons, Peking University, Beijing 100871, China∥College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

*S Supporting Information

ABSTRACT: Magnetic resonance imaging (MRI) compatible neural electrodes areimportant for combining high-resolution electrophysiological measurements with moreglobal MRI mapping of brain activity, which is critical for fundamental neurosciencestudies, as well as clinical evaluation and monitoring. Copper is a favorable material touse 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 vitrocell tests and in vivo histology studies. Local field potentials and single-unit spikes wererecorded from rodent brains with the G-Cu microelectrodes. Notably, the G-Cumicroelectrodes show no image artifacts in a 7.0 T MRI scanner, indicating minimalmagnetic field distortion in their vicinity. This high MRI compatibility of our G-Cuprobes would open up new opportunities for fundamental brain activity studies andclinical applications requiring continuous MRI and electrophysiological recordings.

KEYWORDS: Neural electrodes, magnetic resonance imaging, graphene biosensing, anticorrosion, biocompatibility

Neural interfacing with electrodes constitutes the basis ofelectrophysiological research and many clinical applica-

tions, such as brain-controlled prosthetic devices,1 deep brainstimulation (DBS),2 and preoperative localization of epilepticfoci.3 Magnetic resonance imaging (MRI) compatible neuralelectrodes are important for combining high-resolution electro-physiological measurements with more global MRI mapping ofbrain activity, which is critical for fundamental neurosciencestudies.4 MRI compatible electrodes can also be beneficial inclinical applications, including verification of placement andstability of implanted DBS electrodes5 and long-term epilepsymonitoring, in which repeated electrophysiological measure-ments 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 goodstability and interfacial electrochemical characteristics. How-ever, these materials, even when nonferromagnetic, may inducesevere field distortions due to the mismatch of magneticsusceptibility between the metals and water/tissues, thusproducing image artifacts or blind spots around the electrodesin MRI7 that cause inconvenience or interference foranatomical and functional MRI studies.5,8 Copper (Cu) has amagnetic susceptibility very close to water and should yieldnegligible image artifacts under MRI. However, Cu cannot be

directly used as implantable neural electrodes because of itsknown toxicity to brain tissues.9

In this study, we demonstrate that this challenge can beovercome by encapsulating Cu microwires with graphene fromlow-pressure chemical vapor deposition (CVD) (Figure 1a).The graphene encapsulated Cu (G-Cu) microwires showsignificantly eliminated toxicity to brain tissues, as evidenced bythe in vitro cell tests, in vivo histology, and MRI studies.Distinct from Pt microwires, the G-Cu microwires exhibitnegligible image artifacts under 7.0 T MRI. Local field potential(LFP) and single-unit action potentials of high signal-to-noiseratio were recorded from rat brains using the G-Cumicroelectrodes. These results indicate that the G-Cu can beused as highly MRI compatible implantable neural electrodesthat would be beneficial in various aspects, from neuro-physiological studies of brain activity to clinical diagnoses andmonitoring. Furthermore, the concept of using high-qualitygraphene as a protective or modification layer for biomaterialscould be extended to other biomedical applications, whichcould play an important role in improving biological−materialinteractions for enhanced biosafety and biocompatibility, as

Received: September 12, 2016Revised: October 27, 2016Published: November 1, 2016

Letter

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© 2016 American Chemical Society 7731 DOI: 10.1021/acs.nanolett.6b03829Nano Lett. 2016, 16, 7731−7738

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well as the development and design of new medicallyacceptable materials.The graphene encapsulation layer was deposited directly on

the Cu microwires surface by low-pressure CVD.10,11

Compared to transferring the graphene layer on Cu microwires,

the CVD approach could provide seamless, full coveragecoating of graphene to achieve an impervious barrier for Cucorrosion. A typical scanning electron microscopy (SEM)micrograph of the as-grown G-Cu microwires is presented inFigure 1b, with a magnified image of the dashed box shown in

Figure 1. Graphene encapsulated copper (G-Cu) microwires. (a) Schematic drawing of the G-Cu implanted neural electrodes. (b) A typical SEMmicrograph of the as-grown G-Cu. Inset, magnified image of the dashed box. Scale bar, 50 μm; inset, 10 μm. Red arrowheads refer to graphenewrinkles. The green arrowhead indicates the wrinkle crossing the Cu steps and boundaries. Yellow arrowheads refer to the flakes of graphene withmultiple layers. (c) Raman spectrum and optical image (inset) of the graphene printing transferred to 300 nm Si/SiO2 substrate from Cu microwireusing a 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 (redcurve) microwire samples. The graphene encapsulation layer significantly slowed down Cu corrosion.

Figure 2. In vitro cytotoxicity test. (a) Normalized viability of PC 12 cells cultured with G-Cu and bare Cu microwire samples for various timesmeasured 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 themicrowires. Scale bar, 300 μm.

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the inset. The narrow, dark lines in the SEM image (red arrowheads) are graphene wrinkles, which is a signature feature of theCVD grown graphene layers on Cu.10,12 The wrinkles crossingthe Cu steps and boundaries (green arrow heads) imply thecontinuity of the graphene film. The flakes with darker colors(yellow arrow heads) indicate graphene with multiple layers,with decreasing brightness attributed to monolayer, bilayer, andtrilayer graphene films, respectively. With a PDMS stamp, thegraphene layer on a Cu microwire was printing transferred to a300 nm SiO2/Si substrate,

13 as shown in the optical image inthe inset of Figure 1c. The clear image of the graphene stripproved the successful growth of graphene on Cu microwires.Raman spectrum of the transferred graphene strip shows a Gpeak (at ∼1582 cm−1) and a 2D peak (at ∼2700 cm−1), whichare characteristic of graphene. In addition, the small ratio of theintegrated peak area between the D (at ∼1350 cm−1) and Gbands indicates a low defect level in the graphene strip.14

The electrochemical characterization confirmed the effective-ness of the graphene encapsulation layer as corrosion-inhibitingcoating for Cu microwires. For bare Cu microwires, the cyclicvoltammetry (CV) measurement (blue curve, Figure 1d) inNa2SO4 solution exhibits characteristic anodic and cathodicpeaks that can be attributed to the electrodissolution of Cu andthe electroreduction of Cu ions. In contrast, G-Cu microwireswith the same exposure area in Na2SO4 electrolyte do notfeature any peaks, and exhibit dramatically lower current atpositive potentials (red curve and inset, Figure 1d). SEMimages after the CV scan reveal that there was clear damage tothe bare Cu microwire surface with increased roughness, whilethe 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 thecorrosion rates of the bare Cu and G-Cu microwires. As seenfrom Figure 1e, for the G-Cu microwire sample, the opencircuit potentials (OCPs) where the rates of the anodic andcathodic processes are balanced had a negative shift toapproximately −580 mV, compared to −250 mV for the bareCu microwire sample, concomitant with a sharp decrease in thecathodic reaction rate. We then calculated the corrosion rate forthe 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 inSupporting Information for details).15−17 These results indicatethat the graphene encapsulation layer serves as a barrierbetween the solution and the Cu surface that effectivelyprevents the corrosion of Cu in aqueous media.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 Cumicrowires of the same diameter and length were addedseparately to PC 12 cell cultures of the same seeding densityand same amount of culture medium. After 12, 24, 36, and 48 hincubation, CCK-8 solution was applied, and the resultantsupernatants were assayed by a spectrophotometer. Identicalassays without adding any microwires were conducted as acontrol, to which the cell viability was normalized. The resultsfrom the CCK-8 assay are shown in Figure 2a. It can be seenthat, in contrast to bare Cu microwires which show strongcytotoxic effects, the viability of PC 12 cells cultured togetherwith the G-Cu microwires for different periods of time remains

Figure 3. In vivo electrophysiological recording. (a) A four-channel G-Cu microelectrode array assembly placed next to a ruler. 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 exposedG-Cu (bright core) as the active recording site and the Parylene insulation layer (dark shell). Scale bar, 100 μm. (b) Magnitude and phase ofelectrode impedance recorded in 1× PBS (pH 7.4). (c) Schematic of a G-Cu microelectrode implanted into the CA1 region of the hippocampus of arat brain. (d) Representative acute recording of high-frequency (0.3−6 kHz) electrophysiological signal using a G-Cu microelectrode. (e) Piledsingle-unit neural recordings over 60 s. Colors serve to distinguish the two distinct single units. (f) Mean waveform for each unit spike. (g) Resultsfrom PCA showing two distinct clusters. (h) Raw LFP recorded acutely from the G-Cu microelectrode. (i) Power density spectra across the LFPrange.

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as high as over 93%, indicative of the absence of solublecytotoxic factors from the G-Cu microwires.For the live/dead cell assay, G-Cu and bare Cu microwires

were put into direct contact with PC 12 cells and incubatedunder 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 testedincubation times, the G-Cu microwires (positions marked bythe white dashed lines) are surrounded predominantly by denseliving cells. Only a few sparsely distributed dead cells are visible.

Figure 4. Histological studies of tissue response to chronically implanted G-Cu microelectrodes. (a) Immunofluorescence images of tissue responsesfollowing a 5 week implantation of a G-Cu, Pt, and bare Cu microwires. All microwires were 100 μm in diameter. The Pt and G-Cu microwires wereimplanted 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 centerof 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 over 1 day and 5week implantation. The G-Cu and Pt microwire implants had a significantly decreased “kill zone” size compared to the bare Cu implant for both 1day and 5 week postimplantation (***p < 0.001, n = 5, one-way ANOVA). There was no significant difference in neuron “kill zone” size between theG-Cu and Pt microwire implants for both time points. (d) Fluorescence images of an immuno-stained brain slice sectioned coronally at theimplantation site after 7 days of implantation with a G-Cu microelectrode. The 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 areawhere the G-Cu was exposed to act as the recording site. Scale bar, 300 μm.

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The cell viability and distribution are comparable to the controlsamples in which no microwires were added (SupportingInformation, Figure S2). This result indicates that the G-Cumicrowires show no adverse effect on cell viability. However,for the bare Cu microwire samples, a large number of dead cellscan be found in the area surrounding the wire, which isindicative of high cytotoxicity from the Cu. This toxic effectbecomes more prominent with the increase of incubation time,as shown by the increasing number of dead cells. For theincubation time of 48 h, a large empty area forms surroundingthe bare Cu microwires, where neither living nor dead cells canbe found. The SEM images of the bare Cu microwires after 1day and 7 days incubation with the culture media under normalculture conditions show obviously increased roughness andcracks on the surface, which indicates the profound corrosion ofCu under the culture conditions, while the G-Cu surfaceremains intact, indicative of negligible corrosion (SupportingInformation, Figure S3). All of these results suggest that the G-Cu microwires, with significantly suppressed corrosion inaqueous media, have little effect on cell viability. The non/lowcytotoxicity and high biocompatibility of the G-Cu microwiresmakes them a promising candidate as implantable neuralelectrodes that would be beneficial in some special cases.The G-Cu microwires were assembled into implantable

neural electrodes after being insulated with a ∼10 μm thicklayer of Parylene-C through a vacuum vapor depositionprocess, with the tip area exposed as an electrically active site(Figure 3a). Electrochemical impedance spectroscopy (EIS)measurement gives an average impedance value of ∼150 kΩand phase lag of −71° at 1 kHz for the G-Cu microelectrodeswith a diameter of 100 μm (Figure 3b). In vivo neuralrecording capabilities of the G-Cu microelectrodes were verifiedin the CA1 region of the hippocampus of an anaesthetized rat,as schematically shown in Figure 3c. A representative acuteelectrophysiological recording in the high-frequency range(0.3−6 kHz) is shown in Figure 3d. We were able to detect andisolate two single-unit spikes with peak-to-peak amplitude of 36and 69 μV and signal-to-noise ratio of ∼4 and 7, respectively, asshown by the pile plot (Figure 3e), the mean waveform (Figure3f), and principle component analysis (Figure 3g). The highsignal-to-noise ratio of the single-unit recording suggests a closeproximity between the firing neurons and the G-Cu micro-electrodes. A simultaneously recorded local field potential(LFP) signal and its power spectral density plot are shown inFigure 3h and i, respectively, with the latter data exhibiting abroad maximum at ca. 3 Hz. This broad feature differs from thesharper peaks typically observed in the rat hippocampus,18 andwe believe future studies will be needed to further understandthis feature with our new probes. Importantly, our single-unitrecordings demonstrate that the G-Cu microelectrodes createeffective interfaces capable of recording high quality neuronalactivities. Thus, it is found that G-Cu microwires perform aswell as conventional metal microwires as neural recordingelectrodes, but with the advantage of being much more MRI-compatible, as discussed below.To assess the in vivo biocompatibility of the G-Cu

microelectrodes, we evaluated the brain tissue reaction tochronically implanted G-Cu microwires through histologystudies. Briefly, rat brain tissue was fixed first and thensectioned using standard procedures following the retrieval ofthe implanted microwires (see the Experimental Section inSupporting Information for details). Tissue sections werestained using immunohistochemistry for markers chosen to

visualize the presence of neuronal nuclei (NeuN), astrocytes(GFAP), microglia (Iba1), and cell nuclei (DAPI). Representa-tive confocal microscopy images of stained brain slicessectioned horizontally at cortical depth 5 weeks post-implantation can be found in Figure 4a. Normalizedfluorescence intensity profiles as a function of distance fromthe insertion site (center of the microwire implants) are plottedin Figure 4b. For G-Cu microwire implants, an increase in thenumber of GFAP+ and Iba1+ cells compared to thebackground tissue at the vicinity of the G-Cu implant wasobserved, concurrent with a moderate decrease in the numberof NeuN+ cells. This accumulation of activated microglia andastrocytes (i.e., gliosis), together with the neuronal loss aroundthe neural electrode/tissue interface, is characteristic of braintissue inflammatory response to implanted microelectrodes,which also occurred for other nontoxic metal or siliconimplants.19,20 It is important to note that the extent of thegliosis for the G-Cu microwire implants, in terms of theupregulation level and zone size for activated microglia andastrocytes, is comparable to that for the Pt microwire implantsof the same diameter. A small difference is that the astrocytesand microglia accumulated right at the Pt microwire−tissueinterface, forming a tighter sheath encapsulating the implants.In contrast, for the G-Cu microwires, microglia and astrocytestend to diffuse and distribute in a larger area away from theimplants (Figure 4a and b). We speculate that the antifoulingsurface of graphene accounts for this difference.21 Since thedense cellular sheath that encapsulates the neural electrode andisolates it from the surrounding brain tissue is one of the maincauses of reduced electrode performance,20,22,23 the “diffuse”behavior of astrocytes and microglia around the G-Cumicrowire implants could be beneficial for maintaining thelong-term stability of neural recording.The G-Cu and Pt microwires also showed a similar impact

on the surrounding neuronal population, as revealed by theimmunofluorescence images and intensity profiles of the NeuN+ cells. A quantitative comparison of the size of the neuron “killzone”, which is defined as the area around the implants withsignificantly lowered neuronal density, shows no statisticallysignificant difference between the G-Cu and Pt implants forboth acute and chronic time points (1 day and 5 weeks,respectively; Figure 4c). The fact that the degree of neuron losswas around the same level as the Pt microwire implantssuggests that the neuron degeneration was not due to the toxiceffects of Cu but rather resulted from the chronic inflammatoryresponse of the host brain to implantable microelectrodes,which also occurs for other nontoxic metal or siliconimplantable electrodes. These results indicate that the G-Cumicrowires show negligible toxicity to brain tissues and havebiocompatibility and biosafety that are comparable to Ptmicrowires, at least over early chronic time scales.Conversely, severe necrosis was observed in the vicinity of

the bare Cu microwire implant, which is evidence of cytotoxiceffects from Cu. Representative immunofluorescence imagesand intensity profiles 1 day postimplantation show severaldifferences compared to the G-Cu and Pt microwire implants(Supporting Information, Figure S4): (1) An 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 atthis acute time point is mainly due to the displacement of tissueduring implantation for the G-Cu and Pt implants,24−26 theextended neuron “kill zone” for the bare Cu implant indicates

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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 Cuimplant exhibited a clear astrocyte loss in their close proximity.It is known that astrocytes are intrinsically less capable ofwithstanding toxic products than giant cells and meningealelements.9 This result suggests that the cytotoxic signals fromthe bare Cu are fatal to astrocytes, as well; and (3) Apronounced elevated expression of Iba1 indicates a higheractivation of microglia around the bare Cu microwire implant,although the activated microglia is located further away fromthe bare Cu microwire implants than the G-Cu and Ptmicrowire implants. Microglia is known to form a front line ofdefense during acute and chronic inflammatory responses. Thishigher immunoreactivity of microglia was consistent with themuch higher toxicity of bare Cu than the G-Cu and Ptmicrowires. 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-Cuand Pt microwire implants, suggesting progressive neuronaldegeneration due to continuous exposure to noxious agentsreleased from bare Cu (Figure 4c). Meanwhile, the astrocyteloss zone also progressed substantially at this chronic timepoint, leading to a large hole around the bare Cu implant, incontrast to its accumulation around the G-Cu and Pt microwireimplants (Figure 4a and b). Similar effects have been reportedin earlier studies of toxic metal implants where necrotic tissuesand edema were found to be dominant at the implant−tissueinterface.9 Microglia, on the other hand, survived the cytotoxiceffects from Cu better, although the majority of the microglia inclose proximity to the Cu microwire implants exhibit differentcell morphology compared to the background tissue and thoseat the G-Cu and Pt microwires interface (SupportingInformation, 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 regionof the hippocampus. The microelectrode was insulated with∼10 μm thick Parylene, except for the tip area. An elevatedexpression of Iba1 and GFAP around the implant indicated theformation of gliosis. The neurons in close proximity to theimplant maintained high viability and density along the G-Cumicroelectrodes, including both the Parylene-C insulated areaand the uninsulated tip area at this acute time point (inset ofFigure 4d). This result indicates that, after microelectrodefabrication and assembly, graphene at the uninsulated tip areawhich serves as an active recording site remains intact andeffective as a Cu corrosion barrier, ensuring high biocompat-ibility of the G-Cu microelectrodes. It should be pointed outthat the insulation layer Parylene-C can also act as a barrier forCu corrosion. But it cannot be applied at the exposed recordingsite where the toxicity of Cu will be more fatal for the recordingbecause it causes the degeneration of neurons right around therecording site. Graphene, on the other hand, can be an effectiveanticorrosion layer for Cu microwires without compromisingthe recording capability of the electrode.MRI image artifacts of the G-Cu microwires were studied in

comparison to Pt microwires of the same diameter implantedcontralaterally in rat brain using a 7.0-T MR scanner (BrukerBioSpin MRI, Germany), as schematically shown in Figure 5a.A significant difference in artifact size between the G-Cu and Ptmicrowire implants was observed in both the coronal andhorizontal sections of the T2*-weighted images, as shown inFigure 5b and c. The artifact size of the Pt microwire was ∼2.3mm in comparison to its real diameter of 100 μm. Remarkably,the G-Cu microwire showed a size of ∼150 μm (between oneand two pixels in size), indicating a negligible artifact. In T1-and T2-weighted images, the artifact size of the Pt microwirereduced to ∼1.6 mm and 1.2 mm, respectively. In addition, theG-Cu microwire, with its presence confirmed by the micro-computed tomography (micro CT) scans, was barely visibleunder T1- and T2- weighted sequences (Supporting

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 arrowspoints to the Pt and G-Cu implant, respectively. (d) Schematic diagram of a rat implanted contralaterally with a bare Cu and G-Cu microwire usedfor 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-Cumicrowire. Green and red arrows points to the bare Cu and G-Cu implant, respectively. All microwire implants were the same diameter of ∼100 μm.

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Information, Figure S6). The remarkable difference in artifactsize between the G-Cu and Pt microwires reflects the higherMRI compatibility of the G-Cu microwires.Artifact-free MRI enables anatomical and functional neuro-

imaging of brain tissue surrounding implantable electrodes.Visualization of brain structures where electrophysiologicalsignals are recorded or neural stimulation is applied could bebeneficial to many clinical applications and fundamental studies,such as the localization of epileptic foci,2 efficacy and safetyevaluation of DBS,5 implanted electrode placement and stabilityverification,5,6 and the neurophysiological study of fMRIsignals.3 Metallic implants in the body would becomemagnetized under MRI and perturb the nearby static magneticfields that lead to artifacts. The variation of magneticsusceptibility between the implants and surrounding tissues isusually the dominant cause for the artifacts. The magneticsusceptibility χ of human tissues is usually very close to that ofwater, χ = −9.05 ppm. Cu has a magnetic susceptibility of−9.63 ppm, almost closest to that of water among all theavailable conducting materials (see Table S1 in SupportingInformation for magnetic susceptibility values of commonelectrode materials).7 Significantly, our MRI results show thatthe G-Cu microwires do have negligible artifacts during thevarious imaging sequences used in routine MRI studies underhigh field strength MRI (7.0 T), compared to ∼20× artifact ofthe Pt microwires (χ = 279 ppm). It is noted that the microwireimplants usually have larger artifacts in T2*-weighted imagesthan T1- and T2-weighted images. T2* relaxation refers to thedecay of transverse magnetization caused by a combination ofspin−spin relaxation and magnetic field inhomogeneity.27 So, itis more sensitive to the magnetic susceptibility mismatchbetween the implants and tissues. Since T2* relaxation formsthe basis for many magnetic resonance applications, includingfMRI,27 the use of G-Cu, with its capability of minimizingimage distortion, could play a unique role in functionalneuroimaging for both fundamental brain studies and clinicalapplications. In addition to magnetic susceptibility mismatch,artifacts can also arise due to eddy currents induced inconductive implants by gradient switching and the RF field.28

However, due to the relatively small geometrical size and areareceiving the magnetic flux, the induced eddy currents in G-Cumicrowires should be quite small, ensuring a negligible artifactunder MRI.29

A pairwise comparison between the MRI images of the G-Cuand bare Cu microwire implants 24 h postimplantation (Figure5d−f, and Supporting Information, Figure S7) reveals severalinteresting characteristics. First, the G-Cu and bare Cumicrowire implants showed comparable artifact size, whichsuggests that the graphene encapsulation layer did not make anoticeable change to the magnetic susceptibility, and noadditional field distortion was induced. Second, the high signalsurrounding the bare Cu microwire implant indicates a circularedematous region, approximately 1.2 mm in diameter in theT2*-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-Cumicrowire indicates that it resulted from the cytotoxic effects ofthe bare Cu. This result constitutes additional evidence thatgraphene encapsulation can significantly eliminate the toxicityof Cu to brain tissue.Graphene has several advantages as anticorrosion layer for

Cu microwires for the purpose of making highly MRIcompatible neural electrodes. First, graphene is conductive

and will not compromise the recording capability of theelectrodes. Second, because of the one-atom layer thickness andthe close magnetic susceptibility between carbon and water/tissue, the graphene coating does not make noticeable changeon magnetic susceptibility of the electrodes which is importantto preserve the high MRI compatibility of Cu. As indicated bySEM and Raman spectrum, the graphene we used here ismainly single layer with some small bilayer and trilayer islands.Our in vitro cell cytotoxicity test and in vivo histology studiesshow that single layer graphene is enough to effectively preventthe corrosion of Cu, although we believe that increasing thelayer number of graphene and encapsulation of Cu microwireswith large single crystal graphene30,31 could further increase thebiosafety and biocompatibility of G-Cu microelectrodes to evenlonger time scales.In summary, we achieved a new type of neural micro-

electrode from graphene encapsulated copper that is artifact-free under high field strength (7.0 T) MRI, and has highbiosafety and biocompatibility. The unique MRI compatibilityof this electrode would be beneficial to a wide range ofapplications, from neurophysiological studies of brain activ-ities,32 to patient monitoring which requires continuouselectrophysiological recording and anatomical/functional MRIstudies.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.nano-lett.6b03829.

Experimental details, supporting figures, and table (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: xjduan@pku.edu.cn.Author ContributionsS.Z. and X.D. designed the experiments. S.Z., H.R., B.D., andM. T. performed the graphene growth and characterization.S.Z. and X.L. conducted the in vitro cytotoxicity test. S.Z. andZ.X. did the in vivo electrophysiology recording. S.Z. performedthe histology and MRI studies with the assistance of L.L. andX.F. S.Z., Z.X., X.L., and X.D. analyzed the data and wrote themanuscript. X.D. supervised the project. All of the authorsdiscussed the results and commented on the manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Dr. Shaowu Li and Dr. Song Yang from theFunctional Neuroimage Department, Beijing NeurosurgicalInstitute, for help in magnetic resonance imaging; the CoreFacilities at the School of Life Sciences, Peking University, forassistance with confocal microscopy; and Dr. Yunlong Huo forhelp with micro-CT imaging. X.D. acknowledges support fromthe National Natural Science Foundation of China (No.21422301, 21373013), the National Basic Research Program ofChina (No. 2014CB932500, 2016YFA0200103), and China’s1000 Young Talent Award program.

■ REFERENCES(1) Bouton, C. E.; Shaikhouni, A.; Annetta, N. V.; Bockbrader, M. A.;Friedenberg, D. A.; Nielson, D. M.; Sharma, G.; Sederberg, P. B.;

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Nano Letters Letter

DOI: 10.1021/acs.nanolett.6b03829Nano Lett. 2016, 16, 7731−7738

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