Edward W. Keefer et al- Carbon nanotube coating improves neuronal recordings

8/3/2019 Edward W. Keefer et al- Carbon nanotube coating improves neuronal recordings

1/6

Carbon nanotube coating improves

neuronal recordings

EDWARD W. KEEFER1*, BARRY R. BOTTERMAN2, MARIO I. ROMERO1,3, ANDREW F. ROSSI4

AND GUENTER W. GROSS5

1UTSW Medical School, Department of Plastic Surgery, Dallas, Texas 75390, USA2UTSW Medical School, Department of Cell Biology, Dallas, Texas 75390, USA3Texas Scottish Rite Hospital for Children, Dallas, Texas 75219, USA4Vanderbilt University, Department of Psychology, Nashville, Tennessee 37203, USA5University of North Texas, Department of Biology, Denton, Texas 76203, USA

*e-mail: [email protected]

Published online: 29 June 2008; doi:10.1038/nnano.2008.174

Implanting electrical devices in the nervous system to treat neural diseases is becoming very common. The success of thesebrain machine interfaces depends on the electrodes that come into contact with the neural tissue. Here we show thatconventional tungsten and stainless steel wire electrodes can be coated with carbon nanotubes using electrochemical techniquesunder ambient conditions. The carbon nanotube coating enhanced both recording and electrical stimulation of neurons inculture, rats and monkeys by decreasing the electrode impedance and increasing charge transfer. Carbon nanotube-coatedelectrodes are expected to improve current electrophysiological techniques and to facilitate the development of long-lastingbrainmachine interface devices.

Electrical stimulation of the nervous system is used to ameliorate

conditions such as epilepsy, Parkinson disease, depression,hearing loss and chronic pain. The recent demonstration ofwilful computer cursor movement by a tetraplegic patient1 andremarkable work showing animal neural control of externaldevices25 offer hope that currently intractable clinical conditionscan be treated. However, the efficacy of any of these interventionsis ultimately determined by the quality of the neuronelectrodeinterface. However, creating a universal interface with selectivity,sensitivity, good charge transfer characteristics and long-termchemical and recording stability remains a formidable challenge.

Neurophysiologists have used sharpened wire metal electrodesfor over 50 years to study brain function6,7, and recently, neuralprobes have been fashioned from silicon8,9, ceramic10 and flexiblesubstrates1113. However, in all cases, the final contact between

brain tissue and amplifiers is still a metal surface. The type ofmetal, its area of exposure, and the texture of the metal surfacedetermine the properties of the electrodes and therefore thespecific application. To enhance electrode sensitivity or increaseelectric charge for stimulation, the impedance must belowered7,14. This step generally increases the geometric area of theelectrode tip, with a concomitant loss of selectivity and increasedtissue damage during insertion. Adding a colloidal metal layer toincrease the surface area for a given geometric area has met withstability problems. For example, electrodeposition of platinumblack creates a porous, low-impedance structure, but it ismechanically fragile and degrades over time7. Activated iridiumoxide has been shown to have excellent charge transferproperties when used for stimulation, but the surface ischemically unstable7,15.

Carbon nanotubes (CNTs) have intrinsically large surface areas

(7001,000 m2

g21

) and intriguing electrical and physicalproperties, including extremely high conductance and aspectratios16. These attributes suggest that a microelectrode coatedwith CNTs may have low impedance and high charge transfercharacteristics, and remain chemically inert and biocompatible.In addition, such electrodes could retain a small tip size, andthus high selectivity. We have established techniques for coatingmetal electrodes with CNTs, and tested their function in culturedneuronal networks, the motor cortex of anaesthetized rats, andarea V4 of rhesus macaques performing a visual task. Our resultsshow that CNT-modified electrodes are robust, have greatlydecreased impedances, lower susceptibility to noise, andincreased ability to activate neurons when used forelectrical stimulation.

We first tried electrochemical deposition of an aqueoussuspension of multiwalled CNTs (MWNTs) and potassium-goldcyanide (KAuCN) on indium-tin oxide multi-electrode array(MEA) electrodes17 (Fig. 1a). The rice-like morphology of theCNT coating presumably results from the deposition of bundlesof nanotubes as opposed to single ones; we did not use theprolonged sonication at high power levels required to suspendsingle nanotubes in aqueous solutions18. We confirmed thepresence of CNTs by analysing the surface composition of thecoated electrodes with energy-dispersive X-ray spectrography(EDS) (Fig. 1b). Impedance spectroscopy, which measuresfrequency-dependent changes in impedance, and cyclicvoltammetry, in which changes in current are measured as anapplied voltage pulse is ramped between pre-set limits, were usedto determine if the CNT coating altered the electrochemical

ARTICLES

nature nanotechnology| ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology 1
mailto:[email protected]://www.nature.com/doifinder/10.1038/nnano.2008.174http://www.nature.com/naturenanotechnologyhttp://www.nature.com/naturenanotechnologyhttp://www.nature.com/doifinder/10.1038/nnano.2008.174mailto:[email protected]


2/6

properties. Measurements made before and after coating of theelectrode in Fig. 1a showed decreased impedance at thebiologically relevant frequency of 1 kHz from 940 kV to 38 kV,and an approximately 40-fold increase in charge transfer (Fig. 1c,d respectively) after coating. On average, the CNT/gold

composite coating lowered the impedance of MEA electrodes bya factor of 23 at 1 kHz (Fig. 1e), and increased charge transfer bya factor of 45 (Fig. 1f).

Electrical stimulation experiments with cultured neuronalnetworks grown on 64-electrode MEAs were carried out to test ifthe CNT coatings altered the capacity to activate neurons. Thirty-two of the MEA electrodes were coated with gold only, and theother 32 electrodes with the CNT/gold composite (seeSupplementary Information, Fig. S1a). In agreement with ourprevious study using CNT sheets19, CNT-coated electrodes provideda suitable substrate for neural growth. Dissociated frontal cortexcultures seeded on the CNT-coated electrodes grew vigorously andremained spontaneously active for up to 88 days (oldest time pointtested, median age 37 days in vitro n 7 MEAs). We recorded aminimum of 60 min of spontaneous activity to permit comparison

of the recording characteristics of the different electrode surfaces.The percentage of electrodes with identifiable single units, and thefiring rate of the identified units were nearly identical between thetwo electrode coatings (see Supplementary Information, Fig. S1b, c).Other neuronal properties such as waveform shapes andamplitudes were normal when recorded through CNT-coatedelectrodes (see Supplementary Information, Fig. S1d). From theseobservations, we conclude that electrodeposited CNT coatings arepermissive for neuronal growth and function for at least threemonths, are stable under physiological conditions, and are wellsuited for recording neural activity.

As predicted, stimulus pulses passed through the CNT-coatedelectrodes were much more effective in evoking neuronal responsesthan stimuli introduced through the gold-coated control electrodes.

Figure 2a shows a peri-stimulus-time histogram constructed bystacking 400 consecutive 750-mV biphasic stimulus pulses providedthrough 20 gold-coated and 20 CNT-coated electrodes (10 stimuluspulses/electrode). The colour-coded histogram shows the totalnumber of action potentials recorded from the network (1-ms bins)in the 50-ms intervals immediately before and after the stimuluspulses. The stimulusresponse curves in Fig. 2b summarize sevenseparate MEA stimulation experiments as the stimulus intensitywas varied from 1001,000 mV. The threshold for eliciting at leastone network response to each electrical stimulus was lowered over500 mV on CNT-coated electrodes. Voltage-controlled stimulationusing CNT-coated electrodes was more effective than gold-coatedelectrodes at every voltage tested. The increased neural response tostimulation is presumably mediated by the lowered impedance andincrease in charge transfer through the nanotube coating. Lower

0

400

CNT

stim.

stim.

GoldTrialnumber

50 ms

40

30

20

10

02.0

195 mV 725 mV

2.5 3.0

log Ev (mV)

Responseratio

Figure 2 The functional effect of CNT coatings in vitro. a, Colour-coded

histogram summarizing the neuronal network responses to 400 consecutive

750-mV electrical pulses provided through 20 gold-coated (below horizontal line)

and 20 CNT-coated electrodes. The 50-ms intervals before and after the

stimulus (vertical line) are shown. Network responses to CNT-mediated stimuli

increased by a factor of 40 over the gold-mediated stimuli in this experiment.

b, Stimulusresponse curve summarizing seven separate MEA experiments. The

CNT-coated electrodes lowered the threshold for eliciting at least one network

response for each stimulus input by an average 530 mV. The grey curve shows

results for the gold-coated electrodes and the black curve those for the

CNT-coated electrodes.

I(nA)

Ev

10,187

225

Gold CNT

logC

p(pF)

logZ()

logZ(k)

748 k

32 k

log f(Hz)

Gold CNT

Coated9

7

5

3

1

11

0

0

0

22

34

5

3

1 2 3 4 5 0.8

50

25

0

25

0 0.8

Uncoated

10 m

1 m

2 4 6

1

2C peak

keV

KCNT

Au

W

C

Figure 1 Characterization of CNT-coated MEA electrodes. a, SEM image of

CNT-coated MEA electrode ($20mm diameter). The crater was formed by

ablating the overlying dielectric layer to access the indium-tin oxide conductor.

Inset: high magnification reveals the porous character of the CNT coating.

b, Energy-dispersive X-ray analysis confirms the presence of carbon in the MEAcoating. c, An impedance spectroscopy scan shows the CNT coating led to a

decreased impedance at all frequencies (1 1021 to 1 105 Hz). d, A cyclic

voltammetry scan shows that the CNT coating increases the charge transfer

across the electrode surface (0.1 V s21 scan rate). e,f, The CNT coating led to a

23-fold decrease in impedance (e) and to a 45-fold increase in charge transfer

(f) (+s.e.m., n 20 gold electrodes, n 20 CNT electrodes, 4 MEAs).

Table 1 Electrochemical properties of nanotube modified electrodes.*

Normalized impedance(V cm22)

Normalized capacitance(mF cm22)

CNT/ED 0.16 2.34CNT/CoV 0.075 38CNT/Ppy 0.77 755

*Values are means of 47 different electrodes per coating type, normalized by estimating the geometric area

from scanning electron microscope images.

ARTICLES

nature nanotechnology| ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology2
http://www.nature.com/naturenanotechnologyhttp://www.nature.com/naturenanotechnology


3/6

electrode impedances imply lower noise levels7; we found r.m.s. noiselevels were on average 65% lower on CNT-coated electrodescompared to gold-coated electrodes (see SupplementaryInformation, Fig. S1e). Lower impedances and noise levels shouldalso enhance electrode sensitivity. Some evidence for this is seenwhen we examine the network responses to electrical stimulation

grouped by electrode coating. CNT-coated electrodes recordedneuronal responses to almost twice as many stimulus pulses, evenwhen the stimulus was delivered through a gold electrode (seeSupplementary Information, Fig. S1f). This apparent gain insensitivity did not occur at the expense of selectivity, however, asthe ability to discriminate single neurons was unchanged (seeSupplementary Information, Fig. S1b).

The substrate-embedded MEA electrodes proved to be excellenttools for measuring the effects of different electrode coatings onelectrical stimulation; however, the planar MEA electrodes areunlike the elongated three-dimensional electrodes used in vivo bymost electrophysiologists. It is possible that the flat geometry ofthe MEA electrodes or the indium-tin oxide metal is uniquelysuited for depositing CNTs. Therefore, for our next experiments,

we chose to coat commercially available tungsten and stainlesssteel sharpened wire electrodes by electrochemical deposition ofthe same CNT/KAuCN solution used to modify the MEAelectrodes. Characterization of the successfully modifiedelectrodes showed lowered impedances and increasedcapacitances comparable to those found using MEA electrodes(Table 1).

We also tested different CNT attachment schemes andcombinations of CNTs with other materials on the wireelectrodes. The covalent attachment of CNTs to an amine-functionalized gold-coated tungsten sharpened wire is shown inFig. 3a. As purchased COOH-modified MWNTs werefunctionalized by refluxing with thionyl chloride20. The acylchloride modified nanotubes produced in this reaction were thendeposited on an amine-coated gold electrode surface with

cathodic current. The increase in charge transfer for the electrodeshown in Fig. 3a was greater than 140-fold (Fig. 3b). Using a.c.voltammetry, an electrochemical technique that can revealchanges in charge transfer capacity and relative phase shiftbetween applied voltage and charging current (phase angle)conferred by a particular electrode coating, we see that the

covalent coating decreased the phase angle by 308 (Fig. 3c),implying that the charge transfer mechanism of the CNT-modified surface is more resistive in character than the originaltungsten wire.

Electropolymerization of conductive polymers (CPs) such aspolypyrrole (Ppy) and polythiophene on neural electrodes hasbeen reported2124, and the combination of CNTs and Ppy wasshown to increase charge transfer beyond that seen with CPsalone25,26. These findings prompted us to coat sharp electrodeswith mixtures of CNTs and CPs. The electrodes modified withthis composite material exhibited increases in charge transfergreater than those found with the CNT/gold or covalentattachment schemes (Table 1). The CNT/Ppy coatings alsodecreased impedance values and phase angles. Figure 3d shows a

stainless steel electrode on which we used a UV laser to removeparylene insulation from randomly chosen locations on the sidesof the electrode shaft. We then polymerized a mixture of CNTsdispersed in an aqueous pyrrole (0.5 M) solution on the laser-exposed stainless steel. The inset shows a crater filled with theCNT/Ppy composite. The increase in charge transfer resultingfrom this coating was greater than 1,600-fold (Fig. 3e). Acomplex plane impedance plot reveals the enhanced chargetransfer results from a drop in both real and imaginarycomponents of the impedance (Fig. 3f).

The in vivo recording quality of CNT-coated sharp electrodeswas tested in two different preparations: the motor cortex ofanaesthetized rats and the visual cortex area V4 of monkey. Themotor cortex is the area of the brain that controls planning andinitiation of most voluntary movements, and is the target of

Phaseangle

5 m

Z(104

)

Z(104)

0.50

1

0.5

0

0.5

1.0 0

20

40

60

0.8 0 0.8

2

1.0 1.5 2.0

1 m

Ev

0.8 0 0.8

100 m

5 m

I(A)

I(A)

Ev

1

2

3

0

1

0.8 0 0.8

Ev

Pre CNT

Post CNT

Pre CNT

Post CNT

Pre CNT

Post CNT

Pre CNT

Post CNT

Figure 3 Characterization of sharpened metal electrodes coated with CNTs. a , CNTs covalently attached to a sharp tungsten electrode. b,c, Covalent coating of

CNTs increased the charge transfer (b) and decreased the phase angle (c). d, Parylene insulation on the electrode removed by UV laser. The image shows the

exposed stainless steel shaft. e, The CNTPpy coatings increased charge transfer by a factor of 1,600. f, Complex-impedance plots show that enhanced charge

transfer results from a drop in the real (Z0) and imaginary (Z00) components of the impedance.

ARTICLES



4/6

much research to produce neurally controlled prostheticdevices4,5,27. Area V4 of the primate visual cortex is located onthe cortical surface, and its physiological responses are well

characterized2832

. The efficacy of CNT-coated electrodes forrecording and stimulation of surface structures is essential forboth basic research and clinical applications such as neuralprosthetics, because large areas of primate sensory and motorrepresentations reside on the cortical surface.

For the rat experiments, we chose tungsten wire stereotrodes,parallel sharpened wire electrodes with separation between theelectrode tips of 125 mm. We coated one tip of each stereotrodewith CNT/gold, and the other uncoated tip served as control.The unvarying geometric arrangement of the stereotrode tipsallowed us to make quantitative comparisons between recordingproperties of the electrode surfaces, as the small tip separationensured that both electrodes would monitor virtually the sametissue volume. Figure 4a shows traces of raw data recorded from

one such stereotrode with an uncoated (top trace) and CNT-coated (bottom trace) electrode tip. The data were acquiredunfiltered (18,000 Hz amplifier bandwidth) other than a 60-Hznotch filter to block electric line noise contamination (seeSupplementary Information). The measured impedance of theelectrode used to acquire the top trace was 924 kV, and theelectrode coated with CNT/gold had an impedance of 21 kV(decreased from a measured 1.038 MV before coating). The twotraces oscillate in parallel, reflecting the common source of neuralactivity they recorded. The CNT trace obviously shows largeamplitude, fast events representing single neuron spikes. Closerexamination reveals that the baseline oscillation amplitude of theCNT trace is also greater, showing the increased sensitivity of theCNT-coated electrode for detecting local field potentials (LFPs),the summed activity of multiple neurons entrained in coherent

oscillations. The record from the uncoated electrode containsusable neural derived data, but the difference in informationbetween the two traces can be appreciated by calculating the

power spectra. Figure 4b shows power spectra produced using60 s of data acquired from the same recording session shown inFig. 4a. The black line shows the spectrum of the CNT-coatedelectrode; the red line is that of the bare tungsten wire. The CNTelectrode data has significantly more power at every frequencyfrom 11,000 Hz. Figure 4c summarizes the differences in powerfrom five different stereotrodes coated in a similar manner; theCNT-coated electrodes averaged 14.7, 15.5 and 9.9 dB increasesin the 110, 10100, and 1001,000 Hz frequency bands,respectively (14 independent recordings, 5 stereotrodes).Figure 4d,e shows spectrograms calculated from 4 s of stereotrodedata spanning 1 2,000 Hz. Obviously, the lower spectrogramreflects the increased information content of the CNT acquireddata. We have also used CNT/Ppy-modified single-wire

electrodes to record in the rat with similar good results (seeSupplementary Information, Fig. S2).The covalent attachment scheme described above was used to

modify five stainless steel electrodes used for recording in area V4of monkey cortex. The trained animal was passively fixated on aspot displayed on a computer screen throughout the recordings.An uncoated control electrode and a CNT electrode weremounted 1 mm apart in a single microdrive; both electrodes werethen lowered until they penetrated the dura. All electrodespunctured the dura at a pristine site. Recordings were made aftera 30-min rest period to permit tissue settling. Figure 5a shows500 ms of simultaneously recorded raw LFP traces for bothelectrodes, with the traces overlain. The two recordings show astrong temporal correlation; however, similar to what was seen inthe stereotrode recordings, the amplitude of the CNT-coated

Frequency (Hz)

100 1,000500

dB

180

80

1

2,000

(Hz)

110 101001001,000

Frequency (Hz)

dN

0

20

(s)0 4

1

2,000

(Hz)

(s)

0

WCNT

4

Figure 4 Stereotrode recordings from the rat motor cortex. a, Data recorded

from a bare tungsten (red trace) and CNT/gold-coated (black trace) stereotrodetip over 150 ms. b, Power spectra calculated from 60 s of neural activity. The

CNT-coated electrode (black trace) showed increased power when compared

with the bare electrode (red trace) at all frequencies (11,000 Hz). c, Average

increase in power for five CNT-coated stereotrodes compared to bare tungsten

controls over three different frequency bands (60 Hz notch filter used, 14

separate recordings). d,e, Spectrograms (12,000 Hz) of bare (d) and CNT-

coated (e) stereotrodes over 4 s. The recording quality of the CNT electrode

exceeds the bare tungsten at all time points and all frequencies.

(mV)

60 Hz

(dB)

(Hz)

Control

CNT

10 m

3 m

100 ms

75

105

135

165

60

100

140

0.2

0.1

0

0.1

195100 200 300

Figure 5 CNT-coated electrode recordings in the primate visual cortex.

a, Local field potential traces from bare controls (red trace) and CNT-coated

(black trace) electrodes show correlated activity but larger amplitude responses

from CNT-coated electrodes. b, Representative power spectral density analysis

for the range 1300 Hz. CNT-coated electrodes acquired an average of 7.4 dB

more power (4 coated, 4 control electrodes). Inset: baseline subtracted view of

60-Hz line-noise peak; CNT electrodes recorded 17.3 dB less 60-Hz line noise

than uncoated controls. c, Composite of three scanning electron micrographs

showing the electrode after recording from the monkey visual cortex. The

insulating layer that originally extended to within 20mm of the electrode tip

(white line marked by left arrow) peeled back (right arrow) along the electrode

shaft when the electrode penetrated the dura. Inset: a higher magnification view

of the electrode tip shows that the covalently attached CNTs remained intact

despite the damage to the parylene insulation.

ARTICLES



5/6

electrode recording is increased compared with the control.Power spectra analysis shows that the CNT electrode data hadmore power across the frequency band of 1300 Hz (Fig. 5b).The inset is a baseline-subtracted overlay highlighting the 60 Hznoise peak; the five CNT electrodes averaged 17.4 dB less line-noise contamination, consistent with their lowered impedances.

The durable quality of the CNT coating can be appreciated byexamining Fig. 5c. It shows an image of a covalently modified sharp

electrode composed of three separate SEM micrographs taken afterthe electrode was used for recording from the monkey visual cortex.Before the recording session, the electrode insulation extended towithin 20 mm of the tip. The mechanical stress of penetrating themonkey dura caused the parylene insulation to peel back fromthe electrode tip and roll up the shaft. In contrast, the covalentlyattached CNTs remained intact.

We have shown that CNT-coated electrodes have improvedelectrochemical and functional properties in cultured neurons,rat motor cortex and monkey visual cortex. Controlleddeposition of CNTs on flat MEA electrodes and sharpened wireelectrodes demonstrated that the CNT coatings can be applied toa variety of substrates and geometries. CNT-coated electrodeshad increased sensitivity for recording neurons, decreased

susceptibility to electrical noise, and functioned as broadbanddetectors of neural activity. It was possible to record LFPs, multi-unit activity and neuronal spiking simultaneously with oneelectrode. The efficacy of electrical stimulation was also greatlyincreased by the CNT coatings.

Previous reports have shown the biocompatibility of CNTsubstrates to support hippocampal and dorsal-root ganglionneuron growth for periods of 214 days in vitro3335. In thesestudies, the CNTs were adhered to a surface by air-drying CNTdispersions, creating a CNT mat or felt as a substrate for neuraladhesion. In each case, electrical activity of the neurons wasrecorded by patch-clamp. Some researchers have also used CNTsubstrates for passing electrical stimulation. Others haveproduced CNT MEAs using chemical vapour deposition (CVD)

mediated growth of CNTs in defined locations on siliconwafers3638. Vertical nanotube pillar electrodes about 50 mm indiameter were used to stimulate dissociated hippocampalneurons, and responses were measured optically36. Previously,recordings of spontaneous neural activity with arrays containingsixty 80-mm-diameter electrodes have been made37. Theelectrodes had extremely low impedances and provided aroughened surface that produced excellent cellelectrodecoupling. These characteristics presumably contributed to theextremely high signal-to-noise ratios reported. In otherexperiments, 10-mm-tall carbon nanofibres were used to recordand stimulate from organotypic hippocampal slice cultures38. Thevertical dimension permitted the electrode tip to penetratethrough the layer of cell debris that commonly coats such

cultures. Spontaneous and evoked field potentials with goodSNRs were acquired through the nanofibres, and the calculatedcharge transfer capacity of the vertical nanofibres equalled that ofactivated iridium oxide38.

Our electrodeposition technique allows the placement of CNTsand CNT/CP composites on a variety of substrates, similar to thedispersion drying method3335, but permits the flexibility ofselective localization and patterning potential shown by thereports using CVD-mediated growth. CVD requires hightemperatures (400 900 8C), constraining the choice of electrodematerials and manufacture. Electrodeposition of CNTs can becarried out under ambient conditions in mild solutions, and is aflexible process. The electrochemical properties of the coating canbe manipulated by controlling CNT concentration, depositioncharge, solvent, or co-agents. We show that regardless of the

attachment scheme used, CNT/gold, CNT/Ppy, or covalentlinkage through an amide bond, CNTs improved the recordingand stimulating characteristics of neural electrodes. It seemslikely that the methods we demonstrate can have a significantimpact on a variety of electrophysiological techniques, includingBMI applications requiring bidirectional interaction with thenervous system.

METHODS

MWCNTs and COOHMWCNTs were purchased from Cheap Tubes. MEAswere purchased from the Centre for Network Neuroscience (CNNS). Sharpenedwire electrodes and stereotrodes were from Microprobes. Additional reagents forCNT coatings were from Sigma. All cell culture reagents were also from Sigmaunless otherwise noted.

CNT DEPOSITION

We coated electrodes with CNTs using three different methods. First, carbonnanotubes were deposited from an aqueous solution (0.33 mg ml21) ofMWNTs and 10 mM KAuCN with monophasic voltage pulses (01.2 V, 50%duty cycle, 112 min). Second, acid-chloride-functionalized CNTs wereprepared by refluxing COOHMWNTs with thionyl chloride for 3 h at 80 8C.The modified CNTs were centrifuged at 12,000 r.p.m. for 30 min and residualthionyl chloride removed. The COClMWNTs were diluted indimethylformamide to a concentration of 1 mg ml21. Covalent attachment toamine-modified gold-coated electrode surfaces was performed byelectrodeposition under constant-voltage conditions at 10 V for 7090 min.Third, carboxyl-modified CNTs and the conductive polymer polypyrrole werepolymerized under argon by a constant voltage of 0.75 V from an aqueoussolution of 0.5 M Ppy, 1 mg ml21 COOHCNTs.

PRIMARY NEURON CULTURES

Timed pregnant mice (E16) were killed by exposure to CO2

vapour. Embryoswere removed and decapitated, and the whole brain removed in cold (4 8C)Ca2-free buffer. Frontal cortices were removed, pooled and finely minced. Usinga pipette, the tissue was transferred to a tube containing 5 ml of equilibrated(10% CO

2, 37 8C) neurobasal media (Invitrogen: 21103-049) with B27

(Invitrogen: 17504-044) NB-B27 containing 50 mg ml21 DNase (Sigma: D-4263,50 mg ml21). The tube was then placed in a 37 8C water bath for 15 min before

tissue was dissociated by gentle trituration. The dissociated cells were thencentrifuged at 800 r.p.m. for 3 min. The cell pellet was washed once with 5 ml ofequilibrated NB-B27 and resuspended in NB-B27. The cells were counted on ahaemocytometer and seeded at a density of 1 105 cm22 on MEAs madehydrophilic by flaming followed by the addition of poly-D-lysine (Sigma: P7280,50 mg ml21) and laminin (Roche 1243217, 10 mg ml21). The cells were thenincubated at 37 8C in 10% CO

2. After four days, one-third of the media was

exchanged with fresh, equilibrated NB-B27. On day 7, one-third of the media wasagain exchanged with fresh NB-B27 containing ARA-C (Sigma: C-6645,0.5 uM). On day ten, a complete media change was performed with equilibratedNB-B27. Finally, one-third of the media was exchanged twice a week for theremainder of the cultures life.

MEA RECORDINGS

Multielectrode array recording was performed with the MultichannelAcquisition Processor (MAP) System, a computer-controlled 64-channel

amplifier system (Plexon). Temperature was controlled at 37 8C with a custom-designed heating block; pH was maintained at 7.35 with a constant flow ofhumidified 90% air/10% CO

2. MEA stimulation experiments were enabled by a

custom-designed set of 64 pre-amplifiers allowing computer control of stimuluschannel selection and switching with stimulus artifact rejection circuitry(L. Howard and M. Gosney, Department of Electrical Engineering, SMU, Dallas).

RAT RECORDINGS

Rats were anaesthetized by IP injection, heads fixed in a stereotaxic frame, andthe motor cortex exposed. Sharp electrodes were lowered undermicromanipulator control until neural activity was evident on an oscilloscope,then electrodes were inserted a further 800 mm. Electrodes rested undisturbed for5 min, then spontaneous neural activity was recorded with a 16-channelRecorder System (Plexon). A total of nine rats were used in these experiments,with 514 separate recordings acquired from each rat. All experimentalprocedures were performed in accordance with the National Institutes of Health

ARTICLES



6/6

Guide for the Care and Use of Laboratory Animals, and approved by the UTSWInstitutional Animal Care and Use Committee.

MONKEY RECORDINGS

Recordings were made from cortical area V4 while the monkey was passivelyfixating a spot on a video monitor. A flashing colour square was used to verifythat cortical responses were normal. A CNT-modified electrode and a controlelectrode separated by 1 mm were simultaneously introduced through pristinedura using a single microdrive. After electrode insertion, a period of at least

30 min was allowed for tissue settling before data collection. One monkey wasused in these experiments during two different recording sessions. Allexperimental procedures in non-human primates were performed in accordancewith the National Institutes of Health Guide for the Care and Use of LaboratoryAnimals, and approved by the Vanderbilt Institutional Animal Care andUse Committee.

SCANNING ELECTRON MICROSCOPY

Scanning electron micrographs were acquired on an FEI Quanta 200 ESEMunder low vacuum conditions. Surface composition analysis was carried out witha Genesis XM2 X-ray microanalysis tool (EDAX).

ELECTROCHEMICAL ANALYSIS

Electrochemical evaluation of electrodes was performed with a CHI 6007Cpotentiostat (CH Instruments).

STATISTICSStudent paired and unpaired t-test was used to evaluate the statistical significanceof coating effects on electrode performance, P, 0.05.

Received 10 March 2008; accepted 2 June 2008; published 29 June 2008.

References1. Hochberg, L. R. et al. Neuronal ensemble control of prosthetic devices by a human with tetraplegia.

Nature 442, 164171 (2006).2. Chapin, J. K., Moxon, K. A., Markowitz, R. S. & Nicolelis, M. A. Real-time control of a robot arm

using simultaneously recorded neurons in the motor cortex. Nature Neurosci. 2, 664670 (1999).3. Schwartz, A. B., Cui, X. T., Weber, D. J. & Moran, D. W. Brain-controlled interfaces: movement

restoration with neural prosthetics. Neuron 52, 205220 (2006).4. Schwartz, A. B. Cortical neural prosthetics. Annu. Rev. Neurosci. 27, 487507 (2004).5. Taylor, D. M., Tillery, S. I. & Schwartz, A. B. Direct cortical control of 3D neuroprosthetic devices.

Science 296, 18291832 (2002).6. Hubel, D. H. Tungsten microelectrode for recording from single units. Science 125, 549550 (1957).7. Loeb, G. E., Peck, R. A. & Martyniuk, J. Toward the ultimate metal microelectrode. J. Neurosci.

Methods 63, 175183 (1995).8. Campbell, P. K., Jones, K. E. & Normann, R. A. A 100 electrode intracortical array: structural

variability. Biomed. Sci. Instrum. 26, 161165 (1990).9. Campbell, P. K., Jones, K. E., Huber, R. J., Horch, K. W. & Normann, R. A. A silicon-based,

three-dimensional neural interface: manufacturing processes for an intracortical electrode array.IEEE Trans. Biomed. Eng. 38, 758768 (1991).

10. Moxon, K. A., Leiser, S. C., Gerhardt, G. A., Barbee, K. A. & Chapin, J. K. Ceramic-based multisiteelectrode arrays for chronic single-neuron recording. IEEE Trans. Biomed. Eng. 51, 647656 (2004).

11. Pellinen, D., Moon, T., Vetter, R., Miriani, R. & Kipke, D. Multifunctional flexible parylene-basedintracortical microelectrodes. Conf. Proc. IEEE Eng. Med. Biol. Soc. 5, 52725275 (2005).

12. Jensen, W., Yoshida, K. & Hofmann, U. G. In-vivo implant mechanics of flexible, silicon-basedACREO microelectrode arrays in rat cerebral cortex. IEEE Trans. Biomed. Eng. 53, 934940 (2006).

13. Cheung, K. C., Renaud, P., Tanila, H. & Djupsund, K. Flexible polyimide microelectrode array forin vivo recordings and current source density analysis. Biosens. Bioelectron 22, 17831790 (2007).

14. Robinson, D. The electrical properties of metal electrodes. Proc. IEEE56, 10651071 (1968).

15. Cogan, S. F., Guzelian, A. A., Agnew, W. F., Yuen, T. G. & McCreery, D. B. Over-pulsing degradesactivated iridium oxide films used for intracortical neural stimulation. J. Neurosci. Methods 137,141150 (2004).

16. Ijima, S. Helical microtubes of graphitic carbon. Nature 354, 5658 (1991).17. Gross, G. W., Wen, W. Y. & Lin, J. W. Transparent indium-tin oxide electrode patterns for

extracellular, multisite recording in neuronal cultures. J. Neurosci. Methods 15, 243252 (1985).18. Yu, J., Grossiord, N., Koning, C. E. & Loos, J. Controlling the dispersion of multi-wall carbon

nanotubes in aqueous surfactant solution. Carbon (New York) 45, 618623 (2007).19. Galvan-Garcia, P. et al. Robust cell migration and neuronal growth on pristine carbon nanotube

sheets and yarns. J. Biomater. Sci. Polym. Ed. 18, 12451261 (2007).20. Kim, S. K., Choi, H. Y., Lee, H. J. & Lee, H. Characteristics of electrodeposited single-walled carbon

nanotube films. J. Nanosci. Nanotechnol. 6, 36143618 (2006).

21. Cui, X. et al. Surface modification of neural recording electrodes with conductingpolymer/biomolecule blends. J. Biomed. Mater. Res. 56, 261272 (2001).

22. Cui, X., Wiler, J., Dzaman, M., Altschuler, R. A. & Martin, D. C. In vivo studies ofpolypyrrole/peptide coated neural probes. Biomaterials 24, 777787 (2003).

23. George, P. M. et al. Fabrication and biocompatibility of polypyrrole implants suitable for neuralprosthetics. Biomaterials 26, 35113519 (2005).

24. Ludwig, K. A., Uram, J. D., Yang, J., Martin, D. C. & Kipke, D. R. Chronic neural recordings usingsilicon microelectrode arrays electrochemically deposited with a poly(3,4-ethylenedioxythiophene)(PEDOT) film. J. Neural Eng. 3, 5970 (2006).

25. Hughes, M., Chen, G. Z., Shaffer, M. S. P., Fray, D. J. & Windle, A. H. Electrochemical capacitance ofa nanoporous composite of carbon nanotubes and polypyrrole. Chem. Mater. 14, 16101613 (2002).

26. Nguyen-Vu, T. D. et al. Vertically aligned carbon nanofibre arrays: an advance towardelectricalneural interfaces. Small2, 8994 (2006).

27. Schwartz, A. B. Useful signals from motor cortex. J. Physiol. 579, 581601 (2007).28. Desimone, R. & Schein, S. J. Visual properties of neurons in area V4 of the macaque: sensitivity to

stimulus form. J. Neurophysiol. 57, 835868 (1987).29. Schein, S. J. & Desimone, R. Spectral properties of V4 neurons in the macaque. J. Neurosci.

10, 33693389 (1990).30. Luck, S. J., Chelazzi, L., Hillyard, S. A. & Desimone, R. Neural mechanisms of spatial selective

attention in areas V1, V2, and V4 of macaque visual cortex. J. Neurophysiol. 77, 2442 (1997).

31. Chelazzi, L., Miller, E. K., Duncan, J. & Desimone, R. Responses of neurons in macaque area V4during memory-guided visual search. Cereb. Cortex11, 761772 (2001).32. Ungerleider, L. G., Galkin, T. W., Desimone, R. & Gattass, R. Cortical connections of area V4 in the

macaque. Cereb. Cortex18, 477499 (2008).33. Lovat, V. et al. Carbon nanotube substrates boost neuronal electrical signalling. Nano Lett. 5,

11071110 (2005).34. Mazzatenta, A. et al. Interfacing neurons with carbon nanotubes: electrical signal transfer and

synaptic stimulation in cultured brain circuits. J. Neurosci. 27, 69316936 (2007).35. Liopo, A. V., Stewart, M. P., Hudson, J., Tour, J. M. & Pappas, T. C. Biocompatibility of native and

functionalized single-walled carbon nanotubes for neuronal interface. J. Nanosci. Nanotechnol.6, 13651374 (2006).

36. Wang, K., Fishman, H. A., Dai, H. & Harris, J. S. Neural stimulation with a carbon nanotubemicroelectrode array. Nano Lett. 6, 20432048 (2006).

37. Gabay, T. et al. Electro-chemical and biological propertiesof carbon nanotube based multi-electrodearrays. Nanotechnology18, 035201 (2007).

38. Yu, Z. et al. Vertically aligned carbon nanofibre arrays record electrophysiological signals fromhippocampal slices. Nano Lett. 7, 21882195 (2007).

Supplementary Information accompanies this paper at www.nature.com/naturenanotechnology.

AcknowledgementsThe authors thank L. Howard and M. Gosney of the SMU Department of Electrical Engineering forproviding the custom designed MOSFET pre-amplifiers used in electrical stimulation experiments. Wewish to thank H. Wiggins and C. Patten of Plexon for their rapid response to requests forelectrophysiological equipment modifications.

Author contributionsE.W.K.conceived, designed and performedexperiments,and wrotepaper.B.R.B. and M.I.R. assisted withrodent experiments. A.F.R. provided monkey data. E.W.K. and G.W.G. developed coating techniques.

Author informationReprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/.Correspondence and requests for materials should be addressed to E.W.K.

ARTICLES

http://www.nature.com/naturenanotechnologyhttp://npg.nature.com/reprintsandpermissions/http://npg.nature.com/reprintsandpermissions/http://www.nature.com/naturenanotechnologyhttp://www.nature.com/naturenanotechnologyhttp://npg.nature.com/reprintsandpermissions/http://npg.nature.com/reprintsandpermissions/http://www.nature.com/naturenanotechnology

Edward W. Keefer et al- Carbon nanotube coating improves neuronal recordings

Documents

Transcript of Edward W. Keefer et al- Carbon nanotube coating improves neuronal recordings