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All-carbon-nanotube flexible multi-electrode array
for neuronal recording and stimulation
Moshe David-Pur & Lilach Bareket-Keren & Giora Beit-Yaakov &
Dorit Raz-Prag & Yael Hanein
#
The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Neuro-prosthetic devices aim to restore impaired
function through artificial stimulation of the nervous system.
A lingering technological bottleneck in this field is the reali-
zation of soft, micron sized electrodes capable of injecting
enough charge to evoke localized neuronal activity without
causing neither electrode nor tissue damage. Direct stimula-
tion with micro electrodes will offer the high efficacy needed
in applications such as cochlear and retinal implants. Here we
present a new flexible neuronal micro electrode device,
based entirely on carbon nanotube technology, where both
the conducting traces and the stimulating electrodes consist
of conducting carbon nanotube films embedded in a poly-
meric support. The use of carbon nanotubes bestows the
electrodes flexibility and excellent electrochemical proper-
ties. As opposed to contemporary flexible neuronal elec-
trodes, the technology presented here is both robust and
the resulting stimulating electrodes are nearly purely ca-
pacitive. Recording and stimulation tests with chick retinas
were used to validate the advantageous properties of the
electrodes and demonstrate their suitability for high-efficacy
neuronal stimulation applications.
Keywords Carbon nanotubes
.
Multi electrode array
.
Neuronal recording
.
Neuronal stimulation
.
Flexible
.
Prosthesis
1 Introduction
Flexible neuronal micro electrode technology progressed ex-
tensively over the past several decades hand in hand with the
overall development in the field of neuro-prosthetics. Several
novel fabrication approaches suited for micro electrode appli-
cations were devised. These schemes attempt to achieve flex-
ible electronic technology integration with high surface rough-
ness while maintaining bio-compatibility and durability in
physiological conditions. Commonly, these devices use metal
electrodes such as gold (Sandison et al. 2002;Chenetal.
2009, 2011a; Wester et al. 2009;Lacouretal.2010;Wei
et al. 2011), titanium (Takeuchi et al. 2004), electroplated
platinum black (Adams et al. 2005;Rodgeretal.2008;
Graudejus et al. 2009, 2012; Rui et al. 2011), tungsten (Wei
et al. 2011), platinum (Cheung et al. 2007; Mercanzini et al.
2008; Myllymaa et al. 2009; Viventi et al. 2011) and iridium
(Rodger et al. 2008; Fomani and Mansour 2011)depositedon
various flexible supports such as polyimide (Sandison et al.
2002; Takeuchi et al. 2004; Cheung et al. 2007; Viventi et al.
2011), parylene C (Rodger et al. 2008; Wester et al. 2009)or
poly(dimethylsiloxane) (PDMS) (Graudejus et al. 2009, 2012;
Lacour et al. 2010; Wei et al. 2011
). These metal electrodes
achieve neural stimulation by Faradaic current injection
through the electrode-electrolyte interface. Electron transfer,
associated with the Faradaic charge stimulation, can induce
irreversible reduction and oxidation reactions that can damage
both the electrode and the tissue (Merrill et al. 2005; Cogan
2008). Storage and injection of charge can also occur from
valence changes in multivalent electrode coatings such as
Moshe David-Pur and Lilach Bareket-Keren contributed equally to this
work.
Electronic supplementary material The online version of this article
(doi:10.1007/s10544-013-9804-6) contains supplementary material,
which is available to authorized users.
M. David-Pur
:
L. Bareket-Keren
:
G. Beit-Yaakov
:
Y. Hanein
School of Electrical Engineering, Tel-Aviv University,
Tel-Aviv 6997801, Israel
M. David-Pur
:
L. Bareket-Keren
:
G. Beit-Yaakov
:
D. Raz-Prag
:
Y. H a n e i n ( *)
Tel-Aviv University Center for Nanoscience and Nanotechnology,
Tel-Aviv University, Tel-Aviv 6997801, Israel
e-mail: YaelHa@tauex.tau.ac.il
Biomed Microdevices
DOI 10.1007/s10544-013-9804-6
Iridium oxide (Robblee et al. 1983; Klein et al. 1989)that
undergo reversible reduction-oxidation reactions (Merrill
et al. 2005;Cogan2008). Consequently , capacitive charge
stimulation is preferable for neuronal stimulation, as it
involves only a displacement current associated with
charging and discharging of the electrode-electrolyte dou-
ble layer (Merrill et al. 2005). Common capacitive elec-
trode materials include titanium nitride (TiN), tantalum-
tantalum oxide and the more recently investigated carbon
nanotubes (CNTs) (Rose et al. 1985; Gabay et al. 2007;
Cogan 2008). Conducting polymers, such as polypyrrole
(PPy) and poly(ethylenedioxythiophene) (PEDOT) are mixed
conductors, exhibiting both electron and ion transport within
the polymer film (Ludwig et al. 2006; Abidian et al. 2010;
Blau et al. 2011).
A related key requirement in neuronal electrode technology
is large specific capacitance (C
s
). Large specific capacitance
reduces the electrode impedance, without increasing its geo-
metric area. The reduction in impedance is essential for effi-
cient, high resolution neuronal recording and stimulation
(Robinson 1968;Loebetal.1995; Merrill et al. 2005;
Cogan 2008). One of the best materials to exhibit both large
specific capacitance as well as non-Faradaic behavior is po-
rous TiN with C
s
in the range of 2 mFcm
−2
(Gabay et al.
2007). It was recently demonstrated that pristine CNTs exhibit
similar performances to those of TiN with C
s
values in the
range of 3–10 mFcm
−2
(Gabay et al. 2007). Accordingly,
CNTs have been suggested by several studies as a future
material for neuronal stimulation applications and several
fabrication schemes have been studied. Primarily, direct
growth of CNT electrodes (Wang et al. 2006; Gabay et al.
2007;Suetal.2010) as well as CNT coatings of metal
electrodes by electro-polymerization (Keefer et al. 2008), drop
coating from a solution (Gabriel et al. 2009) and micro-contact
printing (Fuchsberger et al. 2011) on a rigid support were
described. To accommodate flexibility, CNT transfer onto
a polymeric support (Su, Lin et al. 2009;Tsaietal.2009;
Carnahan et al. 2010;Chang-Jianetal.2010) was recently
presented. However, the lack of a simple platform to
allow the realization of fully functional devices consisting of
pristine CNT surfaces has left this technology so far largely
unused.
Here, we present a novel flexible neuronal micro elec-
trode device, based solely on multi-walled CNT (MWCNT)
films embedded in a flexible polymeric support. We dem-
onstrate a new simple and robust fabrication technique to
realize the seamless CNT circuit on the flexible substrate.
Next, t he electrical and electrochemical properties of the
CNT electrodes and of the CNT conducting traces were
studied, using a scheme of specially designed electrode
arrays. Finally, the flexible CNT M EA was applied for
extracellular neuronal recording and stimulation of chick
retinas.
2Methods
2.1 Flexible CNT MEA fabrication
Flexible CNT MEAs were fabricated as follows. First, standard
lithography (AZ1518 photoresist; Clariant) was used to form
the desire d circuit pattern on a Silicon/Silicon dioxide (Si/
SiO
2
) support. A 2.5 nm Ni catalyst layer was deposited using
an e-beam evaporator (VST). A resist lift-off process was then
performed, followed by an oxygen plasma treatment to remove
all photoresist residues. Next, MWCNTs were grown by chem-
ical vapor deposition (CVD) (Lindberg Blue) with ethylene
(20 sccm) and hydrogen (1,000 sccm) at 900 °C. A flexible
substrate, medical adhesive tape, parylene C, polyimide or
poly(dimethylsiloxane) (PDMS), was applied and peeled off
with the CNT pattern. Medical adhesive tape (Steri-Drape,
3 M) was attached to the CNT pattern and pressed lightly.
Parylene C was applied by on the CNT pattern by vapor
deposition. Polyimide, prepared from a poly(p yromellitic
dianhydride-co-4,4′-oxydianiline) 15 wt.% solution in N-
methyl-2-Pyrrolidone (Sigma-Aldrich) was spin coated and
cured at 350 °C under nitrogen atmosphere. Uncured PDMS
(Sylgard 184, Dow Corning), mixed in a 10:1 ratio by weight,
was casted or spin coated and cured at 60 °C. Peeling-off of
very thin PDMS films (~100 μm) re quired the deposition of a
thin Cr layer (2 nm) followed by Au layer (6 nm) using an e-
beam evaporator prior to PDMS application to reduce the
adhesion between the SiO
2
and the PDMS. To guarantee the
final cleanliness of the CNT film, half cured PDMS films
(60°Cfor5min)wereusedinastateofaviscouspolymer
and were applied as an adhe sive film onto the CNTs. The
use of partially cured films substantially reduced wetting of
the CNTs. Finally, a passivation PDMS membrane with
predefined holes and the CNT flexible circuit were bonded.
The PDMS passivation layer was prepared using a SU8-
3050 (MICRO-CHEM) patterned mold (see Supplementary
Fig. 1). PDMS passivation was bonded using a custom made
holder mounted on a microscope stage. PDMS-PDMS bonding
was promoted by oxygen plasma treatment to both films.
Oxidation of PDMS surface exposes silanol groups (Si-OH)
so when the two films are brought together they form covalent
siloxane bonds (Si-O-Si) which provide excellent sealing (Duffy
et al. 1998). Bonding with polyimide and parylene C substrates
was achieved by means of an intermediate thin layer of liquid
PDMS followed by curing at 60 °C. Finally , the medical tape
was bonded with the passivation by exploiting the adhesiveness
of the tape. These processes yielded 30–65 % clean and capac-
itive electrodes utilizing an entirely manual preparation. We
expect that mechanizing the process can dramatically improve
the yield. For electrophysiological experiments the flexib le
CNT MEA was mounted on a PCB (49×49 mm
2
)with60
Au traces and c ontact pads. A glass chamber was mounted
on top of the PCB using uncured PDMS.
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2.2 Electrical resistance measurements of CNT films
Sets of CNT bars with differen t lengths and constant width and
height, were fabricated between TiN pads as follows. A 100 nm
T iN layer was sputtered (MRC RF sputter) on a Si/SiO
2
support
followed by lithography and reactiv e ion etching (Nextral 860) to
pattern the T iN pads. Due to a marked difference between the
diffusion rate of Ni through SiO
2
and TiN at the CNT growth
temperature, two layers of Ni were deposited by an e-beam
evaporator (VST). The first layer (8 nm) was deposited on the
inner half of the TiN pads and the second layer (2.5 nm) between
the T iN pads on the SiO
2
substrate. Finally a CNT f ilm was
grown by CVD (for detailed illustration see Supplementary
Fig. 2). Current versus voltage screen of the dif ferent length
T iN-CNTs-T iN bars was recorded and their electrical resistance
was calculated (for details see Supplementary Fig. 3).
2.3 Electrochemical analysis
The electrochemical properties of the CNT electrode s were char-
acterized by performing cyclic voltammetry (CV) and electro-
chemical impedance spectroscopy (EIS) in PBS. An Ag/AgCl
electrode served as a reference electrode and a platinum wire as a
counter electrode. CV measurements were conducte d using a
potentiostat (263A Princeton Applied Research) under ambient
conditions and record ed using the PowerCV software (Princeton
Applied Research). The DC capacitance was derived from the
oxidation current versus the scan rate data according to the
relation:i=C·dV/dt in which i is the charging current, C is the
DC capacitance and dV/dt is the scan rate. EIS measurements
were conducted under equilibrium conditions by applying small
(10 mV) AC signals over the frequency range of 1 Hz to 10 kHz
using a lock-in amplifier (SR830, Stanford Research Systems)
and a potentiostst (263A, Princeton Applied Research).
2.4 Retina preparation and handling
Embryonic chick retinas (day 14) were isolated and trans-
ferred to the experimental chamber, placed RGC layer down
onto the flexible MEAs. Better coupling between the tissue
and the electrodes was achieved by placing a small piece of
polyestermembranefilter(5μm pores; Sterlitech, Kent, WA,
USA) on the retina followed by a ring weight which served as
a slice anchor holder. Retinas were kept at physiological
conditions according to a previously reported protocol
(Hammerle et al. 1994) with temperature of 34 °C and perfuse
(2–5 ml/min) with oxygenated artificial cerebro-spinal fluid.
2.5 Electrical recording
Neuronal electrical signals were amplified (gain ×1,200,
MultiChannel Systems MEA1060-Inv, Reutlingen, Germany),
digitized using a 128-channel analogue to digital converter
(MultiChannel Systems MC_Card, Reutlingen, Germany) and
recorded (MultiChannel Systems MC_Rack, Reutlingen,
Germany). All additional signal analysis was performed using
Matlab software (MathWorks). Electrically stimulated neuro-
nal activity was digitized at 20 kHz and spikes were detected
by setting a threshold of signal to noise ratio (SNR) SNR>4
(related to the pre-stimulation noise level). Due to amplifier
saturation artifact, the period of 20 ms post stimulation was
ignored. The response of the retinal site to electrical stimula-
tion was defined as the detected spikes count.
2.6 Electrical stimulation
Chick retinas were electrically stimulated using a dedicated
stimulator (STG-1008, Multi-Channel Systems, Reutlingen,
Germany) through one of the MEA electrodes each time
(versus an external reference) with charge-balanced bi-
phasic (cathodic first) current stimulation (pulse width: 1 ms
and pulse amplitude: 1–10 μA). Each stimulation session
included stimulations at the entire intensity range (increased
by 1 μA every 10 s) and was repeated five times. To validate
that the electrical stimulation resulted from synaptic process-
es, synaptic blockers CNQX (Sigma) and APV (Sigma) were
applied (75 μM and 400 μMrespectively).
3Results
3.1 All-CNT flexible MEA fabrication
We investigated a new fabrication technique utilizing a combi-
nation of micro and nano schemes to realize non-Faradaic CNT
basedelectrodeswithveryhighspecificcapacitanceusinga
simple fabrication process. To support a simple and robust
fabrication process, the electrodes are made exclusively of
CNTs so no complex fabrication integration was required . The
general fabrication process, described in Fig. 1a, is based on
loosely-bound MWCNT films grown using CVD process from
athinNilayer(Fig.1a-2). The Ni layer is deposited on a
support Si/SiO
2
substrate (Fig. 1a-1). An uncured polymer
(e.g. PDMS or polyimide) is then casted on the substrate
with the CNT film. After curing, the CNTs are integrated
with the polymer . The polymer and the CNT films can then
be peeled-off from the surface (Fig. 1a-3). Similar results
can be obtained by applying an adhesive tape against the
CNT pattern or by using vapor deposition of Parylene C. The
CNT carrying film and a second layer of holey PDMS
membrane are then bonded together (Fig. 1a-4) to form a
flexible circuit containing passivated CNT conducting tracks
and exposed CNT electrodes. The biocompatibility of PDMS,
parylene C and polyimide is well established. Polyimide and
parylene C have comparable elastic moduli of ~2–4 GPa (two
to three orders of magnitude lower than that of metal and
Biomed Microdevices
silicon), while PDMS elasticity (depending on preparation
conditions) can be further reduced down to ~0.05 MPa
(Rousche et al. 2001; Brown et al. 2005;Rodgeretal.2008;
Meacham et al. 2011). Polyimide can be patterned using
standard microfabrication such as photolithography and reac-
tive ion etching (Cheung et al. 2007;Mercanzinietal.2008)
and parylene C has superior resistance to moisture. Finally, the
adhesive medical tape enables quick and simple fabrication
with well exposed CNT films. Such films may be well suited
for skin-applied electrode arrays.
The process is general enough to include additional layers
for multi-layer stacking, as well as to incorporate additional
elements such as photodiodes. Photodiodes integration with
CNT electrode array would enable neuronal stimulation using
light, a desirable feature in retinal implants aimed at substitut-
ing degenerated photoreceptors.
This scheme has several notable advantages over previously
proposed concepts. Foremost, it is simple for implementation,
requiring only two independent lithographic steps. Unlike dis-
persion methods, the use of standard lithography allows high
resolution patterning of the CNT film and a simple integrating of
the CNT pattern with the polymer substrate. Moreover, the
entire device it based only on very few elementary fabrication
steps. Additionally, the device benefits from strong overall
stability against peeling and degradation due to seamless inte-
gration between the electrodes and conducting traces. Finally
and most importantly, at no stage of the process, the surfaces of
the CNT electrodes a re exposed to any solv ents, photo-resists, or
electro-plating baths rendering the entire process very clean, and
therefore ensuring the non-Faradaic nature of the electrodes.
While the process described above appears to be straight
forward, two critical properties must be carefully maintained to
guarantee proper function of the end device. Foremost, is the
high effective surface area of the electrodes. Clean CNTs have
outstanding electrochemical properties, however, impurities
and polymeric residues can dramatically hamper the proper
operation of the electrodes. Indeed, we have noticed that the
cleanliness of the electrode surface can be compromised if the
polymer (e.g. PDMS) penetrates the CNT film. The second
critical requirement is the electrical conductivity of the CNT
interconnects.
3.2 Characterization of flexible CNT devices
We begin by discussing the cleanliness of the CNT films and
their electrochemical properties. We found that different poly-
mers and deposition methods (e.g. spin coating, applying adhe-
sive tape, and vapor deposition) dramatically affect the extent of
the polymer penetration into the film. Accordingly, careful
validation of the morphological and electrochemical properties
of the electrodes is important. Validation was achieved by using
electrode arrays with different electrode diameter (100, 150,
Fig. 1 All-CNT flexible multi-
electrode arrays. a Electrode
fabrication scheme. (1) The
process is based on a single
photolithographically defined Ni
catalyst layer. (2) The CNT film is
then grown using a CVD process.
(3) Next, the film is transferred to
a polymeric support (e.g. medical
adhesive tape, PDMS, Parylene
C, polyimide). (4) Finally, a
second polymeric layer (PDMS)
with predefined holes is bonded
with the CNT carrying film for
passivation. b Different patterns
of flexible CNT electrode arrays
on different support layers: (1)
PDMS, (2) medical adhesive tape,
(3) Parylene C and (4) polyimide
Biomed Microdevices
200, 250, 300, 350, 400 and 450 μm). Electrode arrays were
realized following the scheme depicted in Fig. 1a and were then
systematically tested. The CNT film cleanliness was first vali-
dated qualitatively using environmental scanning electron mi-
croscopy (ESEM). Figure 2a shows ESEM images of a typical
CNT surface on a medical adhesive tape (Fig. 2a-1) , on Paryle ne
C(Fig.2a-2) and on PDMS (Fig. 2a-3). While part of the CNT
film is embedded in the cured PDMS, the top surface of the
CNTs is clearly exposed (Fig. 2a-3). Apparently clean, highly
intertwined MWCNTs were observed on the medical tape and
on the Parylene C surfaces (Fig. 2a-1 and a-2). Cross section
imageoftheCNTfilmonamedicaladhesivetape(Fig.2a-1,
inset) demonstrates a CNT film on top of the flexible medical
tape substrate. Under proper preparation conditions clean CNT
films were reliably transferred to all different flexible substrates
described above (see Section 2).
The ESEM imaging was followed by electrochemical
characterization using CV that records current resulting from
Fig. 2 Electrochemical and
transport properties of CNT
devices. a An ESEM image of
MWCNTs on a medical adhesive
tape; Inset: a zoom out ESEM
cross section image of a MWCNT
film on a medical adhesive tape
(marked with arrow) (1), Parylene
C (2), and PDMS (3), scale bar:
2 μm; Inset scale bar: 100 μm. b
CV scans of a CNT electrode
(100 μm in diameter) at different
scan rates with blue, red and black
lines corresponding to scan rates
of 15, 50 and 150 mV/s
respectively. c Charging current
versus scan rate of a CNT
electrode (100 μm in diameter),
solid line is a linear fit. d CNT
electrode capacitance versus
electrode surface area, solid line is
a linear fit. Inset: Microscope
image of CNT electrodes (100,
150, 200 and 250 μmin
diameter). Measurements shown
are for a single representative set
of devices. e CNT electrode
(100 μm in diameter) impedance
versus frequency. All
electrochemical measurements
were performed in PBS with an
Ag/AgCl reference electrode. f
Raman spectrum of a MWCNT
film. g CNT film electrical
resistance versus number of
squares. Inset: Microscope image
of different length TiN-CNTs-TiN
bars used to derive film electrical
resistance. Measurements shown
are for a single representative set
of devices
Biomed Microdevices
scanning the applied voltage, and EIS, which measures
frequency-dependent changes in the impedance. CV and EIS
measurements were performed with a three-electrode cell con-
figuration using phosphate buffered saline (PBS) and Ag/AgCl
reference electrode. The CV data (Fig. 2b) is markedly flat,
showing no signs of reactivity, as expected from clean CNT
electrodes (Gabay et al. 2007). Current versus scan-rate plots
show clear linear dependence (Fig. 2c) in accordance with a
double layer capacitor model. Finally, the capacitance of differ-
ent size electrodes was calculated and plotted and the specific
capacitance value was derived, yielding values as high as 2
mFcm
−2
(Fig. 2d). Variation of the impedance with frequency
(1 Hz to 10 kHz) is presented in Fig. 2e. The impedance of a
100 μm diameter CNT electrode (including its long conducting
trace) at biologically relevant frequency for neural recording of
1kHzis55kΩ. The elec trochemical measurements were also
used as a tool to directly quantify the extent of the clean surface.
Sensitive surface analytical methods such as X-ray photoelec-
tron spectroscopy (XPS) could also be used as complementary
tool to electrochemical measurements.
Since the CNT films also constitute the circuit lines of our
devices, their electrical resistance is consequential (Agrawal
et al. 2007). The electrical performances of MWCNTs depend
on many factors such as average length, diameter, wall num-
ber, structural defects, film thickness, and the amount of
amorphous carbon (Ferrari and Robertson 2000). While some
of these parameters can be controlled in the growth process to
optimize the conductivity of the films, CNT films generally
suffer from poor conductivity compared with typical metals.
We note that for our device needs, owing to the large
electrode-solution impedance, exceptionally high trace con-
ductivity is not critically important and values in the order of
several kilo ohms are acceptable.
To validate the CNT film quality and to quantify the
electrical conductivity values, CNT films were characterized
using Raman spectroscopy (RS) and direct electrical measure-
ments respectively. RS was performed to characterize the
nature and the quality of the MWCNT films (Fig. 2f).
Raman spectrum of the CNT films show two distinct peaks
at 1,360 (D-band) and 1,580 cm−1(G-band)(Thomsenand
Reich 2007). We used the ratio between the D and the G band
(I
D
/I
G
) as a crude characterization of the defect density and
each CNT film was measured at 20 different sites. The I
D
/I
G
for all films was higher than one, indicating fairly poor film
quality associated with the highly entangled CNTs. However,
we have extensively used similar films in the past to perform
recording from dissociated neurons (Gabay et al. 2007; Shein
et al. 2009) and from mouse retina (Shoval et al. 2009)with
excellent results. The obtained films are thus very well suited
for neuronal stimulation. To validate the durability of the CNT
films upon mechanical stress we have tested the electrical
properties of the CNT films following repeated cycles (up to
30 cycles) of folding and winding. No significant change in
film resistivity was identified, during or after these manipula-
tions. A major concern when considering the biocompatibility
of the CNT electrodes is Ni traces and we have tested our CNT
electrodes for Ni traces and performed biocompatibility tests
by culturing rat cortical cells on the CNT films (according to a
previously reported protocol (Shein et al. 2009)). We have
conducted energy-dispersive x-ray spectroscopy (EDS) tests
that revealed very small residues of Ni. Apparently Ni is
effectively embedded in the CNTs and has no adverse effects.
Finally, to reliably measure the electrical resistance of the
MWCNT traces, a special testing scheme was implemente d.
Sets of different length MWCNT bars (width and height
remained constant) were fabricated (Supplementary Fig. 2)with
TiN contacts (TiN-CNTs-TiN). The TiN pads are instrumental to
achieve reliable Ohmic contacts to the CNT films, guarantying
consistent measurements. It should be noted that while the
contact resistance of TiN is substantial, TiN is a conducting
material most suitable for CNT growth under the high temper -
ature of the CVD process and therefore is a very convenient
material to perform the film resistance validation discussed here.
Current versus voltage trace for each TiN-CNTs-T iN bar was
recorded and the electrical resistance was calculated. All sam-
ples exhibited an Ohmic behavior with values ranging between
2 and 15 kΩ. To derive their sheet resistance, electrical resistance
values were plotted versus the number of squares in each bar
(Fig. 2g; for explanation on sheet resistance calculations see
Supplementary Fig. 3). Values ranging between 160 and 1,850
Ω/□ for differe nt CVD growth conditions of the MWCNT film
were obtained. Owing to the high electrode-electrolyte imped-
ance values, we conclu de that the CVD grown MWCNT films
are conducting well enough to be readily used as effective
conducting traces for our application.
3.3 Extracellular neuronal recording and stimulation
using the flexible CNT MEA
Having established the electrical as well as the electrochemical
properties of the CNT films, we now turn to describe the electro-
physiological performances of the flexible electrodes. An elec-
trode array compatible with a standard multi-electrode array
recording and stimulation setup was realized on a printed circuit
board (PCB) support (Fig. 3a). The array consists of 16 elec-
trodes on a medical tape support each connected to an external
pad. A top PDMS passivation layer , 150 μm thick and with
50 μm diameter holes, was used to define the effective size of
the electrodes (Fig. 3a, inset). The flexible array was then
mounted onto the PCB carrier to accommodate the link between
the electrodes and external amplifiers. A glass cylinder was
glued to the PCB support to serve as a well for the physiological
medium.
Embryonic chick retina (day 14) was used as a neuronal
model. The retina was extracted and transferred to the medium
chamber under physiological conditions. The retina was then
Biomed Microdevices
flattened on the electrode array (Fig. 3a), with the retinal
ganglion cell (RGC) layer facing down (as in an epi-retinal
implant) and was anchored with a weight. Figure 3b illustrates
a circuit model for extracellular recording and stimulation of
neuronal tissue using the micro electrode array, depicting the
electrochemical interface resistance and the capacitance of the
CNT electrode as well as the solution derived shunt capaci-
tance and the stimulation point.
At day 14 the embryonic retina is still at an early develop-
mental stage and clear spontaneous activity waves were
recorded demonstrating the overall functionality of the device
and the setup. We next tested the CNT electrodes suitability to
evoke electrical activity in the retina tissue. Stimulation was
achieved at currents as low as 4 μA(Fig.4a and b)and
stimulation pulse width of 1 ms. With nearly perfectly capac-
itive electrodes, these values are well within the limits of safe
stimulation. The observed electrical response is typical for
pre-synaptic cells activation. Validation of the synaptic pro-
cesses was achieved with the use of the synaptic blockers 6-
cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-
phosphonovaleric acid (APV). 400 s after the introduction of
the synaptic blockers no retinal ganglion cell activation was
measured (Fig. 4c).
4 Discussion and conclusions
We can now turn to look at how our new CNT electrodes rank
compared with previously reported technologies. Table 1
summarizes specific DC capacitance, stimulation threshold
and SNR values obtained with other CNT and flexible elec-
trode technologies. The table refers to studies that demonstrat-
ed either recording or stimulation of neuronal activity. DC
capacitance values of 1–10 mFcm
−2
were measured from
most CNT electrodes on both rigid and flexible substrates.
The all CNT flexible MEA presented in this study is well
within this range with 2 mFcm
−2
, exceeding both CNT elec-
trodes grown directly on flexible polyimide with 0.1 mFcm
−2
(Hsu et al. 2010;Chenetal.2011b)aswellasPtelectrodes
coated with SWCNT (drop coating) on a rigid Pyrex substrate
with 4.5·10
−6
mFcm
−2
(Gabriel et al. 2009). It should be noted
that SNR and stimulation threshold values depend on the
examined tissue as well as on the size and shape of the
electrode. Therefore they cannot be used as a direct measure
of MEA devices. The SNR in particular, provides only a
validation for the acceptable performance of the electrodes.
A stimulation threshold of 4 nC measured by our flexible
CNT MEA is lower than that reported by other CNT MEA
Fig. 3 Flexible CNT MEA for
extracellular neuronal
recording and stimulation. a
The flexible CNT electrode array
mounted on a PCB support
linking the electrodes to external
amplifiers (scale bar: 5 mm). The
array consists of 16 electrodes and
a top passivation layer with
50 μm-diameter holes which
define the electrode effective size.
An embryonic chick retina (day
14) was flattened on the electrode
array. The edge of the retina is
marked with a dashed line. Inset:
enlargement of the electrodes area
(area marked with a solid line;
scale bar: 200 μm). b Acircuit
model for extracellular recording
and stimulation from a neural
tissue using the flexible electrode
array. The model demonstrates
the electrochemical interface
resistance and capacitance of the
CNT electrode and the solution
derived shunt capacitance as well
as the point of stimulation
Biomed Microdevices
technologies (both rigid and flexible) and is similar to values
obtained with TiN commercial devices, further demonstrating
the overall high quality of our devices.
We have shown that our new flexible all CNT MEA perfor-
mances are equivalent to rigid CNT technologies with the
obvious major advantage of being flexible. Two flexible CNT
technologies used for neuronal recording and stimulation were
reported before. Lin and co-workers fabricated a vertically
aligned CNT (VACNT) MEA embedded in Parylene-C film
(Lin et al. 2009) while Hsu and co-workers used low temper-
ature CVD (i.e. 400 °C) to directly grow CNT MEA on
polyimide (Hsu et al. 2010;Chenetal.2011b). The specific
capacitance of our CNT MEA is significantly higher than that
of the directly grown CNT s on polyimide. Compared with both
rigid and flexible technologies, our device benefits from the
advantages of a very clean and simple fabrication scheme and
most importantly a seamless integra tion between the electrode
and the circuit, ultimately supporting a reliable and scalable
fabrication of state of the art flexible MEAs. To conclude this
discussion, our novel flexible CNT electrodes, as other clean
carbonbasedelectrodes,aredistinguishedbyhavingaclear
capacitive nature. Being produced by a simple, clean and robust
process, these electrodes properties surpass previously de-
scribed technologies.
Notwithstanding these promising results, some improve-
ments in the fabrication scheme are desirable. For exam-
ple, the 150 μm insulation layer locates our electrodes at a
significant distance from the tissue, limiting the spatial reso-
lution of the device as reflected in the relative low amplitude
of the recorded signals (Fig. 4a). Reducing the thickness of the
insulation layer will also improve the adhesion of the tissue to
the electrodes, further promoting the spatial resolution.
To summarize, a new scheme based on CNTs was presented
and demonstrated as an advantageous approach to form high
performance neuronal electrode array devices. The electrodes
gain their performances from the combination of several dif-
ferent CNT properties. Foremost, CNTs films have extremely
large surface area making them very effective electrochemical
electrode with capacitive charge injection mechanism. CNTs
are also inert and strong, making the electrodes stable in
biological conditions. As CNT films are suitable to withhold
bending, they are very well suited for flexible electronic appli-
cations. In the realm of multi-electrode arrays, this feature is
particularly important as flexible MEA devices are of great
interest for implantable applications. Unlike other coatings
that may tend to crack and disconnect from the flexible
substrate during bending, CNT films are durable owing to
their remarkable mechanical properties and the unique struc-
ture of the MWCNTs film. The entangled bundles of tubes,
forming a dense and continuous yet porous film, make these
films particularly optimal for neuronal applications. Additio-
nally, the adhesion between the CNT s and the polymeric
substrate is strong, making the CNT film an integrat ed part
of the substrate. Since CNTs are chemically inert they are
also durable against corrosion, a very common challenge in
conventional metal technology in biological applications. The
circuit structure is seamless and all elements, connecting pads,
conducting traces and electrodes are made of CNT. This is an
enormous advantage for both in vivo and in vitro long term use
since it eliminates delamination of the coatings and the for-
mation of cracks. These cracks result with leakage currents
and failure of the device as often occurs with layering and
connection of different materials. All these properties are
added to the relatively simple and robust fabrication process
discussed above. This fabrication process can be easily ex-
tended to include elements such as photodiodes and allows for
stacking of different functionality layers, make the all-carbon-
Fig. 4 Electrical recording and stimulation of chick retina with
flexible CNT MEA. a Evoked activity using a biphasic cathodic first
pulse (arrowhead). The large signal at t=0 is an artifact of the stimulation.
b Firing rate of evoked activity at different stimulation intensities (3–10
nC). c Firing rate of evoked activity after synaptic blockers CNQX and
APV application (stimulation was applied every 10 s). After 400 s no
retinal ganglion cells activation is observed
Biomed Microdevices
Table 1 Neuronal recording and stimulation multi-electrode technologies
Reference Electrode description In vitro testing scheme Area (μm
2
) Specific DC
capacitance (mFcm
−2
)
Stimulation
threshold
SNR
Rigid CNT
MEAs
Wang et al. (2006) Vertically aligned MWCNT (CVD) MEA on
a quartz substrate with PEGPL coating
Embryonic rat hippocampal cells 2,500–10,000 1.6 10 nC NA
Gabay et al. (2007) MWCNT (CVD) MEA on a Si substrate Rat cortical cultures 5,024 10 NA 135
Commercial TiN MEA; not coated with CNT Rat cortical cultures 706 2.5 NA 4
b
Keefer et al. (2008) ITOMEAcoatedwithMWCNT-Au
(electrochemical deposition)
Mice frontal cortex cultures 314 3.24 195 mV Recording
Gabrieletal.(2009) Pt MEA coated with SWCNT (drop coating) on
aPyrexsubstrate
Isolated rabbit retinas 1,256 0.000045 NA 21
b
Su et al. (2010) Cone-shaped Si MEA coated with MWCNT
(CVD) after O
2
plasma
Crayfish giant neurons 10–2,000 2.5 NA 42.3
Fuchsberger et al. (2011) T iN MEA coated with MWCNT (micro-contact
printing).
Rat postnatal hippocampal
cultures
5,024 2.5
a
NA Recording
Flexible CNT
MEAs
Lin et al. (2009) Vertically aligned CNT electrodes embedded in
Parylene C film
Crayfish nerve cord 1,962 Not available NA 257
Hsu et al. (2010) CNT MEA (CVD) on polyimide after
UV-ozone modification
Crayfish giant neurons 3,600–40,000 0.1 NA 150
Chen et al. (2011b) CNT MEA (CVD) on polyimide after UV-ozone
modification
Crayfish caudal photoreceptor 7,850–125,6 00 0.21 NA 6.2
In vivo, EcoG of rat motor cortex NA 8.68
This paper CNTs on a medical tape Embryonic chick retina 1,962–125,664 2 4 nC 20
Other flexible
MEAs
Blau et al. (201 1) PEDOT :PSS +5 % ethylene glycol (v/v)
electrodes on PDMS
Embryonic rat and mice hearts 1 1,30 4 Not available NA 100
Rat cortico-hippocampl cultures 5
Chen et al. (2011a ) Au MEA on Parylene C reinforced with
SU8 and PEG filled micro channels
Crayfish lateral giant nerve 2,500 Not available NA 32
NA-stimulation/record ing were not applied using the MEA
a
Calculated from EIS at 1 Hz
b
Calculated from data in the article
Biomed Microdevices
nanotube flexible neural electrodes, presented here, a promis-
ing element in future neuro-prosthetic devices.
Acknowledgments The authors thank Nurit Atar for providing the
polyimide substrates. Micro and nano fabrication and characterization were
performed at Tel Aviv University Center for Nanoscience and Nanotech-
nology. We also acknowledge the support of a grant from the Israel
Ministry of Science and T echnology, the Israel Science Foundation and
the European Research Council funding under the European Community’s
Seventh Framework Program (FP7/2007– 2013)/ERC grant agreement
FUNMANIA-306707.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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