Biomed Microdevices
DOI 10.1007/s10544-013-9804-6
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 realization of soft, micron sized electrodes capable of injecting
enough charge to evoke localized neuronal activity without
causing neither electrode nor tissue damage. Direct stimulation 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 polymeric support. The use of carbon nanotubes bestows the
electrodes flexibility and excellent electrochemical properties. As opposed to contemporary flexible neuronal electrodes, the technology presented here is both robust and
the resulting stimulating electrodes are nearly purely capacitive. 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.
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. Hanein (*)
Tel-Aviv University Center for Nanoscience and Nanotechnology,
Tel-Aviv University, Tel-Aviv 6997801, Israel
e-mail: YaelHa@tauex.tau.ac.il
Keywords Carbon nanotubes . Multi electrode array .
Neuronal recording . Neuronal stimulation . Flexible .
Prosthesis
1 Introduction
Flexible neuronal micro electrode technology progressed extensively 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 applications were devised. These schemes attempt to achieve flexible electronic technology integration with high surface roughness while maintaining bio-compatibility and durability in
physiological conditions. Commonly, these devices use metal
electrodes such as gold (Sandison et al. 2002; Chen et al.
2009, 2011a; Wester et al. 2009; Lacour et al. 2010; Wei
et al. 2011), titanium (Takeuchi et al. 2004), electroplated
platinum black (Adams et al. 2005; Rodger et al. 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) deposited on
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
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Iridium oxide (Robblee et al. 1983; Klein et al. 1989) that
undergo reversible reduction-oxidation reactions (Merrill
et al. 2005; Cogan 2008). 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 double layer (Merrill et al. 2005). Common capacitive electrode materials include titanium nitride (TiN), tantalumtantalum 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 (Cs). Large specific capacitance
reduces the electrode impedance, without increasing its geometric area. The reduction in impedance is essential for efficient, high resolution neuronal recording and stimulation
(Robinson 1968; Loeb et al. 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 porous TiN with Cs 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 Cs 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; Su et al. 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; Tsai et al. 2009;
Carnahan et al. 2010; Chang-Jian et al. 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 electrode device, based solely on multi-walled CNT (MWCNT)
films embedded in a flexible polymeric support. We demonstrate a new simple and robust fabrication technique to
realize the seamless CNT circuit on the flexible substrate.
Next, the 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 MEA was applied for
extracellular neuronal recording and stimulation of chick
retinas.
2 Methods
2.1 Flexible CNT MEA fabrication
Flexible CNT MEAs were fabricated as follows. First, standard
lithography (AZ1518 photoresist; Clariant) was used to form
the desired circuit pattern on a Silicon/Silicon dioxide (Si/
SiO2) 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 chemical 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(pyromellitic
dianhydride-co-4,4′-oxydianiline) 15 wt.% solution in Nmethyl-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) required the deposition of a
thin Cr layer (2 nm) followed by Au layer (6 nm) using an ebeam evaporator prior to PDMS application to reduce the
adhesion between the SiO2 and the PDMS. To guarantee the
final cleanliness of the CNT film, half cured PDMS films
(60 °C for 5 min) were used in a state of a viscous polymer
and were applied as an adhesive 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 SU83050 (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 capacitive electrodes utilizing an entirely manual preparation. We
expect that mechanizing the process can dramatically improve
the yield. For electrophysiological experiments the flexible
CNT MEA was mounted on a PCB (49×49 mm2) with 60
Au traces and contact 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 different lengths and constant width and
height, were fabricated between TiN pads as follows. A 100 nm
TiN layer was sputtered (MRC RF sputter) on a Si/SiO2 support
followed by lithography and reactive ion etching (Nextral 860) to
pattern the TiN pads. Due to a marked difference between the
diffusion rate of Ni through SiO2 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 TiN pads on the SiO2 substrate. Finally a CNT film was
grown by CVD (for detailed illustration see Supplementary
Fig. 2). Current versus voltage screen of the different length
TiN-CNTs-TiN 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 electrodes were characterized by performing cyclic voltammetry (CV) and electrochemical 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 conducted using a
potentiostat (263A Princeton Applied Research) under ambient
conditions and recorded 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 transferred 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
polyester membrane filter (5 μm 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 neuronal 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 stimulation 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 biphasic (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 processes, synaptic blockers CNQX (Sigma) and APV (Sigma) were
applied (75 μM and 400 μM respectively).
3 Results
3.1 All-CNT flexible MEA fabrication
We investigated a new fabrication technique utilizing a combination of micro and nano schemes to realize non-Faradaic CNT
based electrodes with very high specific capacitance using a
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
a thin Ni layer (Fig. 1a-2). The Ni layer is deposited on a
support Si/SiO2 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
Fig. 1 All-CNT flexible multielectrode 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
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; Rodger et al. 2008;
Meacham et al. 2011). Polyimide can be patterned using
standard microfabrication such as photolithography and reactive ion etching (Cheung et al. 2007; Mercanzini et al. 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 substituting 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 dispersion 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 integration between the electrodes and conducting traces. Finally
and most importantly, at no stage of the process, the surfaces of
the CNT electrodes are exposed to any solvents, 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 polymers and deposition methods (e.g. spin coating, applying adhesive 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,
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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 validated qualitatively using environmental scanning electron microscopy (ESEM). Figure 2a shows ESEM images of a typical
CNT surface on a medical adhesive tape (Fig. 2a-1), on Parylene
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
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 μm in
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
intertwined MWCNTs were observed on the medical tape and
on the Parylene C surfaces (Fig. 2a-1 and a-2). Cross section
image of the CNT film on a medical adhesive tape (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
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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 configuration 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 different 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
1 kHz is 55 kΩ. The electrochemical measurements were also
used as a tool to directly quantify the extent of the clean surface.
Sensitive surface analytical methods such as X-ray photoelectron 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 number, 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 conductivity 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 measurements 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) (Thomsen and
Reich 2007). We used the ratio between the D and the G band
(ID/IG) as a crude characterization of the defect density and
each CNT film was measured at 20 different sites. The ID/IG
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 manipulations. 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 implemented.
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 temperature 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-TiN bar was
recorded and the electrical resistance was calculated. All samples 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 different CVD growth conditions of the MWCNT film
were obtained. Owing to the high electrode-electrolyte impedance values, we conclude 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 electrophysiological performances of the flexible electrodes. An electrode 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 electrodes 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
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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 A circuit
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
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 capacitance and the stimulation point.
At day 14 the embryonic retina is still at an early developmental 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 capacitive 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 processes was achieved with the use of the synaptic blockers 6cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5phosphonovaleric 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 electrode technologies. The table refers to studies that demonstrated 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 electrodes grown directly on flexible polyimide with 0.1 mFcm−2
(Hsu et al. 2010; Chen et al. 2011b) as well as Pt electrodes
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
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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
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 performances 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 temperature CVD (i.e. 400 °C) to directly grow CNT MEA on
polyimide (Hsu et al. 2010; Chen et al. 2011b). The specific
capacitance of our CNT MEA is significantly higher than that
of the directly grown CNTs 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 integration 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
carbon based electrodes, are distinguished by having a clear
capacitive nature. Being produced by a simple, clean and robust
process, these electrodes properties surpass previously described technologies.
Notwithstanding these promising results, some improvements in the fabrication scheme are desirable. For example, the 150 μm insulation layer locates our electrodes at a
significant distance from the tissue, limiting the spatial resolution 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 different 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 applications. 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 structure 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. Additionally, the adhesion between the CNTs and the polymeric
substrate is strong, making the CNT film an integrated 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 formation 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 extended to include elements such as photodiodes and allows for
stacking of different functionality layers, make the all-carbon-
Biomed Microdevices
Table 1 Neuronal recording and stimulation multi-electrode technologies
Rigid CNT
MEAs
Reference
Electrode description
In vitro testing scheme
Area (μm2)
Specific DC
capacitance (mFcm−2)
Stimulation
threshold
SNR
Wang et al. (2006)
Vertically aligned MWCNT (CVD) MEA on
a quartz substrate with PEGPL coating
MWCNT (CVD) MEA on a Si substrate
Commercial TiN MEA; not coated with CNT
ITO MEA coated with MWCNT-Au
(electrochemical deposition)
Pt MEA coated with SWCNT (drop coating) on
a Pyrex substrate
Cone-shaped Si MEA coated with MWCNT
(CVD) after O2 plasma
TiN MEA coated with MWCNT (micro-contact
printing).
Vertically aligned CNT electrodes embedded in
Parylene C film
CNT MEA (CVD) on polyimide after
UV-ozone modification
CNT MEA (CVD) on polyimide after UV-ozone
modification
Embryonic rat hippocampal cells
2,500–10,000
1.6
10 nC
NA
Rat cortical cultures
Rat cortical cultures
Mice frontal cortex cultures
5,024
706
314
10
2.5
3.24
NA
NA
195 mV
135
4b
Recording
Isolated rabbit retinas
1,256
0.000045
NA
21b
Crayfish giant neurons
10–2,000
2.5
NA
42.3
Rat postnatal hippocampal
cultures
Crayfish nerve cord
5,024
2.5a
NA
Recording
1,962
Not available
NA
257
Crayfish giant neurons
3,600–40,000
0.1
NA
150
Crayfish caudal photoreceptor
In vivo, EcoG of rat motor cortex
Embryonic chick retina
Embryonic rat and mice hearts
Rat cortico-hippocampl cultures
Crayfish lateral giant nerve
7,850–125,600
0.21
1,962–125,664
11,304
2
Not available
NA
NA
4 nC
NA
2,500
Not available
NA
6.2
8.68
20
100
5
32
Gabay et al. (2007)
Keefer et al. (2008)
Gabriel et al. (2009)
Su et al. (2010)
Fuchsberger et al. (2011)
Flexible CNT
MEAs
Lin et al. (2009)
Hsu et al. (2010)
Chen et al. (2011b)
Other flexible
MEAs
This paper
Blau et al. (2011)
CNTs on a medical tape
PEDOT:PSS+5 % ethylene glycol (v/v)
electrodes on PDMS
Chen et al. (2011a)
Au MEA on Parylene C reinforced with
SU8 and PEG filled micro channels
NA-stimulation/recording 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 promising 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 Nanotechnology. We also acknowledge the support of a grant from the Israel
Ministry of Science and Technology, 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.
References
M.R. Abidian, J.M. Corey et al., Conducting-polymer nanotubes improve electrical properties, mechanical adhesion, neural attachment, and neurite outgrowth of neural electrodes. Small 6(3), 421–
429 (2010)
C. Adams, K. Mathieson et al., Development of flexible arrays for in vivo
neuronal recording and stimulation. Nucl. Inst. Methods Phys. Res.
A 546(1–2), 154–159 (2005)
S. Agrawal, M. S. Raghuveer et al., Defect-induced electrical conductivity increase in individual multiwalled carbon nanotubes. Appl. Phys.
Lett. 90(19) (2007)
A. Blau, A. Murr et al., Flexible, all-polymer microelectrode arrays for
the capture of cardiac and neuronal signals. Biomaterials 32(7),
1778–1786 (2011)
X.Q. Brown, K. Ookawa et al., Evaluation of polydimethylsiloxane
scaffolds with physiologically-relevant elastic moduli: interplay
of substrate mechanics and surface chemistry effects on vascular
smooth muscle cell response. Biomaterials 26(16), 3123–3129
(2005)
D. Carnahan, D. Esposito et al., Direct growth of individual carbon
nanotubes on a flexible plastic substrate via selective heating. Nanotechnology 21(22) (2010)
S.K. Chang-Jian, J.R. Ho et al., A flexible carbon nanotube field emitter
fabricated on a polymer substrate by a laser separation method. Solid
State Commun. 150(13–14), 666–668 (2010)
Y.Y. Chen, H.Y. Lai et al., Design and fabrication of a polyimide-based
microelectrode array: application in neural recording and repeatable
electrolytic lesion in rat brain. J. Neurosci. Methods 182(1), 6–16
(2009)
C.H. Chen, S.C. Chuang et al., A three-dimensional flexible microprobe
array for neural recording assembled through electrostatic actuation.
Lab Chip 11(9), 1647–1655 (2011a)
Y. C. Chen, H. L. Hsu et al., An active, flexible carbon nanotube
microelectrode array for recording electrocorticograms. J. Neural
Eng. 8(3) (2011b)
K.C. Cheung, P. Renaud et al., Flexible polyimide microelectrode array
for in vivo recordings and current source density analysis. Biosens.
Bioelectron. 22(8), 1783–1790 (2007)
S.F. Cogan, Neural stimulation and recording electrodes. Annu. Rev.
Biomed. Eng. 10, 275–309 (2008)
D.C. Duffy, J.C. McDonald et al., Rapid prototyping of microfluidic
systems in poly(dimethylsiloxane). Anal. Chem. 70(23), 4974–
4984 (1998)
A.C. Ferrari, J. Robertson, Interpretation of Raman spectra of disordered
and amorphous carbon. Phys. Rev. B 61(20), 14095–14107 (2000)
A.A. Fomani, R.R. Mansour, Fabrication and characterization of the
flexible neural microprobes with improved structural design. Sensors Actuators B Phys. 168(2), 233–241 (2011)
K. Fuchsberger, A. Le Goff et al., Multiwalled carbon-nanotubefunctionalized microelectrode arrays fabricated by microcontact
printing: platform for studying chemical and electrical neuronal
signaling. Small 7(4), 524–530 (2011)
T. Gabay, M. Ben-David et al., Electro-chemical and biological properties
of carbon nanotube based multi-electrode arrays. Nanotechnology
18(3) (2007)
G. Gabriel, R. Gomez et al., Easily made single-walled carbon nanotube
surface microelectrodes for neuronal applications. Biosens.
Bioelectron. 24(7), 1942–1948 (2009)
O. Graudejus, Z. Yu et al., Characterization of an elastically stretchable
microelectrode array and its application to neural field potential
recordings. J. Electrochem. Soc. 156(6), 85–94 (2009)
O. Graudejus, B. Morrison et al., Encapsulating elastically stretchable
neural interfaces: yield, resolution, and recording/stimulation of
neural activity. Adv. Funct. Mater. 22(3), 640–651 (2012)
H. Hammerle, U. Egert et al., Extracellular recording in neuronal networks with substate integrated microelectrode arrays. Biosens.
Bioelectron. 9(9–10), 691–696 (1994)
H.L. Hsu, I.J. Teng et al., Flexible UV-ozone-modified carbon nanotube
electrodes for neuronal recording. Adv. Mater. 22(19), 2177–2181
(2010)
E.W. Keefer, B.R. Botterman et al., Carbon nanotube coating improves
neuronal recordings. Nat. Nanotechnol. 3(7), 434–439 (2008)
J.D. Klein, S.L. Clauson et al., Morphology and charge capacity of sputtered
iridium oxide films. J. Vac. Sci. Technol., A 7(5), 3043–3047 (1989)
S.P. Lacour, S. Benmerah et al., Flexible and stretchable micro-electrodes
for in vitro and in vivo neural interfaces. Med. Biol. Eng. Comput.
48(10), 945–954 (2010)
C.M. Lin, Y.T. Lee et al., Flexible carbon nanotubes electrode for neural
recording. Biosens. Bioelectron. 24(9), 2791–2797 (2009)
G.E. Loeb, R.A. Peck et al., Toward the ultimate metal microelectrode. J.
Neurosci. Methods 63(1–2), 175–183 (1995)
K.A. Ludwig, J.D. Uram et al., Chronic neural recordings using silicon
microelectrode arrays electrochemically deposited with a poly(3,4ethylenedioxythiophene) (PEDOT) film. J. Neural Eng. 3(1), 59–70
(2006)
K.W. Meacham, L. Guo, S.P. De Weerth, P. Hochman, Selective stimulation of the spinal cord surface using a stretchable microelectrode
array. Front. Neuroeng. 4, 12 (2011)
A. Mercanzini, K. Cheung et al., Demonstration of cortical recording
using novel flexible polymer neural probes. Sensors Actuators B
Phys. 143(1), 90–96 (2008)
D.R. Merrill, M. Bikson et al., Electrical stimulation of excitable tissue:
design of efficacious and safe protocols. J. Neurosci. Methods
141(2), 171–198 (2005)
S. Myllymaa, K. Myllymaa et al., Fabrication and testing of polyimidebased microelectrode arrays for cortical mapping of evoked potentials. Biosens. Bioelectron. 24(10), 3067–3072 (2009)
L.S. Robblee, J.L. Lefko et al., Activated IR - an electrode suitable for
reversible charge injection in saline solution. J. Electrochem. Soc.
130(3), 731–733 (1983)
D. Robinson, The electrical properties of metal electrodes. Proc. IEEE
56(6), 7 (1968)
D.C. Rodger, A.J. Fong et al., Flexible parylene-based multielectrode
array technology for high-density neural stimulation and recording.
Sensors Actuators B Chem. 132(2), 449–460 (2008)
T.L. Rose, E.M. Kelliher et al., Assesment of capacitor electrodes for
intracortical neural stimulation. J. Neurosci. Methods 12(3), 181–
Biomed Microdevices
193 (1985)
P.J. Rousche, D.S. Pellinen et al., Flexible polyimide-based intracortical
electrode arrays with bioactive capability. IEEE Trans. Biomed.
Eng. 48(3), 361–371 (2001)
Y.F. Rui, J.Q. Liu et al., Parylene-based implantable Pt-black coated
flexible 3-D hemispherical microelectrode arrays for improved neural interfaces. Microsyst. Technol. 17(3), 437–442 (2011)
M. Sandison, A.S.G. Curtis et al., Effective extra-cellular recording from
vertebrate neurons in culture using a new type of micro-electrode
array. J. Neurosci. Methods 114(1), 63–71 (2002)
M. Shein, A. Greenbaum et al., Engineered neuronal circuits shaped and
interfaced with carbon nanotube microelectrode arrays. Biomed.
Microdevices 11(2), 495–501 (2009)
A. Shoval, C. Adams et al., Carbon nanotube electrodes for effective
interfacing with retinal tissue. Frontiers in Neuroengineering 2 (2009)
W. S. Su, C. M. Lin et al., Patterning and growth of carbon nanotubes on a
highly structured 3D substrate surface. J. Micromech. Microeng. 19(10)
(2009)
H.C. Su, C.M. Lin et al., A cone-shaped 3D carbon nanotube probe for
neural recording. Biosens. Bioelectron. 26(1), 220–227 (2010)
S. Takeuchi, T. Suzuki et al., 3D flexible multichannel neural probe array.
J. Micromech. Microeng. 14(1), 104–107 (2004)
C. Thomsen, S. Reich, Raman scattering in carbon nanotubes in Light
Scattering in Solids Ix: Novel Materials and Techniques, ed. by M.
Cardona, R. Merlin, (2007), 108, 115–235
T. Y. Tsai, C. Y. Lee et al., Transfer of patterned vertically
aligned carbon nanotubes onto plastic substrates for flexible
electronics and field emission devices. Appl. Phys. Lett.
95(1) (2009)
J. Viventi, D.H. Kim et al., Flexible, foldable, actively multiplexed, highdensity electrode array for mapping brain activity in vivo. Nat.
Neurosci. 14(12), 1599–1605 (2011)
K. Wang, H.A. Fishman et al., Neural stimulation with a carbon nanotube
microelectrode array. Nano Lett. 6(9), 2043–2048 (2006)
P. Wei, R. Taylor et al., Stretchable microelectrode array using roomtemperature liquid alloy interconnects. J. Micromech. Microeng.
21(5) (2011)
B. A. Wester, R. H. Lee et al., Development and characterization of in vivo
flexible electrodes compatible with large tissue displacements. J. Neural
Eng 6(2) (2009)