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Semiconductor Nanorod−Carbon Nanotube Biomimetic Films for
Wire-Free Photostimulation of Blind Retinas
Lilach Bareket,
†,‡
Nir Waiskopf,
§,∥
David Rand,
†,‡
Gur Lubin,
†,‡
Moshe David-Pur,
†,‡
Jacob Ben-Dov,
†,‡
Soumyendu Roy,
†,‡
Cyril Eleftheriou,
#
Evelyne Sernagor,
#
Ori Cheshnovsky,
⊥,‡
Uri Banin,
§,∥
and Yael Hanein*
,†,‡
†
School of Electrical Engineering,
‡
Tel Aviv University Center for Nanoscience and Nanotechnology, and
⊥
School of Chemistry, Tel
Aviv University, Tel Aviv 69978, Israel
§
Institute of Chemistry and
∥
Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904,
Israel
#
Institute of Neuroscience, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom
*
SSupporting Information
ABSTRACT: We report the development of a semiconductor
nanorod-carbon nanotube based platform for wire-free, light
induced retina stimulation. A plasma polymerized acrylic acid
midlayer was used to achieve covalent conjugation of
semiconductor nanorods directly onto neuro-adhesive, three-
dimensional carbon nanotube surfaces. Photocurrent, photo-
voltage, and fluorescence lifetime measurements validate
efficient charge transfer between the nanorods and the carbon
nanotube films. Successful stimulation of a light-insensitive
chick retina suggests the potential use of this novel platform in
future artificial retina applications.
KEYWORDS: Neural photostimulation, carbon nanotubes, quantum rods, retinal implant, neural prosthesis, plasma polymerization
Awide range of medical conditions is associated with
dysfunctional neuronal connectivity and sensory informa-
tion transfer to the brain. Neuroprosthetic systems attempt to
treat these conditions, and several devices are already in
medical use to treat deafness, Parkinson’s disease, and chronic
pain, to name just a few.
1−3
Visual prosthetic devices are
presently developed as an approach to treat blindness. In
particular, there is a dire need to help the ever growing number
of patients suffering from age-related macular degeneration in
which the photoreceptors in the retina degenerate. A particular
challenge is the need for high-resolution stimulation, along with
effective interfacing with the remaining neurons in the retina.
Contemporary approaches to address these challenges are
based on metallic electrodes and are typified by relatively low
spatial resolution, rigidity, and cumbersome wiring.
4,5
Neuronal
activation with optical stimulation of photoresponsive surfaces
6
offers an alternative, wire-free route to address the need for
artificial vision.
7−9
Several approaches have been recently
proposed including conducting polymers (CPs)
10−12
and
quantum dot (QD) films.
13−15
QDs directly interfacing the
cell membrane were also suggested.
16
Recent studies employing
CPs as a photoactive layer have attracted considerable attention
as they offer advantages such as simple fabrication and
mechanical flexibility.
17
The major disadvantages of CPs are
their low stability under continuous stimulation, exposure to
ultraviolet (UV) light or heat which may gradually degrade
their properties,
18,19
as well as the risk of toxic residues. These
issues have yet to be tested to fully explore the potential of
these materials in retinal implant applications.
An additional emerging approach to vision restoration is
optogenetics, most commonly including the introduction of
bacterial opsins into neurons through viral transfection.
20
Recent studies have demonstrated the use of optogentics for
restoring light-sensitivity to residual cells in a degenerate
retina.
21,22
The optogenetics approach offers high temporal
resolution and cell specificity, and it is minimally invasive.
Nevertheless, there are still many challenges that have to be
overcome to make this technology suitable for application in
vision restoration. Foremost is the long-term expression of light
sensitive proteins to avoid the need for repeated injections,
controlling and sustaining gene expression and the efficiency of
transfection methods. Semiconductor nanocrystal (SCNC)
systems are particularly attractive for neuronal stimulation
applications due to their tunable optical and electronic
properties, photostability, and chemical interfacing diversity.
However, despite extensive efforts, efficient SCNC mediated
neuronal stimulation with ambient light intensities has not been
demonstrated yet. Here, we introduce a novel approach for
Received: September 7, 2014
Revised: October 25, 2014
Published: October 28, 2014
Letter
pubs.acs.org/NanoLett
© 2014 American Chemical Society 6685 dx.doi.org/10.1021/nl5034304 |Nano Lett. 2014, 14, 6685−6692
This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited.
wire-free retinal photostimulation, based on a combination of
two nanomaterial systems ideally suited for neurostimulation:
semiconductor nanorods (NRs) and carbon nanotubes
(CNTs) (Figure 1a). The nanorod geometry enables efficient
light absorbance, followed by effective charge separation at the
NR−CNT interface.
23
CNTs are particularly suitable for this
application having superior neuronal recording and stimulation
properties
24,25
originating from their high surface rough-
ness
26,27
and biomimetic nature.
28,29
The use of highly porous
three-dimensional CNTs as SCNC carrying microelectrodes
therefore supports high SCNC loading along with the
formation of an electrochemically safe interface, with excellent
coupling with the neuronal tissue.
30
To achieve clean and effective NR−CNT conjugation, we
developed a special covalent bonding scheme based on plasma-
polymerized acrylic acid (ppAA) coated CNT films, amine
modified NRs, and carbodiimide chemistry (Figure 1a). High-
density CNT films were first grown on titanium nitride (TiN)
coated silicon/silicon dioxide (Si/SiO2) substrates using
chemical vapor deposition (CVD) which supports CNT
patterning, film porosity, and cleanliness. CNT films were
then coated with a ppAA coating featuring carboxylic functional
groups (needed for the conjugation step) using a plasma
polymerization process. The plasma polymerization offers high
conformity, strong adhesion, and surfaces containing no
contaminates.
31,32
X-ray photoelectron spectroscopy (XPS)
data of the ppAA coated CNT films show a clear carboxylic
group (OC−OH) peak component, which is absent in
pristine CNT films, validating the buildup of a ppAA (Figure
1b).
Scanning electron microscopy (SEM) imaging of the CNT
surface after plasma polymerization reveals that the CNT films
Figure 1. NR−CNT films and electrode arrays. (a) Schematic representation of the photoactive electrode preparation. NR conjugation onto a CNT
film is based on covalent binding enabled by a ppAA coating of the CNTs. Light is absorbed by the film, followed by charge separation at the NR−
CNT interface which elicits a neuronal response. (b) XPS chemical analysis of pristine (continuous line) and ppAA coated CNT film (dashed line)
demonstrating the formation of carboxylic groups. (c) SEM image of a ppAA coated CNT film demonstrating preservation of porous structure; the
scale bar is 200 nm. (d) Absorption (continuous line) and emission (dashed line) spectrum of the CdSe/CdS NRs; Inset: a TEM image of the
CdSe/CdS NRs; the scale bar is 20 nm. (e) SEM image of a NR−CNT film; the scale bar is 100 nm. NRs appear as bright elongated elements on the
CNTs. (f) An optical microscope image of a CNT multi electrode array (MEA); the scale bar is 200 μm. (g) CNT electrode array on a PDMS
flexible support; the scale bar is 1 mm.
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retained their porous and entangled surface (Figure 1c). This
high porosity is important to maintain the CNT’s superior
electrochemical features and their adhesiveness to neuronal
tissue.
25,30
Prototypical cadmium selenide/cadmium sulfide (CdSe/
CdS; core/shell) NRs were used as a model system for
SCNCs. NRs were synthesized in a seeded growth approach
using a modification of a previously described synthesis
procedure.
33
Figure 1d shows the absorption and emission
spectra and a transmission electron microscopy (TEM) image
of these NRs. The absorption spectrum (continuous line)
manifests a weak first exciton peak at 600 nm related to the
CdSe seed and a sharp increase in absorption below 470 nm
mainly due to the contribution of the CdS shell. The emission
spectrum (dashed line) shows a peak at 610 nm. TEM imaging
of these CdSe/CdS NRs reveals a homogeneous size
distribution, 40 ±5 nm in length and 5 ±0.8 nm in diameter
(Figure 1d, inset).
To render the NRs soluble in an aqueous solution and to
support their conjugation with the CNT films, organic ligands
from the synthesis stage were replaced with the antioxidant,
tripeptide−glutathione (GSH), using a ligand exchange
process.
34
The GSH coating also helps in stabilizing and
protecting the SCNCs as will be elaborated later in the text.
GSH coated CdSe/CdS NRs (CdSe/CdS−GSH NRs) were
covalently conjugated to the ppAA coated CNT film using
carbodiimide chemistry
35
with 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfo-
succinimide (sulfo-NHS). SEM imaging validates effective NRs
conjugation across the CNT surface without NR aggregation
(Figure 1e), as well as NR penetration into the three-
dimensional CNT matrix (see Figure S1). Having established
a successful loading of NRs onto CNT/ppAA film, this
Figure 2. Embryonic chick retina stimulation with NR−CNT electrodes. (a) Photovoltage (negative polarity) of a CdSe/CdS−GSH NR−CNT
electrode versus time (electrode diameter 210 μm). Electrodes were illuminated with a violet light source (405 nm) for 100 ms at intensity of 70
mW/cm2. Inset: Photocurrent versus time for the same electrode under the same illumination conditions. (b) A chick retina (E14) placed on a
CdSe/CdS−GSH NR−CNT MEA; the scale bar is 500 μm. (c) Extracellular voltage trace (top) recorded from a chick retina following 100 ms light
stimulation (405 nm; pulse interval of 30 ms) at different intensities (1.2, 3, 6, and 12 mW/cm2; stimulation pulses are marked by blue arrow heads).
The bottom panel is an enlargement of the spike burst following a light pulse of 3 mW/cm2. (d) Extracellular voltage traces recorded from a chick
retina under illumination with 3 mW/cm2and 30 ms pulses (405 nm; marked by blue arrow heads) with different interpulse intervals (2 s, 1 s, 500
ms, 250 ms; repeated five times for each interval) when maintained in a control medium (2.5 mM Ca2+; top), low Ca2+ medium (0.5 mM Ca2+;
middle), and measured again in a medium with normal Ca2+ concentration (2.5 mM Ca2+; bottom).
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approach was used to deposit SCNCs on patterned CNT/
ppAA multielectrode arrays (CNT MEAs) (Figure 1f).
Moreover, patterned CNT/ppAA/NR electrodes can be
transferred onto a flexible support such as poly-
(dimethylsiloxane) (PDMS) for implantation purposes (Figure
1g).
36
CNT MEA fabrication process is described at length in
Gabay et al. (2007)
24
and CNT pattern transfer to PDMS films
in David-Pur et al. (2014).
37
To validate the effectiveness of the NR−CNT film for optical
stimulation of a light-insensitive neuronal tissue, photovoltage
and photocurrent measurements were conducted using a
modulated light source. CdSe/CdS−GSH NR−CNT electro-
des (210 μm in diameter) were immersed in phosphate
buffered saline (PBS). Open-circuit voltage (Figure 2a) and
short-circuit current (Figure 2a, inset) were measured using a
high surface area platinum mesh as a reference electrode.
Photovoltage traces (Figure 2a) are typified by a voltage
increase during illumination (to negative values), followed by a
discharge when light is turned off. Such characteristics can be
explained by an equivalent simplified circuit consisting of a
current source and a diode coupled with the electrochemical
interface of the CNT system (see Figure S2a). The rate of
charging−discharging of a capacitor through passive and
resistive circuit elements is inversely proportional to the
electrochemical electrode capacitance. Since CNT films have
a large surface area, they also have a large capacitance at the
CNT−electrolyte interface. During illumination the electrons
generated by photoexcitation accumulate at this capacitor. After
light is turned off, the capacitor discharges slowly, and hence
the recorded voltage also drops slowly.
Light-insensitive embryonic chick retinas at day 14 of
development (E14) were used as a neuronal model. Waves of
spontaneous activity originating from retinal ganglion cells
(RGCs) at E13−18,
38
and the acquirement of photosensitivity
by photoreceptors as well as by intrinsically photosensitive
retinal ganglion cells (ipRGCs; the melanopsin dependent non-
image-forming pathway) typifies the development of these
retinas. At E14, retinal cells are at early maturation stage, and
photoreceptors are not yet developed. The mRNA of the long
wave cone opsins only begins to appear at E14, and transcripts
of rhodopsin and of short waves cones opsins begin to appear
at E15.
39
Photoreceptor electrical activity in response to light is
not detected before E17.
40
Although melanopsin expression
starts as early as E4,
41
ipRGCs are less sensitive to light than
photoreceptors and require light stimulation of several seconds
to evoke a response.
42
We have validated the light insensitivity
of our neuronal model by demonstrating that photostimulation
of E14 retinas (n= 19), placed on commercial TiN MEA
(MultiChannel Systems), did not elicit neuronal activity
12
(see
also Figure S3a). Retinas (n= 4) were placed on CdSe/CdS−
GSH NR−CNT MEAs, which were used for both optical
stimulation and simultaneous electrical recording, with the
RGC layer facing down (Figure 2b). Extracellular electrical
signals of retina activity were recorded following pulsed
photostimulation using different pulse durations and intensities
of violet light (405 nm).
The data in Figure 2c and d show a pronounced electrical
response to 100 ms photostimulation. Excitation intensities
were varied within the range of 1.2−12 mW/cm2(Figure 2c,
top) (corresponding with ambient light intensities), revealing
an intensity threshold of 3 mW/cm2. Repetitive pulses of
subthreshold stimulus (30 ms at 3 mW/cm2) resulted in a
delayed response (Figure 2d, top), indicating a charge buildup
in the NR−CNT interface owing to the slow discharge kinetics.
A closer inspection of the data reveals two time scales
(Figure 2c, bottom): A short latency burst with rapid activity
and a long latency response typified by a tonic spiking activity.
This time separation is conspicuously similar to the separation
observed in conventional electrical stimulation where direct
(RGC) and indirect (bipolar cell) activations are typically
discernible.
43
The presence of axon collaterals projecting back
to the inner plexiform layer in embryonic chick retina may also
be responsible for these late responses.
44
Owing to the low
current generated by the NR−CNT electrodes, the typical
stimulation times are long, yielding markedly longer latency
values than commonly obtained in electrical stimulation (∼1
ms) for direct response. Moreover, while direct activation is
highly synchronized in electrical stimulation, here it appears
sparse with little overlap between different activated elements.
This effect is likely a direct result of the slow activation, causing
different elements to respond at different times. The biological
nature of this light induced electrical activation of the retina was
further revealed using a low calcium (Ca2+) medium (using 0.5
mM of CaCl2and additional 2 mM of MgCl2), in which
synaptic activity is hampered (Figure 2d). Under these
conditions, the long latency response is markedly reduced,
indicating the synaptic nature of the indirect response, while
the short latency and rapid response remains intact.
Retinas were left on the NR−CNT MEAs for as long as 24 h
and retained their spontaneous activity, validating their
physiological viability on these surfaces (see also Figure S3c).
Additionally, a cell viability assay was conducted, testing the
viability of primary neurons cultured on CdSe/CdS−GSH
NR−CNT patterns compared with neurons cultured on poly-D-
lysine coating (a standard culturing environment for embryonic
rat neurons). Cell viability was monitored at 7, 14, and 21 days
in vitro. No significant differences were found in cell viability
under normal culturing conditions (see Figure S4).
The CdSe/CdS−GSH NR deposition protocol described
above is the obtained optimum after experimenting with several
alternative materials and procedures. Various conjugation
procedures and SCNC configurations were systematically
explored to better understand the underlying principles until
highly efficient surfaces were realized. Three different particle
architectures were compared: CdSe QDs with a diameter of 3.7
±0.1 nm, CdSe/CdS QDs with a diameter of 7 ±0.8 nm, and
CdSe/CdS NRs with a diameter of 5 ±0.8 nm and length of 40
±5 nm (Figure 3a), all coated with GSH (CdSe−GSH, CdSe/
CdS−GSH, and CdSe/CdS NR−GSH; see also Figure S5 for
SCNC optical characterization). First, the particle loading,
derived from inductively coupled plasma mass spectrometry
(ICP-MS) analysis, was investigated.
The amount of CdSe−GSH QDs deposited on the CNT
films was found to be, on average ∼1014 particles/cm2, an order
of magnitude larger than CdSe/CdS−GSH QDs and CdSe/
CdS−GSH NRs with ∼1013 particles/cm2(Figure 3b, top). As
CdSe−GSH QDs are smaller, compared with the CdSe/CdS−
GSH QDs, this effect may be attributed to the size of the
SCNCs. It is also important to note that, to accommodate good
solubility, the maximum concentrations achievable without
discernible SCNC aggregation were used (300−900 nM, 100
nM, and 20−40 nM for CdSe−GSH QDs, CdSe/CdS−GSH
QDs, and CdSe/CdS−GSH NRs, respectively).
Loading efficiency provides important information, yet it
does not reveal whether the loaded SCNC contribute to
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efficient charging of the interface. Poor charge transfer may be
associated with SCNC intrinsic properties such as the number
of surface traps and volume, or system properties such as the
formation of SCNC aggregates, poor SCNC−CNT coupling,
and differences in band offsets between the SCNCs and the
CNTs, which hinder light induced charge transfer. To
investigate which of the films described above yielded good
electrical properties, photocurrent measurements of coated
CNT films (0.46 cm2,defined by illumination spot size) were
measured. Peak photocurrent values were extracted for each
sample from the photocurrent traces (measured at 30 mW/
cm2; see also Figure S2). As can be seen in Figure 3b, while
CdSe−GSH QDs were effectively deposited on the CNT films,
they yielded poor photocurrent values. The best photocurrent
values were obtained with CdSe/CdS−GSH NRs. The band
diagram describing CdSe/CdS NR−CNT interface presented
in the inset of Figure 3b supports a mechanism of electron
transfer from the excited NRs to the CNTs. Based on these
results, CdSe/CdS−GSH NR were chosen as the CNT coating
material for retina stimulation.
The spectral response of the CdSe/CdS−GSH NR−CNT
films 530, 660, and 850 nm shows almost no photocurrent (see
Figure S2b). This result is consistent with the NR absorption
spectrum (Figure 1d), confirming the role of the NRs in
facilitating light induced charge separation at the NR−CNT
interface. Furthermore, the spectral sensitivity demonstrates the
tunability of the system, which may be exploited for future
development of color selective films. We note the increase in
photocurrent with increased intensity (Figure S2c) and the
reliability of the photocurrent response with repetitive
stimulations (Figure S2c, inset). Additionally, we validated
that the covalent conjugation contributes to improved photo-
current compared with physical adsorption (see Figure S6,
bottom).
Finally, we compared fluorescence lifetime measurements of
dry CdSe/CdS−GSH NR−CNT films to results from CdSe/
CdS−GSH NRs deposited on SiO2(CdSe/CdS−GSH NR−
SiO2) (Figure 3c). It is clearly apparent that the proximity of
the CNTs shortens the CdSe/CdS−GSH NR fluorescence
lifetime, consistent with charge separation at the NR−CNT
interface introducing a competing mechanism to the
fluorescent recombination of charge carriers. These findings
are further supported by previous studies demonstrating light
induced charge transfer between QDs and CNTs.
45,46
More-
over, in fluorescence imaging of patterned CNT films on SiO2
substrate, with NRs conjugated to the surface (Figure 3c, inset),
NR−CNT regions appear dark compared to the fluorescence of
the NR−SiO2background. This effect is consistent with the
existence of a charge transfer from the CdSe/CdS−GSH NRs
to the CNTs. Fully understanding the underlying mechanisms
of light harvesting, charge separation, and photocurrent
generation in our novel nanostructured device remains a
challenge, under ongoing study in our laboratories.
Indeed, to fit in vivo applications, the system must retain its
inertness and its performance without causing toxic effects.
SCNCs, especially free in solution, may induce adverse
cytotoxicity effects
47,48
originating from their toxic composition
and from their nanoscale size.
49
The most predominant cause
for SCNC cytotoxicity is the generation of reactive oxygen
species and/or release of cadmium ions to the solution upon
photoexcitation.
49,50
These may interact with proteins or DNA,
leading to mutations and cell death. The retina stimulation
experiments presented here validated the short-term bio-
compatibility (up to 24 h) of these systems (see also Figure
S3). Long-term in vitro stability (up to 21 days) was validated
using viability tests of dissociated cortical neurons (as described
earlier in this text; see also Figure S4). It is important to note
Figure 3. SCNC loading on CNT films. (a) Schematic drawing of the
SCNCs compared: CdSe−GSH QDs (left), CdSe/CdS−GSH QDs
(center), and CdSe/CdS−GSH NRs (right). (b) Loading yield
derived from ICP-MS analysis of different SCNCs (top). Average
photocurrent for different SCNCs recorded following an excitation
pulse of 30 mW/cm2for 100 ms with a 405 nm illumination source
(bottom). Highest photocurrents were obtained with CdSe/CdS−
GSH NRs. Inset: A schematic band diagram of the CdSe/CdS NR−
CNT system. (c) Photoluminescence lifetime data of CdSe/CdS−
GSH NR conjugated to CNTs (solid line) and to SiO2(dotted line).
Inset: Fluorescence microscope image of CNT pattern on SiO2
substrate. CNTs and SiO2substrate are coated with CdSe/CdS-
GSH NRs; the scale bar is 200 μm.
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that the coating material plays a crucial role in stabilizing and
protecting SCNC from degradation and is therefore an
important parameter in determining the safety and stability of
the CdSe/CdS NR−CNT system. GSH is a well-known natural
antioxidant molecule that can contribute also to an improved
biocompatibility.
51
Our investigation has indeed validated the
in vitro biocompatibility of these systems. Further optimization
of the stability of the system toward implementation in long-
term in vivo applications will be needed and is beyond the
scope of this investigation. By effectively incorporating SCNCs
onto CNT surfaces, highly efficient photoresponsive porous
films were realized, demonstrating for the first time a
nanomaterial based approach for retinal photostimulation
with better performances and a path toward further
optimization.
Most notably, not only our nano interfaces provide highly
efficient photosensitivity, but equally important, they form a
truly three-dimensional interface with optimized binding
between the biological tissue and the optoelectronic device.
27,30
The use of a three-dimensional matrix as well as an optimized
selection of SCNCs, their surface coating, and conjugation
procedure contributes to the superior properties of these films.
Several pioneering studies reported the use of QDs and CPs to
elicit a neuronal response. In the only report to date to achieve
neuronal activation with QDs, a 800 mW/cm2intensity was
used
15
(compared with 3 mW/cm2reported here). Membrane
hyperpolarization or depolarization was achieved using an
intensity of ∼0.36 mW/cm2.
13
Polymeric systems have
achieved stimulation with values ranging from 0.03 to 1500
mW/cm2.
10−12
The lowest value was achieved in a subretinal
configuration using a retina with residual light sensitivity,
making the exact comparison to our system cumbersome.
Compared with previously reported neural photostimulation
technologies utilizing CPs and QDs, our CNT−NR interfaces
have an improved efficiency (i.e., lower threshold for evoking
action potentials), durability, flexibility, and the demonstrated
capacity to elicit localized stimulation. The stable CNT
interface, with its capacitive charge transfer mechanism and
low impedance, is overall an optimal platform for efficient
neuronal light induced stimulation.
24,37
NRs embedded onto
CNT films offer several advantages compared with a photo-
conductive semiconductor substrate. Silicon devices developed
for artificial vision are rigid and inherently nontransparent,
necessitating a complicated implantation procedure and an
external power source. Polymeric based systems, which are
emerging as an alternative for silicon, will have to overcome
long-term stability issues typifying such systems. Both silicon
and polymeric based solution do not offer the special tissue
binding properties of CNTs. Furthermore, compared with
utilizing an injected SCNC system our NR−CNT platform
contributes to increased charge separation, as demonstrated by
fluorescence lifetime results (see Figure 3c), and helps
anchoring the SCNC to a substrate thus preventing them
from uncontrolled and possible harmful migration in the tissue.
The use of a bottom up approach implies that the technology
can be further improved, owing to massive activity in this field.
The CdSe/CdS−GSH NR−CNT photoresponsive electrodes
presented here represent a major step toward achieving a wire-
free retinal prosthesis and a paradigm shift from two-
dimensional to significantly superior three-dimensional bio-
mimetic opto-electrical interfacing.
Experimental Section. SCNC Preparation and Coating.
CdSe QDs were synthesized by fast injection of selenium
dissolved in trioctylphosphine (TOP) solution into a four-
necked flask containing cadmium oxide (CdO) in trioctylphos-
phine oxide (TOPO) and n-octadecylphosphonic acid (ODPA)
at 350 °C under argon atmosphere. The crude solution was
then washed with methanol to remove excess ligands. CdSe/
CdS QDs and NRs were synthesized following previously
described protocols based on a seeded growth approach.
52
CdSe SCNCs were mixed with elemental sulfur dissolved in
TOP. This solution was rapidly injected into a four-neck flask
containing TOPO, ODPA, and CdO for the synthesis of CdSe/
CdS QDs, or TOPO, ODPA, CdO, and hexylphosphonic acid
(HPA) for the synthesis of the CdSe/CdS NRs at 360 °C. After
cooling, the crude solution was dissolved in toluene, and
methanol was added in order to precipitate the NCs and
remove excess precursors and ligands. GSH coating was used to
render SCNCs with amine functionality and soluble in an
aqueous solution. The SCNC surface coating was modified
using previously described process.
53
200 μL of a GSH solution
containing of 0.459 mmol of GSH and 100 mg of potassium
hydroxide in 1 mL of methanol were mixed with 1 mL of
SCNCs in chloroform. Basic triple-distilled water (TDW) (2
mL, pH 11−12) was added, and a water phase containing
SCNCs coated with GSH was obtained. After phase transfer, an
excess of ligands or polymer were washed by filtration using
100 kDa cellulose membrane filters (Invitrogen Corp.).
Conjugating SCNCs to High-Density CNT Patterns. CNT
films were then coated with acrylic acid (Sigma-Aldrich) by
plasma polymerization (Pico-RF, Diener) using an input power
of 40 W and acrylic acid vapor pressure of 0.2 mbar for 2 min.
ppAA-coated CNTs were then incubated in a solution of 0.1 M
EDC (Sigma-Aldrich) and 5 mM sulfo-NHS (PIERCE) in PBS
for 30 min at room temperature. Following the 30 min
activation, samples were washed with deionized water (DI), and
SCNCs were added. The CNT substrate and SCNC solution
were then incubated at 50 °C overnight. Finally, samples were
washed three times with DI.
XPS measurements were performed using a 5600 Multi-
Technique System (Physical Electronics).
Structural characterization of CdSe/CdS−GSH NR−CNT
films was performed using SEM (MagellanTM 400L) and TEM
(Tecnai G2Spirit Twin T-12).
SCNC Optical Characterization. Absorbance spectra were
measured using a UV−vis−near−IR spectrophotometer
(JASCO V-570). Photoluminescence spectra were measured
using a fluorimeter (Varian Inc.), and fluorescence lifetime
measurements were carried out using a fluorescence
spectrometer (Edinburgh Instruments FLS920).
ICP-MS Measurements. For ICP-MS, patterns of 0.5 cm ×
0.5 cm CNT films, loosely attached to the Si/SiO2substrate,
were conjugated with SCNCs. CNT−SCNCs were peeled off
the substrate and etched overnight in 1 mL of 69% nitric acid.
Following sonication, 100 μL of the CNT−SCNCs solution
was mixed with 3.35 mL of TDW and analyzed by ICP-MS
(cx7500, Agilent) for Cd. The quantity of Cd in each solution
was calculated using external calibration with standard Cd
solutions.
Photoresponse. The illumination unit consisted of a light-
emitting diode (LED) with a peak wavelength of 405 nm
(Thorlabs) mounted on an upright metallurgical microscope
(Meiji), using a 4×or a 40×water immersion objective,
resulting in illumination intensities within the range of 0.6−70
mW/cm2. LEDs with a peak wavelength at 530, 660, and 850
nm (Thorlabs) were used to study spectral response. The
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measurement unit consisted of a current amplifier (model
1212; DL Instruments) or voltage amplifier (model 1800; A-M
Systems). A photogenerated current or voltage was measured
between the illuminated electrode and a reference electrode
(platinum mesh) in PBS.
Electrical Recordings from Retinas. Coupling between the
tissue and the electrodes was improved by placing a small piece
of polyester membrane filter (5 μm pores; Sterlitech) and a ring
weight on the retina. The filter was removed before light
stimulation to minimize scattering. Retinas were kept at
physiological conditions,
54
at a temperature of 34 °C, and
perfused (2−5 mL/min) with oxygenated (95% O2,5%CO
2)
Tyrode’s solution (5 mM KCl, 25 mM NaHCO3,9mM
glucose, 1.2 mM MgSO4, 1.2 mM HEPES, 0.5 mM glutamine,
2.5 mM CaCl2). Neuronal signals were amplified (gain ×1200
MEA1060-Inv; MultiChannel Systems), digitized using a 64-
channel analogue to digital converter (MC_Card; Multi-
Channel Systems), and recorded (MC_Rack; MultiChannel
Systems).
■ASSOCIATED CONTENT
*
SSupporting Information
Figure S1 showing cross section SEM imaging of CdSe/CdS−
GSH NR−CNT film, Figure S2 showing photocurrent
characterization and electrical circuit model of CdSe/CdS−
GSH NR−CNT films, Figure S3 showing chick retina
spontaneous activity and photostimulation, Figure S4 showing
in vitro cell viability assay results, Figure S5 showing optical
properties of SCNCs used, and Figure S6 showing loading yield
of and average photocurrent of CdSe/CdS−GSH NR−CNT
films using covalent conjugation and physical adsorption. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■AUTHOR INFORMATION
Corresponding Author
*E-mail: yaelha@tauex.tau.ac.il.
Author Contributions
L.B., N.W., and D.R. contributed equally to this work. L.B.,
N.W., D.R., Y.H., U.B., and O.C. conceived and designed the
experiments.L.B.,N.W.,D.R.,G.L.,M.D.P.,andJ.B.D.
performed the experiments and analysis. E.S and C.E. advised
with retina experiments. S.R. helped with electrical modeling.
L.B. and Y.H. wrote the manuscript, and all authors reviewed
and discussed it. Y.H., U.B., and O.C. supervised the project.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
The authors thank Larisa Burstein for XPS measurements, Inna
Brainis and Gilad Cohen for assistance with the neuronal cell
cultures, Qlight Nanotech for some of the SCNCs, and Shlomo
Yitzhaik for many useful discussions. The work described in this
paper was partially supported by a grant from the Israel
Ministry of Science and Technology (O.C., U.B., and Y.H.), by
a grant from the European Research Council funding under the
European Community’s Seventh Framework Program (FP7/
2007−2013)/ERC grant agreement FUNMANIA-306707
(Y.H.) and by a grant from the Biotechnology and Biological
Sciences Research Council (BBSRC BB/1023526/1) (E.S.).
U.B. thanks the Alfred & Erica Larisch memorial chair. N.W.
was supported by a Clara Robert Einstein Scholarship.
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