Neural Stimulation with a Carbon Nanotube Microelectrode Array

into electrodes for neural stimulation. Noble metals, such as platinum, have a long history as neural electrodes. However, the charge injection limit ...
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NANO LETTERS

Neural Stimulation with a Carbon Nanotube Microelectrode Array

2006 Vol. 6, No. 9 2043-2048

Ke Wang,*,† Harvey A. Fishman,‡,§ Hongjie Dai,| and James S. Harris†,⊥ Department of Applied Physics, Department of Ophthalmology, Department of Chemistry, and Department of Electrical Engineering, Stanford UniVersity, Stanford, California 94305 Received May 31, 2006; Revised Manuscript Received August 9, 2006

ABSTRACT We present a novel prototype neural interface using vertically aligned multiwalled carbon nanotube (CNT) pillars as microelectrodes. Functionalized hydrophilic CNT microelectrodes offer a high charge injection limit (1−1.6 mC/cm2) without faradic reactions. The first repeated in vitro stimulation of hippocampal neurons with CNT electrodes is demonstrated. These results suggest that CNTs are capable of providing far safer and more efficacious solutions for neural prostheses than previous metal electrode approaches.

Electrical stimulation of nerve cells is widely employed in neural prostheses (for hearing,1 vision,2 and limb movement restoration3), in clinical therapies (treating Parkinson’s disease, dystonia, and chronic pain4), and in basic neuroscience studies. In all of these applications, an implanted microelectrode array stimulates the neurons and modulates their behavior. An ideal stimulating array needs to be both efficacious and safe.5 However, functional stimulation (especially in the central nervous system) often demands small electrodes with high current density, which conflicts with the electrochemical safety requirements. Undesirable electrochemical change not only damages the electrodes but also causes abnormalities in neural function and cell structure.6 A variety of metals and metal alloys have been fabricated into electrodes for neural stimulation. Noble metals, such as platinum, have a long history as neural electrodes. However, the charge injection limit of bare platinum is low (100-300 µC/cm2, geometrical area);7 hence only large electrodes with a low current density can be safely used. Activated iridium oxide offers a significant improvement in the charge injection limit (2-3 mC/cm2 geometrical area), with a reversible faradic reaction (Ir3+ T Ir4+ + e-).8 Yet it has been reported that activated iridium oxide delaminates under high current pulsing and deposits particles into the surrounding tissue.9 Capacitor electrodes based on tantalum oxide, titanium oxide,10 or silicon transistors11 have been developed that operate more safely by avoiding faradic reactions. However, * Corresponding author, [email protected]. † Department of Applied Physics. ‡ Department of Ophthalmology. § Present address: Plager Vision Center, Santa Cruz, CA. | Department of Chemistry. ⊥ Department of Electrical Engineering. 10.1021/nl061241t CCC: $33.50 Published on Web 08/26/2006

© 2006 American Chemical Society

because of the low capacitance, their charge injection limits are still not comparable to iridium oxide. Carbon nanotubes (CNTs) have intriguing electrochemical, mechanical, and chemical properties for neural electrodes. For example, Young’s modulus of a CNT exceeds 1 TPa, about five times stronger than steel.12 Yet CNTs can be bent and twisted into large angles without breaking.13 This combination of strength and flexibility is highly desirable for penetrating electrodes in neural prostheses. The application of CNTs in neural stimulation and recording has been proposed by our group14 and recently also by other researchers.15 In this work, we present the first demonstration of a functioning neural interface using multiwalled CNTs as microelectrodes, which also can enhance efficacy and satisfy the safety requirements. Critical electrochemical properties of the device are described, followed by the first demonstration of neural stimulation. The chip consists of an array of electrically conductive CNTs integrated onto substrates with prepatterned microcircuitry. Each electrode is an ensemble of multiwalled CNTs and is individually addressable. A cross-sectional schematic of the device is illustrated in Figure 1A. The fabrication processes have been described in greater detail elsewhere.14 In brief, quartz substrates were used because of their optical transparency, thermal stability, and electrical insulation. In situ phosphorus-doped polysilicon was deposited by lowpressure chemical vapor deposition as the conductive layer and then patterned by plasma etching (SF6/CHClF5). The sheet resistance of the 5000 Å thick layer was ∼10 Ω/square. We chose heavily doped polysilicon instead of standard metals in order to minimize the thermal stress between different layers. Significant mismatch of the thermal expansion coefficients causes microscopic cracks and leakage in

Figure 1. CNT pillar microelectrodes: (A) schematic of the cross section (not to scale); (B) a 6 × 6 array of 30 µm × 30 µm electrodes; (C) a 50 µm diameter electrode.

the insulating layers. The leakage current after CNT growth was reduced by 3 orders of magnitude when doped polysilicon replaced platinum as the conductive layer. Silicon also serves as a better substrate for CNT growth than metals. The top insulating layer is an oxide-nitride-oxide sandwich structure. The 2 µm thick silicon dioxide layer effectively reduces capacitive current, the dense silicon nitride layer (1000 Å) blocks ionic current flow, and the top oxide layer (1500 Å) provides a more hydrophilic surface to promote cell adhesion. Plasma etching (CHF3/O2) opened windows through the insulating layers for electrode and contact pad formation. Polysilicon and silicon nitride deposited on the backside of the wafer were also etched away for higher optical transparency. Finally, 60 Å of chromium and 500 Å of platinum were patterned as contact pads, and 20 Å of iron was patterned at the electrode sites, using standard UV photolithography, electron-beam evaporation, and resist liftoff techniques. The wafers were cleaned thoroughly in solvent and deionized water to remove chemical traces from processing before proceeding to CNT synthesis. Multiwalled CNTs were synthesized using a catalytic thermal chemical vapor deposition system.16 The substrates were heated to a growth temperature of 700 °C in oxygenfree argon. Precursor ethylene gas (500 sccm) carried by 500 sccm hydrogen was then fed into the tube for 1-10 min. When the desired growth time was reached, ethylene was switched off and the chamber temperature was ramped down in flowing hydrogen and argon. The nanotubes self-assemble into uniform pillars projecting orthogonally from the surface. Final devices after growth are shown in parts B and C of Figure 1. The size, geometry, and location of the CNT pillars are precisely defined by lithographic patterning of the catalyst. CNTs grown at lower temperatures are less dense and form entangled quasi-planar mats instead, which can be 2044

useful for some applications in which planar electrodes are preferred. The CNT synthesis has very high yield and is exceptionally robust. Similar results were obtained using a wide range of catalyst thicknesses (20-50 Å) and on a variety of substrates (doped and undoped silicon, silicon dioxide, quartz, and platinum). X-ray photoelectron spectroscopy analysis demonstrates that the CNT synthesis follows a base-growth mode, with minimal pyrolytic amorphous carbon formation. The protruding geometry is advantageous in layered tissues, such as the retina and the cortex. The surgically accessible locations for a retinal implant are typically a hundred micrometers or farther from the target neurons, thus higher excitation current is required for planar electrodes. In comparison, either protruding electrodes can be inserted into the tissue or nerve cells can be directed17 to grow conformally into the three-dimensional device,18 achieving better proximity between the electrodes and the targets. Proximity is the key to safer and more efficacious neural stimulation because less current is required.19 This allows for the use of smaller electrodes at a higher density, localizes the stimulation, reduces tissue injury, and lowers power consumption and heat dissipation. Although the high CNT synthesis temperature currently impedes the integration of CNT microelectrodes with soft permeable polymer substrates, which are more compatible with the retinal tissue, it has been shown that this temperature can be significantly reduced if plasma-enhanced chemical vapor deposition techniques are employed.20 After CNT growth, a chamber for electrolytes and cell culture media was formed by bonding an acrylic ring onto the chip with PDMS (poly(dimethylsiloxane)). Cured PDMS is sufficiently biocompatible for this experiment21 and provides long-term bonding without leakage. A home-built electrical connector was used for signal transmission between the chip and external units. The chip was placed into an acrylic holder and good electrical connection between the contact pads on the chip and a printed circuit board was achieved with low resistance ZEBRA elastomeric connectors (Fujipoly, NJ). A two-electrode system was used for all electrochemical measurements. The CNT microelectrode array served as the working electrode, a large Ag/AgCl coil as the reference and counter electrode, and phosphate-buffered saline (PBS, pH ) 7.4) as the electrolyte. The CNT electrodes were first characterized by cyclic voltammetry, using a potentiostat (Gamry FAS2 Femtostat, Gamry Instruments, PA) and Gamry software (Framework and Echem Analyst). Oxygen and hydrogen evolve at +1.0 and -1.5 V (vs Ag/AgCl), respectively, defining a wide operational potential window (Figure 2). Except for a small peak at -0.4 V, which is most likely due to the reduction of adsorbed oxygen,22 the voltammogram is basically featureless within the window, indicating that the current is delivered primarily through charging and discharging the interfacial double layer instead of through faradic reactions. Capacitive charge injection is an ideal mechanism for neural stimulation because no chemical change occurs to either the tissue or the electrode. Nano Lett., Vol. 6, No. 9, 2006

Figure 2. Cyclic voltammogram of a CNT electrode (geometrical area 5 × 10-4 cm2, scan rate 200 mV/s). The arrows indicate the direction of voltage scan.

However, a major problem of capacitive electrodes has been their low charge storage capacity, which can be described as: Qmax ) CVmax ) 0r(A/d)Vmax. For CNT electrodes in physiological solutions, the double layer at the electrodeelectrolyte interface serves as the capacitor. Therefore, the values of the relative permittivity r (≈80), “dielectric” layer thickness d (≈10 Å if the ion concentration is about 10-1 M, such as in saline23), and “safe” potential range Vmax (+1.0 and -1.5 V) are all determined. To increase Qmax, the only adjustable parameter is the surface area A. Since the size of the electrode is usually restricted by the application, a reasonable approach is to increase the ratio of “effective” area (i.e., accessible surface area that contributes to the double-layer capacitance) to geometrical area. The CNT electrode is an ensemble of individual MWNTs (typical diameter 30-50 nm) with relatively large spacing between the nanotubes (on the order of tens of nanometers). For an aqueous electrolyte, pores as small as 0.5 nm should be sufficient to contribute to the double-layer capacitance.24 Thus the CNT electrode inherently has a large surface area. However, the surface consists primarily of the graphitic basal plane. The nonpolar basal plane makes as-grown CNTs highly hydrophobic, most of its large surface area is inaccessible in aqueous solutions and cannot contribute to charge injection. Different surface modification techniques were used to enhance the hydrophilicity of the CNT microelectrodes. Heating the electrode array at 400 °C in air for an hour turns it hydrophilic, by forming surface oxides at the ends and defect sites of the CNT. The thermal oxidation also removes the amorphous carbon deposited between neighboring nanotubes and further increases the accessible surface area. However, the mechanical stability of the device is compromised during this thermal process. Heated CNT electrodes often detach from the substrate during electrochemical cycles. Surface oxides may also change the electronic structure of the CNT and reduce its electrical conductivity. Alternatively, hydrophobic CNT electrodes can be wetted by organic solvents, such as ethanol and isopropyl alcohol. Unfortunately, a significant amount of solvent molecules, which are extremely cytotoxic, are still Nano Lett., Vol. 6, No. 9, 2006

trapped between the porous nanotubes, even after extensive rinsing. It is known that a variety of molecules readily adsorb onto a CNT surface through noncovalent interactions.25 The amphiphilic poly(ethylene glycol)-lipid conjugates (PEGPL) bind strongly to CNTs via van der Waals and hydrophobic interactions between the two lipid alkyl chains and the nanotube sidewall, while the PEG chain extends into water.26,27 Upon incubation with 0.1 mg/mL PEG-PL (Avanti Polar Lipids, AL) for 2-20 h at room temperature, the CNT microelectrodes turned more hydrophilic. A similar hydrophobic-to-hydrophilic transition was also observed after the CNT microelectrodes were incubated with cell culture medium (for example, Neurobasal with B27 supplement). The transition was attributed to nonspecific adsorption of proteins in the culture medium to the nanotube surface. Noncovalent binding is one of the least invasive methods to modify the CNT electrodes without significant disturbance of either their structure or electronic properties. It is a particularly appealing approach because the modification can be designed to meet specific surface requirements, such as promoting or preventing cell adhesion. The accessible surface area of the treated hydrophilic CNT microelectrodes is not directly measurable. However, the interfacial double-layer capacitance, Cd, is proportional to the surface area and can be measured by electrochemical impedance spectroscopy (EIS). The EIS spectra of a representative CNT microelectrode after noncovalent surface treatment are shown in parts A and B of Figure 3. The data can be fitted to an equivalent circuit model (Figure 3C), where the interface is represented by a constant phase element (CPE, with impedance Z ) (1/Cd(jω)R)) in parallel with the faradic impedance, Zf, and Rs is the series solution spreading resistance. Parameter fitting results in a value of 0.97 for R, very close to an ideal capacitor; the faradic impedance, Zf, is 65 MΩ, much higher than the capacitive impedance at all tested frequencies; the spreading resistance, Rs, is 2.9 kΩ. The double-layer capacitance of the functionalized hydrophilic CNT electrode is 1600 µF/cm2 geom (geometrical area is calculated including both the top and sidewall of the pillar). Measured Cd for untreated hydrophobic CNT electrodes is only 5.4 µF/cm2 geom (in good agreement with the graphitic basal plane28); therefore the effective surface area of the CNT electrodes has increased about 300 times after the surface treatment. The charge injection limit is defined as the maximum quantity of charge that an electrode can inject before reaching the water electrolysis potential. It directly limits the minimum electrode size that can be safely used. The charge injection limit was determined experimentally by potential transient measurements. A symmetric biphasic current pulse was applied between a CNT electrode and an Ag/AgCl coil in PBS. The electrode potential excursion was monitored on a digital oscilloscope (Figure 4A). The injected charge was increased gradually (by increasing the amplitude and/or the duration of the current pulse) until the electrode potential reached the hydrolysis limit. After the initial ohmic voltage step (∆VR ) iRs) was subtracted, the resulting curve (Figure 2045

Figure 4. Measurement of the charge injection limit. (A) Voltage excursion of a functionalized CNT electrode (geometrical area ) 5.7 × 10-5 cm2), under anodic-first symmetric biphasic current pulses (80 µA, 1 ms) and (B) with Rs subtracted.

Figure 3. (A) Bode plot and (B) Nyquist plot of a functionalized CNT electrode (geometrical area ) 5 × 10-4 cm2). dc bias, 0 V; sinusoidal ac oscillation, 10 mV. Data fit to the equivalent circuit model illustrated in (C).

4B) represents the potential at the electrode surface. It is a simple saw tooth curve reflecting the charging/discharging of the double-layer capacitor under constant current. The charge injection limit of the functionalized CNT electrodes is 1-1.6 mC/cm2 geom, a significant improvement from CNTs without any treatment (∼20 µC/cm2). To test the durability of the CNT device, the microelectrodes were pulsed (20 µA, 1 ms, biphasic) at 10 Hz continuously for 6 h. No change was observed in the potential transient curve. A comparison of the electrochemical properties of CNT, bare Pt, and IrOx is listed in Table 1. The charge injection ability of a CNT is significantly higher than that of bare platinum but still not as high as that of iridium oxide. However, the large surface area of CNT electrodes has not yet been fully exploited. On the basis of the diameter and packing density of the CNTs, the theoretical limit of the area 2046

enhancement factor (defined as the ratio of accessible surface area to geometrical area of the CNT pillar) is estimated to exceed 103, much higher than the current values (∼300). By tailoring the synthesis and surface modification techniques, it is plausible that a higher charge injection limit can be reached. On the other hand, since the damage threshold for biological tissue is ∼1 mC/cm2,29 practically it is not necessary to increase the electrode charge injection limit much further. As a proof of concept, we demonstrate in vitro stimulation of embryonic rat hippocampal neurons with the CNT microelectrode array. The arrays were sterilized by overnight UV exposure and coated with poly-D-lysine (50 µg/mL, 6 h at room temperature) to enhance cell adhesion to the substrate. When used for the first time, devices were also soaked in cell culture medium (Neurobasal/B27/glutamine/ glutamate) overnight to improve CNT hydrophilicity and to outgas. Neurons were harvested from embryonic day18 rat hippocampal brain tissue (Brainbits, IL) with standard protocols.30 The cells were diluted in culture media and then plated onto the CNT microelectrode arrays, typically at a density of about 100 cells per mm2. After 4 days in culture (37 °C, 5% CO2), the cells were well differentiated and ready for stimulation (Figure 5A). Both the viability and neurite outgrowth were comparable to cultures on plastic Petri dish controls. Action potentials were optically detected by observing intracellular Ca2+ level change with a calcium indicator, Fluo-4 (Molecular Probes, CA).31 Fluo-4 can be loaded into cells and exhibits a large fluorescence intensity increase on binding of free Ca2+. Prior to stimulation, cells were incubated with 2 µM Fluo-4 AM ester (Invitrogen, Molecular Probes, CA) in Ringer’s solution (2.5 mM CaCl2/1.3 mM MgCl2/10 mM HEPES in Hank’s Balanced Salt Solution, Nano Lett., Vol. 6, No. 9, 2006

Table 1. Electrochemical Properties of Several Neural Electrode Materials

potential window (V) charge injection limit (mC/cm2) charge injection mechanism

CNT

bare Pt

IrOx

2.5 1-1.6 capacitive

1.5 0.1-0.3 faradaic, pseudocapacitive

1.5 2-3 faradaic

pH adjusted to 7.4) at room temperature for 30 min. Cells were then rinsed thoroughly with indicator-free Ringer’s solution and further incubated for another 30 min at 37 °C. Stimulation was usually carried out within an hour after loading. Optical recording of the cell stimulation was done using an inverted microscope (Nikon Eclipse TE300) with a xenon source (75 W) and a filter set (470/515 nm, Chroma Technology Corp, VT), connected to a digital camera (Hamamatsu ORCA-ER). Image acquisition (at a rate of 3 frames/s) was automated by a computer and MetaMorph software (Molecular Devices, CA), which also processed and analyzed the data. The ambient temperature was controlled at 35 °C with a home-built thermostat. CNT microelectrodes of 50 µm × 50 µm × 40 µm and 100 µm × 100 µm × 40 µm were used for stimulation tests. Current pulses were applied between a CNT electrode and four return electrodes placed at the corners of the chip. Since the CNT electrodes operate primarily through capacitive current, charge-unbalanced stimulation protocols can also be used, so long as enough time is given for the capacitor electrode to discharge. The resulting voltage excursion was

Figure 5. Representative example of hippocampal cell stimulation with a CNT microelectrode array. (A) Cells cultured on an array of 100 µm × 100 µm CNT electrodes (40 µm tall). The white arrow points to the electrode to which current pulses (cathodic, ∼40 µA, 1 ms) were applied. The black arrowheads point to cells that are shown in (B). (B) Fluo-4 images before and after a stimulus. (C) Fluorescence intensity change with repeated cell stimulation. Nano Lett., Vol. 6, No. 9, 2006

monitored by a digital oscilloscope to ensure that the electrode potential did not exceed the electrochemical safety limits. Among nine different devices tested, seven successfully excited the neurons in a reliable way. Figure 5B shows the fluorescence level increase in the hippocampal cells after a stimulus was applied. To maximize the detection dynamic range, digital contrast and brightness were reduced until the cells were barely visible before stimulation. More importantly, the neurons can be stimulated repeatedly with the CNT electrodes. An example is shown in Figure 5C. A series of 1-ms monophasic cathodic pulses were applied with intervals of 4-5 s, to ensure that the fluorescence intensity returned to the resting level and the electrode discharged completely after each pulse (it takes a few hundred milliseconds with the oscilloscope connected). Fluorescence intensity change is calculated as ((F′ - B′) (F0 - B0)/(F0 - B0)) × 100%, where F0 and F′ represent the cell fluorescence intensity at the resting state and upon stimulation, respectively, while B0 and B′ represent the background fluorescence level before and after stimulation, respectively. Each fluorescence intensity peak represents one or a few action potentials. The multiple peaks show that the cell responded consistently to the stimulating pulses. The stimulation was repeated over 100 times. This repeated excitation response indicates good cell physiological condition and normal CNT electrode functionality, even under prolonged, charge-unbalanced stimulation. Furthermore, the cells were still able to be chemically excited by glutamate (8 µL, 10 mM) after the electrical stimulation. To determine the stimulation threshold, 1-min intervals were given between the stimuli. Fluorescent imaging was also paused during the interval to avoid photobleaching the Fluo-4 molecules. Then images were captured again and a current pulse of slightly higher amplitude (or duration) was applied. These procedures were repeated until a transient increase in cell fluorescence level was observed and confirmed with quantitative analysis of the fluorescence intensity. The stimulation threshold was 10-20 µA for 1 ms single cathodic pulses. It should be noticed that the cell-electrode distance was relatively large in this experimental setup using inverted fluorescence microscopy. The 200 µm × 200 µm polysilicon pad underlying each CNT pillar was less transmissive at the working wavelength of Fluo-4, virtually no fluorescent signal could be detected within that region. CNTs were also nontransparent; hence only cells 50 µm or further from the electrode edge could be seen in the fluorescence images. We expect that if upright fluorescence microscopy (or smaller polysilicon pads) is used instead, the stimulation threshold will drop significantly since cells closer to the electrodes can be studied as well. 2047

In summary, a prototype neural interface using CNT arrays as microelectrodes has been developed. We have demonstrated the first electrical stimulation of primary neurons using CNT electrodes. The neurons can grow and differentiate on the device and can be repeatedly excited even with charge-unbalanced stimulation protocols. We also show that the CNT microelectrodes have superior electrochemical properties, which can be further enhanced by surface modification. The CNT electrodes operate predominantly with capacitive current (ideal for neural stimulation), while offering a high charge injection ability of 1-1.6 mC/cm2. Therefore, small electrodes can be used without electrochemical hazards. This is an exciting advance toward the realization of both efficacious and safe electrical stimulation of neurons, which is critical for a variety of prosthetic and therapeutic applications. These CNT electrodes may also act as recording units to sense electrical activities in the nervous system. Acknowledgment. This work was supported by the Department of Ophthalmology at Stanford, Stanford Center for Integrated Systems research grant, and VISX, Inc. Device fabrication was performed at the Stanford Nanofabrication Facility. The authors thank Dr. Rainer Fasching for assistance with the electrochemical experiments. Supporting Information Available: EIS Bode plots of CNT electrodes before and after surface treatments, potential transient demonstrating the durability of CNT electrodes under continuous pulsing, and an image of a CNT microelectrode array with a bonded chamber. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Wilson, B. S.; Lawson, D. T.; Muller, J. M.; Tyler, R. S.; Kiefer, Annu. ReV. Biomed. Eng. 2003, 5, 207. (2) Weiland, J. D.; Liu, W.; Humayun, M. S. Annu. ReV. Biomed. Eng. 2005, 7, 361. (3) Navarro, X.; Krueger, T. B.; Lago, N.; Micera, S.; Stieglitz, T.; Dario, P. J. Peripher. NerV. Syst. 2005, 10, 229. (4) Benabid, A. L. Curr. Opin. Neurobiol. 2003, 13, 696. (5) Merrill, D. R.; Bikson, M.; Jefferys, J. G. J. Neurosci. Methods 2005, 141, 171.

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NL061241T

Nano Lett., Vol. 6, No. 9, 2006