IEEE TRANSACTIONS ON NUCLEAR SCIENCE, VOL. 51, NO. 5, OCTOBER 2004
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Large-Area Microelectrode Arrays for Recording of Neural Signals K. Mathieson, S. Kachiguine, C. Adams, W. Cunningham, D. Gunning, V. O’Shea, K. M. Smith, E. J. Chichilnisky, A. M. Litke, A. Sher, and M. Rahman, Member, IEEE
Abstract—To understand the neural code, that the retina uses to communicate the visual scene to the brain, large-area microelectrode arrays are needed to record retinal signals simultaneously from many recording sites. This will give a valuable insight into how large biological neural networks (such as the brain) process information, and may also be important in the development of a retinal prosthesis as a potential cure for some forms of blindness. We have used the transparent conductor indium tin oxide to fabricated electrode arrays with approximately 500 electrodes spaced at 60 m. The fabrication procedures include photolithography, electron-beam lithography, chemical etching and reactive-ion etching. These arrays have been tested electrically using impedance measurements over the range of frequencies important when recording extracellular action potentials (0.1–100 kHz). The data has been compared to a circuit model of the electrode/electrolyte interface. One type of array (512 electrodes) behaves as theory would dictate and exhibits an impedance of 200 k at 1 kHz. The other array (519 electrodes) has an impedance of 350 k at this frequency, which is higher than predicted by the models. This can perhaps be attributed to the difference in fabrication techniques. The 512-electrode array has been coupled to low-noise amplification circuitry and has recorded signals from a variety of retinal tissues. Example in vitro recordings are shown here.
Index Terms—Fabrication, in vitro recording, microelectrode arrays, retinal signals.
I. INTRODUCTION
R
ECORDING signals from retinal tissue has historically been performed on a single cell basis. However more recent results from multineuronal recordings [1], [2] suggest that neurons do not act as independent sources of information but instead signal in a concerted fashion. In order to examine the effects of connectivity on the processing of neural signals, highdensity microelectrode arrays are needed. Current commercially available systems range from 64 to 128 electrodes. To probe a significant area of the retina we have developed fabrication processes [3] for a multielectrode array consisting of around 500 electrodes spaced at 60 m. When manufacturing
Manuscript received November 18, 2003; revised May 18, 2004. This work was supported by Engineering and Physical Sciences Research Council and the National Institutes of Health. K. Mathieson, C. Adams, W. Cunningham, D. Gunning, M. Rahman, V. O’Shea, and K. M. Smith are with the Department of Physics and Astronomy, University of Glasgow, Glasgow G128QQ, U.K. (e-mail: K.Mathieson@ physics.gla.ac.uk). S. Kachiguine, A. M. Litke, and A. Sher are with the Santa Cruz Institute for Particle Physics, University of California, Santa Cruz, CA 95064 USA (e-mail:
[email protected]). E. J. Chichilnisky is with the Salk Institute, La Jolla, CA 92037 USA. Digital Object Identifier 10.1109/TNS.2004.835873
this number of electrodes, at these densities, the difficulty of a reliable large area lift-off process begins to pose a problem. Here we have developed a scalable process using a 150–300 nm thick layer of the transparent conductor indium tin oxide (ITO) on a glass substrate, thereby avoiding the use of the lift-off process. The ITO transparency permits accurate alignment of the cells to the electrodes. Two seperate methods of producing these arrays have been pursued. The first uses photolithography and chemical (or wet) etching to pattern the ITO into a 512-electrode array. This type of etching, while offering a quick, inexpensive and stable process, is isotropic and so restrictive if higher-density arrays are required. The second method uses electron-beam (e-beam) lithography and reactive-ion (or dry) etching which, though more expensive and time consuming, is highly directional and offers increased resolution. This permits larger area arrays and reduced electrode spacings to be considered. The 512-electrode array has been coupled to low-noise highdensity microelectronics for amplification of signals. The whole system has been used in recording data in vitro, from live retinal tissue in experiments conducted at the Salk Institute for Biological Studies. The 519-electrode array has been fabricated and will be installed in the same readout system, undergoing retinal experiments later this year. A review of this system is given in [4]. Here, we describe the fabrication procedures needed to realize these arrays and test them electrically, comparing their performance to a simple circuit model. For the 512-electrode array, the model represents the experimental data well and is used to explain the effect of electroplating the electrodes with platinum. The model highlights a variation with expected theory when compared to the 519-electrode array. This may be attributed to the differing fabrication procedures and requires further study. The 512-electrode array has an impedance of 200 k at 1 kHz, while the 519-electrode has an impedance of 350 k at the same frequency. The performance of the 512 system shows the validity of using such arrays in retinal experiments. II. MICROELECTRODE ARRAY FABRICATION The fabrication procedures for two types of electrode array are outlined here. The first is a 512-electrode array with an electrode spacing of 60 m. The electrodes have a hexagonal close-packed layout but cover a rectangular area. The preferred geometry would be a hexagonal coverage by these electrodes, but routing 512 electrodes at 60 m spacing with these line widths dictates the rectangular geometry. This is due to the fabrication technology giving a minimum line width of 4 m. A
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Fig. 1. Layout of the 512-electrode array. The ITO wires are 4 m wide and the electrodes have not been platinised.
IEEE TRANSACTIONS ON NUCLEAR SCIENCE, VOL. 51, NO. 5, OCTOBER 2004
Fig. 2. Layout of the 519-electrode array. The ITO wires are 2 m wide and the electrodes have not been platinised.
change in the processing technology permits the overall hexagonal geometry. This gives 519-electrode arrays with the additonal benefit of being further scalable to higher-density arrays. A. Patterning the 512-Electrode Array This type of array has an electrode spacing of 60 m and has 128 bond pads per side. An illustration of the electrode layout is shown in Fig. 1. These bond pads have a pitch of 200 m and facilitate wirebonding to the readout electronics. The arrays are fabricated on 4-in wafers with a 150 nm ITO layer having a sheet resistivity of 17 /square. Photolithographic methods are used to pattern the ITO layer. Firstly a layer of Molybdenum (Mo) is deposited on top of the ITO layer. Then a 1 m thick layer of Shipley-3612 resist is spun onto the Mo layer. Exposure of the resist by UV light gives the 512-electrode pattern. The resist acts as a mask for the plasma etching of the Mo using F-155 (Freon). A flow rate of 70 sccm, a pressure of 100 mtorr and a rf power of 55 W was used. The resist and Molybdenum mask the ITO, which is etched for 8 min in concentrated hydrochloric acid. The rest of the Mo layer is then removed again by repeating the previous plasma etch step, leaving only nontransparent alignment marks and patterned ITO. B. Patterning the 519-Electrode Array Due to the hexagonal layout of the electrode pattern, this microelectrode array requires line widths of 2 m (see Fig. 2). This is approaching the limit of our photolithography and so here we use electron-beam (e-beam) lithography to realize the array. Fig. 3 is a schematic illustrating the principal fabrication steps required. These arrays are fabricated on 40 40 mm glass plates with a 300 nm ITO coating. The sheet resistivity of this coating is 13 /square. 1) E-Beam Patterning: A 100 nm layer of titanium is electrodeposited onto the ITO. Then a layer of e-beam resist (58% Shipley UVIII diluted with ethyl lactate) was spun on to the titanium layer at 3000 rpm for 1 min. This gives a resist layer thickness of 300 nm. The resist is patterned using electron-beam exposure with a base dose of 20 C/cm being increased by up to 150%. This variation in e-beam exposure compensates
Fig. 3. Schematic illustrating the fabrication steps for the 519-electrode array.
for back-scattered electrons which would otherwise over-expose the high-resolution areas. The resist is developed in CD-26 for 60 s and given an oxygen plasma clean for 60 s. 2) Reactive-ion Etching: This resist acts as a mask for the titanium which is selectively removed using reactive-ion etching techniques (RIE). The gas used is SiCl with an etch pressure of 9 mtorr, an RF power of 250 W and a flow rate of 18 sccm. An etch time of 9.5 min removes the titanium layer whilst leaving a thinned layer of the UVIII mask. The patterned titanium is then used as a mask for the patterning of the ITO. The ITO is etched using CH H RIE with an RF power of 100 W, a pressure of 11 mtorr and a flow rate of 5/25 sccm CH H . A 15 min etch ensures good ITO removal by giving a 25% over-etch. The with an UVIII and titanium layers are then removed using RF power of 120 W, a pressure of 2–3 mtorr and a flow rate of 10 sccm. Twelve minutes strips the sample back to the ITO layer resulting in a highly-transparent array of 519 ITO wires. C. Passivation Layer In order to electrically isolate the ITO wires a layer of lowstress Si N was plasma deposited on top of the electrode structure. The 512-electrode array has a Si N layer of thickness of 2 m. The 519-electrode array has a 1 m low-stress layer of Si N and was plasma deposited in two steps. The quality of
MATHIESON et al.: LARGE-AREA MICROELECTRODE ARRAYS FOR RECORDING OF NEURAL SIGNALS
this passivation layer has to be high, since any pores may result in signals coming from more than one point in any particular channel. Patterning of the Si N layer is necessary to open vias down to the end of the ITO wires and also to allow electrical connection to the bondpads. A negative photoresist (Futurrex NR73000P) was used to pattern the Si N layer of the 512-electrode array. This resist acts as a good etch mask for the etching of the Si N using Freon. Shipley S1818 photoresist was used as a with mask for the 519-electrode array which was RIE using an RF power of 120 W, a pressure of 2–3 mtorr and a flow rate of 10 sccm. Fifteen minutes allows a 30% over etch to ensure all Si N is removed. Mask removal is completed by a 10 mins ultrasound clean in warm acetone. A 1 m layer of aluminum is then sputtered onto the ITO bond pads to ensure good electrical contact for subsequent wire bonding.
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Fig. 4. Left: Section of the 512-electrode array after electroplating with platinum black. Right: A close-up of a single platinised electrode, illustrating the granular structure of the electrode (diameter of electrode 5 m).
D. Electrode Platinization Platinum electrodes are often used in biological experiments. They are well suited due to two properties. First, platinum is a relatively inert metal and so does not react with the biological tissue. Second, electroplated platinum allows the formation of electrodes with a granular structure, which has the effect of increasing the area of the electrode and reducing the impedance at the cell-electrode interface. This type of platinum is known as platinum black. To form platinum electrodes a solution of 1% platinic chloride, 0.08% lead acetate, and 98.92% RO water is deposited on the region above the electrode array. A platinum wire is placed in this solution and kept at a positive potential with respect to the electrodes. Each electrode is supplied with a current density of 4 nA m . The electrodes are platinised for approximately 20 s, which forms platinum electrodes with a diameter matching that of the vias in the passivation layer (5 m). Fig. 4 shows a section of the 512-electrode array after electrode platinization, with a close-up of one of the electrodes. The underlying ITO trace has a a square electrode site measuring 8 m a side. The opening in the passivation layer limits the platinum electrode to a diameter of 5 m, giving a geometric area of 19.6 m . These electrodes are robust enough to last for the duration of a retinal experiment (up to 15 h). However, after removal of the retinal tissue and the subsequent cleaning some of the platinum is removed. This only affects the platinum electrodes, the rest of the array has remained to this date (tens of experiments) unaffected. A feature of the readout circuitry is an application specific integrated circuit (ASIC) containing a current generator for every electrode. This enables easy re-platinization of the electrode array after every experiment. The readout circuitry is detailed in [8]. In this way, the array is restored to the same condition as before the experiment. III. IMPEDANCE MEASUREMENTS In order to characterize the electrical response of the microelectrode arrays we have performed impedance measurements on the 512 and 519-electrode arrays. A circuit model for this type of electrode/electrolyte interface has been proposed by Kovacs et al. [5] and is adapted and compared to the experimental
Fig. 5. Circuit diagram representing the electrode/electrolyte interface (Kovacs [5]). C is the interfacial capacitance, R is the charge transfer resistance, R is the Warburg resistance, C is the Warburg capacitance, and R is the spreading resistance.
data recorded here. The experimental results were obtained under the following conditions. The electrode array area was covered with 0.1 molar saline solution and a platinum wire electrode was used to supply a sinusoidal ac voltage of 100 mV peak to peak. A needle probe made contact with the bond pad of the electrode under study. A Hewlett Packard 4274A LCR meter was used to vary the frequency of the ac voltage signal and record the impedance of the electrode. Ten electrodes were each measured 5 times and the averaged reading taken. A. Circuit Model of Electrode/Electrolyte Interface By modeling the electrode/electrolyte interface and comparing with experimental data, it is possible to obtain an increased understanding of the processes at the electrode interface. This is important for fully understanding recorded signals, the design of future arrays and can offer some insights if the arrays are used to stimulate biological cells. Kovacs [5], proposed a circuit model to represent the interface between an electrode and an electrolyte. Fig. 5 shows a schematic of this circuit. The electrode interface is represented by a capacitor ( —the interfacial capacitance) and resistor ( —the charge transfer resistance) connected in parallel. The interfacial capacitance represents a layer of positive ions surrounding a negatively charged metal interface. The impedance of this term is inversely proportional to the frequency of the ac signal and the surface area of the platinised
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IEEE TRANSACTIONS ON NUCLEAR SCIENCE, VOL. 51, NO. 5, OCTOBER 2004
electrode. The charge transfer resistance is the dc current path across the electrode interface and also depends inversely on the surface area of the platinised electrode. The two parallel Warand ) are a related to diffusion effects and burg terms ( become important only at high frequencies outwith the range considered here (0.1–100 kHz) and so will be ignored in the following discussion. is a resistive term caused by The spreading resistance the spreading of current from a conductive solution into a small electrode. It depends on the geometrical area of the electrode and so is determined by the size of the vias etched through the Si N layer to the ITO. The coupling capacitance to the neighand through the passivation layer are boring channel calculated values from [6]. These values are the same for both the 512-electrode array and the 519-electrode array, because although the track width is reduced by a factor 2 for the 519 array, the thickness of the ITO layer is a factor of 2 greater. The ITO traces also have a resistance associated with them. This can be calculated using the sheet resistivity of the ITO layers (13 /square for the 519-electrode arrays and 17 /square for the 512-electrode arrays) and estimating the number of squares over the surface area of the ITO trace. Multiplying these two values together gives an estimate of the 5 k . The major part of which resistance of the ITO trace, is due to the narrow traces at the centre of the device. This has been verified by the fabrication of test ITO traces, 0.5 mm long with a width of 2 m and a thickness of 300 nm. The resistance of this ITO trace is 2 k , and is indicitive of the resistance of the central wires of the full scale arrays. This implies that the ITO traces could be represented in a circuit model as a 5 k resistor. This value is significantly lower than the other terms discussed above and so it effect on the impedance of the array can be ignored. However, future arrays may require narrower ITO traces in which case this resistive term may become significant and limit the trace width of these ITO arrays. 1) Circuit Simulations: The PSPICE circuit simulator was used to model the circuit diagram illustrated in Fig. 5. The values for the components were adapted from [7] and are shown in Table I. is the only The surface area of the platinum electrodes unknown parameter. It affects only the interfacial capacitance and the charge transfer resitance , since the geometry of the arrays gives the other parameters. IV. RESULTS A. Impedance Results Fig. 6 shows a comparison between the PSPICE simulations and the experimental data. The values are the theoretical values and depend upon the surproposed in [5]–[7]. However, face area of the fractal-like platinum deposits. It is the surface area parameter which is allowed to vary in order to fit the simulated to the experimental data. For the 512-electrode array this times the geometrical area of method gives an increase of the electrodes (19.6 m ). The simulated curve with this value is shown as the dotted line in Fig. 7 and gives a good repreof sentation of the experimental data. The same method of analysis does not give a satisfactory fit to the experimental data from the
TABLE I CAPACITIVE AND RESISTIVE VALUES FOR PSPICE SIMULATION. A SURFACE AREA OF THE PLATINUM ELECTRODES
IS THE
Fig. 6. Impedance as a function of frequency. The 512 electrode array experimental data fits the model (dashed line) well. The 519-electrode does not fit the model well and so no fit is shown. array data
(1)
()
519-electrode array. This indicates that there are some further circuit elements needed to fully represent this type of array. B. Retinal Recordings A 512-electrode array was used to record retinal impulses from a guinea pig retina. The array was placed into the readout system, where each channel was wire bonded to the front end of an ASIC. This ASIC was developed by Dabrowski et al. [8]. It has 64 channels of differential amplification, bandpass filtering, and output amplification, followed by a 64:1 analogue multiplexer. The bandpass is typically 80–2000 Hz, with an equivalent rms input noise of 5 V. This rises to 7 V when the electrodes are immersed in saline solution. The system was operated at 20 kHz readout rate on each channel, with the whole array being read out simultaneously. The ASIC also ac-couples the electrode array from the amplification stage through 150 pF capacitors. This coupling means that the dc-offset seen in Fig. 7 does not arise from any electrode or biological sources. The offset is a feature of the ASIC and can be filtered out during data analysis. The retinal tissue is placed ganglion cell side down on the microelectrode array in a physiological saline solution at 37 C to keep the tissue alive. A computer driven display focussed onto the top of the tissue can stimulate the retina. The resulting voltage spikes are recorded by the microelectrode array and a charge coupled device (CCD) camera placed below the transparent array allows the spatial information to be correlated with each spike train.
MATHIESON et al.: LARGE-AREA MICROELECTRODE ARRAYS FOR RECORDING OF NEURAL SIGNALS
Fig. 7. Single response from an electrode showing extracellular action potentials from a guinea pig retina. Obtained using a 512-electrode array.
Fig. 7 shows a voltage pulse recorded from the output (ganglion cell) of guinea-pig retinal tissue. The stimulus is a black moving bar pattern on a grey background. The clear signal definition illustrates that the 512-electrode array operates extremely well as a multi-site recording device for neural signals. V. DISCUSSION AND CONCLUSION We have described the fabrication methods needed to realize high-density transparent electrode arrays for neuronal recordings. The 512-electrode array has been shown to behave as theory would predict if the platinum electrodes give a surface area increase of approximately 100 times the geometrical area. It has been reported in reference [9] that a surface increase of 80–130 times the geometric area can result from electroplating with platinum black. So the area increase observed for the 512-electrode array is in agreement with this. 200 k at 1 kHz freA typical electrode impedance of quency was obtained using this array. This measurement is important for the design of stimulation electrodes, since it governs the amount of voltage needed to supply a given current to a cell. Though more important criteria are the charge delivery capacity of the electrode and its electrochemical stability. The use of these electrodes as stimulating and recording electrodes opens up more applications in the study of large-area biological networks. The 519-electrode array does not fit well with the theoretical circuit model. The difference could be due to fabrication techniques involved (RIE of semiconductor materials is known to sometimes introduce electrical damage). For this array the effect is not thought to be significant, as the impedance is still within acceptable limits. However, this requires further experimental investigation, as it may become an issue for future electrode arrays. The 519-electrode array fabrication technique utilizes e-beam lithography and dry-etch techniques, which are scalable to higher densities. This allows a 519-electrode array with 30 m electrode spacing to be fabricated by halving the track width to 1 m. Alternatively, by keeping the electrode
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Fig. 8. ITO traces with 1 m width and separation. The electrode spacing is 30 m.
spacing at 60 m, 1 m track widths allow an array with 2053 electrodes (covering an area of 7.3 mm) to be fabricated. These changes will allow neuroscientists to explore interesting aspects of cell connectivity at smaller inter-cell dimensions or over an increased area of the retina. Development of these arrays has already begun and Fig. 8 shows some ITO wires with 1 m width and separation and an inter-electrode spacing of 30 m. ACKNOWLEDGMENT The authors would like to thank the Technical Staff at the Detector Development Laboratory, the Staff at the Nanoelectronics Research Centre, and the Dry-Etch Laboratory—all at the University of Glasgow; and C. Storment for discussions on 512-electrode array fabrication. REFERENCES [1] M. Meister, L. Lagnado, and D. A. Baylor, “Concerted signalling by retinal ganglio-cells,” Science, vol. 270, pp. 1207–1210, Nov. 1995. [2] M. Meister and M. J. Berry II, “The neural code of the retina,” Neuron, vol. 22, no. 3, pp. 435–450, Mar. 1999. [3] K. Mathieson, W. Cunningham, J. Marchal, J. Melone, M. Horn, D. Gunning, R. Tang, C. Wilkinson, V. O’Shea, K. M. Smith, A. Litke, E. J. Chichilnisky, and M. Rahman, “Detection of retinal signals using position sensitive microelectrode arrays,” Nucl. Instr. Meth. A, vol. 513, pp. 51–56, 2003. [4] A. M. Litke, E. J. Chichilnisky, W. Dabrowski, A. A. Grillo, P. Grybos, S. Kachiguine, M. Rahman, and G. Taylor, “Large-scale imaging of retinal output activity,” Nucl. Instr. Meth. A, vol. 501, pp. 298–307, 2003. [5] G. T. A. Kovacs, “Introduction to the theory, design, and modeling of thin-film microelectrodes for neural interfaces,” in Enabling Technologies for Cultured Neural Networks, D.A. Stenger and T.M. McKenna, Eds. New York: Academic, 1994, pp. 121–165. [6] T. Sakarai and K. Tamaru, “Simple formulae for two- and three-dimensional capacitances,” IEEE Trans. Electron Devices, vol. 30, pp. 183–185, Jan. 1983. [7] D. A. Borkholder, “Cell based biosensors using microelectrodes,” Ph.D. dissertation, Stanford Univ., Stanford, CA, 1998. [8] W. Dabrowski, P. Grybos, P. Hottowy, A. Skoczen, K. Swientek, N. Bezayiff, A. A. Grillo, S. Kachiguine, A. M. Litke, and A. Sher, “Development of integrated circuits for readout of microelctrode arrays to image neuronal activity in live retinal tissue,” in IEEE Nuclear Scienc Symp. Conf. Rec., 2003. [9] C. S. Kim and S. M. Oh, “Enzyme sensors by electrodeposition on platinized platinum electrodes,” Electrochim. Acta., vol. 41, no. 15, pp. 2433–2439, 1996.
