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Fabricating high-density microarrays for retinal recording
Microelectronic Engineering 67–68 (2003) 520–527 www.elsevier.com / locate / mee
Fabricating high-density microarrays for retinal recording K. Mathieson a , *, W. Cunningham a , J. Marchal a , J. Melone a , M. Horn a , V. O’Shea a , K.M. Smith a , A. Litke b , E.J. Chichilnisky c , M. Rahman a a
Department of Physics and Astronomy, University of Glasgow, Glasgow G12 8 QQ , Scotland, UK b SCIPP University of California Santa Cruz, Santa Cruz, CA 95604, USA c The Salk Institute for Biological Studies, San Diego, CA 92037 -1099, USA
Abstract Understanding how the retina encodes the visual scene is a problem, which requires large area, high-density microelectrode arrays to solve. The correlated signals that emerge from the output (ganglion) cells of the retina form a code, which is not well understood. We use a combination of electron beam lithography, photolithography and dry-etch pattern transfer to realise a 519-electrode array in the transparent conductor indium tin oxide (ITO). The electrodes are spaced at 60 mm in a hexagonal close-packed geometry. A mix and match lithography procedure is utilised, whereby the high-density inner region is fabricated using electron beam lithography whilst the outer sections are realised by photolithography. Reactive ion etching (RIE), using CH 4 / H 2 , of the ITO forms the array structure and SF 6 RIE allows resist removal and patterning of vias through a plasma deposited Si 3 N 4 protective layer. The electrical properties of the ITO layer are unaffected by the etching procedures. A reliable method for achieving low-impedance electroplated platinum electrodes has been employed to yield electrode impedances of | 20 kV. An array fabricated using these dry-etch techniques is shown to record action potentials from live retinal tissue in neurophysiological experiments. 2003 Elsevier Science B.V. All rights reserved. Keywords: Microelectrode arrays; Retinal recording; ITO; Dry-etching
1. Introduction Recording 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, high-density microelectrode arrays are needed. Current systems are limited to 64 electrodes, though recently a 128-electrode system has appeared. * Corresponding author. E-mail address: [email protected] (K. Mathieson). 0167-9317 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0167-9317(03)00109-6
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To probe a significant area of the retina we are developing fabrication processes for a multielectrode array consisting of around 500 electrodes spaced at 60 mm. When manufacturing this number of electrodes the difficulty of a reliable large area lift-off process begins to pose a problem for arrays made using metals such as gold. Here we have developed a scalable process using a 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. The array is formed by CH 4 / H 2 reactive ion etching of the ITO. A 1-mm thick layer of silicon nitride is deposited onto the ITO and provides a protective coating. An SF 6 RIE allows the formation of vias through this coating to the ITO layer, after which the electrodes may be platinised. A reliable process has been developed for the fabrication of a 61-electrode array that uses photolithography for the masking steps. These arrays have been used to record signals from live retinal tissue at the Salk Institute for Biological Studies in San Diego. A 519-electrode array has been fabricated using the same procedure but with additional steps for the high-density central region which requires e-beam lithography. We use Shipley UVIII as an electron beam (e-beam) resist and dry-etch mask. After the CH 4 / H 2 etch the mask is removed using an SF 6 RIE. These arrays require a 519-channel electronic read-out system, which is nearing completion and will allow testing in retinal tissue experiments. The process is scalable to approximately 2000 electrodes by halving the track width. This will provide an excellent opportunity to study a relatively large area (2 3 2 mm 2 ) of the retina in detail. The fabrication steps for the 61-electrode array have been reported in detail in Ref. [3] and so will not be covered here. However, we do report upon the performance of this array showing, for the first time, how an array fabricated using these RIE techniques behaves in neurophysiological experiments. The etching processes used for the production of the 61-electrode array are also employed in the manufacture of the 519-electrode array. We exploit a mix-and-match lithography, which combines photolithography with e-beam lithography, to fabricate a 519-electrode array. The photolithographic steps are used to fabricate the outer regions, where the line width remains above the limits of our photolithography resolution ( | 1 mm). The densely packed inner section has line widths of 1 mm, which we do with e-beam lithography. This permits ready scaling for larger array sizes.
2. Fabrication
2.1. Overview The fabrication steps needed to realise a 519-electrode array include photolithography, electron beam lithography, dry etch techniques and plasma deposition of silicon nitride (Si 3 N 4 ). The complete process flow is summarized in Fig. 1, with Fig. 1(a) illustrating the new development needed for the 519-electrode array. Fig. 1(b) and (c) shows the photolithographic, RIE and deposition processes adapted from Ref. [3]. The fabricated structure (outer region fabricated by photolithography), with a close up of the central region (requires e-beam lithography) and the ITO wires is shown in Fig. 2. The two lithography steps (e-beam and photolithography) are aligned through Ti:Au (30 nm:130 nm) alignment marks deposited onto the ITO layer using an electron beam evaporator.
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Fig. 1. Process flow diagram showing the steps needed to fabricate a 519-electrode array. (a) Pattern transfer using e-beam lithography; (b) pattern transfer using photolithography and (c) Si 3 N 4 deposition and patterning.
Fig. 2. Left: design of the 519-electrode array, each side measures 3.2 cm and has |130 bond-pads. Right upper: central region, which requires e-beam lithography to realise. Right lower: close-up of fabricated ITO wires in the high-density region, where line spacing and line width are reduced to 1 mm.
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2.2. High-density inner section The first area to be fabricated in this 519-channel retinal sensor is the high-density inner section. First the glass–ITO plate was baked at 180 8C to remove any humidity and allowed to cool for 20 s. Then hexamethyldisilizane (HMDS) was spun onto the ITO layer at 3000 rpm for 30 s and baked at 80 8C for 20 min. HMDS acts as a primer for the application of Shipley UVIII, a chemically amplified deep ultraviolet (DUV) resist, which we use as a fast e-beam resist and dry-etch mask. A 49% solution of 1 mm UVIII, diluted using ethyl lactate, was spun at 3000 rpm for 60 s. The sample then underwent a soft-bake at 130 8C for 60 s on a vacuum hot plate. A 5-nm layer of Ni was then evaporated onto the surface of the UVIII. This Ni layer acts as a discharging layer since the glass substrate and UVIII resist electrically isolate the sample. This can lead to the sample charging and discharging in time and may result in stitching errors and pattern distortion (see Fig. 3). The Ni layer was kept to a 5-nm thickness to avoid any scattering of the electron beam during exposure. The e-beam exposure was done in a Leica lithography systems electron beam pattern generator (EBPG 5HR 100) using a beam voltage of 50 kV. Dose tests were performed over the range 20–50 mC / cm 2 with 40 mC / cm 2 producing the best results. The sample was then given a postbake of 135 8C for 60 s after which the Ni layer was removed by rinsing in RO water–HCl (100:1) until the sample became clear indicating removal of the Ni layer. To develop the sample we used undiluted CD-26 developer for 60 s, with RO water rinse and nitrogen blow dry. A 30-s oxygen descum was necessary to remove any residual resist.
2.2.1. ITO patterning UVIII has been shown to have good dry etch resistance [4], which is similar to that of standard photoresists. In an earlier paper [3] we developed etch parameters for the patterning of ITO using Shipley S1818 photoresist. We use the same etch to produce wires in ITO here and show that UVIII has good resistance to this etch. The sample was etched using CH 4 / H 2 RIE with an rf power of 100 W, a pressure of 11 mTorr and a flow-rate of 5 / 25 sccm CH 4 / H 2. This gives an etch rate of | 25 nm / min and offers smooth vertical walls with good uniformity. An etch time of 15 min was used giving a 25% over-etch to ensure good ITO removal. The problem with this etch and masking procedure is that CH 4 / H 2 tends to polymerize the UVIII resist. This results in a mask that can be very difficult to remove after the CH 4 H 2 RIE. An extended acetone clean had no effect on removing the UVIII mask. Instead we developed a RIE technique for the removal of polymerized UVIII. The sample was etched using SF 6 with an rf power of 120 W, a pressure of 2–3 mTorr and a flow-rate of 10 sccm. A time of 12 min gave satisfactory UVIII removal. The sample appears transparent after the SF 6 etch whereas beforehand the UVIII gave a slight discolouration. The electrical properties of the ITO were checked using a HP4145A semiconductor parameter analyser. The I–V measurements demonstrated that the UVIII had been removed successfully and that the electrical properties of the ITO were not affected. Fig. 4 shows an optical comparison of the ITO wires before and after the UVIII has been removed with the SF 6 etch. 2.3. Low-density outer section To write the whole array using e-beam lithography would be both costly and time-consuming. By contrast photolithography can pattern large areas in a very short time and has a lower relative cost.
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Fig. 3. Effects of resist charging on the fabrication of the inner section. Thin lines can be seen shorting the ITO wires together. These artefacts are removed when a 5-nm discharging layer was deposited before pattern exposure.
Fig. 4. Left: ITO wires after the CH 4 / H 2 etch, the glass substrate has a discolouration due to the presence of the UVIII. Right: ITO wires after the UVIII has been removed using the SF 6 etch.
The main drawback of photolithography is the resolution of the pattern definition. However, in this array, the dimensions of the line widths and spacings widen and at a certain distance from the centre, photolithography becomes preferable. To fabricate the ITO wires in the outer section we used the sample with the central high-density area already completed by e-beam and dry-etch techniques. Shipley S1818 photoresist was spun on to the sample at 4000 rpm for 30 s. The resist then had a 30-min bake at 90 8C followed by exposure in UV light for 11.5 s. The mask used covered the central area (already processed with e-beam lithography) and transferred the rest of the pattern shown in Fig. 2. The sample was developed out using a solution of Microposit concentrate developer–RO water (1:1) for 75 s under agitation, and then nitrogen blow-dried. A 5-min postbake at 120 8C was found previously [3] to give the resist mask good dry-etch resistance but still permitted mask removal using warm acetone. The RIE parameters are the same as described previously for the CH 4 / H 2 etch step.
2.4. Passivation layer After the ITO wire fabrication a passivation layer is required to isolate the wires and protect them during biological experiments. We used a 1-mm low-stress Si 3 N 4 layer for this purpose. The layer is deposited in two steps since it was found that this improved the quality of the film. In previous arrays we used a one-step deposition, which resulted in micropores in the Si 3 N 4 layer. If a pore is located above an ITO wire then it will electroplate during the platinisation procedure creating a secondary electrode in an undefined position. These false electrodes introduce an uncertainty in the assignment of signals to a particular position and so it is crucial that these micropores are not present. It was supposed that the micropores are due to small debris on the sample and so we adopted the following solution. The sample underwent a 10-min acetone clean in ultrasound followed by an isopropanol rinse and 5 min in RO water. The cleaning continued with a 30-min oxygen plasma ash in a
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Plasmafab 505 barrel asher. The first layer of Si 3 N 4 was deposited to a thickness of 500 nm and the previous cleaning steps were repeated before the next 500-nm layer of Si 3 N 4 was deposited. This two-step deposition means that if any pores are created in the first layer the secondary layer will cover them. Patterning of the Si 3 N 4 layer is necessary to open vias down to the end of the ITO wires and also to allow electrical connection to the bondpads. This is done by using Shipley S1818 photoresist as a mask (same parameters as before) and RIE using SF 6 with an rf power of 120 W, a pressure of 2–3 mTorr and a flow-rate of 10 sccm; 15 min allows a 30% over-etch to ensure all Si 3 N 4 is removed. Mask removal is completed by a 10-min ultrasound clean in warm acetone.
2.5. Platinisation of electrodes Platinum electrodes are often used in biological experiments. They are well suited due to two properties. Firstly platinum is a relatively inert metal and so does not react with the biological tissue. Secondly electroplated platinum allows the formation of electrodes with a granular structure, which has the effect of increasing the area of the electrode and reduces the impedance at the cell–electrode interface. To form platinum electrodes each contact is connected to a voltage supply, set at 22 V, through a 10-MV resistor. A drop of 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 drop and kept at a positive potential with respect to the electrodes. The 22 V supply is kept on for 60 s and the electrodes are kept under observation in a microscope. The arrays are platinised using a variation of the procedure outlined in Ref. [5]. Here the authors describe a method for preparing strongly adherent platinum electrodes by electroplating whilst agitating in ultrasound. The nature of our arrays means that electroplating in ultrasound is difficult. Instead we electroplate first then clean with acetone under ultrasound agitation. Any poor quality electrodes are removed with only the well-adhering platinum remaining. The array is then electroplated once again to complete the platinisation procedure. The impedance of the platinised electrodes was measured using a HP 4192A LF impedance analyser. The measurements took place in 0.1 M NaCl in water with a platinum wire supplying an AC voltage of 0.2 V. For an electrode diameter of 2 mm an impedance of 20 kV was measured at 10 kHz. This compared with an unplatinised impedance of 0.6 MV. An example of the platinised electrodes is shown in Fig. 5, where the granular structure can be seen.
3. Retinal recording using a dry-etched multielectrode array The readout system for a 61-electrode array has been developed [6,7] and is being used in biological experiments. However, the system for recording signals from the 519-electrode array is currently under development, so no biological studies have yet been preformed. The dry-etch techniques used here to fabricate a 519-electrode array have been used to fabricate a 61-electrode array [3] in the same materials (glass substrate, ITO array). The performance of this array in biological experiments has not been reported and is included here to demonstrate the viability of using RIE to form high-density arrays for retinal studies.
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Fig. 5. Picture of the platinised electrodes. The inter-electrode separation is 60 mm and the granular structure of the electrodes can be seen.
A schematic of the experimental set-up is shown in Fig. 6. The retinal tissue is placed ganglion cell side down on the microelectrode array in a physiological saline solution at 35 8C to keep the tissue alive. A computer driven display focused onto the top of the tissue can stimulate the retina. The resulting voltage spikes are recorded by the microelectrode array and a CCD camera placed below the transparent array allows the spatial information to be correlated with each spike train.
Fig. 6. Experimental set-up used to measure spike trains from retinal tissue. The transparency of the array allows the signals to be correlated with image and electrode position.
Fig. 7. Retinal signals recorded using a 61-electrode array fabricated by the dry-etching of ITO.
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Fig. 7 shows a voltage pulse recorded from the output (ganglion cell) of some retinal tissue by a dry-etched microelectrode array. The background photon absorption rates for the long, middle and short wavelength sensitive cones were approximately equal to the absorptions that would have been caused by spatially uniform monochromatic lights of wavelength 561, 530 and 430 nm and intensity 9000, 9000 and 5000 photons / mm 2 s, respectively, incident on the photoreceptors.
4. Conclusions We have developed a process using mix-and-match lithography to enable the fabrication of a 519-electrode array. The array is fabricated in the transparent conductor ITO to enable accurate alignment of cells and electrodes. Consecutive platinisation steps with intermediate ultrasound agitation allow the formation of high-quality platinum electrodes. The dry-etch steps, plasma deposited Si 3 N 4 and the platinisation procedure has produced microelectrode arrays that can detect the output signals of the retina.
Acknowledgements The authors thank the staff of the Detector Development group, the Nanoelectronics Research Centre and the Dry-etch group, all at Glasgow University. The microelectrode array development is funded by EPSRC (UK) under their Life Sciences Interface.
References [1] M. Meister, L. Lagnado, D.A. Baylor, Science 270 (1995) 1207–1210. [2] M. Meister, M.J. Berry II, Neuron 22 (1999) 435–450. [3] W. Cunningham, K. Mathieson, F.A. McEwan, A. Blue, R. McGeachy, J.A. McLeod, C. Morris-Ellis, V. O’Shea, K.M. Smith, A. Litke, M. Rahman, J. Phys. D: Appl. Phys. 34 (2001) 2804–2809. [4] D. MacIntyre, S. Thoms, Microelectron. Eng. 35 (1997) 213–216. [5] C.A. Maresse, Anal. Chem. 59 (1987) 217–218. [6] A.M. Litke, Nucl. Instr. Meth. A 418 (1998) 203–209. [7] A.M. Litke, Nucl. Instr. Meth. A 435 (1999) 242–249.
Fabricating high-density microarrays for retinal recording K. Mathieson a , *, W. Cunningham a , J. Marchal a , J. Melone a , M. Horn a , V. O’Shea a , K.M. Smith a , A. Litke b , E.J. Chichilnisky c , M. Rahman a a
Department of Physics and Astronomy, University of Glasgow, Glasgow G12 8 QQ , Scotland, UK b SCIPP University of California Santa Cruz, Santa Cruz, CA 95604, USA c The Salk Institute for Biological Studies, San Diego, CA 92037 -1099, USA
Abstract Understanding how the retina encodes the visual scene is a problem, which requires large area, high-density microelectrode arrays to solve. The correlated signals that emerge from the output (ganglion) cells of the retina form a code, which is not well understood. We use a combination of electron beam lithography, photolithography and dry-etch pattern transfer to realise a 519-electrode array in the transparent conductor indium tin oxide (ITO). The electrodes are spaced at 60 mm in a hexagonal close-packed geometry. A mix and match lithography procedure is utilised, whereby the high-density inner region is fabricated using electron beam lithography whilst the outer sections are realised by photolithography. Reactive ion etching (RIE), using CH 4 / H 2 , of the ITO forms the array structure and SF 6 RIE allows resist removal and patterning of vias through a plasma deposited Si 3 N 4 protective layer. The electrical properties of the ITO layer are unaffected by the etching procedures. A reliable method for achieving low-impedance electroplated platinum electrodes has been employed to yield electrode impedances of | 20 kV. An array fabricated using these dry-etch techniques is shown to record action potentials from live retinal tissue in neurophysiological experiments. 2003 Elsevier Science B.V. All rights reserved. Keywords: Microelectrode arrays; Retinal recording; ITO; Dry-etching
1. Introduction Recording 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, high-density microelectrode arrays are needed. Current systems are limited to 64 electrodes, though recently a 128-electrode system has appeared. * Corresponding author. E-mail address: [email protected] (K. Mathieson). 0167-9317 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0167-9317(03)00109-6
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To probe a significant area of the retina we are developing fabrication processes for a multielectrode array consisting of around 500 electrodes spaced at 60 mm. When manufacturing this number of electrodes the difficulty of a reliable large area lift-off process begins to pose a problem for arrays made using metals such as gold. Here we have developed a scalable process using a 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. The array is formed by CH 4 / H 2 reactive ion etching of the ITO. A 1-mm thick layer of silicon nitride is deposited onto the ITO and provides a protective coating. An SF 6 RIE allows the formation of vias through this coating to the ITO layer, after which the electrodes may be platinised. A reliable process has been developed for the fabrication of a 61-electrode array that uses photolithography for the masking steps. These arrays have been used to record signals from live retinal tissue at the Salk Institute for Biological Studies in San Diego. A 519-electrode array has been fabricated using the same procedure but with additional steps for the high-density central region which requires e-beam lithography. We use Shipley UVIII as an electron beam (e-beam) resist and dry-etch mask. After the CH 4 / H 2 etch the mask is removed using an SF 6 RIE. These arrays require a 519-channel electronic read-out system, which is nearing completion and will allow testing in retinal tissue experiments. The process is scalable to approximately 2000 electrodes by halving the track width. This will provide an excellent opportunity to study a relatively large area (2 3 2 mm 2 ) of the retina in detail. The fabrication steps for the 61-electrode array have been reported in detail in Ref. [3] and so will not be covered here. However, we do report upon the performance of this array showing, for the first time, how an array fabricated using these RIE techniques behaves in neurophysiological experiments. The etching processes used for the production of the 61-electrode array are also employed in the manufacture of the 519-electrode array. We exploit a mix-and-match lithography, which combines photolithography with e-beam lithography, to fabricate a 519-electrode array. The photolithographic steps are used to fabricate the outer regions, where the line width remains above the limits of our photolithography resolution ( | 1 mm). The densely packed inner section has line widths of 1 mm, which we do with e-beam lithography. This permits ready scaling for larger array sizes.
2. Fabrication
2.1. Overview The fabrication steps needed to realise a 519-electrode array include photolithography, electron beam lithography, dry etch techniques and plasma deposition of silicon nitride (Si 3 N 4 ). The complete process flow is summarized in Fig. 1, with Fig. 1(a) illustrating the new development needed for the 519-electrode array. Fig. 1(b) and (c) shows the photolithographic, RIE and deposition processes adapted from Ref. [3]. The fabricated structure (outer region fabricated by photolithography), with a close up of the central region (requires e-beam lithography) and the ITO wires is shown in Fig. 2. The two lithography steps (e-beam and photolithography) are aligned through Ti:Au (30 nm:130 nm) alignment marks deposited onto the ITO layer using an electron beam evaporator.
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Fig. 1. Process flow diagram showing the steps needed to fabricate a 519-electrode array. (a) Pattern transfer using e-beam lithography; (b) pattern transfer using photolithography and (c) Si 3 N 4 deposition and patterning.
Fig. 2. Left: design of the 519-electrode array, each side measures 3.2 cm and has |130 bond-pads. Right upper: central region, which requires e-beam lithography to realise. Right lower: close-up of fabricated ITO wires in the high-density region, where line spacing and line width are reduced to 1 mm.
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2.2. High-density inner section The first area to be fabricated in this 519-channel retinal sensor is the high-density inner section. First the glass–ITO plate was baked at 180 8C to remove any humidity and allowed to cool for 20 s. Then hexamethyldisilizane (HMDS) was spun onto the ITO layer at 3000 rpm for 30 s and baked at 80 8C for 20 min. HMDS acts as a primer for the application of Shipley UVIII, a chemically amplified deep ultraviolet (DUV) resist, which we use as a fast e-beam resist and dry-etch mask. A 49% solution of 1 mm UVIII, diluted using ethyl lactate, was spun at 3000 rpm for 60 s. The sample then underwent a soft-bake at 130 8C for 60 s on a vacuum hot plate. A 5-nm layer of Ni was then evaporated onto the surface of the UVIII. This Ni layer acts as a discharging layer since the glass substrate and UVIII resist electrically isolate the sample. This can lead to the sample charging and discharging in time and may result in stitching errors and pattern distortion (see Fig. 3). The Ni layer was kept to a 5-nm thickness to avoid any scattering of the electron beam during exposure. The e-beam exposure was done in a Leica lithography systems electron beam pattern generator (EBPG 5HR 100) using a beam voltage of 50 kV. Dose tests were performed over the range 20–50 mC / cm 2 with 40 mC / cm 2 producing the best results. The sample was then given a postbake of 135 8C for 60 s after which the Ni layer was removed by rinsing in RO water–HCl (100:1) until the sample became clear indicating removal of the Ni layer. To develop the sample we used undiluted CD-26 developer for 60 s, with RO water rinse and nitrogen blow dry. A 30-s oxygen descum was necessary to remove any residual resist.
2.2.1. ITO patterning UVIII has been shown to have good dry etch resistance [4], which is similar to that of standard photoresists. In an earlier paper [3] we developed etch parameters for the patterning of ITO using Shipley S1818 photoresist. We use the same etch to produce wires in ITO here and show that UVIII has good resistance to this etch. The sample was etched using CH 4 / H 2 RIE with an rf power of 100 W, a pressure of 11 mTorr and a flow-rate of 5 / 25 sccm CH 4 / H 2. This gives an etch rate of | 25 nm / min and offers smooth vertical walls with good uniformity. An etch time of 15 min was used giving a 25% over-etch to ensure good ITO removal. The problem with this etch and masking procedure is that CH 4 / H 2 tends to polymerize the UVIII resist. This results in a mask that can be very difficult to remove after the CH 4 H 2 RIE. An extended acetone clean had no effect on removing the UVIII mask. Instead we developed a RIE technique for the removal of polymerized UVIII. The sample was etched using SF 6 with an rf power of 120 W, a pressure of 2–3 mTorr and a flow-rate of 10 sccm. A time of 12 min gave satisfactory UVIII removal. The sample appears transparent after the SF 6 etch whereas beforehand the UVIII gave a slight discolouration. The electrical properties of the ITO were checked using a HP4145A semiconductor parameter analyser. The I–V measurements demonstrated that the UVIII had been removed successfully and that the electrical properties of the ITO were not affected. Fig. 4 shows an optical comparison of the ITO wires before and after the UVIII has been removed with the SF 6 etch. 2.3. Low-density outer section To write the whole array using e-beam lithography would be both costly and time-consuming. By contrast photolithography can pattern large areas in a very short time and has a lower relative cost.
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Fig. 3. Effects of resist charging on the fabrication of the inner section. Thin lines can be seen shorting the ITO wires together. These artefacts are removed when a 5-nm discharging layer was deposited before pattern exposure.
Fig. 4. Left: ITO wires after the CH 4 / H 2 etch, the glass substrate has a discolouration due to the presence of the UVIII. Right: ITO wires after the UVIII has been removed using the SF 6 etch.
The main drawback of photolithography is the resolution of the pattern definition. However, in this array, the dimensions of the line widths and spacings widen and at a certain distance from the centre, photolithography becomes preferable. To fabricate the ITO wires in the outer section we used the sample with the central high-density area already completed by e-beam and dry-etch techniques. Shipley S1818 photoresist was spun on to the sample at 4000 rpm for 30 s. The resist then had a 30-min bake at 90 8C followed by exposure in UV light for 11.5 s. The mask used covered the central area (already processed with e-beam lithography) and transferred the rest of the pattern shown in Fig. 2. The sample was developed out using a solution of Microposit concentrate developer–RO water (1:1) for 75 s under agitation, and then nitrogen blow-dried. A 5-min postbake at 120 8C was found previously [3] to give the resist mask good dry-etch resistance but still permitted mask removal using warm acetone. The RIE parameters are the same as described previously for the CH 4 / H 2 etch step.
2.4. Passivation layer After the ITO wire fabrication a passivation layer is required to isolate the wires and protect them during biological experiments. We used a 1-mm low-stress Si 3 N 4 layer for this purpose. The layer is deposited in two steps since it was found that this improved the quality of the film. In previous arrays we used a one-step deposition, which resulted in micropores in the Si 3 N 4 layer. If a pore is located above an ITO wire then it will electroplate during the platinisation procedure creating a secondary electrode in an undefined position. These false electrodes introduce an uncertainty in the assignment of signals to a particular position and so it is crucial that these micropores are not present. It was supposed that the micropores are due to small debris on the sample and so we adopted the following solution. The sample underwent a 10-min acetone clean in ultrasound followed by an isopropanol rinse and 5 min in RO water. The cleaning continued with a 30-min oxygen plasma ash in a
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Plasmafab 505 barrel asher. The first layer of Si 3 N 4 was deposited to a thickness of 500 nm and the previous cleaning steps were repeated before the next 500-nm layer of Si 3 N 4 was deposited. This two-step deposition means that if any pores are created in the first layer the secondary layer will cover them. Patterning of the Si 3 N 4 layer is necessary to open vias down to the end of the ITO wires and also to allow electrical connection to the bondpads. This is done by using Shipley S1818 photoresist as a mask (same parameters as before) and RIE using SF 6 with an rf power of 120 W, a pressure of 2–3 mTorr and a flow-rate of 10 sccm; 15 min allows a 30% over-etch to ensure all Si 3 N 4 is removed. Mask removal is completed by a 10-min ultrasound clean in warm acetone.
2.5. Platinisation of electrodes Platinum electrodes are often used in biological experiments. They are well suited due to two properties. Firstly platinum is a relatively inert metal and so does not react with the biological tissue. Secondly electroplated platinum allows the formation of electrodes with a granular structure, which has the effect of increasing the area of the electrode and reduces the impedance at the cell–electrode interface. To form platinum electrodes each contact is connected to a voltage supply, set at 22 V, through a 10-MV resistor. A drop of 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 drop and kept at a positive potential with respect to the electrodes. The 22 V supply is kept on for 60 s and the electrodes are kept under observation in a microscope. The arrays are platinised using a variation of the procedure outlined in Ref. [5]. Here the authors describe a method for preparing strongly adherent platinum electrodes by electroplating whilst agitating in ultrasound. The nature of our arrays means that electroplating in ultrasound is difficult. Instead we electroplate first then clean with acetone under ultrasound agitation. Any poor quality electrodes are removed with only the well-adhering platinum remaining. The array is then electroplated once again to complete the platinisation procedure. The impedance of the platinised electrodes was measured using a HP 4192A LF impedance analyser. The measurements took place in 0.1 M NaCl in water with a platinum wire supplying an AC voltage of 0.2 V. For an electrode diameter of 2 mm an impedance of 20 kV was measured at 10 kHz. This compared with an unplatinised impedance of 0.6 MV. An example of the platinised electrodes is shown in Fig. 5, where the granular structure can be seen.
3. Retinal recording using a dry-etched multielectrode array The readout system for a 61-electrode array has been developed [6,7] and is being used in biological experiments. However, the system for recording signals from the 519-electrode array is currently under development, so no biological studies have yet been preformed. The dry-etch techniques used here to fabricate a 519-electrode array have been used to fabricate a 61-electrode array [3] in the same materials (glass substrate, ITO array). The performance of this array in biological experiments has not been reported and is included here to demonstrate the viability of using RIE to form high-density arrays for retinal studies.
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Fig. 5. Picture of the platinised electrodes. The inter-electrode separation is 60 mm and the granular structure of the electrodes can be seen.
A schematic of the experimental set-up is shown in Fig. 6. The retinal tissue is placed ganglion cell side down on the microelectrode array in a physiological saline solution at 35 8C to keep the tissue alive. A computer driven display focused onto the top of the tissue can stimulate the retina. The resulting voltage spikes are recorded by the microelectrode array and a CCD camera placed below the transparent array allows the spatial information to be correlated with each spike train.
Fig. 6. Experimental set-up used to measure spike trains from retinal tissue. The transparency of the array allows the signals to be correlated with image and electrode position.
Fig. 7. Retinal signals recorded using a 61-electrode array fabricated by the dry-etching of ITO.
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Fig. 7 shows a voltage pulse recorded from the output (ganglion cell) of some retinal tissue by a dry-etched microelectrode array. The background photon absorption rates for the long, middle and short wavelength sensitive cones were approximately equal to the absorptions that would have been caused by spatially uniform monochromatic lights of wavelength 561, 530 and 430 nm and intensity 9000, 9000 and 5000 photons / mm 2 s, respectively, incident on the photoreceptors.
4. Conclusions We have developed a process using mix-and-match lithography to enable the fabrication of a 519-electrode array. The array is fabricated in the transparent conductor ITO to enable accurate alignment of cells and electrodes. Consecutive platinisation steps with intermediate ultrasound agitation allow the formation of high-quality platinum electrodes. The dry-etch steps, plasma deposited Si 3 N 4 and the platinisation procedure has produced microelectrode arrays that can detect the output signals of the retina.
Acknowledgements The authors thank the staff of the Detector Development group, the Nanoelectronics Research Centre and the Dry-etch group, all at Glasgow University. The microelectrode array development is funded by EPSRC (UK) under their Life Sciences Interface.
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