Fabrication of Subretinal 3D Microelectrodes with Hexagonal Arrangement

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Article Fabrication of Subretinal 3D Microelectrodes with Hexagonal Arrangement

Hee Won Seo , Namju Kim and Sohee Kim * Department of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea; [email protected] (H.W.S.); [email protected] (N.K.) * Correspondence: [email protected]; Tel.: +82-53-785-6217

Received: 29 March 2020; Accepted: 27 April 2020; Published: 29 April 2020

Abstract: This study presents the fabrication of three-dimensional (3D) microelectrodes for subretinal stimulation, to accommodate adjacent return electrodes surrounding a stimulating electrode. For retinal prosthetic devices, the arrangement of return electrodes, the electrode size and spacing should be considered together, to reduce the undesired dissipation of electric currents. Here, we applied the hexagonal arrangement to the microelectrode array for the localized activation of retinal cells and better visual acuity. To provide stimuli more eﬃciently to non-spiking neurons, a 3D structure was created through a customized pressing process, utilizing the elastic property of the materials used in the fabrication processes. The diameter and pitch of the Pt-coated electrodes were 150 µm and 350 µm, respectively, and the height of the protruded electrodes was around 20 µm. The array consisted of 98 hexagonally arranged electrodes, supported by a ﬂexible and transparent polydimethylsiloxane (PDMS) base, with a thickness of 140 µm. Also, the array was coated with 2 µm-thick parylene-C, except the active electrode sites, for more focused stimulation. Finally, the electrochemical properties of the fabricated microelectrodes were characterized, resulting in the mean impedance of 384.87 kΩ at 1 kHz and the charge storage capacity (CSC) of 2.83 mC cm 2. · − The fabricated microelectrodes are to be combined with an integrated circuit (IC) for additional in vitro and in vivo experiments.

Keywords: retinal implant; subretinal stimulation; 3D electrodes; hexagonal electrodes; transparent base

1. Introduction Retinal degenerative diseases are one of the causes that impair the vision of patients. Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are the diseases caused by the degeneration of photoreceptors, which ﬁnally lead to visual loss [1–4]. Photoreceptor cells consist of rod and cone cells in the retina, and these cells convert light entering the eye into electrical signals. However, if photoreceptors are lost because of AMD and RP, these signals cannot be generated and transmitted to non-spiking neurons in the inner nuclear layer (INL) [5]. Thus, this results in no responses of the retinal ganglion cells (RGCs) that transmit the signals to the optic nerve. Hence, to restore the vision in patients with such diseases, various retinal prostheses have been developed, which can replace the function of lost photoreceptors [5–13]. The retinal prosthesis, including stimulating electrodes, can partially restore the patient’s vision by stimulating live RGCs using a variety of surgical methods [14]. Depending on the surgical procedure, there are epiretinal electrodes placed on the retinal surface to directly stimulate RGCs [6,11,15–18], subretinal electrodes implanted between the outer nuclear layer (ONL) and the retinal pigment epithelium (RPE) [7–10,19–24], and suprachoroidal electrodes implanted between the choroid and the sclera [12]. Mostly, the stimulating electrodes have been developed to stimulate retinal cells epiretinally or subretinally [6–11], which diﬀer in their stimulation thresholds to electrically elicit the responses of

Micromachines 2020, 11, 467; doi:10.3390/mi11050467 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 467 2 of 12

RGCs. The stimulation threshold can vary depending on the type of pulse applied to the electrode

(monophasicMicromachines or 2020 biphasic),, 11, x pulse polarity (cathodic or anodic), and pulse duration [25–29].2 Typically, of 12 the epiretinal electrodes, to elicit direct RGC responses, have low stimulation thresholds with pulses of shorterRGCs. durations The stimulation of around threshold 0.5 ms, can whereas vary depending the subretinal on the electrodes, type of pulse which applied avoid to the direct electrode activation of RGCs,(monophasic have relatively or biphasic), low pulse stimulation polarity (cathodic thresholds, or anodic), with pulses and pulse of longer duration durations [25–29]. Typically, ranging from the epiretinal electrodes, to elicit direct RGC responses, have low stimulation thresholds with pulses 1 to 10 ms [8,25,29]. For subretinal electrodes, stimulating pulses are transmitted to non-spiking of shorter durations of around 0.5 ms, whereas the subretinal electrodes, which avoid direct neurons in INL (horizontal, bipolar and amacrine cells), and due to the slow nature of these neurons, activation of RGCs, have relatively low stimulation thresholds, with pulses of longer durations stimuliranging with from moderately 1 to 10 ms long [8,25,29]. pulse For durations subretinal (1 electrodes, to 10 ms) stimulating help reduce pulses the thresholdsare transmitted [5,25 to]. non- Previously,spiking neurons we in focused INL (horizontal, on the subretinal bipolar and approach amacrine that cells), can and deliver due to su theffi slowcient nature stimulation of these to the cellsneurons, in the INL, stimuli and with demonstrated moderately along three-dimensional pulse durations (1 subretinal to 10 ms) help electrode reduce array,the thresholds based on [5,25]. a flexible and transparentPreviously, base we [30 focused], which, on the however, subretinal had approach a small that number can deliver of electrodes; sufficient upstimulation to 25 in to an the area of 2.8 mmcells in2.8 the mm INL,. Here, and demonstrated we present a a subretinal three-dimensiona electrodel subretinal array with electrode an increased array, based number on a offlexible electrodes × to anticipateand transparent better visualbase [30], acuity. which, For however, that, a had large a smal numberl number of electrodes of electrodes; need up to 25 be in accommodated an area of within2.8 the mm limited × 2.8 sizemm. ofHere, the device.we present In addition, a subretinal when electrode an electric array field with is generatedan increased by number the stimulating of electrodes to anticipate better visual acuity. For that, a large number of electrodes need to be electrodes, the arrangement of return electrodes, the electrode size and spacing should be considered accommodated within the limited size of the device. In addition, when an electric field is generated together to reduce undesired dissipation of electric currents, i.e., crosstalk [31,32]. Based on such by the stimulating electrodes, the arrangement of return electrodes, the electrode size and spacing previousshould results, be considered we aimed together to implement to reduce the undesired hexagonal dissipation return electrodes of electric surroundingcurrents, i.e., acrosstalk stimulating electrode[31,32]. in Based center, on tosuch achieve previous the results, localized we ai activationmed to implement of RGCs the on hexagonal stimulating return sites. electrodes Moreover, by adaptingsurrounding the hexagonala stimulating electrode electrode arrangement, in center, to advantages achieve the such localized as more activation correct of identification RGCs on of symbolsstimulating [33] and sites. a higher Moreover, electrode by adapting density the withinhexagonal the electrode limited devicearrangement, area [advantages34] can be such anticipated. as Finally,more we correct fabricated identification a three-dimensional of symbols [33] (3D) and microelectrodea higher electrode array density with within 98 electrodes the limited based device on the transparentarea [34] base can andbe anticipated. characterized Finally, the electrochemicalwe fabricated a three-dimensional properties of the fabricated(3D) microelectrode electrodes, array prior to in vitrowith and 98 electrodes in vivo experiments. based on the transparent base and characterized the electrochemical properties of the fabricated electrodes, prior to in vitro and in vivo experiments. 2. Materials and Methods 2. Materials and Methods 2.1. Design of Subretinal Electrode Array 2.1. Design of Subretinal Electrode Array Figure1 shows the schematic illustrations of the 3D microelectrodes for subretinal stimulation. Figure 1 shows the schematic illustrations of the 3D microelectrodes for subretinal stimulation. The microelectrodes,The microelectrodes, which which can be can combined be combined with anwith integrated an integrated circuit (IC),circuit are (IC), subretinally are subretinally implantable, as shownimplantable, in Figure as 1showna. The in electrode Figure 1a. array The consists electrode of 98array hexagonally consists of arranged98 hexagonally electrodes, arranged and the sensedelectrodes, light can and be the converted sensed light into can the be converted electrical into signal the byelectrical the photodiodes signal by the inphotodiodes the IC that in the is to be integratedIC that with is to the be electrodes.integrated with For thethe photodiodeselectrodes. For to the detect photodiodes light directly, to detect polydimethylsiloxane light directly, (PDMS)polydimethylsiloxane was used as the (PDMS) transparent was used base as of the the transparent electrodes, base as of shown the electrodes, in Figure as1 shownb. The in thickness Figure of PDMS1b. was The chosen thickness to beof 140PDMSµm. was The chosen distance to be between 140 μm. the The centers distance of twobetween neighboring the centers electrodes of two was set toneighboring be 350 µm, electrodes and the heightwas set ofto thebe 350 3D μ electrodesm, and the height was set of tothe be 3D around electrodes 20 µwasm. set to be around 20 μm.

Figure 1. Schematic illustrations of microelectrodes for subretinal stimulation. (a) The subretinal Figure 1. Schematic illustrations of microelectrodes for subretinal stimulation. (a) The subretinal implantimplant is placed is placed between between the the outer outer nuclear nuclear layerlayer (ONL) (ONL) and and the the retinal retinal pigment pigment epithelium epithelium (RPE) (RPE) to to stimulatestimulate non-spiking non-spiking neurons. neurons. (b ()b Stimulation) Stimulation pulses are are generated generated when when photodiodes photodiodes in the in implant the implant convertconvert light light into into electrical electrical signals. signals.

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2.2. Generation of Silicon Pillars and Transparent BaseBase A p-type silicon wafer was used to fabricate the 3D microelectrodes. The diameter and thickness of the used silicon wafer were 6-inch and 675 μµm, respectively. First, Ti and Pt were deposited on the silicon waferwafer using using a a sputtering sputtering process process (SRN-110, (SRN-110 Sorona,, Sorona, Anseong, Anseong, Korea), Korea), which which served served as the as metal the layermetal of layer the electrodes,of the electrodes, as shown as inshown Figure in2 Figurea. Ti and 2a Pt. Ti layers and Pt were layers deposited were deposited with thicknesses with thicknesses of 50 nm andof 50 200 nm nm, and respectively, 200 nm, respectively, and patterned and patterned using a photoresist using a photoresist (AZ5214, MicroChemicals,(AZ5214, MicroChemicals, Ulm, Germany). Ulm, Germany).

Figure 2. Schematic diagram of the main processes to fabricate the 3D microelectrodes in a Figure 2. Schematic diagram of the main processes to fabricate the 3D microelectrodes in a hexagonal hexagonal arrangement. arrangement. After patterning the metal layer, 3D pillars were created in the silicon wafer by using a deep reactive ion etchingAfter patterning (DRIE) process the metal (LPX layer, PEGASUS, 3D pillars SPTS, were Allentown, created in PA, the USA), silicon along wafer with by theusing designed a deep electrodereactive ion patterns, etching as (DRIE) shown inprocess Figure (LPX2b. An PEGASU 8 µm-thickS, SPTS, photoresist Allentown, mask PA, (AZ9260, USA), MicroChemicals, along with the Ulm,designed Germany) electrode was usedpatterns, for DRIE. as shown The height in Figure of the 2b. silicon An pillars8 μm-thick was chosen photoresist to be 160maskµm. (AZ9260, In order toMicroChemicals, easily fill the transparent Ulm, Germany) PDMS was between used the for fabricated DRIE. The silicon height pillars, of the thesilicon silicon pillars wafer was was chosen cut into to ninebe 160 chips μm. using In order a dicing to easily process fill (DAD3240, the transparent Disco, Tokyo,PDMS Japan).between A chipthe fabricated contained 20silicon electrode pillars, arrays the insilicon a dimension wafer was of 35cut mm into nine35 mm, chips as using shown a indicing Figure process3. In the (DAD3240, chip, the barDisco, patterns Tokyo, surrounding Japan). A chip the × electrodescontained were20 electrode also created arrays to in guide a dimension the cutting of of35 the mm chip × 35 into mm, the as electrode shown in arrays Figure for 3. the In finalthe chip, step. the barNext, patterns to create surrounding the transparent the electrodes and flexible were base, also PDMS created (Sylgard to guide™ 184, the Dow cutting Corning, of the Midland,chip into MI,the USA)electrode was arrays filled betweenfor the final the siliconstep. pillars. After oxygen plasma (CUTE-B, Femtoscience, Hwaseong, Korea) was treated on the chip to enhance the adhesion between the PDMS to be filled and the chip surface, liquid PDMS was filled into the space generated between the pillars and the ‘lid’, as shown in Figure2c. The PDMS was mixed with curing agent at a 10:1 weight ratio. The lid, which covered the top of the silicon pillars, was made of the same PDMS mixture coated with 3 µm-thick parylene-C. In this process, the elastic PDMS lid was uniformly pressured using binder clips to realize a 3D electrode shape. A slide glass was used on the lid for the uniform distribution of pressure by the binder clips, and the lid evenly covered the top of the silicon pillars. After the PDMS was filled into the space between the chip and the lid, microbubbles contained in the liquid PDMS were removed using a vacuum desiccator (SW-V200D, Samwoo Engineering, Seoul, Korea). Then, the chip was cured in an oven at 60 ◦C for 4 h, and separated from the lid, as shown in Figure2d. As a result, a 3D structure by

Micromachines 2020, 11, 467 4 of 12 the silicon electrodes in combination with the PDMS base was created by the pressure applied to the Micromachineselastic lid. 2020, 11, x 4 of 12

Figure 3. (Left)(Left) Photograph Photograph of of aa chip chip containing containing 20 20 arrays arrays of of circular circular silicon silicon pillars, pillars, in ina dimension a dimension of 35of 35mm mm × 3535 mm. mm. (Right) (Right Scanning) Scanning electron electron microscopic microscopic (SEM) (SEM) images images of of silicon silicon pillars pillars after after deep × reactive ion etching. The scale bars are 100 μµm.

2.3. GenerationNext, to create of Connection the transparent Pads and and Parylene flexible Insulation base, PDMS (Sylgard™ 184, Dow Corning, Midland, MI, USA)To completely was filled reveal between the PDMS the silicon on the oppositepillars. After side ofoxygen the chip, plasma the silicon (CUTE-B, on the Femtoscience, backside was Hwaseong,removed. First, Korea) the was bulk treated of the silicon on the was chip removed to enhance physically the adhesion through between a grinding the processPDMS to (DA08AG, be filled andKTL, the Anseong, chip surface, Korea), liquid leaving PDMS only was 50 filledµm-thick into the silicon. space Angenerated isotropic between wet etching the pillars process andwas the ‘lid’, as shown in Figure 2c. The PDMS was mixed with curing agent at a 10:1 weight ratio. The lid, performed to etch the remaining silicon, where a mixture of HF (48%) and HNO3 (69%) in a 1:3 volume whichratio was covered used [the35]. top The of solution the silicon was pillars, agitated was at made 500 rpm of the for 10same min PDMS using mixture a magnet coated stirrer with (WiseStir 3 μm-® thickMSH-20D, parylene-C. DAIHAN In this Scientific, process, Wonju,the elastic Korea). PDMS After lid was the uniformly chip was fixedpressured to a custom-designedusing binder clips jig, to realizeit was immersed a 3D electrode in the shape. solution. A slide As theglass first was step, used the on silicon the lid surface for the was uniform etched distribution until the PDMS of pressure began byto appear,the binder and clips, the etchingand the timelid evenly was about covered 7 min, the top with of no the agitation silicon pillars. of the After solution. the PDMS Next, thewas mixed filled into the space between the chip and the lid, microbubbles contained in the liquid PDMS were solution of HF and HNO3 with a 1:5 volume ratio was used for the second etching step. The solution wasremoved changed using to a createvacuum the desiccator pads with (SW-V200D, a relatively Sa slowmwoo chemical Engineering, reaction, Seoul, and Korea). the wet Then, etching the chip was performedwas cured untilin an theoven height at 60 of °C the for pads 4 hours, reached and the se PDMSparated base. from In the this lid, step, as theshown etching in Figure time was 2d. about As a result,4 min, a with 3D structure no agitation by the of thesilicon solution. electrodes To remove in combination the residual with solution, the PDMS the base chip was was created washed by with the pressure applied to the elastic lid. deionized water for 2 min and baked on a hotplate at 110 ◦C for 10 min [30]. The transparent base of the electrodes was completely exposed, as shown in Figure2e. 2.3. GenerationNext, an insulating of Connection layer Pads was and coated Parylene on the Insulation topside of the microelectrode array for more focused stimulation.To completely As shown reveal in Figurethe PDMS2f, 2 onµm-thick the opposite parylene-C side of was the coated chip, the (NRPC-500, silicon on Nuritech, the backside Incheon, was removed.Korea). An First, adhesion the bulk promoter of the silicon (Silquest was A-174,removed Merck, physically Darmstadt, through Germany) a grinding was process used to(DA08AG, enhance KTL,the adhesion Anseong, between Korea), parylene-C leaving only and the50 topsideμm-thick of thesilicon. microelectrodes. An isotropic Then, wet aetching dry etching process process was wasperformed performed to etch to expose the remaining the electrode silicon, sites, where using a amixture patterned of maskHF (48%) made and of aHNO photoresist3 (69%) (AZ9260, in a 1:3 volumeMicroChemicals, ratio was Ulm,used Germany),[35]. The solution with a thicknesswas agitated of 11 atµ 500m. Usingrpm for a reactive10 minutes ion using etching a processmagnet (VITA,stirrer (WiseStir Femtoscience,® MSH-20D, Hwaseong, DAIHAN Korea), Scientific, parylene-C Wonju, on the Korea). electrode After sites the waschip removedwas fixed with to a custom- a power designedof 200 W andjig, it an was oxygen immersed gas flow in ofthe 100 solution. sccm for As 15 th min,e first as step, shown the in silicon Figure surface2g. was etched until the PDMSTo create began the connectionto appear,pads and forthe the etching electrodes, time metalwas about layers 7 were minutes, deposited with on no the agitation backside of of the solution.electrodes, Next, so that the the mixed microelectrodes solution of couldHF and be HNO electrically3 with connecteda 1:5 volume with ratio the IC.was First, used the for chip the wassecond cut etchinginto individual step. The electrode solution arrays was changed using a cutter to create (DN-52, the pads Dorco, with Seoul, a relatively Korea) for slow an accuratechemical sputtering reaction, and the wet etching was performed until the height of the pads reached the PDMS base. In this step, the etching time was about 4 minutes, with no agitation of the solution. To remove the residual solution, the chip was washed with deionized water for 2 minute and baked on a hotplate at 110 °C for 10 minutes [30]. The transparent base of the electrodes was completely exposed, as shown in Figure 2e.

Micromachines 2020, 11, 467 5 of 12 process. To deposit the metal layers only on the etched silicon surface, not on the PDMS surface, the sputtering process was performed using a patterned mask. The used mask was a 40 µm-thick polyethylene terephthalate (PET) ﬁlm patterned by a UV laser machine (ALM100, Sejoong Information Technology, Seoul, Korea). The patterned mask was manually aligned on the backside of the electrodes under a microscope (SZMN45TR-STL1, Sunny Optical Technology, Yuyao, China). Using a sputtering process (DKC161125, Daeki Hi-Tech, Daejeon, Korea), Ti and Au layers were deposited with thicknesses of 50 nm and 200 nm, respectively, on the silicon surface. Figure2h shows the connection pads created on the opposite side of the electrodes.

2.4. Electrochemical Characterization Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed to investigate the electrical properties of the fabricated microelectrodes. The electrodes were connected using a custom-designed parylene-C cable fabricated based on a previous study [36]. To connect the pads of the electrode array with the parylene-C cable, conductive epoxy (Duralco™ 125-2, Contronics, Brooklyn, NY, USA) was used, and the assembled device was cured in an oven at 35 ◦C for 16 h. Next, the parylene-C cable was connected to a printed circuit board (PCB), through a zero-insertion-force (ZIF) connector. As the ZIF connector (502598-3393, Molex, Lisle, IL, USA) had only 33 channels, all 98 electrodes could not be connected to the connector at the same time. Thus, we designed the parylene-C cable to have three parts of connection pads: the first part had 32 electrical lines and pads to connect 32 electrodes in the center of the array, while the second and third parts had 33 electrical lines and pads each, to connect the remaining electrodes. For EIS, a potentiostat (Reference 600+, Gamry Instruments, Warminster, PA, USA) and a custom-designed faraday cage with a dimension of 30 cm 38 cm 52 cm were used. The fabricated electrode array, an Ag/AgCl electrode, and a × × platinum wire were used as the working electrodes, the reference electrode, and the counter electrode, respectively. The prepared electrodes were immersed in phosphate buffer saline (PBS) solution (PBS 10010023, Thermo Fisher Scientific, Waltham, MA, USA), and the frequency was swept from 10 Hz to 1 MHz with a sinusoidal AC input voltage of 30 mVrms. For CV, the potential limits were set from 0.6 V to 0.8 V, at a scan rate of 50 mV s 1 [30,37]. − · − 3. Results and Discussion The 3D microelectrode array with 98 hexagonally arranged electrodes was fabricated using micro-electro-mechanical systems (MEMS) technologies. Figure4 shows the photographs of the completed 3D microelectrodes. 98 electrodes were arranged in a dimension of 4.4 mm 3.8 mm × within an array, and the outermost electrodes were designed to be used as return electrodes only. If an electrode is selected as the stimulating electrode except for the outermost electrodes, six surrounding return electrodes can guard the stimulating electrode, resulting in 64 effective pixels in the vision. The completed electrodes had the diameter and pitch of 150 µm and 350 µm, respectively, supported by a 140 µm-thick PDMS base, and the topside of the array, except the electrode sites, was coated with 2 µm-thick parylene-C. Figure5 shows the scanning electron microscopic (SEM) images (FlexSEM 1000, Hitachi, Tokyo, Japan) of the microelectrodes. The Pt-coated electrodes protruded around 20 µm from the PDMS base, as shown in Figure5a. The height of the protruded electrodes can be controlled by both the pressing strength of the binder clips and the strength of the PDMS lid. The higher the pressure of the binder clips, the stronger the elastic PDMS lid can press the PDMS between the silicon pillars, resulting in more protruded electrodes. On the other hand, if the pressure of the binder clips is constant, the height of the protruded electrodes can be varied according to the elasticity of the lid itself. The elasticity of the lid may depend on the mixing ratio, curing temperature, and curing time [38,39]. Figure5b shows the Au-coated connection pads on the backside of the microelectrodes. Although the diameter of the patterned mask was set to be the same as the diameter of the pads, the metal layers were deposited slightly wider than the pad area. Since the mask physically covered the pads, resulting in a little gap, Micromachines 2020, 11, 467 6 of 12 metal ions spread beyond the pad area. However, it can be easily corrected by reducing the diameter of theMicromachines masking pattern.2020, 11, x 6 of 12

Micromachines 2020, 11, x 7 of 12

In our previous work [30], we fabricated 3D electrodes with a grid arrangement in an area of 2.8 mm × 2.8 mm, based on the dicing of bulk silicon. Such physical removal of silicon, however, has limitations in fabricating the array with an increased number of electrodes within the limited device area, and in that electrode arrangement other than grid or rectangular arrangement is not possible. On the contrary, the current study suggests that the new fabrication method can overcome such limitations. Through the chemical etching of silicon, it was possible to accommodate more electrodes with smaller sizes within the same area of the device, particularly in any arrangement, such as grid, rectangular, hexagonal, and octagonal arrangements. We successfully realized 98 hexagonally arranged electrodes in an area of 4.4 mm × 3.8 mm using the proposed fabrication method, while only 40 electrodes could have been constructed if they were arranged in a grid and fabricated using the previous method published in [30]. Nevertheless, compared to the previous studies by others [7,44], the number of our fabricated electrodes is relatively insufficient, although we have increased the number of electrodes from 25 in our previous study to 98 electrodes in the current study. In [7], silicon-based electrode arrays with 250 pixels were fabricated, which had a height of 10 μm and a diameter of 14 μm. To achieve better visual acuity, our array should be improved with a much increased number of electrodes within the limited device area, such as those reported in the previous studies [7,22,44]. Using the proposed fabrication method, we anticipate that it would be possible to decrease the electrode size down to 70 μm, increasing the number of electrodes up to 260 within the same area of the device. The hexagonal electrode arrangement allows higher electrode densities compared to the rectangular arrangement [34]. Thus, the hexagonal arrangement not only makes it possible to allow a higher resolution of the phosphene image in the same area allowed for a device, but would also have slight advantages in acuity tasks [33,34]. A simulation study reported that by using guarded hexagonal return electrodes, effective retinal stimulation can be ensured, if the diameter of the electrodes under 100 μm is used and the distance between the electrodes and the target neurons is FigureFigure 4. Photographs 4. Photographs of of the the fabricated fabricated 3D3D microelectrode array: array: (a) (topa) topview view of the of array the array in a in a far enough [31]. The effectiveness of the hexagonal return electrodes in minimizing crosstalk between dimensiondimension of 4.4 of mm4.4 mm3.8 × 3.8 mm mm and and ( (bb,–cc)) its its sideside views views showing showing (b ()b the) the topside topside containing containing stimulating stimulating pixels and localized activation× of retinal cells, however, needs to be investigated in in vitro as well as electrodeelectrode sites sites and and (c) the(c) the backside backside containing containing thethe connection pads. pads. The The scale scale bars bars are are1 mm. 1 mm. in vivo environments, using the fabricated electrodes. Figure 5 shows the scanning electron microscopic (SEM) images (FlexSEM 1000, Hitachi, Tokyo, Japan) of the microelectrodes. The Pt-coated electrodes protruded around 20 μm from the PDMS base, as shown in Figure 5a. The height of the protruded electrodes can be controlled by both the pressing strength of the binder clips and the strength of the PDMS lid. The higher the pressure of the binder clips, the stronger the elastic PDMS lid can press the PDMS between the silicon pillars, resulting in more protruded electrodes. On the other hand, if the pressure of the binder clips is constant, the height of the protruded electrodes can be varied according to the elasticity of the lid itself. The elasticity of the lid may depend on the mixing ratio, curing temperature, and curing time [38,39]. Figure 5b shows the Au-coated connection pads on the backside of the microelectrodes. Although the diameter of the patterned mask was set to be the same as the diameter of the pads, the metal layers were deposited slightly wider than the pad area. Since the mask physically covered the pads, resulting in a little gap, metal ions spread beyond the pad area. However, it can be easily corrected by reducing the diameter of the masking pattern. Figure 6 shows the parylene-C-coated microelectrode array with the exposed electrode sites, where parylene-C was removed. As shown in Figure 6, parylene-C residue on the electrode site was completely removed. Parylene has been suggested as a suitable coating material due to the great barrier property [40,41]. Previously reported studies investigated the biocompatibility of parylene implanted in the retina [42,43]. In [43], the biocompatibility of a parylene-based retinal device implanted subretinally was investigated for three months, in which reactions such as fibrosis and tissue damage did not occur around the implant. However, as an encapsulation material, the very FigureFigure 5. SEM 5. SEM images images of theof the microelectrodes. microelectrodes. ((a) Three-dimensionally prot protrudedruded electrodes electrodes created created long-term in vivo performance of parylene needs to be evaluated for chronic implants. by theby pressingthe pressing process process using using an an elasticelastic lid. lid. (b ()b Au-coated) Au-coated connection connection pads padson the on opposite the opposite side of the side of the electrodes.electrodes.

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Figure6 shows the parylene-C-coated microelectrode array with the exposed electrode sites, where parylene-C was removed. As shown in Figure6, parylene-C residue on the electrode site was completely removed. Parylene has been suggested as a suitable coating material due to the great barrier property [40,41]. Previously reported studies investigated the biocompatibility of parylene implanted in the retina [42,43]. In [43], the biocompatibility of a parylene-based retinal device implanted subretinally was investigated for three months, in which reactions such as ﬁbrosis and tissue damage did not occur around the implant. However, as an encapsulation material, the very long-term in vivo performance ofMicromachines parylene needs2020, 11 to, x be evaluated for chronic implants. 8 of 12

FigureFigure 6.6. SEM images of the electrode arrayarray insulatedinsulated byby parylene-C,parylene-C, excludingexcluding thethe electrodeelectrode sites.sites.

InFigure our previous7 shows the work assembled [30], we fabricateddevice for 3Delectroc electrodeshemical with characterization a grid arrangement and the in results. an area The of 2.8 mm 2.8 mm, based on the dicing of bulk silicon. Such physical removal of silicon, however, electrode× array combined with the parylene-C cable was connected to a printed circuit board (PCB) hasfor the limitations impedance in fabricating and CV measurement, the array with as anshown increased in Figure number 7a. Overall, of electrodes 32 microelectrodes within the limited were deviceconnected area, to andthe ZIF in that connector electrode through arrangement the first conne otherction than pads grid of or the rectangular parylene-C arrangement cable. The second is not possible.and third Onparts the of contrary, connection the pads current were study not used suggests in the that initial the electrode new fabrication characterization, method can but overcome reserved suchfor the limitations. next experiments Through using the all chemical electrodes etching in the ofnear silicon, future. it The was impedance possible to of accommodate the microelectrodes more electrodeswas measured with smallerto be 384.87 sizes within ± 52.11 the k sameΩ at area1 kHz of the(Figure device, 7b). particularly In the array, in any the arrangement, area of an exposed such as grid,electrode rectangular, was about hexagonal, 0.018 mm and2, and octagonal the measured arrangements. impedance We successfully at 1 kHz was realized 21.4 M 98Ω hexagonally·mm−2 when arranged electrodes in an area of 4.4 mm 3.8 mm using the proposed fabrication method, while only converted to a value per unit area. In the× previous studies [30,37], the unit-area impedances of Pt- 40coated electrodes 3D electrodes could have were been 30.3 constructed MΩ·mm−2 and if they 62.5were MΩ·mm arranged−2, respectively. in a grid andThe fabricatedobtained impedance using the previoususing the method fabricated published electrodes in [ 30was]. Nevertheless,comparable to compared those values. to the The previous CV of studiesthe electrodes by others was [7 ,also44], themeasured, number as of shown our fabricated in Figure electrodes7c. The calculated is relatively CSC was insu 2.83ﬃcient, ± 0.22 although mC·cm−2 we and have comparable increased to thethe numbervalues reported of electrodes in the fromprevious 25 in studies our previous [30,37], ranging study to from 98 electrodes 1.24 mC·cm in−2the to 4.4 current mC·cm study.−2. In [7], µ silicon-basedAlthough electrode Pt-coated arrays electrodes with with 250 pixelsimpedances were fabricated,in such a range which were had demonstrated a height of 10 to bem andable µ ato diameter stimulate of the 14 retinalm. To cells achieve [30], electrode better visual materials acuity, capable our array of inje shouldcting behigher improved charge withare desirable a much increasedto elicit RGC number responses of electrodes securely within and to the minimize limited ti devicessue damage area, such in the as thosestimulated reported area. in Typically, the previous the studieselectrode [7 ,materials22,44]. Using with the low proposed impedance fabrication show high method, charge we injection anticipate capacity that it (CIC), would and be possiblehigher CIC to µ decreasecan lead to the higher electrode current size density down to in 70 safem, voltag increasinge limits, the without number undesired of electrodes electrochemical up to 260 within reactions the same[37]. For area this, of the it would device. be better to change the electrode material to the one that can result in lower impedance in the same area. For example, activated iridium oxide film (AIROF) or sputtered iridium oxide film (SIROF) can lower the impedance of the electrodes further down to tens of kΩ [37,45,46]. In previously reported studies [7,11,21,22,47,48], various types of retinal electrodes were fabricated using AIROF or SIROF to achieve the low electrode impedance and high CIC. A previous study investigated the threshold charge density according to electrode sizes [49]. Low threshold charge densities were obtained using a large stimulation field of electrodes (≥ 0.078 mm2), coated with SIROF. Assuming the same experimental condition that iridium oxide were used as the electrode material, the fabricated electrodes (with an area of 0.018 mm2) were expected to have the threshold charge density of about 50 μC·cm−2 based on the results presented in [49]. As further studies, we are planning to change the material of the electrodes and verify the subretinal stimulation of the fabricated electrodes in in vitro and in vivo environments.

Micromachines 2020, 11, 467 8 of 12

The hexagonal electrode arrangement allows higher electrode densities compared to the rectangular arrangement [34]. Thus, the hexagonal arrangement not only makes it possible to allow a higher resolution of the phosphene image in the same area allowed for a device, but would also have slight advantages in acuity tasks [33,34]. A simulation study reported that by using guarded hexagonal return electrodes, effective retinal stimulation can be ensured, if the diameter of the electrodes under 100 µm is used and the distance between the electrodes and the target neurons is far enough [31]. The effectiveness of the hexagonal return electrodes in minimizing crosstalk between pixels and localized activation of retinal cells, however, needs to be investigated in in vitro as well as in vivo environments, using the fabricated electrodes. Figure7 shows the assembled device for electrochemical characterization and the results. The electrode array combined with the parylene-C cable was connected to a printed circuit board (PCB) for the impedance and CV measurement, as shown in Figure7a. Overall, 32 microelectrodes were connected to the ZIF connector through the first connection pads of the parylene-C cable. The second and third parts of connection pads were not used in the initial electrode characterization, but reserved for the next experiments using all electrodes in the near future. The impedance of the microelectrodes was measured to be 384.87 52.11 kΩ at 1 kHz (Figure7b). In the array, the area of an exposed electrode ± was about 0.018 mm2, and the measured impedance at 1 kHz was 21.4 MΩ mm 2 when converted · − to a value per unit area. In the previous studies [30,37], the unit-area impedances of Pt-coated 3D electrodes were 30.3 MΩ mm 2 and 62.5 MΩ mm 2, respectively. The obtained impedance using the · − · − fabricated electrodes was comparable to those values. The CV of the electrodes was also measured, as shown in Figure7c. The calculated CSC was 2.83 0.22 mC cm 2 and comparable to the values ± · − reportedMicromachines in the 2020 previous, 11, x studies [30,37], ranging from 1.24 mC cm 2 to 4.4 mC cm 2. 9 of 12 · − · −

Figure 7. a Figure( 7.) Completed(a) Completed electrode electrode array array integratedintegrated with with parylene-C parylene-C cable cable and and printed printed circuit circuit board board (PCB),(PCB), for electrochemicalfor electrochemical characterization. characterization. ( (bb)) Measured Measured impedance impedance of fabricated of fabricated microelectrodes. microelectrodes. MeansMeans and and standard standard deviations deviations for for magnitude magnitude and phase phase are are shown. shown. (c) (Cyclicc) Cyclic voltammogram voltammogram curves curves of theof Pt-coatedthe Pt-coated electrodes electrodes measured measured at at a a scanscan rate of of 50 50 mV·s mV −s1. 1. · − 4. Conclusions We have developed a hexagonally arranged 3D microelectrode array for subretinal prostheses. The base of the array was made of transparent PDMS, so that the photodiodes of the IC connected to the electrodes can sense light directly. By a customized pressing process utilizing the elastic property of the used material, a 3D structure of the electrode array was created, resulting in the electrodes that protruded around 20 μm from the PDMS base. The height of the protruded electrodes can be controlled by varying the pressing strength of the PDMS filled between the silicon pillars. The hexagonal arrangement of electrodes was realized through the chemical etching of silicon, with which any arrangement of electrodes other than hexagonal arrangement is also possible. Such hexagonally arranged electrodes are expected to stimulate retinal cells with a larger number of electrodes in a limited device area, compared to the rectangular arrangement of electrodes. By considering the pitch and size of the electrodes, the 3D microelectrodes can be expanded to have more pixels for even higher resolution if desired. In the future, in vitro and in vivo experiments will be conducted to evaluate the stimulating performance of the fabricated 3D microelectrodes; in particular, the effectiveness of hexagonal return electrodes.

Funding: This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation, funded by the Korean government (No. NRF-2017M3A9E2056463).

Micromachines 2020, 11, 467 9 of 12

Although Pt-coated electrodes with impedances in such a range were demonstrated to be able to stimulate the retinal cells [30], electrode materials capable of injecting higher charge are desirable to elicit RGC responses securely and to minimize tissue damage in the stimulated area. Typically, the electrode materials with low impedance show high charge injection capacity (CIC), and higher CIC can lead to higher current density in safe voltage limits, without undesired electrochemical reactions [37]. For this, it would be better to change the electrode material to the one that can result in lower impedance in the same area. For example, activated iridium oxide film (AIROF) or sputtered iridium oxide film (SIROF) can lower the impedance of the electrodes further down to tens of kΩ [37,45,46]. In previously reported studies [7,11,21,22,47,48], various types of retinal electrodes were fabricated using AIROF or SIROF to achieve the low electrode impedance and high CIC. A previous study investigated the threshold charge density according to electrode sizes [49]. Low threshold charge densities were obtained using a large stimulation field of electrodes ( 0.078 mm2), coated with SIROF. Assuming the same experimental ≥ condition that iridium oxide were used as the electrode material, the fabricated electrodes (with an area of 0.018 mm2) were expected to have the threshold charge density of about 50 µC cm 2 based on the · − results presented in [49]. As further studies, we are planning to change the material of the electrodes and verify the subretinal stimulation of the fabricated electrodes in in vitro and in vivo environments.

4. Conclusions We have developed a hexagonally arranged 3D microelectrode array for subretinal prostheses. The base of the array was made of transparent PDMS, so that the photodiodes of the IC connected to the electrodes can sense light directly. By a customized pressing process utilizing the elastic property of the used material, a 3D structure of the electrode array was created, resulting in the electrodes that protruded around 20 µm from the PDMS base. The height of the protruded electrodes can be controlled by varying the pressing strength of the PDMS ﬁlled between the silicon pillars. The hexagonal arrangement of electrodes was realized through the chemical etching of silicon, with which any arrangement of electrodes other than hexagonal arrangement is also possible. Such hexagonally arranged electrodes are expected to stimulate retinal cells with a larger number of electrodes in a limited device area, compared to the rectangular arrangement of electrodes. By considering the pitch and size of the electrodes, the 3D microelectrodes can be expanded to have more pixels for even higher resolution if desired. In the future, in vitro and in vivo experiments will be conducted to evaluate the stimulating performance of the fabricated 3D microelectrodes; in particular, the eﬀectiveness of hexagonal return electrodes.

Author Contributions: Conceptualization, H.W.S. and S.K.; methodology, H.W.S. and N.K.; investigation and validation, H.W.S.; visualization, H.W.S. and N.K.; writing—original draft preparation, H.W.S. and S.K.; writing—review and editing, H.W.S., N.K. and S.K.; supervision, S.K.; project administration, S.K.; funding acquisition, S.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation, funded by the Korean government (No. NRF-2017M3A9E2056463). Acknowledgments: The authors would like to thank staff members in the Center for Core Research Facilities at DGIST for their technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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