An open-source transparent microelectrode array

Our goal was to develop a transparent MEA that combines high performance with low cost. We sought to keep raw-material cost below $10 per device and maintain compatibility with academic cleanroom facilities. To keep with these goals, MEA fabrication was designed to use basic microelectronic photolithographic techniques [15]. For transparency, glass was used as the base substrate. For patterning the electrical components of the array, we considered many materials that would maintain transparency. We excluded certain materials, such as graphene [16], which require advanced fabrication, and thus may pose a challenge for open-source assembly. Conversely, we excluded insulator materials such as nitride or oxide owing to the specialized equipment required to deposit such materials and the additional steps needed to pattern them [17]. We ultimately selected indium tin oxide (ITO) as the conductor and SU-8 as the insulator due to their transparency, electrical conductivity, biocompatibility, and ease of fabrication [18, 19].

Fabrication was performed at the Shared Instrumentation Materials Facility cleanroom at Duke University. ITO-coated borosilicate glass wafers (100 mm diameter, ∼10 Ω sq⁻¹, University Wafer) were used as base substrates (figure 1(A)). P20 adhesion promoter (Shin-Etsu Micro Si) was flash deposited while each wafer was spinning on the spin coater (Headway) then Shipley S1813 positive photoresist (Kayaku Advanced Materials) was deposited and spun at 3000 rpm for 30 s, and baked for 1 min at 115 °C (figure 1(B)). Wafers were exposed on a mask aligner (MA/BA6, Karl Suss) for a 120 mJ cm⁻² ultraviolet (UV) exposure developed in MIF 319 developer (Kayaku Advanced Materials) for 60 s with mild agitation, and then thoroughly cleaned with dionzed (DI) water (figure 1(C)). After inspecting the lithography of each wafer, the exposed ITO was etched by submerging samples into a strongly acidic solution (4:2:1 HCl:H₂O:HNO₂ by volume); etch time varied from 3 to 5 min depending on the potency of the acidic solution, which decreased exponentially over time due to the reaction of HCl and HNO₂ to form aqua regia (figure 1(D)). To ensure that the ITO had been etched sufficiently, the sheet resistances of etched areas were measured using a four-point probe and confirmed to be at least 100 × 10⁶ Ω sq⁻¹ before proceeding. After etching, remaining photoresist was removed by sonicating each sample in acetone for 10 min, exposing the patterned ITO conductive material (figure 1(E), blue).

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SU-8 (Kayaku Advanced Materials) insulator was deposited onto the samples (figure 1(F)) by spin coating at 2000 rpm for 30 s (for a thickness of 2 µm), then baked at 65 °C for 1 min, followed by 95 °C for 1 min. This temperature ramp was important as it minimized the mechanical stress applied to the SU-8 layer, which can crack if heated too quickly. The wafers were then allowed to cool slowly to ambient room temperature. To prepare the wafers for exposure, opaque wafer dicing tape (Ultron Corporation) was placed on the back of the wafers to prevent unwanted reflection of UV light during exposure of 100 mJ cm⁻². The exposed samples were then baked in the same step-wise fashion as previously described before developing for 90 s with fast agitation. The samples were cleaned with isopropanol, inspected using green light, and baked for 30 min at 150 °C. Thus, the insulator was removed from the electrode pads (i.e. the recording surface) while retaining insulator over the wiring (figure 1(G)).

After baking, the wafers were inspected to ensure that all 20 µm diameter pads had been fully cleared of SU-8 insulator. The wafers were then diced into four MEA devices. For the culture well, oval chambers (6 mm tall × 23 mm long × 16 mm wide with 2 mm wall thickness) were laser cut from an acrylic sheet (8560K358, McMaster Carr). After cutting, the wells were cleaned thoroughly to remove any charring. For final assembly of each device, the bottom of an acrylic well was coated in a thin layer of polydimethylsiloxane (PDMS) polymer (761036, Millipore Sigma) and center-placed atop a MEA device. The assembled device is baked at 80 °C for 1 h to cure the PDMS. The final device has a 24 mm × 40 mm footprint (figure 3(B)).

The PCB was designed as an interface between the MEA and the OpenEphys acquisition system. Our goals were to minimize electrical noise, maximize mechanical stability with repeated use, and to make it easy to quickly connect or remove a sample. To achieve the mechanical goals, the PCB uses spring-loaded pins to make contact with the MEA, enabling reliable contact and easy loading of MEAs with minimal physical wear on the PCB (figure 2(A)). For compatibility with the OpenEphys system, the 64 channel board connects directly to two 32-channel RHD 32 headstages (C3314, Intan Technologies) via industry-standard Omnetics connectors, immobilized with cyanoacrylate glue for physical stability. For electrical considerations, we incorporated a large ground plane that can be connected to a surrounding Faraday cage.

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Ease of integration was important when designing the interface with existing inverted microscopes. Here we detail two versions of the stage interface: the first designed specifically to fit into an ASI AZ-2000FT stage, and the second to fit into a 24-well plate cut out for compatibility with most microscopes. Both designs use a clamshell base to interface with the microscope stage, and a clamshell top affixed to the PCB. The design allows an MEA to be easily inserted within the clamshell, ensuring reliable contact between the MEA and spring-loaded pins of the PCB. A cut-out in the base allows for optical access from below.

To assemble the system, the clamshell base is attached to the microscope stage, then the PCB is bolted to the clamshell top. Next, an MEA is centered in the clamshell base cut-out, and the clamshell top tightened down using wing nuts. The RHD 32 headstages are then attached to the board and a silver chloride reference electrode is added to the well and connected to the reference pin on the board (figure 2(B)).

To increase conductivity of the 20 µm diameter MEA pads, we polymerized PEDOT:PSS (poly ethylenedioxythiophene:poly styrene sulfonate) via standard protocols [20]. Briefly, a solution of 10 µM EDOT and 100 µM PSS was prepared in deionized H₂O, applied to the MEA surface, and connected to a Keithley 2450 source measure unit (Keithley 2450-EC, Tektronix, Inc.) such that the anode corresponded to the ITO pads of the MEA, and the cathode was a silver chloride wire in solution [21]. Electropolymerization was driven by holding a potential of 1 V for 10 min (figures 3(A) and (B)). The bare ITO 20 µm diameter MEA pads were found to have a measured impedance of 1.15 ± 0.19 MΩ at 1 kHz. After polymerization, the PEDOT coated ITO electrode sites were found to have a measured impedance of 92.75 ± 2.97 KΩ at 1 kHz. These values are consistent with previous studies [22].

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Devices were coated with high molecular weight poly-d-lysine (PDL) to improve neuronal attachment and biocompatibility [23]. One milliliter of PDL (P7405, Sigma) diluted to 0.5 mg ml⁻¹ was added to the well and incubated for 1 h at room temperature with mild agitation. The well was washed ten times with water, followed by an ethanol wash, then left to dry.

Primary hippocampal neurons were isolated from postnatal rat pups using established protocols [24], then electroporated (V4SP-3096, LONZA) with genetic constructs containing ChR2-mScarlet (opsin that drives action potentials in response to light fused to a red fluorescent protein) and GCaMP6s (green fluorescent calcium indicator) [25]. Approximately one million cells were seeded onto each device and flooded with NbActiv4 media (NB4, BrainBits). Samples were maintained in a 37 °C, 5% CO₂ incubator for 2 weeks, with half of the media replaced at DIV3 then weekly thereafter. Mature cells were healthy on the surface of the devices (figure 3(B), right).

Samples were prepared for recording by gradually replacing NB4 culture media with Tyrode's solution to minimize shear stress. The MEA devices were then mounted onto an inverted fluorescent microscope (IX83 with 10× APO air objective, Olympus Lifescience) using the ASI-stage clamshell design and connected via dual Intan RHD 32-channel headstages to the OpenEphys acquisition system. Samples were centered on the stage and focused under at 580 nm excitation (mScarlet). For optogenetic induction of action potentials, samples were illuminated with 470 nm blue light. Electrical recordings were collected in the OpenEphys software environment and analyzed offline in Matlab.