Fabrication of SU-8-Based Microelectrode Arrays

The surface MEA chip and flexible MEAs were fabricated following previous procedures with small modifications [34 (link)]. A Si wafer with a 1µm thick SiO₂ layer (University Wafer Inc., South Boston, MA, USA) was first cleaned by sonicating in acetone, isopropanol, and DI water sequentially for 5 min each step. The wafer was then dried on a hot plate at 150 °C for 3 min, then the surface was cleaned and activated by O₂ plasma using a reactive ion etcher (RIE, Trion Phantom III LT, Clearwater, FL, USA) for 120 s at 200 mTorr pressure and 150 Watts power. The wafer was then spin-coated with SU-8 2015 (MicroChemicals, Ulm, Germany) at 5000 rpm for 1 min and soft baked at 65 °C for 3 min and 95 °C for 5 min to evaporate solvent, then the wafer was exposed using a maskless aligner (MLA, MLA100, Heidelberg Instruments, Heidelberg, Germany) with a dose of 400 mJ/cm². After exposure, the SU-8 first layer was post-baked at 65 °C for 3 min and 95 °C for 5 min, developed using SU-8 developer (MicroChemicals, Germany) for 1 min, and cleaned by isopropanol and DI water, Then, the wafer was hard baked at 200 °C, 180 °C, and 150 °C for 5 min each for SU-8 curing and allowed to cool down below 95 °C (step skipped for in vitro MEA fabrication). The wafer was then treated with O2 plasma to clean, activate, and roughen the SU-8 with RIE for 75 s at a pressure of 200 mTorr and 150 W power. The treated wafer was then spin-coated with AZ P4620 photoresist (MicroChemicals, Germany) at 5000 rpm for 1 min and baked at 105 °C for 5 min for resist curing. After baking, the wafer was exposed using MLA with a dose of 700 mJ/cm², then developed using AZ400k 1:4 developer (MicroChemicals, Germany), cleaned by water rinse, and dried with N2 gas flow. A mild 120 s RIE O2 plasma treatment at pressure 600 mTorr and 60 W power was performed to clean the SU-8 before metal deposition. A 10 nm Ti adhesion layer and 100 nm Au layer were evaporated on the wafer using an Electron Beam Evaporator Plassys MEB550S (Marolles-en-Hurepoix, France). The metal was then lifted off in acetone overnight. The next day, the wafer was first rinsed with water, dried under N₂ flow, and cleaned by O2 plasma for 60 s at 600 mTorr and 60 W, then spin-coated with SU-8 2015 for insulation layer at 5000 rpm for 1 min and soft baked at 65 °C for 2 min and 95 °C for 5 min. The wafer was then exposed using MLA with a dose of 400 mJ/cm², post-backed, and developed with an SU-8 developer. The wafer was then cleaned with isopropanol and water, and was hard baked at 200 °C, 180 °C, and 150 °C for 5 min each to fully cure SU-8 and allowed to cool down below 95 °C. For in vitro use, the wafer was then cut into rectangular pieces (1.5 × 2 cm) containing an MEA and used for in vitro experiments. For in vivo use, the flexible MEAs were released from the wafer using buffered oxide etchant (1:7) (MicroChemicals, Germany) in an acid hood for 8 h to etch away the SiO₂ layer. Customized PCBs were mounted with ZIF and Omnetics connectors. ZIF connectors were used to interface with flexible MEAs, and Omnetics connectors were used to interface with the potentiostat and electrophysiology recording system.