S1818

To analyze the behaviour of a single cells transfected with full length WT and P229S and to see the effect of the mutation on a single cell in detail, we used the method of photoresist lift-off assisted patterning of ECM proteins (LOP). Previously, Moeller et al. described the detailed protocol for this method (67 (link)). Shortly, the coverslips (18 × 18 mm Paul Marienfeld GmbH & Co. KG Lauda-Königshofen Germany) were cleaned using acetone, isopropanol and MQH₂O, dried and treated with oxygen plasma for 2 min (Vision 320 RIE, Advanced Vacuum). The photoresist S1818 (Microchem, Westborough, Massachusetts, USA) was spin-coated on the coverslips using the standard contact photolithography and 2 μm thick resist layer was photopatterned (5.6–7.5 mW/cm² at 365 nm, OAI instruments). The ice-cream patterns were designed using a chrome mask. The S1818 structure on the coverslip was treated with oxygen plasma for 15 s at 80 W (PICO plasma cleaner; diener) followed by incubating the S1818 structure with 0.1 mg/ml PLL-g-PEG in PBS solution for 30 min at room temperature. The coverslip was then rinsed with MQ and placed in a previously cleaned beaker for the lift-off procedure, protein coating and cell culturing. The lift-off and cell culturing procedures are described in the Supplementary Material.

Borosilicate coverslips (22 × 32 mm) intensely cleaned with acetone were sputtered with Cr and Au to the thickness of 30 nm and 200 nm, respectively. The ion beam sputtering system (EIS-220, Elionix, Tokyo, Japan) was used for sputtering. The typical sputtering time was 20 min for each process. Photolithography was then performed to form the electrode lines and connection pads by using the photoresist S1818 (MicroChem, MA, USA). After cleaning with piranha solution, to form the patterned windows for Pt/Pd islands, the substrate was subject to secondary lithography with the photoresist SU8-3005 (MicroChem, MA, USA). Pt/Pd was then sputtered to ~ 100 nm using the ion sputter (E-1030, Hitachi, Japan). The sputtering time was typically 180 s. To peel off the SU8 layer of the windows, the substrate was treated with N-Methyl-2-pyrrolidone (TCI, Tokyo, Japan) at 72 ºC for several hours After cleaning with piranha solution, the third lithography was done to form the insulator layer as well as the circular electrode openings with a diameter of 50 μm. A silicone washer with an inner diameter of 6 mm and a height of 1 mm was glued with poly-dimethylsiloxane to form the recording chamber. The whole device was kept in a desiccator and treated with plasma cleaning before use (PDC-32G, Harrick Plasma, N.Y., USA).

The fabrication steps of the soft liquid-metal neural probe are as follows: (1) a Si wafer (Dasom RMS, Republic of Korea) was spin-coated by a LOR 3 A lift-off resist (MicroChem) as a sacrificial layer. (2) A 1 µm-thick parylene-C layer was deposited as the bottom layer of a neural probe using a parylene coating system (NRPC-500, Nuritech Co. Ltd., Republic of Korea). (3) EGaIn was printed to 5-µm-wide lines on this parylene-C layer. (4) Another 2.5-µm-thick parylene-C layer was deposited as the top layer of a neural probe. (5) The photoresist S1818 (MicroChem) was spin-coated and then photolithographically patterned for defining the probe shape with opening the tip area of electrodes. (6) The areas of parylene-C where the S1818 layer did not cover was etched away by O₂ plasma using a reactive ion etching (RIE) system (LAT Co. Ltd., Republic of Korea), and then the S1818 photoresist was dissolved out using acetone. (7) The S1818 photoresist was spun again and then photolithographically patterned for opening the electrode pads, and procedure (6) was repeated. (8) The resulting probes were lifted off using a remover PG solution (MicroChem) by dissolving the sacrificial layer (the LOR 3A lift-off resist) and by releasing the neural probes from the Si wafer. (9) The released neural probes were rinsed with deionized (DI) water.