Additional file 1 of Optimizing the fabrication of a 3D high-resolution implant for neural stimulation
Creators: name: Shpun, Gal
name: Farah, Nairouz
name: Chemla, Yoav
name: Markus, Amos
name: Leibovitch, Tamar Azrad
name: Lasnoy, Erel
name: Gerber, Doron
name: Zalevsky, Zeev
name: Mandel, Yossi
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Computational Biology

description: Additional file 1: S1. UV dose effect on AZ positive photoresist patterning. a) Photoresist residues inside the AZ pattern as the result of underexposure. b) A clean AZ pattern following a sufficient exposure dose. Scale bar-50 µm. S2. Resolution marks on SU-8 layers used to optimize the UV exposure dose for each layer. The required dose depends on the light source power, the features’ density, and size as well as the photoresist’s properties and thickness. a) SU-8 3005 (5µm in height), UV dose 200mJ/cm2 with a resolution down to 3µm (5.5 11.5µm in height). b) SU-8 3025 (11.5µm in height), UV dose 300mJ/cm2 with a resolution down to 20µm. Scale bar-20µm. Fig. S3. Electrode detachment due to poor metal-SU-8. Fig. S4. Strong attachment of relief gold between two electrodes due to surface tension. a) SEM image top view, Scale bar-5µm. b) Zoom-in obtained through FIB cross sectioning of the area demarcated with a white rectangle in a. Scalebar 3µm. Fig. S5. Resolving the Thermal stress effect on the SU-8 surface. (a) Cracks in the SU-8 surface (black arrows) resulting from thermal stress as is visualized by bright-field imaging. (b) Gradual curing and the addition of a material with nickel as an intermediate CTE value resolved the thermal stress effect. Scale bar-200µm. Fig. S6. Resolving the “stream-line” effects on the gold coating of the AZ photoresist in a multi-layer configuration. Images of the AZ photoresist patterned with dots sized 20-80µm and coated with Cr/Au (20/200nm). a-b) The “stream-lined” effect is clearly visible (white arrows), where an AZ line splits the circular implant, after it is concentrated in the external ring that defines the implant). c-) Good uniformity of AZ patterning is obtained by the additional steps of slow spin rate coating and full manual deposition. Scale bar - 0.5mm. Fig. S7. Optimization of implant release by wet etching of a sacrificial layer while preventing electrode detachment. a) A representative image of an SU-8 implant demonstrating electrode detachment following FeCl3 etching at room temperature for 5h. b) Electrodes are preserved intact following an optimal release process, where Nickel (Ni, 200nm) served as a sacrificial layer, Chromium (Cr, 10nm) as an adhesion layer, and HNO3 as an etchant at room temperature overnight. Scale bar-200nm. Fig. S8. XPS survey spectrum of the modified gold surface revealing the presence of RGD. In the inserts, a zoom-in on the N1s and S2p peaks show the presence of the amin (NH3) and thiol (SH) groups from the peptide on the gold surface, respectively. Fig. S9. SU-8 surface treatments. a-c) Contact angle measurements for various SU-8 surface treatments. a) Untreated cured SU-8 used as a control, 80o C. b) N2 plasma-treated cured SU-8 (150W, 2min), 40oC) O2 plasma-treated cured SU-8 (150W, 2min), 7.5oC. d-i) PRP cell growth affected by surface treatment. d-f) 1-day post-seeding. g-i) 4 days post-seeding. a-g) Cured untreated SU-8 used as a control. c-d) N2 plasma (100W, 2min). e-f) O2 plasma (100W, 2 min). Acquired by a bright-field microscope. Scale bar-100μm. Fig. S10. Retinal response for current stimulation. Representative average fluorescence change highlighting successful robust stimulation induced by repetitive current stimuli administered at 0.2 Hz. The disappearance of these responses 1min after the addition of verapamil Ca+2 blocker to the medium (denoted by a black arrow) and their recovery following washout can be readily seen. Red stems denote the stimulation time. Scale bar-100µm. Fig. S11. Retinal response to high-resolution electrical stimulation. (a) Localized fluorescence changes in response to electrical stimulation of a RGC-GCaMP6f-labelled retina imaged using a 40x objective; scale bar=10µm. (b) A representative average fluorescence change (in the area denoted in red in a) induced by increasing the current amplitudes indicating an activation threshold of 0.8mC/cm2. (c) The overall strength-duration curve of the investigated retinal tissue. (red asterisk) and the Lapicque fit (the solid blue line). Fig S12. Device Biocompatibility. Cell viability, defined as the ratio between the dead cells (visualized using the proTUNNEL staining) and the overall counted nuclei (visualized using Hoechst), of ARPE cells seeded on a control surface (cover glass) and ARPE cells seeded on an implant.