Fabrication of phototransistor arrays for the factitious retina
The gadget consists of Si channels (340 nm), Cr (5 nm)/Au (100 nm)/Pt (30 nm) supply (S)/drain (D)/interconnect electrodes, SiO2 dielectric layer (500 nm) and indium tin oxide gate (G) electrode (150 nm). The Si channel, which is a consultant photoabsorbing semiconductor, was chosen as a proof of idea, however it may be simply changed by different alternate options with greater sensitivity and adaptability (that’s, two-dimensional supplies) to additional improve the optoelectronic efficiency of synthetic retinas. For the fabrication of those phototransistor arrays, first, an array of single-crystalline Si, which serves because the channel of the transistor, was photolithographically patterned utilizing a constructive photoresist (S1818, MicroChem) on a silicon-on-insulator wafer (340 nm boron-doped p-type Si with a resistivity of 8.5 Ω cm on 400 nm buried oxide; Soitec). This transistor array was fabricated on a skinny and clear polyimide movie (thickness, 8 μm). The Si channels had been etched with an RIE system with sulfur hexafluoride (SF6) plasma (SF6 25 s.c.c.m./Ar 55 s.c.c.m.; 300 W/40 s), finishing the channel isolation course of. Any subsequently remaining photoresist residue was eliminated utilizing a piranha resolution (10 min). To separate the Si channel from the silicon-on-insulator wafer, the buried oxide layer was etched in a 50% hydrogen fluoride resolution for 18 min. Second, the sample of Si channels was transferred from a silicon-on-insulator wafer onto the versatile and clear polyimide movie (8 μm) utilizing a polydimethylsiloxane stamp (SYLGARD 184, 10:1 weight ratio of base and curing agent). Cr 5 nm/Au 100 nm had been deposited utilizing an electron-beam evaporator and had been photolithographically patterned to kind a supply (S) electrode, a drain (D) electrode and interconnects41,42,43. Then, a sacrificial layer (LOR 3A photoresist, Kayaku) was spun on the substrate and photolithographically patterned, and this was adopted by the deposition of 30-nm-thick Pt on the opened space with an electron-beam evaporator. This steel layer was deposited to forestall the penetration of gallium atoms to the Au drain electrodes. Subsequently, silicon dioxide (SiO2) was deposited with a thickness of 500 nm at 150 °C utilizing plasma-enhanced chemical vapour deposition, and it was photolithographically patterned as a dielectric layer. Then, for the patterning of the gate (G) electrode, a sacrificial layer (LOR 3A photoresist, Kayaku) was spun on the substrate and photolithographically patterned. Indium tin oxide was deposited as a gate electrode with a thickness of 150 nm at room temperature by radio-frequency magnetron sputtering, and it was immersed in mr-Rem 700 (lift-off resolution, micro resist know-how) at 60 °C for 30 min to soften the sacrificial layer. As a biocompatible encapsulation layer, a 1-μm-thick layer of parylene C was deposited and photolithographically patterned by dry etching with RIE (O2 40 s.c.c.m., 100 W/240 s) to open the world for the direct printing of 3D LM microelectrodes.
Fabrication of 3D LM microelectrodes
The important thing steps within the fabrication of the 3D LM microelectrodes are as follows:
(1) Direct printing of 3D pristine EGaIn electrodes: the direct printing system consists of a capillary nozzle linked to an ink reservoir; a pneumatic stress controller and a six-axis stage with automated actions within the x, y and z axes; two tilting axes within the x and y axes; and rotation within the x–y airplane. First, a pipette puller (P-1000, Sutter Instrument) was used to make a glass capillary (Sutter Instrument) as a nozzle with interior diameters of 5 to 50 μm. Then, a nozzle was mounted onto a syringe-type reservoir, and a substrate was positioned on the six-axis stage. The entire LM printing steps had been recorded by the microscope digicam (QImaging MicroPublisher 5.0 with real-time viewing, Teledyne Photometrics) to manage the nozzle from the substrate utilizing the six-axis stage (H-820 6-Axis Hexapod, Physik Instrumente) through the printing course of. The space between the tip of the nozzle and the substrate was managed to be within the vary of two–16 μm in line with the diameter of the nozzle, and the pneumatic stress (∼50 psi) was utilized to ship the EGaIn ink (75.5% gallium and 24.5% indium alloy by weight; Changsha Santech Supplies) from a reservoir onto the substrate by means of the nozzle. After we managed the z axis of the six-axis stage to make contact between EGaIn and the opened space of the drain electrode, the ink was instantly printed in a round form on the highest floor of the drain to exhibit a thicker base of the 3D micropillar for its structural stability. By adjusting the printing movement alongside the z axis at a velocity within the vary of 1 to 500 μm s–1, the 3D pillar of EGaIn with a uniform diameter (besides the round base half) could be printed (Supplementary Video 1). On publicity to air, EGaIn instantaneously types a skinny strong layer (∼1 nm) of gallium oxide on its floor beneath atmospheric oxygen ranges to keep up its vertical 3D construction of EGaIn. This oxide pores and skin is skinny sufficient to keep away from considerably damaging the mobile interfaces, and it’s strong sufficient to keep up its 3D form towards gravity and floor stress.
(2) Selective opening of 3D electrode suggestions: after the printing of the 3D pristine EGaIn electrodes, extra parylene C (thickness, 1 μm) was deposited on the whole gadget, together with the 3D electrodes for the passivation of their sidewalls. Solely their suggestions had been selectively opened utilizing anisotropic O2 RIE (100 W/240 s), as the extra parylene C encapsulating layer served as a protecting layer of the primary parylene C encapsulation layer in addition to the encapsulation layer of the sidewalls of the 3D micropillars.
(3) Deposition of Pt nanoclusters: to organize 50 ml of an electroplating resolution, we combined 50 ml of deionized water, 10 mg of lead acetate trihydrate (Sigma-Aldrich) and 0.5 g of platinum tetrachloride (Sigma-Aldrich) at room temperature. This electroplating resolution was stirred for 20 min by ultrasonic vibration. The electroplating was carried out by ion switch between the cathode and anode within the Pt electroplating resolution. After mounting the gadget to a multichannel recording interface (MZ-60, Tucker-Davis Applied sciences), a cathode (the 3D pristine EGaIn microelectrode that’s to be electroplated) and an anode (Ti/Pt electrode) had been immersed on this electroplating resolution, and every electrode was linked to a supply meter (Keithley 2400, Tektronix). {An electrical} present of 0.1 mA was utilized for 60 s to generate the electroplating response (Supplementary Fig. 25). As a consequence of potential variations in currents beneath gentle publicity, we carried out the electroplating of Pt nanoclusters at midnight state.
(3) Rinsing strategy of the factitious retina: earlier than the implantation of the gadget, we rinsed the factitious retina by gently immersing the gadget in 70% ethanol resolution (15 min) and deionized water (15 min) adopted by ultraviolet publicity (30 min).
Ex vivo animal experiments
Ex vivo experiments had been carried out based mostly on the rules and had been authorised by the Institute of Animal Care and Use Committee of Yonsei College (IACUC-A-201911-985-01, IACUC-202011-1164-05, Yonsei IACUC). The recording concerned the retinas of 5 mice for each WT and rd1 sort, and for the recording of retinal responses (that’s, visually or electrically evoked retinal spike potentials and firing price of the spikes); every recording electrode was positioned adjoining to every stimulation electrode (pitch between the stimulating and recording electrodes, 40 μm). The retinas of each WT mice (C57BL/6J, Japan SLC) and rd1 mice (C3H/HeJ, Japan SLC) had been explanted, and small items (∼4 × 4 mm) had been remoted and transferred to the factitious retina with the phosphate-buffered saline medium by RGCs going through the gadget, and a heating pad was used to keep up the temperature of the retinas at 37 °C. The animal was instantly sacrificed after extraction. The remoted retinas from WT and rd1 mice had been instantly positioned on our gadget (consisting of 36 stimulating and recording electrode pairs), and our 3D electrodes had been directed in direction of the RGC aspect of the retina in phosphate-buffered saline media. Instantly earlier than implanting this gadget into the retina, the gadget pattern was immediately frozen to show the liquid-phase EGaIn right into a strong by leaving it in chilly storage (beneath the melting level of EGaIn, ∼15.7 °C). Then, the protrudent pillar form of the 3D electrodes returned to a liquid part and didn’t collapse even after being implanted into the retina.
Since mice are dichromatic mammals having solely two cone varieties (blue and inexperienced gentle delicate)42,43, the blue gentle (wavelength, 470 nm) was used for publicity on this experiment. For electrical stimulation, the transistor of our gadget was operated with a selected situation (VG, d.c. bias of 5 V; VD: pulsed bias of 1 V with a length of 1 ms and frequency of 10 Hz) and the recordings had been carried out with the adjoining recording electrodes. Electrophysiological recordings of the retina had been carried out by multielectrode array recording and multichannel stimulation (PZ5 and Topic Interface, Tucker-Davis Applied sciences) and a knowledge processor with a real-time controller (RZ2 BioAmp Processor, Tucker-Davis Applied sciences). We recorded the VEP and EEP alerts at a 25 kHz sampling price utilizing a 300 Hz low-pass filter and three,000 Hz high-pass filter. The experimental information had been processed additional by making use of a band-pass filter with MATLAB R2021a (MathWorks). No information factors had been excluded from the analyses.
In vivo implantation
The rd1 mice (n = 3) had been anaesthetized with an intraperitoneal injection of a mix of tiletamine and zolazepam (1:1, 15 mg kg–1 physique weight) and xylazine hydrochloride (10 mg kg–1 physique weight). The pupils of the mice had been dilated with eye drops that contained 0.5% phenylephrine and 0.5% tropicamide. The physique temperatures of the mice had been maintained at 37 °C with a heating pad.
For the surgical procedures, the mouse was positioned in a head holder to keep up the top in a hard and fast place and to permit entry to the attention. The top holder was positioned beneath an optical microscope with an illuminator. A transparent 2.2 mm corneal knife (KAI MEDICAL; CCR-22AGF) was used to make a 1.5 mm incision within the space of the pars plana. Instantly earlier than implanting this gadget into the retina, the gadget pattern was immediately frozen to show the liquid-phase EGaIn right into a strong by leaving it in chilly storage (beneath the melting level of EGaIn, that’s, ∼15.7 °C). Then, it was implanted into the vitreous cavity (that’s, hooked up to the retinal surfaces) by way of the incision that was made earlier. For stopping cataracts throughout a steady practical evaluation, a ten g drop of hypromellose (Hycell oph soln) was utilized to the floor of the cornea. Throughout this in vivo experiment, a hydrogel-based synthetic vitreous physique was initially stuffed within the vitreous cavity of the mouse eye to forestall undesired uncomfortable side effects, comparable to hypotony (low intraocular stress). After the experiment, the mice had been instantly euthanized by carbon dioxide inhalation in a carbon dioxide chamber. No statistical strategies had been used to predetermine the pattern sizes, however our pattern sizes are just like these reported in earlier publications9,11.
In vivo animal experiments
In vivo experiments had been carried out based mostly on the rules of the Institute of Animal Care and Use Committee of Yonsei College (IACUC-A-202205-1478-01, Yonsei IACUC). Contemplating the dimensions of the eyeball of a mouse (diameter, ∼3 mm), we fabricated a synthetic retina built-in with 6 × 6 arrays of phototransistors (pixel pitch, 200 μm; gadget width, 2 mm) with 3D LM microelectrodes (top, 60 μm; diameter, 20 μm). This synthetic retina was implanted into the innermost retinal floor of the rd1 mouse epiretinal, with exterior gadget interconnections. All of the units and animals examined had been randomly chosen. The recording strains had been linked to the glass pad with interconnect electrodes after which patterned with photolithography and moist etching after the deposition of Cr/Au (10/100 nm) by an electron-beam evaporator. The interconnect pad was inserted into the multielectrode array recorder with a multichannel stimulator (PZ5 and Topic Interface, Tucker-Davis Applied sciences) and a knowledge processor with a real-time controller (RZ2 BioAmp Processor, Tucker-Davis Applied sciences). The multichannel experimental information of the spike sign and firing price had been obtained and exported by evaluation software program (Synapse Suite model 94, Tucker-Davis Applied sciences). Then, the info had been processed and mapped with MATLAB R2021a (MathWorks) and Origin 2022b software program. Full-field gentle illumination (470 nm, TouchBright T1 with BN470 band-pass filter, Dwell Cell Instrument) or a laser (wavelength, 415 nm) by means of an ellipsoidal sample of a shadow masks was utilized to the fundus of the mouse’s eye for gentle publicity (length, 5 s). No information factors had been excluded from the analyses. Additionally, information met the assumptions of the statistical assessments used.
Statistical evaluation
All information had been offered as imply ± customary deviation (s.d.). Statistical calculations of P worth had been carried out utilizing an open-source code of MATLAB R2021a. Significance was calculated utilizing an unpaired one-tailed t-test.
Reporting abstract
Additional info on analysis design is offered within the Nature Portfolio Reporting Abstract linked to this text.
