MicroElectroThermoForming (μETF): One-step Versatile 3D Shaping of Flexible Microelectronics for Enhanced Neural Interfaces

MicroElectroThermoForming (μETF): One-step Versatile 3D Shaping of Flexible Microelectronics for Enhanced Neural Interfaces | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MicroElectroThermoForming (μETF): One-step Versatile 3D Shaping of Flexible Microelectronics for Enhanced Neural Interfaces Joonsoo Jeong, Dong Hyeon Lee, Younghoon Park, Yoon Seo, Hyunbeen Jeong, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4924574/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2025 Read the published version in npj Flexible Electronics → Version 1 posted 13 You are reading this latest preprint version Abstract Increasing the proximity of microelectrode arrays (MEA) to targeted neural tissues is crucial for establishing efficient neural interfaces for both recording and stimulation applications. This has been achieved by constructing protruding three-dimensional (3D) structures on top of conventional planar microelectrodes via additional micromachining steps. However, this approach adds fabrication complexities and limits the 3D structures to certain shapes. We propose a one-step fabrication of versatile microscopic 3D structures for thin-film MEAs via “microelectrothermoforming (µETF)” of thermoplastics, by utilizing 3D-printed molds to locally deform planar MEAs into desired protruding and recessing shapes. Electromechanical optimization of the µETF process enabled a 3D MEA with 80 µm protrusions and/or recession for 100 µm diameter. Its versatile shaping capabilities are demonstrated by simple and simultaneous forming of diverse 3D structures on a single MEA. The benefits of 3D MEA are evaluated in retinal stimulation through numerical simulations and ex vivo experiments, confirming a threshold lowered by 1.7 times and spatial resolution enhanced by 2.2 times. Physical sciences/Engineering/Electrical and electronic engineering Biological sciences/Biotechnology/Nanobiotechnology/Bionanoelectronics microelectrode arrays liquid crystal polymer neural interface microthermoforming 3D electrode Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction High proximity of electrodes to neural cells plays a critical role in achieving efficient neural interfaces for various recording and stimulation applications 1 – 4 . Improved signal quality in neural recordings can result from intimate contact between the electrode surface and targeted tissues 1 , 3 , 5 , 6 , whereas in neural stimulation, lower stimulation thresholds and higher spatial resolution can be achieved through reduced current spreading 2 , 4 , 7 , 8 . Thus far, improved electrode–cell proximity has typically been achieved using microelectromechanical system (MEMS) technologies, either by directly structuring rigid materials into needle-like shapes, such as Utah arrays, or by building various three-dimensional (3D) microstructures on top of thin film-based microelectrode arrays (MEAs), such as pillars 1 , 4 , 5 , 9 – 11 , mushrooms 2 , 7 , 12 , hemispheres 13 , sheaths 14 , and arrowheads 15 . However, despite their effectiveness, rigid 3D electrodes may exhibit mechanical mismatch with soft neural tissues. Furthermore, the creation of 3D microstructures on top of thin-film MEAs using conventional MEMS technologies requires additional fabrication steps, such as photolithography, vacuum deposition, electroplating, and wet and dry etching 16 – 18 . These steps increase the complexity of the fabrication process, the degree of which usually scales with the complexity of the 3D structures. Moreover, it is challenging to fabricate 3D features with different shapes and heights within a single MEA, owing to the inherent 2D nature of the traditional microfabrication process. To address these problems, we propose a novel “microelectrothermoforming (µETF)” process for producing versatile 3D microstructures on a polymer-based MEA through a simple one-step thermal pressing of conventional planar arrays. This method leverages a well-established thermoforming technique used in the plastic industry, in which a thermoplastic sheet is heated above the glass transition temperature (T g ) and pressed against a metal mold to create 3D structures in a simple manner 19 . The microelectronic adaptation of thermoforming in this study enables the creation of microscopically protruding and recessed versatile 3D structures with embedded electrical functionalities, facilitating the development of tailored structures for optimized electrode–cell interfaces. Thermoforming and microthermoforming for biomedical applications have been explored by Truckenmüller et al. 19 and in subsequent studies, in which heated thermoplastic sheets were pneumatically or mechanically pressed against perforated metal molds to generate 3D shapes, such as microwell platforms 20 , 21 , cell culture chips 22 , 23 , and microchannels 24 , 25 . However, the methods demonstrated in those studies primarily formed bulk polymer sheets that lacked active components such as electrical contacts and interconnections. Additionally, they required specialized tooling to exert pneumatic pressure, and their formable 3D structures were commonly limited to hemispherical shapes. The proposed µETF process effectively addresses the challenges associated with fabrication complexity and structural restrictions in known 3D microstructures for enhanced neural interfaces. Microelectrothermoforming involves the one-step thermal pressing of a conventionally prepared thin-film planar MEA against a mold carrying the desired 3D structures. Therefore, two distinct advantages can be achieved compared to existing MEMS-based 3D forming: 1) process simplicity and 2) shape versatility. The proposed approach simplifies 3D fabrication by adding only one pressing step to the fabrication of a conventional planar thin-film MEA. Moreover, the use of 3D printing technology for preparing molds enables the formation of virtually any 3D microstructures, allowing heterogeneous structures of diverse shapes and heights to be created within a single electrode array via the same one-step µETF process. This simplicity and flexibility in forming 3D microstructures can expand design possibilities, facilitating the development of optimized electrode–neuron interfaces that align with the anatomical and neurophysiological features of a targeted nervous system. Liquid crystal polymer (LCP) film was used as a thermoplastic substrate in this study, primarily owing to its mechanical strength, chemical inertness, and biocompatibility. Additionally, the low water absorption rate of LCP can contribute to the long-term reliability of chronically implanted devices 26 . However, µETF can be utilized for other thermoplastics commonly used in biomedical applications. This study introduces a one-step µETF process for generating versatile protruding and recessed 3D structures on LCP-based MEAs (LCP MEA) with mechanical considerations for preserving the electrical properties of MEA. As a proof of concept, the 3D MEA was optimized for retinal stimulation, the benefits of which were assessed via both computational analysis and ex vivo experiments in mouse model. A variety of 3D structures, such as wells, domes, walls, and triangles, were constructed on LCP MEAs, demonstrating the potential utility of the µETF in a wide range of biomedical applications. Results mETF for simple and versatile 3D structuring Representative benefits of 3D MEA are illustrated in Fig. 1 a. The protruding structures enable closer proximity to target cells, establishing more localized neural interfaces for both neural recording and stimulation 27–30 . This is in contrast to conventional planar MEAs, in which the electrode surfaces inherently lie below the top surface 31–33 . As outlined in Fig. 1b, the proposed one-step mETF process locally deforms a planar MEA into a 3D MEA with microscopic protruding and/or recessed structures. A planar 25-channel LCP MEA prepared via a conventional microfabrication process 32 (see Supplementary Fig. S1) is thermally pressed (> T g ) against a 3D-printed mold within a set of metal jigs and elastomer layers for alignment (see Supplementary Fig. S2–3 for details), replicating the protruding and/or recessed 3D microstructures of the mold to the MEA, as shown in Fig. 1c. Additionally, the microelectrothermoformed 3D MEA can be grossly deformed into nonplanar shapes to conform to surrounding tissues, such as eye curvature for retinal electrodes, through a similar “macro” electrothermoforming step (mETF, see Supplementary Fig. S4). Using a 3D mold with 80 mm-height pillars (Supplementary Fig. S5), the schematics (top row) and photographs (bottom row) in Fig. 1d show the evolution of a (i) 40-mm-thick planar LCP MEA to a (ii) protruding or (iii) recessed mETF MEA, and subsequently to a (iii) mETF + mETF MEA, achieving both high proximity to target cells and conformability to target tissues. The recessed structures in (iii) can be easily generated using the identical processes and tools to those for the protrusions, simply by flipping the planar array upside down in the fixture, offering a convenient approach to create well-like structures for highly localized electrode–cell environments 29 . Scanning electron microscopy (SEM) images of individual electrode sites before and after mETF are presented in Fig. 1e, with their cross-sectional profiles shown in Fig. 1f. Each channel site with a diameter of 200 mm was selectively elevated or lowered by a height of 80 mm, which represents an optimized height for subretinal electrode arrays as a proof-of-concept application of mETF (details in subsequent sections). The optical surface profiles of the protruding (top) and recessed (bottom) channel sites are shown in Fig. 1g. The mETF produced 3D structures that replicated the original mold structures, exhibiting a slightly widened base diameter (~150%) and sloped sidewalls (~70°). The electrode diameter as small as 100 mm was also successfully thermoformed using the same mETF configuration (Supplementary Fig. S6), which was utilized for following ex vivo experiments. Similarly, versatile protruding and recessed structures of diverse heights and shapes can be created without adding fabrication complexities. Figure 2 a–f present 25-channel LCP MEAs formed with varying protrusion/recessed heights from 80 to 200 mm using a 3D mold (Supplementary Fig. S7). Their SEM images (Fig. 2a, b), cross-sectional images (Fig. 2c, d), and optical 3D profiles (Fig. 2e, f) confirm the faithful replication of the 3D mold structures onto the LCP MEAs. Furthermore, the versatility of the microthermoforming process was demonstrated with various microstructures, including polygons (triangles, rectangles, and hexagrams), ovals (sunken, plateau, and walled), domes, and S-shaped walls, as shown in Fig. 2g and h; their SEM images and optical profiles are shown in Fig. 2i and j, respectively. All distinct structures were created via one-step mETF using corresponding 3D molds shown in Supplementary Fig. S7. Such versatility and consequent design flexibility can be leveraged to create optimized 3D structures tailored for various in vivo and ex vivo neural interfacing applications. Electromechanical considerations for mETF Despite the simplicity and versatility of the mETF process, it is important to consider the tensile stress exerted on the thin gold layers, which may lead to cracks or disconnections. Therefore, the mETF process was optimized with respect to the 3D mold design and pattern layout. Fig. 3 a presents a finite element analysis (FEA) of the mechanical stress induced within the embedded thin gold layer during an 80-mm-height mETF. Based on the relative stress distribution for the top, bottom, and neutral planes within a gold layer (Fig. 3b), the highest tensile stresses are expected at the top and bottom corners of the sidewall (insets in Fig. 3a). On the other hand, the top protruding area remained relatively stress-free because of the pillar-shaped mold with a flat top surface. The resulting plateau-like structure of the mETF electrode ensured minimal damage to the circular electrode area, which were designed to be 10 mm smaller than the mold top diameter, as outlined on the transversal stress distribution in Fig. 3c. This is in contrast to forming a 3D structure using a dome-shaped mold, in which electrode sites are subjected to highest tensile stress, leading to significant cracks on the gold electrode surface after mETF (Supplementary Fig. S8). The mechanical strength of microelectronic tracks was enhanced by electroplating the gold patterns up to 4 mm thickness ( t ). Additionally, the interconnection lines traversing the region under the greatest stress is configured with serpentine shapes for enhanced robustness against elongation, as shown in Fig. 3c and inset ( r = 20 mm, d = 40 mm, and q = 180˚). Typical serpentines adopted in stretchable electronics exploit global out-of-plane buckling of thin gold tracks embedded within freestanding polymer layers 34,35 . However, the gold serpentine layer in mETF is subject to in-plane constraint as they are pressed between jig and mold. Therefore, the stretchability of such in-plane serpentine gold interconnections was evaluated in comparison with the conventional out-of-plane wavy lines via FEA simulation under the maximum local elongation during 80 mm-height mETF (ε = 15%, red area in Fig. 3c). As shown in Fig. 3d and e, both in-plane and out-of-plane serpentines exhibited reduced maximum stress with lower w and greater q (more details in Supplementary Fig. S9). Although in-plane restriction resulted in 33% higher peak stress than out-of-plane buckling serpentines with w = 10 mm, the wavy patterns are still effective in relieving the tensile stress of gold lines during mETF, by reducing the peak stress by 90% from the straight line. These design considerations were experimentally validated using patterns with varying shapes (straight and wavy) and thicknesses ( t = 300 nm to 4 mm) in 80-mm-height mETF. The representative SEM images in Fig. 3f demonstrate that damages to the gold layers were caused by two distinct mechanisms, as quantified by yield analysis after mETF in Fig. 3g. The “electrode cracks” occurred mostly in the thin gold patterns ( t = 300 nm and 2 mm) with relatively lower mechanical strength, resulting in a distributed crack-formation throughout the circular electrode sites. On the other hand, circular electrodes with higher gold thickness (4 mm) remained intact, while focused mechanical stress caused a single spot of line disconnection, which corresponds to the location of the highest stress estimated in Fig. 3c and d. The wavy shapes with w = 10 mm and t = 4 mm (III) was adopted as the optimized parameters throughout this study for the 80 mm-height-mETF, because they secured 100% tolerance (Fig. 3g). Thicker patterns than 4 mm did not provide mechanical enhancement (Supplementary Fig. S10). Electrochemical and mechanical analysis of mETF MEA The intactness of the neural interfaces during mETF was confirmed via electrochemical analyses, including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) of MEAs before and after mETF. Additionally, iridium oxide (IrOx) layer was electrodeposited on top of the gold channels before and after deformation to demonstrate the compatibility of mETF process with nanoporous surface functionalization techniques widely used for enhancing the charge transfer capabilities of neural interfaces 36,37 . As shown in Fig. 4a and b, the impedance magnitude and phase at 1 kHz showed no significant changes after mETF for both gold and EIROF electrodes (gold: 32.3 ± 10.8 to 27.3 ± 14.1 kW, IrOx: 4.1 ± 0.5 to 3.7 ± 0.4 kW; see Supplementary Fig. S11a and b for EIS spectra). Figure 4c presents the cathodic charge storage capacities (CSC C ), which also confirmed that mETF did not result in any significant degradation (gold: 0.24 ± 0.02 to 0.18 ± 0.02 mC/cm 2 , IrOx: 38.8 ± 8.2 to 35.1 ± 11.7 mC/cm 2 ; see Supplementary Fig. S11c for CV curves). The mechanical resilience of mETF protruding structures against compression was evaluated to assess the potential deformation of the mETF LCP structures under physiological or surgical conditions. As shown in Fig. 4d, the mETF MEA with a height of 80 mm were subjected to increasing compression using a motorized force meter. The magnified plot in Fig. 4e suggests that the 3D electrodes are compressed by no greater than 2.2 mm under the normal range of pressures experienced in physiological conditions, including (i) intraocular pressure (1.3–2.8 kPa 38 ) and (ii) intracranial pressure (0.9–2 kPa 39 ), as described in Fig. 4f. Even after the mETF MEA was completely flattened under a load pressure of 150 kPa, the original height and mechanical properties were recovered after the force is released, as demonstrated by the three cycles of full compression and relaxation (Fig. 4d). These results suggest that the proposed mETF with proper designs enables the reliable production of 3D structures without compromising the physical and electrochemical properties of neural interfaces. Benefits of mETF MEA for subretinal stimulation in FEA study The benefits of mETF MEA with protruding structures were evaluated for subretinal stimulation of blind patients through FEA in a human retinal model 40,41 . A subretinal electrode is implanted under the retina, facing toward the bipolar cells and retinal ganglion cells (RGCs) (see Supplementary Fig. S12 and Table S1 for retinal model). Given the distance between the electrode surface and targeted bipolar cells in the inner nuclear layer (INL), subretinal stimulation is expected to benefit from protruding 3D structures which would create more focused current distribution at the target cells. Improvement in neural interfaces enabled by 3D subretinal electrodes was quantified in terms of stimulation threshold ( I th ), dynamic range (DR), and spatial resolution, as shown in Fig. 5 . The I th and DR were evaluated based on the E-field distribution at INL generated by current injection from a 3×3 subretinal electrode array with varying protruding heights from 0 to 100 mm. The I th is defined as the current intensity required to induce an electric field (E-field) exceeding 3,000 V/m at the plane of the INL to activate bipolar cells 27,42,43 . The DR is a current range from I th to the maximum current ( I max ), beyond which inter-channel interference occurs between adjacent electrodes. The representative E-field distributions at INL from planar and 80-mm-protruding MEA are presented with increasing stimulation currents from planar in Fig. 5a and b. Notably, the 80-mm-protruding electrodes lowered the I th to 4.5 mA from 6 mA of the planar electrodes. Increasing current beyond the I max began to induce inter-channel interference, at which the INL area activated by a channel (contoured by white dashed lines in Fig. 5b) overlapped with the area activated by adjacent channels. The onset of interferences defines the upper boundary of the stimulation DR, which was extended from 14.5 mA for the planar array to 18.9 mA for the 80-mm-protruding array. A similar analysis for varying protruding heights from 0 to 100 mm (see Supplementary Fig. S13) suggested that the higher protrusions resulted in lower I th and wider DR, as plotted in Fig. 5c. Such enhancement of 3D electrodes can be explained by more focused current distribution at INL, as shown in Fig. 5d (I = I th ). The MEA with higher protrusion generated higher contrast in the E-field profiles between the targeted area and untargeted area. However, the 100-mm-protruding array induced an uneven E-field distribution, presumably due to an unmitigated edge effect from an excessively close electrode–cell distance. Therefore, we concluded that an 80 mm protrusion is the optimum height for efficient subretinal stimulation, which was adopted for the proof-of-concept 25-channel 3D array presented in the previous sections. The protruding MEA is also predicted to enhance the spatial resolution of retinal stimulation, which was quantified using the Michelson contrast (MC), measuring the ratio of (E max − E min ) to (E max + E min ) (more details in the Methods), as shown in Fig. 5e. Higher protrusion of the electrodes led to a higher E-field contrast (Supplementary Fig. S14) and correspondingly higher MC values across the entire range of channel pitches from 250 to 600 mm. Benefits of mETF MEA for subretinal stimulation in ex vivo experiments The effectiveness of mETF MEA for enhanced neural interfaces in subretinal stimulation were evaluated through ex vivo retina experiments, by comparing the protruding mETF MEA and planar MEA in terms of stimulation threshold and spatial resolution. Activation of RGCs was monitored by imaging the calcium transients in response to electrical subretinal stimulation, using a custom-built fluorescence microscopy setup (Fig. 6a). As shown in Fig. 6b, the MEA placed under the mouse retina patch included both planar and 80-μm-mETF protruding electrodes with a diameter of 100 mm (Supplementary Fig. S6). A genetically-encoded calcium indicator, sRGECO, was introduced to the retina via adeno-associated viral (AAV) vectors and its expression in RGC layer was confirmed three weeks after the injection (Fig. 6c). A typical calcium transient, in response to biphasic current pulses, is represented by normalized changes in fluorescence, D F / F 0 = ( F peak – F 0 )/ F 0 , as shown in Supplementary Fig. S15. The stimulation threshold was evaluated by quantifying D F / F 0 of RGCs placed within the diameter of stimulating electrode (Fig. 6d) while increasing the current injection. As shown in Fig. 6e, the resulting responses were fitted into sigmoidal functions, from which the threshold was defined as the stimulation current at the half of the maximum D F / F 0 . The protruding electrode significantly lowered the median stimulation threshold to 0.91 mA, compared to 1.55 mA of the planar electrode, as shown in Fig. 6f. This suggests that the closer proximity of protruding structure to bipolar cells and RGCs allowed for neural activation with a lower current. The spatial extent of electrical activation was quantified by D F / F 0 of RGCs depending on their distances from the center of stimulating electrode (Fig. 6d) at a fixed current of I = 20 mA, as shown in Fig. 6g. The response curves were fitted by Gaussian functions to determine the spatial extents, defined as the half-width at half maximum (HWHM) of D F / F 0 . The protruding electrode produced more focused retinal activation by reducing the HWHM from 94.8 to 43.7 mm, which well agrees with the FEA estimation in Fig. 5d. Stimulation with I = 10 mA resulted in consistent outcome (99.7 to 39.2 mm), as shown in Supplementary Fig. S16. The HWHM of the protruding electrode comparable to its radius (50 mm) indicates that the mETF MEA activates the retina with high contrast and minimized inter-channel interference, potentially providing artificial vision with higher spatial resolution. Previous reports have indicated that retinal cells gradually migrate into the voids around the pillars of protruding electrodes after six weeks of in vivo implantation 2,44 . Such migration reduces the separation between the electrodes and target cells with preserved axonal network 27 , suggesting the benefits of 3D mETF MEA may also be applicable to in an in vivo retinal environment. Discussion In this study, we presented a novel µETF process for creating simple and versatile 3D microstructures on thin-film polymer-based neural interfaces using 3D-printed molds to locally deform planar MEAs into desired protruding or recessing shapes. This approach builds upon the widely used polymer thermoforming technology in the plastic industry, but adapts it for microelectronic 3D structuring to create neural interfaces that are intimate with the nervous system. Our proposed technology uses one-step pressing of conventionally prepared planar MEAs to enable virtually any 3D microstructure to be created on microelectrode arrays without requiring additional fabrication steps. This implies that complex structures with different shapes and heights can be created within a single array using the same one-step thermoforming process. This contrasts with previous 3D-shaping techniques for neural interfaces, which require complicated fabrication steps and are limited in their ability to create different structural profiles on the same array, primarily because of the nature of the MEMS process. The simplicity and versatility of our proposed method in forming 3D microstructures expand design possibilities, enabling optimized electrode–neuron interfaces that reflect the anatomical and neurophysiological characteristics of various nervous systems. Additionally, by combining gross macrothermoforming and local microthermoforming, it allowed for an even wider range of customized macrostructures and microstructures beyond the 3D structures presented in this study. We expect that the proposed technique will have potential applications in areas such as wearable electronics, microfluidic systems, and cell culture platforms, where the integration of 3D microstructures and microelectrodes can contribute to achieving optimal performance. Besides, the µETF is compatible with various thermoplastic materials commonly used in implantable and wearable biomedical applications, including parylene-C 14 , cyclic olefin copolymer 45 , perfluoroalkoxy alkane 46 , thermoplastic polyurethane 47 , polymethyl methacrylate 48 , polycarbonate 22 , 49 , and polyvinyl alcohol 50 . The µETF produces 3D structures with inherent round edges and gently sloped sidewalls, which help minimize tissue damage during and after surgical implantation. Mechanical stress within MEA and surrounding tissue was assessed via FEA during forced sliding of an 80-µm height 3D structure into viscoelastic tissue, as shown in Supplementary Fig. S17. The µETF LCP electrode with native round edges was estimated to reduce the maximum stress within the electrode and on tissue by more than 60%, compared to LCP MEA with sharp edges. The stress mitigation of the µETF LCP structure was more pronounced when comparison was made against silicon-based 3D structure with the same shapes. Although degenerated retinas are known to undergo structural changes over time 51 – 53 , a normal human retinal model was employed in this study for the computational simulation of subretinal stimulation. This was primarily due to a lack of consistent structural information available for degenerated retinas in humans. While discrepancies may exist between computational simulations and clinical applications, it is anticipated that the benefits of protruding electrodes predicted in a normal retinal model can also be applied to degenerated retinas, primarily because of the reduced distance between the target cells and electrode surfaces. The accuracy and minimum feature size of the µETF structures are associated with two factors: the precision of the 3D-printed mold, and the mold–MEA alignment. In this study, a printing resolution of 2 µm for the horizontal plane and 5 µm for the vertical axis was employed. Although this resolution is generally suitable for a wide range of neural interfacing applications, recent rapid advancements in 3D printing suggest the potential future availability of molds with even higher precisions. The mold was aligned with the planar MEAs by matching the laser-drilled holes on the arrays with corresponding pins on the metal jig, ensuring a minimum in-plane feature size of approximately 100 µm. However, this approach may be susceptible to mismatches caused by different thermal expansion ratios between the metal jig, epoxy mold, and polymer array. To enhance the accuracy, the 3D microstructures and aligned pins could be printed monolithically, and error-compensating laser drilling could be employed to further reduce the impact of thermal expansion mismatches. Finally, the thermoplastic LCP substrate employed in this study is known to exhibit an exceptionally low water absorption rate (< 0.04%), potentially ensuring the long-term stability of the 3D MEA 32 , 54 . Therefore, combining versatile 3D forming with durable LCP packaging is considered a suitable approach for various neural interface applications. Methods Microfabrication of LCP MEA A planar LCP MEA was fabricated using a previously reported procedure based on conventional microfabrication technologies for thin-film polymer–metal–polymer structures 26 , 32 , 54 , as illustrated in Supplementary Fig. S1 . Briefly, Ti/Au seed layers (50 nm/50 nm) evaporated on a 25-µm-thick LCP film (Vecstar CTQ-25, Kuraray) underwent photolithography to define negative patterns (8 µm thickness, AZ P4620, Merck), followed by gold electroplating. After and seed layer removal (gold etchant and buffered oxide etchant 10:1, Sigma-Aldrich), a 25 µm-thick LCP cover layer (Vecstar CTF-25, Kuraray) was thermally laminated at 285°C with a pressure of 4 kgf/cm 2 for 30 min using a heating press (Model 381, Carver). 3D Mold preparation for µETF A master mold carrying microscopic 3D structures and alignment keys was created via precision 3D printing (microArch S130, Boston Micro Fabrication) and then replicated using a high-temperature epoxy to tolerate the thermoforming process at 200°C. The replication was performed via double casting using polydimethylsiloxane (PDMS) as the negative mold, as illustrated in Supplementary Fig. S2. High-temperature-resistant epoxy (Duralco 4460, Cotronics) was poured over the PDMS negative mold, which was cured in two steps: first at 120°C for 4 h, followed by post-curing at 230°C for 16 h to reinforce its thermal and mechanical tolerance. Alignment and pressing setup for µETF The alignment and pressing setup consisted of a pair of top and bottom metal jigs; a rubber sheet (RBSM5-100, Misumi) and a rubber frame for uniform pressure; and a cylindrical rubber stamp and a rubber frame for pressing the electrode array against the mold (see Supplementary Fig. S3 for more details). The epoxy mold was assembled onto the bottom metal jig, on which the planar MEA array was loaded. Precise alignment was achieved by matching the laser-drilled align holes on the electrode layer with the align pins on the mold. The µETF was performed at a pressure of 6.4 kgf/cm 2 applied at 200°C for 30 min. A UV laser cutter (Samurai UV marking system, DPSS Lasers Inc.) was used for site opening and outlining. Oxygen plasma cleaning (CIONE 4, Femto Science) was performed at 80 W and 80 sccm for 30 min to remove the laser burrs. Electromechanical considerations of 3D MEAs with varying pattern designs The 25-channel MEAs were prepared with varying gold thicknesses and line shapes. The 300-nm-thick gold patterns were prepared via evaporation of Ti/Au layers, photolithography with a negative photoresist (NR9-3000py, Futurrex), and wet etching. The MEAs were not encapsulated by cover layers to facilitate observation of cracks in the gold patterns. The failures of gold patterns after µETF were categorized into two mechanisms. The “electrode crack” is defined as a failure due to distributed cracks within the gold electrode sites, whereas “line disconnection” refers to a disconnection of gold lines. Number of samples for analyzing the failure rate are: 20 for (I)-straight, 20 for (I)-wavy, 40 for (II)-straight, 50 for (II)-wavy, 50 for (III)-straight, and 50 for (III)-wavy. IrOx electrodeposition The laser-opened gold electrodes were electrodeposited with IrOx, following the previously reported protocol 36 , 37 using a potentiostat (CompactStat, Ivium Technologies). The electrodes were subjected to four cleaning cycles of voltage sweep in 1 M sulfuric acid from − 0.4 V to 1.4 V at 50 mV/s. Triangular potentials (0 to 0.55 V) were then iterated for 200 cycles at 50 mV/s, followed by 2,000 cycles of rectangular pulses (0.55 V, 0.5 s and 1 Hz). Electrochemical characterization The electrochemical characterizations were performed with a three-electrode system in the frequency range of 1 Hz to 100 kHz using the potentiostat and phosphate-buffered saline (1X, PH 7.4, Gibco). The CV curves were measured at a scan rate of 50 mV/s in the voltage range of − 0.6 V to 0.8 V, from which the CSC C was calculated using the time integral of the cathodically enclosed area in Eq. (1): $$\:{\text{C}\text{S}\text{C}}_{\text{C}}=\:\frac{1}{vA}{\int\:}_{{E}_{c}}^{{E}_{a}}\left|i\right|dE\:\:\:\:\:[\text{m}\text{C}/{\text{c}\text{m}}^{2}],\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(1\right)$$ where v is the scan rate, A is the geometric area of microelectrode, i is the current, and E a and E c are the anodic and cathodic potential limits, respectively 11 . Number of samples for comparing EIS of the MEAs before and after µETF are: 56 for planar gold, 31 for protruding gold, 43 for planar IrOx, and 63 for protruding IrOx. Number of samples for comparing CSC C of the MEAs before and after µETF are: 12 for planar gold, 16 for protruding gold, 10 for planar IrOx, and 15 for protruding IrOx. FEA simulations Mechanical stress induced in the gold traces was computed using Structural Mechanics module in COMSOL Multiphysics 6.0. The in-plane serpentines were implemented by applying a constraint in gold layers to only deform in the axial direction, while no constraints were applied to the out-of-plane serpentines allowing both axial elongation and 3D buckling. The I th , DR, and spatial resolution in subretinal stimulation were numerically simulated using AC/DC module in COMSOL Multiphysics 6.0 based on a human retinal model 40 , 41 (see Supplementary Fig. S12 and Table S1 ). The stimulation threshold was defined as the stimulating current that generates an electric field of 3,000 V/m at the location of the bipolar cells in the INL, which is the target layer for subretinal stimulation 27 , 42 , 43 . The spatial resolution estimates the extent of inter-channel interference as evaluated by the Michelson contrast (MC) 11 . The MC measures the ratio of the minimum and maximum electric field strengths at the plane of the INL upon simultaneous current injection from neighboring channels using Eq. (2): $$\:\text{M}\text{C}=\:\frac{{\left|E\right|}_{max}-{\left|E\right|}_{min}}{{\left|E\right|}_{max}+{\left|E\right|}_{min}}\:.\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(2\right)$$ Modeling of insertion of MEA in Fig. S17 employed the retina and electrode modeled as linear elastic materials, as described in Table S2. Fluorescent Calcium imaging system A custom-built optical system incorporated both fluorescence and bright field microscopes. The bright field microscope utilized a Köhler illumination configuration, consisting of a near-infrared light emitting diode (LED) at λ = 780 nm (M780L3, Thorlabs), a collector lens, a condenser lens, and two iris diaphragms to ensure uniform illumination of the sample. The fluorescent microscope involved a 565 nm LED (SOLIS-565C, Thorlabs), a dichroic mirror in combination with excitation and emission filters (MDF-MCHA, Thorlabs) to separate the excitation light from emission light. Images were focused onto the CMOS camera using a 40X objective lens (LUMPLFLN40XW, Olympus) with a numerical aperture of 0.8, and a tube lens with a focal length of 200 mm. AAV-meditated gene transfection and retinal preparation All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC; PNU-2023-3237) of Pusan National University. Male C57BL/6 mice (10-week-old, 20–25 g) were housed in a cage under a 12 h light/dark cycles with controlled temperature (22 ± 2°C) and humidity (55% ± 5%), and ad libitum access to food and water. The AAV vectors were used to transfect mouse retinal neurons with sRGECO (AAV8-Ef1a-sRGECO; 2×10 13 GC/mL in HBSS, plasmid number of 137125; Addgene). The mice were anesthetized with avertin (250 mg/kg, Sigma-Aldrich) and placed on a heating pad at 37°C. A 30 G syringe was used to puncture a hole around the limbus. The AAV vectors carrying the genetically encoded calcium indicator were injected into the vitreous for 10 s using a 33G blunt needle (World Precision Instruments) 55 , 56 . After 3 weeks of injection 55 , the retina was prepared following the previously reported protocol by authors 57 . Detached retina was transferred into the electrode-mounted chamber (RC-27L, Warner Instruments LLC) with the photoreceptor facing down, and immobilized by a nylon-mesh anchor (HSG, Ala Scientific Instruments). Total four retinas were used in this study. Ex vivo retinal experiments During the procedures, the prepared chamber was continuously perfused by oxygenated Ames' medium (A1420, Sigma-Aldrich) containing a synaptic blocker cocktail of CNQX (25µM; Hello Bio) and D-AP5 (25µM; Hello Bio) at 34°C with a constant flow rate of 5 mL/min 56,58 . The stimulating MEA was connected to a stimulus generator (STG4004, Multi-Channel Systems) with a reference platinum wire electrode (MW-4310, BASI Research Product). Electrical stimulation was delivered in a burst of seven cathodic-first biphasic pulses at a frequency of 60 Hz, duration of 60 µs and inter-phase delay of 100 µs with varying amplitudes (Supplementary Fig. S15). Data processing for ex vivo experiments The stimulation threshold was determined by fitting Δ F / F 0 = ( F peak – F 0 )/ F 0 versus current amplitudes, using a modified sigmoid function, S ( x ) in Eq. ( 3 ): $$\:S\left(x\right)=\frac{a}{a-\frac{a}{1+{exp}\left(b\times\:c\right)}}\times\:\left(\frac{a}{1+{exp}\left(-b\times\:\left(x-c\right)\right)}-\frac{a}{1+{exp}\left(b\times\:c\right)}\right),$$ 3 where x is the stimulation current, and a , b , and c are fitting parameters. The threshold is defined as the current corresponding to 0.5 F max . The spatial extents were determined by fitting Δ F / F 0 versus distance from the electrode center using a Gaussian function, \(\:G\left(x\right)\) in Eq. ( 4 ): $$\:G\left(x\right)=a\times\:exp\left(-b\times\:{x}^{2}\right)$$ 4 , where x is the distance away from the electrode center, and a and b are fitting parameters. The spatial extent was defined as half of HWHM. All the fittings were performed through nonlinear least-squares methods in Matlab. Declarations Competing interests All authors declare no competing interests. Author Contribution D. H. L. and J. J. conceived and planned the experiments. D. H. L. carried out the experiments and simulations. Y. P. and K.E. designed the optical system for calcium imaging and conducted ex vivo experiments. D. H. L., Y. S., H. J., J. -M. S., M. -H. S., and J. J. contributed to sample preparation, measurements, and interpretation of the results. D. H. L. and J. J. took the lead in writing the manuscript. J. J. supervised the project. All authors helped shape the research and revise the manuscript. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2022R1C1C1010422, RS-2023-00217893, and NRF 2020R1C1C1010505). Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Steins, H. et al. A flexible protruding microelectrode array for neural interfacing in bioelectronic medicine. Microsystems Nanoeng. 8, (2022). Flores, T. et al. 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Supplementary Files 08063DthermoformingsuppleSUBMIT.docx Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2025 Read the published version in npj Flexible Electronics → Version 1 posted Editorial decision: Revision requested 24 Sep, 2024 Reviews received at journal 22 Sep, 2024 Reviews received at journal 11 Sep, 2024 Reviews received at journal 08 Sep, 2024 Reviews received at journal 29 Aug, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers invited by journal 26 Aug, 2024 Editor assigned by journal 26 Aug, 2024 Submission checks completed at journal 26 Aug, 2024 First submitted to journal 16 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4924574","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":358445332,"identity":"25dadbba-8718-4683-a53b-56cd211f2f9c","order_by":0,"name":"Joonsoo Jeong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACxgYURgVCxoBILWeI0IJmQBsRWpjbm489/FFhA2F8nXcncX772QMMP2oYjM0bsGth7DmWbsxzJg3CkN32LHHDmbwEIIfBTOYADi0zcsykGdsOQxiS2w4nbmDIMWDgbWCwkcDleqBKyZ8gLfPfALXMOZw4v/+NAeNfAlokeMG28JhJfmw4nNhwI8eAGWiLGU4tPcfSpCF+SUuTZjj2zHjDjTcGh2WOSRjj0mIIDChJUIgZth8GMmruyM7vzzF8+KbGxnAGLi0NELp+I5DBzMNwAMwDkrjsYGCQR2Yw/oBqGQWjYBSMglGADABvMFpuXbv8LwAAAABJRU5ErkJggg==","orcid":"","institution":"Pusan National University","correspondingAuthor":true,"prefix":"","firstName":"Joonsoo","middleName":"","lastName":"Jeong","suffix":""},{"id":358445333,"identity":"00785702-b105-47f6-b24d-3bb6823ccee6","order_by":1,"name":"Dong Hyeon Lee","email":"","orcid":"","institution":"Pusan National University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Hyeon","lastName":"Lee","suffix":""},{"id":358445334,"identity":"d3709c67-1e16-46f2-b683-c1dd2bd82154","order_by":2,"name":"Younghoon Park","email":"","orcid":"","institution":"Pusan National University","correspondingAuthor":false,"prefix":"","firstName":"Younghoon","middleName":"","lastName":"Park","suffix":""},{"id":358445335,"identity":"f48a1205-f699-4b76-95ec-165f0a4f654f","order_by":3,"name":"Yoon Seo","email":"","orcid":"","institution":"Pusan National University","correspondingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"","lastName":"Seo","suffix":""},{"id":358445336,"identity":"09b789e6-5cb6-4320-90df-bc00515a4586","order_by":4,"name":"Hyunbeen Jeong","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Hyunbeen","middleName":"","lastName":"Jeong","suffix":""},{"id":358445337,"identity":"f862cde9-3627-4849-85c6-f6acc7c8dae5","order_by":5,"name":"Jong-Mo Seo","email":"","orcid":"","institution":"Seoul National University / Seoul National University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jong-Mo","middleName":"","lastName":"Seo","suffix":""},{"id":358445338,"identity":"cded2346-3d6a-4289-9199-4bd5785e916d","order_by":6,"name":"Min-Ho Seo","email":"","orcid":"","institution":"Pusan National University","correspondingAuthor":false,"prefix":"","firstName":"Min-Ho","middleName":"","lastName":"Seo","suffix":""},{"id":358445339,"identity":"d4127d79-fb71-4792-b426-717b558b5124","order_by":7,"name":"Kyungsik Eom","email":"","orcid":"","institution":"Pusan National University","correspondingAuthor":false,"prefix":"","firstName":"Kyungsik","middleName":"","lastName":"Eom","suffix":""}],"badges":[],"createdAt":"2024-08-16 10:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4924574/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4924574/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41528-024-00378-0","type":"published","date":"2025-01-22T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65267566,"identity":"bcfbafd5-ff33-4774-8dce-6cc010752cfc","added_by":"auto","created_at":"2024-09-25 12:21:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":496988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOne-step mETF of LCP MEA to create microscopic protruding and recessed 3D structures for enhanced neural interfaces.\u003c/strong\u003e a) Benefits of localized neural interface (e.g., lower threshold and inter-channel interference) achieved by 3D structures. b) Schematic illustration of mETF process to transfer 3D structures of 3D mold onto planar LCP MEA. c) Cross-sectional illustration of subsequent micro- and macro-thermoforming processes for achieving both high proximity to target cells and conformability to surrounding tissues. d) Schematics (top row) and photographs (bottom row) of LCP MEA (i) before mETF, after ii) protruding or iii) recessed 80-mm-height mETF, and (iv) after macro-ETF to fit eye curvature. Scale bars: 1 mm. e) SEM images and f) cross-sectional images of (i) planar, (ii) 80-mm-protruding, and (iii) 80-mm-recessed electrode sites of mETF LCP MEA. Scale bars: 100 mm. g) Optical profiles of protruding (top) and recessed (bottom) electrode sites.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/39ff09a7f71a1ccd5b0bb774.png"},{"id":65268593,"identity":"99f3d247-c194-4fde-822b-ad044c48af25","added_by":"auto","created_at":"2024-09-25 12:29:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":515197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVersatile 3D structures with diverse shapes and heights created by one-step mETF process.\u003c/strong\u003e Protruding and recessed 3D LCP MEA with varying heights observed using a-b) SEM images (scale bars: 100 mm), c-d) cross-sectional photographs, and e-f) optical profiles. Versatile 3D structures including polygons, ovals, domes, and walls on g) 3D-printed mold transferred onto h) 25-channel LCP MEA, and assessed using i) SEM images and j) optical profiles. Scale bars: 1 mm in photograph and 200 mm in SEM images.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/cd02aea78d9a5d72ff82f5ac.png"},{"id":65268960,"identity":"71fbe4f2-9dfb-4bd8-8edf-38dd1f8b34ef","added_by":"auto","created_at":"2024-09-25 12:37:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":480103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectromechanical considerations of MEA for 80-mm-height mETF.\u003c/strong\u003e a) FEA analysis for cross-sectional stress distribution within deformed gold layers during mETF, at b) top, bottom, and neutral planes. c) Layout of gold electrode and interconnection overlaid on transversal stress distribution. Inset represents geometric parameters of wavy lines. d) Stress distribution and e) comparison of peak stress values induced on interconnections of in-plane and out-of-plane serpentines with varying line widths under e = 15%. T: top view; S: side view. f) SEM images and g) line failure rates after 80-mm mETF with respect to line thickness and shape, suggesting two distinct failure modes of electrode cracks and line disconnection. Scale bars: 200 mm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/5c2e3607bb51e358908e8874.png"},{"id":65268959,"identity":"2bbf40c0-ef4c-401a-88ae-298106178161","added_by":"auto","created_at":"2024-09-25 12:37:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":356985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrochemical and mechanical analysis of MEA for 80-mm-height mETF.\u003c/strong\u003e Comparison of a) EIS magnitude and b) phase at 1 kHz, and c) CSC\u003csub\u003eC\u003c/sub\u003e of gold and IrOx electrodes before and after 80-mm mETF. Box plots represent median and 50% range. d) Mechanical resilience of 80-mm-protruding mETF MEA during three cycles of full compression and relaxation. e) Magnified view showing mETF structures compressed by 2.2 mm under 2.2 kPa load of normal physiological pressures within f) (i) intraocular and (ii) intracranial environment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/bc8565e6905a6cb83d16841e.png"},{"id":65268595,"identity":"8e45551c-d178-49cb-9c0f-0a1e315b3e6d","added_by":"auto","created_at":"2024-09-25 12:29:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":693021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFEA study for estimating benefits of mETF MEA for subretinal stimulation.\u003c/strong\u003e a) Linear (A-A’) and b) transversal E-field distributions at INL level for planar and 80-mm-protruding MEA at: thresholds (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e) of (i) protruding and (ii) planar MEAs, and onset of inter-channel interference (\u003cem\u003eI\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) for (iii) protruding and (iv) planar MEAs. White dashed lines indicate contour of suprathreshold (activated) area. c) I\u003csub\u003eth\u003c/sub\u003e and DR of MEA with increasing heights of protrusion. d) Linear (A-A’) E-field profiles at INL induced by MEA with varying protrusion heights, all injecting \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e\u003cem\u003e. \u003c/em\u003ee)\u003cem\u003e \u003c/em\u003eStimulation resolution represented by Michelson Contrasts depending on protruding heights and electrode pitches.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/d7861010dfbc18bea8a4bc61.png"},{"id":65267568,"identity":"a43afce8-f387-4093-9f92-d925c33f3610","added_by":"auto","created_at":"2024-09-25 12:21:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":545373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e retinal experiments for evaluating effectiveness of mETF MEA for subretinal stimulation.\u003c/strong\u003ea) Schematic illustration of calcium imaging\u003cem\u003e \u003c/em\u003esetup for monitoring retinal activation, and b) its side and top views of the retina chamber with MEAs. c) Expression of genetically-encoded calcium indicatorconfirmed by immunofluorescence images of sRGECO (mCherry), DAPI, and merged one. Scale bar: 20 mm. d) Relative location of RGCs around stimulation electrode for evaluation of stimulation threshold and stimulation resolution. e) Retinal responses in response to increasing electrical stimulation from protruding and planar electrodes. f) Comparison of stimulation thresholds of protruding and planar electrodes, using Wilcoxon rank-sum test (\u003cem\u003ep\u003c/em\u003e = 0.0175). g) Spatial extent of electrical activation depending on distances from center of stimulating electrode (\u003cem\u003eI\u003c/em\u003e = 20 mA). The calcium intensity was normalized to maximum fluorescence at electrode center.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/73274c8cd1c2606e487c9c12.png"},{"id":74859048,"identity":"c47707b8-98d7-4cd2-a4ce-6ad59609b96c","added_by":"auto","created_at":"2025-01-27 16:13:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4165156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/f0b36b0b-35fa-4a03-b675-0ab0e426706c.pdf"},{"id":65267572,"identity":"c1fbe3f8-a593-4eeb-878d-2223230da0d9","added_by":"auto","created_at":"2024-09-25 12:21:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9619968,"visible":true,"origin":"","legend":"","description":"","filename":"08063DthermoformingsuppleSUBMIT.docx","url":"https://assets-eu.researchsquare.com/files/rs-4924574/v1/b256ca4098fdc352033bf83a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MicroElectroThermoForming (μETF): One-step Versatile 3D Shaping of Flexible Microelectronics for Enhanced Neural Interfaces","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHigh proximity of electrodes to neural cells plays a critical role in achieving efficient neural interfaces for various recording and stimulation applications\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Improved signal quality in neural recordings can result from intimate contact between the electrode surface and targeted tissues\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, whereas in neural stimulation, lower stimulation thresholds and higher spatial resolution can be achieved through reduced current spreading\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Thus far, improved electrode\u0026ndash;cell proximity has typically been achieved using microelectromechanical system (MEMS) technologies, either by directly structuring rigid materials into needle-like shapes, such as Utah arrays, or by building various three-dimensional (3D) microstructures on top of thin film-based microelectrode arrays (MEAs), such as pillars\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, mushrooms\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, hemispheres\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, sheaths\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and arrowheads\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, despite their effectiveness, rigid 3D electrodes may exhibit mechanical mismatch with soft neural tissues. Furthermore, the creation of 3D microstructures on top of thin-film MEAs using conventional MEMS technologies requires additional fabrication steps, such as photolithography, vacuum deposition, electroplating, and wet and dry etching\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These steps increase the complexity of the fabrication process, the degree of which usually scales with the complexity of the 3D structures. Moreover, it is challenging to fabricate 3D features with different shapes and heights within a single MEA, owing to the inherent 2D nature of the traditional microfabrication process.\u003c/p\u003e \u003cp\u003eTo address these problems, we propose a novel \u0026ldquo;microelectrothermoforming (\u0026micro;ETF)\u0026rdquo; process for producing versatile 3D microstructures on a polymer-based MEA through a simple one-step thermal pressing of conventional planar arrays. This method leverages a well-established thermoforming technique used in the plastic industry, in which a thermoplastic sheet is heated above the glass transition temperature (T\u003csub\u003eg\u003c/sub\u003e) and pressed against a metal mold to create 3D structures in a simple manner\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The microelectronic adaptation of thermoforming in this study enables the creation of microscopically protruding and recessed versatile 3D structures with embedded electrical functionalities, facilitating the development of tailored structures for optimized electrode\u0026ndash;cell interfaces. Thermoforming and microthermoforming for biomedical applications have been explored by Truckenm\u0026uuml;ller et al.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and in subsequent studies, in which heated thermoplastic sheets were pneumatically or mechanically pressed against perforated metal molds to generate 3D shapes, such as microwell platforms\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, cell culture chips\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and microchannels\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, the methods demonstrated in those studies primarily formed bulk polymer sheets that lacked active components such as electrical contacts and interconnections. Additionally, they required specialized tooling to exert pneumatic pressure, and their formable 3D structures were commonly limited to hemispherical shapes.\u003c/p\u003e \u003cp\u003eThe proposed \u0026micro;ETF process effectively addresses the challenges associated with fabrication complexity and structural restrictions in known 3D microstructures for enhanced neural interfaces. Microelectrothermoforming involves the one-step thermal pressing of a conventionally prepared thin-film planar MEA against a mold carrying the desired 3D structures. Therefore, two distinct advantages can be achieved compared to existing MEMS-based 3D forming: 1) process simplicity and 2) shape versatility. The proposed approach simplifies 3D fabrication by adding only one pressing step to the fabrication of a conventional planar thin-film MEA. Moreover, the use of 3D printing technology for preparing molds enables the formation of virtually any 3D microstructures, allowing heterogeneous structures of diverse shapes and heights to be created within a single electrode array via the same one-step \u0026micro;ETF process. This simplicity and flexibility in forming 3D microstructures can expand design possibilities, facilitating the development of optimized electrode\u0026ndash;neuron interfaces that align with the anatomical and neurophysiological features of a targeted nervous system.\u003c/p\u003e \u003cp\u003eLiquid crystal polymer (LCP) film was used as a thermoplastic substrate in this study, primarily owing to its mechanical strength, chemical inertness, and biocompatibility. Additionally, the low water absorption rate of LCP can contribute to the long-term reliability of chronically implanted devices\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, \u0026micro;ETF can be utilized for other thermoplastics commonly used in biomedical applications.\u003c/p\u003e \u003cp\u003eThis study introduces a one-step \u0026micro;ETF process for generating versatile protruding and recessed 3D structures on LCP-based MEAs (LCP MEA) with mechanical considerations for preserving the electrical properties of MEA. As a proof of concept, the 3D MEA was optimized for retinal stimulation, the benefits of which were assessed via both computational analysis and \u003cem\u003eex vivo\u003c/em\u003e experiments in mouse model. A variety of 3D structures, such as wells, domes, walls, and triangles, were constructed on LCP MEAs, demonstrating the potential utility of the \u0026micro;ETF in a wide range of biomedical applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003emETF for simple and versatile 3D structuring \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative benefits of 3D MEA are illustrated in \u003cstrong\u003eFig. 1\u003c/strong\u003ea. The protruding structures enable closer proximity to target cells, establishing more localized neural interfaces for both neural recording and stimulation\u003csup\u003e27\u0026ndash;30\u003c/sup\u003e. This is in contrast to conventional planar MEAs, in which the electrode surfaces inherently lie below the top surface\u003csup\u003e31\u0026ndash;33\u003c/sup\u003e. As outlined in Fig. 1b, the proposed one-step mETF process locally deforms a planar MEA into a 3D MEA with microscopic protruding and/or recessed structures. A planar 25-channel LCP MEA prepared via a conventional microfabrication process\u003csup\u003e32\u003c/sup\u003e (see Supplementary Fig. S1) is thermally pressed (\u0026gt; T\u003csub\u003eg\u003c/sub\u003e) against a 3D-printed mold within a set of metal jigs and elastomer layers for alignment (see Supplementary Fig. S2\u0026ndash;3 for details), replicating the protruding and/or recessed 3D microstructures of the mold to the MEA, as shown in Fig. 1c.\u003c/p\u003e\n\u003cp\u003eAdditionally, the microelectrothermoformed 3D MEA can be grossly deformed into nonplanar shapes to conform to surrounding tissues, such as eye curvature for retinal electrodes, through a similar \u0026ldquo;macro\u0026rdquo; electrothermoforming step (mETF, see Supplementary Fig. S4). Using a 3D mold with 80 mm-height pillars (Supplementary Fig. S5), the schematics (top row) and photographs (bottom row) in Fig. 1d show the evolution of a (i) 40-mm-thick planar LCP MEA to a (ii) protruding or (iii) recessed mETF MEA, and subsequently to a (iii) mETF + mETF MEA, achieving both high proximity to target cells and conformability to target tissues. The recessed structures in (iii) can be easily generated using the identical processes and tools to those for the protrusions, simply by flipping the planar array upside down in the fixture, offering a convenient approach to create well-like structures for highly localized electrode\u0026ndash;cell environments\u003csup\u003e29\u003c/sup\u003e. Scanning electron microscopy (SEM) images of individual electrode sites before and after mETF are presented in Fig. 1e, with their cross-sectional profiles shown in Fig. 1f. Each channel site with a diameter of 200 mm was selectively elevated or lowered by a height of 80 mm, which represents an optimized height for subretinal electrode arrays as a proof-of-concept application of mETF (details in subsequent sections). The optical surface profiles of the protruding (top) and recessed (bottom) channel sites are shown in Fig. 1g. The mETF produced 3D structures that replicated the original mold structures, exhibiting a slightly widened base diameter (~150%) and sloped sidewalls (~70\u0026deg;). The electrode diameter as small as 100 mm was also successfully thermoformed using the same mETF configuration (Supplementary Fig. S6), which was utilized for following \u003cem\u003eex vivo\u003c/em\u003e experiments.\u003c/p\u003e\n\u003cp\u003eSimilarly, versatile protruding and recessed structures of diverse heights and shapes can be created without adding fabrication complexities. \u003cstrong\u003eFigure 2\u003c/strong\u003ea\u0026ndash;f present 25-channel LCP MEAs formed with varying protrusion/recessed heights from 80 to 200 mm using a 3D mold (Supplementary Fig. S7). Their SEM images (Fig. 2a, b), cross-sectional images (Fig. 2c, d), and optical 3D profiles (Fig. 2e, f) confirm the faithful replication of the 3D mold structures onto the LCP MEAs. Furthermore, the versatility of the microthermoforming process was demonstrated with various microstructures, including polygons (triangles, rectangles, and hexagrams), ovals (sunken, plateau, and walled), domes, and S-shaped walls, as shown in Fig. 2g and h; their SEM images and optical profiles are shown in Fig. 2i and j, respectively. All distinct structures were created via one-step mETF using corresponding 3D molds shown in Supplementary Fig. S7. Such versatility and consequent design flexibility can be leveraged to create optimized 3D structures tailored for various \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e neural interfacing applications.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eElectromechanical considerations for \u003c/strong\u003e\u003cstrong\u003emETF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite the simplicity and versatility of the mETF process, it is important to consider the tensile stress exerted on the thin gold layers, which may lead to cracks or disconnections. Therefore, the mETF process was optimized with respect to the 3D mold design and pattern layout.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Fig. 3\u003c/strong\u003ea presents a finite element analysis (FEA) of the mechanical stress induced within the embedded thin gold layer during an 80-mm-height mETF. Based on the relative stress distribution for the top, bottom, and neutral planes within a gold layer (Fig. 3b), the highest tensile stresses are expected at the top and bottom corners of the sidewall (insets in Fig. 3a). On the other hand, the top protruding area remained relatively stress-free because of the pillar-shaped mold with a flat top surface. The resulting plateau-like structure of the mETF electrode ensured minimal damage to the circular electrode area, which were designed to be 10 mm smaller than the mold top diameter, as outlined on the transversal stress distribution in Fig. 3c. This is in contrast to forming a 3D structure using a dome-shaped mold, in which electrode sites are subjected to highest tensile stress, leading to significant cracks on the gold electrode surface after mETF (Supplementary Fig. S8).\u003c/p\u003e\n\u003cp\u003eThe mechanical strength of microelectronic tracks was enhanced by electroplating the gold patterns up to 4 mm thickness (\u003cem\u003et\u003c/em\u003e). Additionally, the interconnection lines traversing the region under the greatest stress is configured with serpentine shapes for enhanced robustness against elongation, as shown in Fig. 3c and inset (\u003cem\u003er \u003c/em\u003e= 20 mm, \u003cem\u003ed\u003c/em\u003e = 40 mm, and \u003cem\u003eq\u003c/em\u003e = 180˚). Typical serpentines adopted in stretchable electronics exploit global out-of-plane buckling of thin gold tracks embedded within freestanding polymer layers\u003csup\u003e34,35\u003c/sup\u003e. However, the gold serpentine layer in mETF is subject to in-plane constraint as they are pressed between jig and mold. Therefore, the stretchability of such in-plane serpentine gold interconnections was evaluated in comparison with the conventional out-of-plane wavy lines via FEA simulation under the maximum local elongation during 80 mm-height mETF (\u0026epsilon; = 15%, red area in Fig. 3c). As shown in Fig. 3d and e, both in-plane and out-of-plane serpentines exhibited reduced maximum stress with lower \u003cem\u003ew\u003c/em\u003e and greater \u003cem\u003eq \u003c/em\u003e(more details in Supplementary Fig. S9). Although in-plane restriction resulted in 33% higher peak stress than out-of-plane buckling serpentines with \u003cem\u003ew\u003c/em\u003e = 10 mm, the wavy patterns are still effective in relieving the tensile stress of gold lines during mETF, by reducing the peak stress by 90% from the straight line.\u003c/p\u003e\n\u003cp\u003eThese design considerations were experimentally validated using patterns with varying shapes (straight and wavy) and thicknesses (\u003cem\u003et\u003c/em\u003e = 300 nm to 4 mm) in 80-mm-height mETF. The representative SEM images in Fig. 3f demonstrate that damages to the gold layers were caused by two distinct mechanisms, as quantified by yield analysis after mETF in Fig. 3g. The \u0026ldquo;electrode cracks\u0026rdquo; occurred mostly in the thin gold patterns (\u003cem\u003et \u003c/em\u003e= 300 nm and 2 mm) with relatively lower mechanical strength, resulting in a distributed crack-formation throughout the circular electrode sites. On the other hand, circular electrodes with higher gold thickness (4 mm) remained intact, while focused mechanical stress caused a single spot of line disconnection, which corresponds to the location of the highest stress estimated in Fig. 3c and d. The wavy shapes with \u003cem\u003ew\u003c/em\u003e = 10 mm and \u003cem\u003et\u003c/em\u003e = 4 mm (III) was adopted as the optimized parameters throughout this study for the 80 mm-height-mETF, because they secured 100% tolerance (Fig. 3g). Thicker patterns than 4 mm did not provide mechanical enhancement (Supplementary Fig. S10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrochemical and mechanical analysis of \u003c/strong\u003e\u003cstrong\u003emETF MEA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intactness of the neural interfaces during mETF was confirmed via electrochemical analyses, including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) of MEAs before and after mETF. Additionally, iridium oxide (IrOx) layer was electrodeposited on top of the gold channels before and after deformation to demonstrate the compatibility of mETF process with nanoporous surface functionalization techniques widely used for enhancing the charge transfer capabilities of neural interfaces\u003csup\u003e36,37\u003c/sup\u003e. As shown in Fig. 4a and b, the impedance magnitude and phase at 1 kHz showed no significant changes after mETF for both gold and EIROF electrodes (gold: 32.3 \u0026plusmn; 10.8 to 27.3 \u0026plusmn; 14.1 kW, IrOx: 4.1 \u0026plusmn; 0.5 to 3.7 \u0026plusmn; 0.4 kW; see Supplementary Fig. S11a and b for EIS spectra). Figure 4c presents the cathodic charge storage capacities (CSC\u003csub\u003eC\u003c/sub\u003e), which also confirmed that mETF did not result in any significant degradation (gold: 0.24 \u0026plusmn; 0.02 to 0.18 \u0026plusmn; 0.02 mC/cm\u003csup\u003e2\u003c/sup\u003e, IrOx: 38.8 \u0026plusmn; 8.2 to 35.1 \u0026plusmn; 11.7 mC/cm\u003csup\u003e2\u003c/sup\u003e; see Supplementary Fig. S11c for CV curves).\u003c/p\u003e\n\u003cp\u003eThe mechanical resilience of mETF protruding structures against compression was evaluated to assess the potential deformation of the mETF LCP structures under physiological or surgical conditions. As shown in Fig. 4d, the mETF MEA with a height of 80 mm were subjected to increasing compression using a motorized force meter. The magnified plot in Fig. 4e suggests that the 3D electrodes are compressed by no greater than 2.2 mm under the normal range of pressures experienced in physiological conditions, including (i) intraocular pressure (1.3\u0026ndash;2.8 kPa\u003csup\u003e38\u003c/sup\u003e) and (ii) intracranial pressure (0.9\u0026ndash;2 kPa\u003csup\u003e39\u003c/sup\u003e), as described in Fig. 4f. Even after the mETF MEA was completely flattened under a load pressure of 150 kPa, the original height and mechanical properties were recovered after the force is released, as demonstrated by the three cycles of full compression and relaxation (Fig. 4d).\u003c/p\u003e\n\u003cp\u003eThese results suggest that the proposed mETF with proper designs enables the reliable production of 3D structures without compromising the physical and electrochemical properties of neural interfaces.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eBenefits of \u003c/strong\u003e\u003cstrong\u003emETF MEA for subretinal stimulation in FEA study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe benefits of mETF MEA with protruding structures were evaluated for subretinal stimulation of blind patients through FEA in a human retinal model\u003csup\u003e40,41\u003c/sup\u003e. A subretinal electrode is implanted under the retina, facing toward the bipolar cells and retinal ganglion cells (RGCs) (see Supplementary Fig. S12 and Table S1 for retinal model). Given the distance between the electrode surface and targeted bipolar cells in the inner nuclear layer (INL), subretinal stimulation is expected to benefit from protruding 3D structures which would create more focused current distribution at the target cells. Improvement in neural interfaces enabled by 3D subretinal electrodes was quantified in terms of stimulation threshold (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e), dynamic range (DR), and spatial resolution, as shown in \u003cstrong\u003eFig. 5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e and DR were evaluated based on the E-field distribution at INL generated by current injection from a 3\u0026times;3 subretinal electrode array with varying protruding heights from 0 to 100 mm. The \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e is defined as the current intensity required to induce an electric field (E-field) exceeding 3,000 V/m at the plane of the INL to activate bipolar cells\u003csup\u003e27,42,43\u003c/sup\u003e. The DR is a current range from \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e to the maximum current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e), beyond which inter-channel interference occurs between adjacent electrodes. The representative E-field distributions at INL from planar and 80-mm-protruding MEA are presented with increasing stimulation currents from planar in Fig. 5a and b. Notably, the 80-mm-protruding electrodes lowered the \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e to 4.5 mA from 6 mA of the planar electrodes. Increasing current beyond the \u003cem\u003eI\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e began to induce inter-channel interference, at which the INL area activated by a channel (contoured by white dashed lines in Fig. 5b) overlapped with the area activated by adjacent channels. The onset of interferences defines the upper boundary of the stimulation DR, which was extended from 14.5 mA for the planar array to 18.9 mA for the 80-mm-protruding array. A similar analysis for varying protruding heights from 0 to 100 mm (see Supplementary Fig. S13) suggested that the higher protrusions resulted in lower \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e and wider DR, as plotted in Fig. 5c. Such enhancement of 3D electrodes can be explained by more focused current distribution at INL, as shown in Fig. 5d (I = I\u003csub\u003eth\u003c/sub\u003e). The MEA with higher protrusion generated higher contrast in the E-field profiles between the targeted area and untargeted area. However, the 100-mm-protruding array induced an uneven E-field distribution, presumably due to an unmitigated edge effect from an excessively close electrode\u0026ndash;cell distance. Therefore, we concluded that an 80 mm protrusion is the optimum height for efficient subretinal stimulation, which was adopted for the proof-of-concept 25-channel 3D array presented in the previous sections.\u003c/p\u003e\n\u003cp\u003eThe protruding MEA is also predicted to enhance the spatial resolution of retinal stimulation, which was quantified using the Michelson contrast (MC), measuring the ratio of (E\u003csub\u003emax\u003c/sub\u003e \u0026minus; E\u003csub\u003emin\u003c/sub\u003e) to (E\u003csub\u003emax\u003c/sub\u003e + E\u003csub\u003emin\u003c/sub\u003e) (more details in the Methods), as shown in Fig. 5e. Higher protrusion of the electrodes led to a higher E-field contrast (Supplementary Fig. S14) and correspondingly higher MC values across the entire range of channel pitches from 250 to 600 mm.\u003c/p\u003e\n\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBenefits of \u003c/strong\u003e\u003cstrong\u003emETF MEA for subretinal stimulation in \u003cem\u003eex vivo\u003c/em\u003e experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effectiveness of mETF MEA for enhanced neural interfaces in subretinal stimulation were evaluated through \u003cem\u003eex vivo\u003c/em\u003e retina experiments, by comparing the protruding mETF MEA and planar MEA in terms of stimulation threshold and spatial resolution. Activation of RGCs was monitored by imaging the calcium transients in response to electrical subretinal stimulation, using a custom-built fluorescence microscopy setup (Fig. 6a). As shown in Fig. 6b, the MEA placed under the mouse retina patch included both planar and 80-\u0026mu;m-mETF protruding electrodes with a diameter of 100 mm (Supplementary Fig. S6). A genetically-encoded calcium indicator, sRGECO, was introduced to the retina via adeno-associated viral (AAV) vectors and its expression in RGC layer was confirmed three weeks after the injection (Fig. 6c). A typical calcium transient, in response to biphasic current pulses, is represented by normalized changes in fluorescence, D\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0 \u003c/sub\u003e= (\u003cem\u003eF\u003c/em\u003e\u003csub\u003epeak \u003c/sub\u003e\u0026ndash; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, as shown in Supplementary Fig. S15.\u003c/p\u003e\n\u003cp\u003eThe stimulation threshold was evaluated by quantifying D\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0 \u003c/sub\u003eof RGCs placed within the diameter of stimulating electrode (Fig. 6d) while increasing the current injection. As shown in Fig. 6e, the resulting responses were fitted into sigmoidal functions, from which the threshold was defined as the stimulation current at the half of the maximum D\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e. The protruding electrode significantly lowered the median stimulation threshold to 0.91 mA, compared to 1.55 mA of the planar electrode, as shown in Fig. 6f. This suggests that the closer proximity of protruding structure to bipolar cells and RGCs allowed for neural activation with a lower current.\u003c/p\u003e\n\u003cp\u003eThe spatial extent of electrical activation was quantified by D\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0 \u003c/sub\u003eof RGCs depending on their distances from the center of stimulating electrode (Fig. 6d) at a fixed current of \u003cem\u003eI\u003c/em\u003e = 20 mA, as shown in Fig. 6g. The response curves were fitted by Gaussian functions to determine the spatial extents, defined as the half-width at half maximum (HWHM) of D\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e. The protruding electrode produced more focused retinal activation by reducing the HWHM from 94.8 to 43.7 mm, which well agrees with the FEA estimation in Fig. 5d. Stimulation with \u003cem\u003eI\u003c/em\u003e = 10 mA resulted in consistent outcome (99.7 to 39.2 mm), as shown in Supplementary Fig. S16. The HWHM of the protruding electrode comparable to its radius (50 mm) indicates that the mETF MEA activates the retina with high contrast and minimized inter-channel interference, potentially providing artificial vision with higher spatial resolution.\u003c/p\u003e\n\u003cp\u003ePrevious reports have indicated that retinal cells gradually migrate into the voids around the pillars of protruding electrodes after six weeks of \u003cem\u003ein vivo\u003c/em\u003e implantation\u003csup\u003e2,44\u003c/sup\u003e. Such migration reduces the separation between the electrodes and target cells with preserved axonal network\u003csup\u003e27\u003c/sup\u003e, suggesting the benefits of 3D mETF MEA may also be applicable to in an \u003cem\u003ein vivo \u003c/em\u003eretinal\u003cem\u003e \u003c/em\u003eenvironment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we presented a novel \u0026micro;ETF process for creating simple and versatile 3D microstructures on thin-film polymer-based neural interfaces using 3D-printed molds to locally deform planar MEAs into desired protruding or recessing shapes. This approach builds upon the widely used polymer thermoforming technology in the plastic industry, but adapts it for microelectronic 3D structuring to create neural interfaces that are intimate with the nervous system. Our proposed technology uses one-step pressing of conventionally prepared planar MEAs to enable virtually any 3D microstructure to be created on microelectrode arrays without requiring additional fabrication steps. This implies that complex structures with different shapes and heights can be created within a single array using the same one-step thermoforming process. This contrasts with previous 3D-shaping techniques for neural interfaces, which require complicated fabrication steps and are limited in their ability to create different structural profiles on the same array, primarily because of the nature of the MEMS process.\u003c/p\u003e \u003cp\u003eThe simplicity and versatility of our proposed method in forming 3D microstructures expand design possibilities, enabling optimized electrode\u0026ndash;neuron interfaces that reflect the anatomical and neurophysiological characteristics of various nervous systems. Additionally, by combining gross macrothermoforming and local microthermoforming, it allowed for an even wider range of customized macrostructures and microstructures beyond the 3D structures presented in this study. We expect that the proposed technique will have potential applications in areas such as wearable electronics, microfluidic systems, and cell culture platforms, where the integration of 3D microstructures and microelectrodes can contribute to achieving optimal performance. Besides, the \u0026micro;ETF is compatible with various thermoplastic materials commonly used in implantable and wearable biomedical applications, including parylene-C\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, cyclic olefin copolymer\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, perfluoroalkoxy alkane\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, thermoplastic polyurethane\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, polymethyl methacrylate\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, polycarbonate\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and polyvinyl alcohol\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u0026micro;ETF produces 3D structures with inherent round edges and gently sloped sidewalls, which help minimize tissue damage during and after surgical implantation. Mechanical stress within MEA and surrounding tissue was assessed via FEA during forced sliding of an 80-\u0026micro;m height 3D structure into viscoelastic tissue, as shown in Supplementary Fig. S17. The \u0026micro;ETF LCP electrode with native round edges was estimated to reduce the maximum stress within the electrode and on tissue by more than 60%, compared to LCP MEA with sharp edges. The stress mitigation of the \u0026micro;ETF LCP structure was more pronounced when comparison was made against silicon-based 3D structure with the same shapes.\u003c/p\u003e \u003cp\u003eAlthough degenerated retinas are known to undergo structural changes over time\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, a normal human retinal model was employed in this study for the computational simulation of subretinal stimulation. This was primarily due to a lack of consistent structural information available for degenerated retinas in humans. While discrepancies may exist between computational simulations and clinical applications, it is anticipated that the benefits of protruding electrodes predicted in a normal retinal model can also be applied to degenerated retinas, primarily because of the reduced distance between the target cells and electrode surfaces.\u003c/p\u003e \u003cp\u003eThe accuracy and minimum feature size of the \u0026micro;ETF structures are associated with two factors: the precision of the 3D-printed mold, and the mold\u0026ndash;MEA alignment. In this study, a printing resolution of 2 \u0026micro;m for the horizontal plane and 5 \u0026micro;m for the vertical axis was employed. Although this resolution is generally suitable for a wide range of neural interfacing applications, recent rapid advancements in 3D printing suggest the potential future availability of molds with even higher precisions. The mold was aligned with the planar MEAs by matching the laser-drilled holes on the arrays with corresponding pins on the metal jig, ensuring a minimum in-plane feature size of approximately 100 \u0026micro;m. However, this approach may be susceptible to mismatches caused by different thermal expansion ratios between the metal jig, epoxy mold, and polymer array. To enhance the accuracy, the 3D microstructures and aligned pins could be printed monolithically, and error-compensating laser drilling could be employed to further reduce the impact of thermal expansion mismatches.\u003c/p\u003e \u003cp\u003eFinally, the thermoplastic LCP substrate employed in this study is known to exhibit an exceptionally low water absorption rate (\u0026lt;\u0026thinsp;0.04%), potentially ensuring the long-term stability of the 3D MEA\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Therefore, combining versatile 3D forming with durable LCP packaging is considered a suitable approach for various neural interface applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMicrofabrication of LCP MEA\u003c/h2\u003e \u003cp\u003eA planar LCP MEA was fabricated using a previously reported procedure based on conventional microfabrication technologies for thin-film polymer\u0026ndash;metal\u0026ndash;polymer structures\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, as illustrated in Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Briefly, Ti/Au seed layers (50 nm/50 nm) evaporated on a 25-\u0026micro;m-thick LCP film (Vecstar CTQ-25, Kuraray) underwent photolithography to define negative patterns (8 \u0026micro;m thickness, AZ P4620, Merck), followed by gold electroplating. After and seed layer removal (gold etchant and buffered oxide etchant 10:1, Sigma-Aldrich), a 25 \u0026micro;m-thick LCP cover layer (Vecstar CTF-25, Kuraray) was thermally laminated at 285\u0026deg;C with a pressure of 4 kgf/cm\u003csup\u003e2\u003c/sup\u003e for 30 min using a heating press (Model 381, Carver).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3D Mold preparation for \u0026micro;ETF\u003c/h2\u003e \u003cp\u003eA master mold carrying microscopic 3D structures and alignment keys was created via precision 3D printing (microArch S130, Boston Micro Fabrication) and then replicated using a high-temperature epoxy to tolerate the thermoforming process at 200\u0026deg;C. The replication was performed via double casting using polydimethylsiloxane (PDMS) as the negative mold, as illustrated in Supplementary Fig. S2. High-temperature-resistant epoxy (Duralco 4460, Cotronics) was poured over the PDMS negative mold, which was cured in two steps: first at 120\u0026deg;C for 4 h, followed by post-curing at 230\u0026deg;C for 16 h to reinforce its thermal and mechanical tolerance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAlignment and pressing setup for \u0026micro;ETF\u003c/h2\u003e \u003cp\u003eThe alignment and pressing setup consisted of a pair of top and bottom metal jigs; a rubber sheet (RBSM5-100, Misumi) and a rubber frame for uniform pressure; and a cylindrical rubber stamp and a rubber frame for pressing the electrode array against the mold (see Supplementary Fig. S3 for more details). The epoxy mold was assembled onto the bottom metal jig, on which the planar MEA array was loaded. Precise alignment was achieved by matching the laser-drilled align holes on the electrode layer with the align pins on the mold. The \u0026micro;ETF was performed at a pressure of 6.4 kgf/cm\u003csup\u003e2\u003c/sup\u003e applied at 200\u0026deg;C for 30 min. A UV laser cutter (Samurai UV marking system, DPSS Lasers Inc.) was used for site opening and outlining. Oxygen plasma cleaning (CIONE 4, Femto Science) was performed at 80 W and 80 sccm for 30 min to remove the laser burrs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eElectromechanical considerations of 3D MEAs with varying pattern designs\u003c/h2\u003e \u003cp\u003eThe 25-channel MEAs were prepared with varying gold thicknesses and line shapes. The 300-nm-thick gold patterns were prepared via evaporation of Ti/Au layers, photolithography with a negative photoresist (NR9-3000py, Futurrex), and wet etching. The MEAs were not encapsulated by cover layers to facilitate observation of cracks in the gold patterns. The failures of gold patterns after \u0026micro;ETF were categorized into two mechanisms. The \u0026ldquo;electrode crack\u0026rdquo; is defined as a failure due to distributed cracks within the gold electrode sites, whereas \u0026ldquo;line disconnection\u0026rdquo; refers to a disconnection of gold lines. Number of samples for analyzing the failure rate are: 20 for (I)-straight, 20 for (I)-wavy, 40 for (II)-straight, 50 for (II)-wavy, 50 for (III)-straight, and 50 for (III)-wavy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIrOx electrodeposition\u003c/h2\u003e \u003cp\u003eThe laser-opened gold electrodes were electrodeposited with IrOx, following the previously reported protocol\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e using a potentiostat (CompactStat, Ivium Technologies). The electrodes were subjected to four cleaning cycles of voltage sweep in 1 M sulfuric acid from \u0026minus;\u0026thinsp;0.4 V to 1.4 V at 50 mV/s. Triangular potentials (0 to 0.55 V) were then iterated for 200 cycles at 50 mV/s, followed by 2,000 cycles of rectangular pulses (0.55 V, 0.5 s and 1 Hz).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eElectrochemical characterization\u003c/h2\u003e \u003cp\u003eThe electrochemical characterizations were performed with a three-electrode system in the frequency range of 1 Hz to 100 kHz using the potentiostat and phosphate-buffered saline (1X, PH 7.4, Gibco). The CV curves were measured at a scan rate of 50 mV/s in the voltage range of \u0026minus;\u0026thinsp;0.6 V to 0.8 V, from which the CSC\u003csub\u003eC\u003c/sub\u003e was calculated using the time integral of the cathodically enclosed area in Eq.\u0026nbsp;(1):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\text{C}\\text{S}\\text{C}}_{\\text{C}}=\\:\\frac{1}{vA}{\\int\\:}_{{E}_{c}}^{{E}_{a}}\\left|i\\right|dE\\:\\:\\:\\:\\:[\\text{m}\\text{C}/{\\text{c}\\text{m}}^{2}],\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ev\u003c/em\u003e is the scan rate, \u003cem\u003eA\u003c/em\u003e is the geometric area of microelectrode, \u003cem\u003ei\u003c/em\u003e is the current, and \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sub\u003e are the anodic and cathodic potential limits, respectively\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Number of samples for comparing EIS of the MEAs before and after \u0026micro;ETF are: 56 for planar gold, 31 for protruding gold, 43 for planar IrOx, and 63 for protruding IrOx. Number of samples for comparing CSC\u003csub\u003eC\u003c/sub\u003e of the MEAs before and after \u0026micro;ETF are: 12 for planar gold, 16 for protruding gold, 10 for planar IrOx, and 15 for protruding IrOx.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFEA simulations\u003c/h2\u003e \u003cp\u003eMechanical stress induced in the gold traces was computed using Structural Mechanics module in COMSOL Multiphysics 6.0. The in-plane serpentines were implemented by applying a constraint in gold layers to only deform in the axial direction, while no constraints were applied to the out-of-plane serpentines allowing both axial elongation and 3D buckling.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eI\u003c/em\u003e\u003csub\u003eth\u003c/sub\u003e, DR, and spatial resolution in subretinal stimulation were numerically simulated using AC/DC module in COMSOL Multiphysics 6.0 based on a human retinal model\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (see Supplementary Fig. S12 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The stimulation threshold was defined as the stimulating current that generates an electric field of 3,000 V/m at the location of the bipolar cells in the INL, which is the target layer for subretinal stimulation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The spatial resolution estimates the extent of inter-channel interference as evaluated by the Michelson contrast (MC)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The MC measures the ratio of the minimum and maximum electric field strengths at the plane of the INL upon simultaneous current injection from neighboring channels using Eq.\u0026nbsp;(2):\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{M}\\text{C}=\\:\\frac{{\\left|E\\right|}_{max}-{\\left|E\\right|}_{min}}{{\\left|E\\right|}_{max}+{\\left|E\\right|}_{min}}\\:.\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eModeling of insertion of MEA in Fig. S17 employed the retina and electrode modeled as linear elastic materials, as described in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent Calcium imaging system\u003c/h2\u003e \u003cp\u003eA custom-built optical system incorporated both fluorescence and bright field microscopes. The bright field microscope utilized a K\u0026ouml;hler illumination configuration, consisting of a near-infrared light emitting diode (LED) at λ\u0026thinsp;=\u0026thinsp;780 nm (M780L3, Thorlabs), a collector lens, a condenser lens, and two iris diaphragms to ensure uniform illumination of the sample. The fluorescent microscope involved a 565 nm LED (SOLIS-565C, Thorlabs), a dichroic mirror in combination with excitation and emission filters (MDF-MCHA, Thorlabs) to separate the excitation light from emission light. Images were focused onto the CMOS camera using a 40X objective lens (LUMPLFLN40XW, Olympus) with a numerical aperture of 0.8, and a tube lens with a focal length of 200 mm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAAV-meditated gene transfection and retinal preparation\u003c/h2\u003e \u003cp\u003e All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC; PNU-2023-3237) of Pusan National University. Male C57BL/6 mice (10-week-old, 20\u0026ndash;25 g) were housed in a cage under a 12 h light/dark cycles with controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (55% \u0026plusmn; 5%), and ad libitum access to food and water. The AAV vectors were used to transfect mouse retinal neurons with sRGECO (AAV8-Ef1a-sRGECO; 2\u0026times;10\u003csup\u003e13\u003c/sup\u003e GC/mL in HBSS, plasmid number of 137125; Addgene). The mice were anesthetized with avertin (250 mg/kg, Sigma-Aldrich) and placed on a heating pad at 37\u0026deg;C. A 30 G syringe was used to puncture a hole around the limbus. The AAV vectors carrying the genetically encoded calcium indicator were injected into the vitreous for 10 s using a 33G blunt needle (World Precision Instruments)\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. After 3 weeks of injection\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, the retina was prepared following the previously reported protocol by authors\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Detached retina was transferred into the electrode-mounted chamber (RC-27L, Warner Instruments LLC) with the photoreceptor facing down, and immobilized by a nylon-mesh anchor (HSG, Ala Scientific Instruments). Total four retinas were used in this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eretinal experiments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDuring the procedures, the prepared chamber was continuously perfused by oxygenated Ames' medium (A1420, Sigma-Aldrich) containing a synaptic blocker cocktail of CNQX (25\u0026micro;M; Hello Bio) and D-AP5 (25\u0026micro;M; Hello Bio) at 34\u0026deg;C with a constant flow rate of 5 mL/min\u003csup\u003e56,58\u003c/sup\u003e. The stimulating MEA was connected to a stimulus generator (STG4004, Multi-Channel Systems) with a reference platinum wire electrode (MW-4310, BASI Research Product). Electrical stimulation was delivered in a burst of seven cathodic-first biphasic pulses at a frequency of 60 Hz, duration of 60 \u0026micro;s and inter-phase delay of 100 \u0026micro;s with varying amplitudes (Supplementary Fig. S15).\u003c/p\u003e \u003cp\u003e \u003cb\u003eData processing for\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003eexperiments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe stimulation threshold was determined by fitting Δ\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e = (\u003cem\u003eF\u003c/em\u003e\u003csub\u003epeak\u003c/sub\u003e \u0026ndash; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e versus current amplitudes, using a modified sigmoid function, \u003cem\u003eS\u003c/em\u003e(\u003cem\u003ex\u003c/em\u003e) in Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e3\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:S\\left(x\\right)=\\frac{a}{a-\\frac{a}{1+{exp}\\left(b\\times\\:c\\right)}}\\times\\:\\left(\\frac{a}{1+{exp}\\left(-b\\times\\:\\left(x-c\\right)\\right)}-\\frac{a}{1+{exp}\\left(b\\times\\:c\\right)}\\right),$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ex\u003c/em\u003e is the stimulation current, and \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e, and \u003cem\u003ec\u003c/em\u003e are fitting parameters. The threshold is defined as the current corresponding to 0.5\u003cem\u003eF\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e. The spatial extents were determined by fitting Δ\u003cem\u003eF\u003c/em\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e versus distance from the electrode center using a Gaussian function, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:G\\left(x\\right)\\)\u003c/span\u003e\u003c/span\u003e in Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e4\u003c/span\u003e):\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:G\\left(x\\right)=a\\times\\:exp\\left(-b\\times\\:{x}^{2}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ex\u003c/em\u003e is the distance away from the electrode center, and \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e are fitting parameters. The spatial extent was defined as half of HWHM. All the fittings were performed through nonlinear least-squares methods in Matlab.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eD. H. L. and J. J. conceived and planned the experiments. D. H. L. carried out the experiments and simulations. Y. P. and K.E. designed the optical system for calcium imaging and conducted ex vivo experiments. D. H. L., Y. S., H. J., J. -M. S., M. -H. S., and J. J. contributed to sample preparation, measurements, and interpretation of the results. D. H. L. and J. J. took the lead in writing the manuscript. J. J. supervised the project. All authors helped shape the research and revise the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2022R1C1C1010422, RS-2023-00217893, and NRF 2020R1C1C1010505).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSteins, H. \u003cem\u003eet al.\u003c/em\u003e A flexible protruding microelectrode array for neural interfacing in bioelectronic medicine. Microsystems Nanoeng. 8, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlores, T. \u003cem\u003eet al.\u003c/em\u003e Optimization of pillar electrodes in subretinal prosthesis for enhanced proximity to target neurons. 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Commun. 4, 1\u0026ndash;9 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-flexible-electronics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjflexelectron","sideBox":"Learn more about [npj Flexible Electronics](http://www.nature.com/npjflexelectron/)","snPcode":"41528","submissionUrl":"https://submission.springernature.com/new-submission/41528/3","title":"npj Flexible Electronics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"microelectrode arrays, liquid crystal polymer, neural interface, microthermoforming, 3D electrode","lastPublishedDoi":"10.21203/rs.3.rs-4924574/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4924574/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncreasing the proximity of microelectrode arrays (MEA) to targeted neural tissues is crucial for establishing efficient neural interfaces for both recording and stimulation applications. This has been achieved by constructing protruding three-dimensional (3D) structures on top of conventional planar microelectrodes via additional micromachining steps. However, this approach adds fabrication complexities and limits the 3D structures to certain shapes. We propose a one-step fabrication of versatile microscopic 3D structures for thin-film MEAs via \u0026ldquo;microelectrothermoforming (\u0026micro;ETF)\u0026rdquo; of thermoplastics, by utilizing 3D-printed molds to locally deform planar MEAs into desired protruding and recessing shapes. Electromechanical optimization of the \u0026micro;ETF process enabled a 3D MEA with 80 \u0026micro;m protrusions and/or recession for 100 \u0026micro;m diameter. Its versatile shaping capabilities are demonstrated by simple and simultaneous forming of diverse 3D structures on a single MEA. The benefits of 3D MEA are evaluated in retinal stimulation through numerical simulations and \u003cem\u003eex vivo\u003c/em\u003e experiments, confirming a threshold lowered by 1.7 times and spatial resolution enhanced by 2.2 times.\u003c/p\u003e","manuscriptTitle":"MicroElectroThermoForming (μETF): One-step Versatile 3D Shaping of Flexible Microelectronics for Enhanced Neural Interfaces","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-25 12:21:28","doi":"10.21203/rs.3.rs-4924574/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-24T12:16:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-22T07:44:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-11T16:27:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-08T14:06:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-29T05:57:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138835271646575162458917675778855181351","date":"2024-08-27T02:47:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67616209302047527921014471292037934367","date":"2024-08-26T23:55:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188554685886651528172738970951359381060","date":"2024-08-26T23:51:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71035285868020987273526688512560741986","date":"2024-08-26T21:45:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-26T20:12:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-26T09:41:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-26T09:39:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Flexible Electronics","date":"2024-08-16T10:51:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-flexible-electronics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjflexelectron","sideBox":"Learn more about [npj Flexible Electronics](http://www.nature.com/npjflexelectron/)","snPcode":"41528","submissionUrl":"https://submission.springernature.com/new-submission/41528/3","title":"npj Flexible Electronics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b8c3f2a-7ee8-4d8e-bdbe-9f9fb680dc2b","owner":[],"postedDate":"September 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38135372,"name":"Physical sciences/Engineering/Electrical and electronic engineering"},{"id":38135373,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Bionanoelectronics"}],"tags":[],"updatedAt":"2025-01-27T16:09:03+00:00","versionOfRecord":{"articleIdentity":"rs-4924574","link":"https://doi.org/10.1038/s41528-024-00378-0","journal":{"identity":"npj-flexible-electronics","isVorOnly":false,"title":"npj Flexible Electronics"},"publishedOn":"2025-01-22 15:57:30","publishedOnDateReadable":"January 22nd, 2025"},"versionCreatedAt":"2024-09-25 12:21:28","video":"","vorDoi":"10.1038/s41528-024-00378-0","vorDoiUrl":"https://doi.org/10.1038/s41528-024-00378-0","workflowStages":[]},"version":"v1","identity":"rs-4924574","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4924574","identity":"rs-4924574","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}