Biomimetic Microelectronics for Regenerative Neuronal Cuff Implants

Smart biomimetics, a unique class of devices combining the mechanical adaptivity of soft actuators with the imperceptibility of microelectronics, is introduced. Due to their inherent ability to self-assemble, biomimetic microelectronics can firmly yet gently attach to an inorganic or biological tissue enabling enclosure of, for example, nervous fibers, or guide the growth of neuronal cells during regeneration. Living species possess the ability to adapt their shape during the life cycle, e.g., through growth, replication, healing, or motion. Imitating this behavior of nature archetypes, synthetic systems attain adaptability to environmental changes (e.g., artificial irises1 adjust to the light intensity) as well as possibility to interact with the environment mechanically2, 3 or chemically.4, 5 Mimicking the mechanics of the plant cell swelling process in osmosis, the shape of soft objects can be tailored by using stimuli-responsive polymers.6 In hydrogel composites,7 a reversible shape transformation including elongation, twisting, or folding8 is achieved upon external chemical or thermal stimulation rendering these biomimetic devices to be mechanically active. Although mechanically adaptive to the environment, these soft actuators do not carry active electronics to assess and communicate the environmental changes. Alternatively, there are ultrathin and lightweight mechanically flexible and even imperceptible electronics,9-12 which are electrically active but lack of reversible self-actuation. Combining mechanical adaptivity of soft actuators with the imperceptibility of microelectronics paves the way toward an entirely new class of devices–smart biomimetics. Smart bio­mimetics can mechanically adapt to and deterministically impact the environment electrically or mechanically. Foreseeable applications of this technology are far reaching. At the large scale, biomimetic microelectronics can assess the electrical activity of the brain13 and the heart14 in conformable and portable NeuroGrids, as well as the electrophysiological processes acting as a component of catheters11 and artificial skins.10, 15 At the microscale, smart biomimetics bear great potential to impact the neurology and regenerative medicine by offering neuronal cuff-type implants16, 17 with unmatched functionalities. Due to the inherent ability to self-assemble, biomimetic microelectronics can be firmly yet gently attached to a biological tissue enabling enclosure of, e.g., nervous fibers, or guiding the growth of neuronal cells during regeneration (Figure 1a). The integrated electronics enables stimulating the biological tissue18 and monitoring the healing process.19 Upon external stimulation, e.g., after healing, the cuff can be opened, releasing thus the nervous tissue with minimal invasivity. Here, we realize mechanically adaptive microchannels of cuff-type implant with integrated high-performance micro­electronics including signal amplifiers and logic circuitry based on indium gallium zinc oxide (IGZO) transistors.12 The active electronic devices are fabricated on a polyimide support capping a hydrogel-based stimuli-responsive layer. The swelling state of the hydrogel can be affected by tailoring the environmental conditions, including solution composition and pH. This provides full control over the shape of the device, which can be deterministically set in a planar or bent state, as well as self-assembled into a Swiss roll-like microtube (Figure 1b) with a tunable dia­meter (Figure 1c) ranging from 50 μm (Figure 1c, top inset) to 1 mm (Figure 1c, bottom inset). Inorganic rolled-up microtubes less than 10 μm in diameter have previously been shown to act as ultracompact microfluidic channels with fully integrated electrodes and field effect transistors, able to detect polar and ionic fluids down to subnanomolar concentrations, sense single cancer cells, and guide neuronal outgrowth.20-22 Medical applications of such tubular architectures have been envisioned for topographically mediated nerve growth, tissue engineering, and regeneration.22 The opportunity to open/close such microscale devices upon external stimulation brings these applications closer to reality and is particularly appealing for neuronal cuff implant applications to enclose and guide the growth of nervous fibers with a typical size of 10–50 μm.18 The achieved device diameters of 50 μm are at least two orders of magnitude smaller compared to the state-of-the-art neuronal cuff implants.23 We prove biocompatibility of the polymeric platform and demonstrate differentiation and guided outgrowth of neural stem cells from the cortex region of E14 mouse embryonic brains in the channels formed by the microtubes (Figure 1d and Figure 2). By patterning the polymeric spacer between neighboring windings of the Swiss roll like architecture, we achieve regular openings with a cross-section area of 3 × 6 μm2 (Figure 1e) potentially enabling the guidance of individual neuronal fiber and single axons, which possess typical diameters in the micrometer range.18 At the same time, the elasticity of the polymeric layer forming the interior of the channel is about 17 MPa, which matches the elasticity of the protective tissues of the central nervous system, in line with the requirements for in vivo implants.3, 23 The total radial pressure imposed by the device upon the self-assembly process is about 600 Pa, which is well below the harm limit of 1300 Pa characteristic for nerves and axons.24 The Swiss roll geometry with multiple windings allows the device to adjust to the dimensions of the nerves during their life circle, reducing the probability of a compression trauma.25, 26 The small thickness of the polymers of less than 1 μm prevents the IGZO electronics from degradation upon severe mechanical deformations, e.g., bending to a curvature radius of 25 μm. This is by far the smallest reported bending radius imposed on the entire circuit containing amplifiers and logic devices. Even in the most bent state, the signal amplifiers and the advanced logic including inverters (NOT gates) and universal (NAND) gates remain fully intact and maintain their functionality. The threshold voltages of individual transistors remain below 1 V and the maximum saturation current approaches remarkably high values of 1 mA. In the assembled state, analog amplifiers possess a unity gain frequency of 30 kHz. We applied the integrated electronics to detect ionic signals from 10 pL of phosphate buffered saline (PBS) solution repeatedly pushed in the channel, demonstrating mV polarization response of the IGZO transistor. With this performance, the platform can be readily used for monitoring the action potential of ionic axons. Furthermore, the demonstrated possibility to integrate the high-performance amplifiers at the location where the signals of interest are acquired allows increasing the signal-to-noise ratio, which is of strong advantage for the diagnostic of neuronal activity.27-29 Fabrication of microchannels with integrated electronics: To realize mechanically active, stimuli-responsive, and high-performance IGZO microelectronics, we formulated and synthesized novel photopatternable polymers allowing a full-scale microelectronic fabrication process including multistep litho­graphy, depositions, as well as dry and wet etching. The polymeric layer stack includes a water-soluble metal–organic sacrificial layer (thickness: 175 nm), a highly cross-linked hydrogel-based swelling layer (thickness: 200 nm), as well as a stiff polyimide film (thickness: 500 nm) acting as a single layer reinforcement for the stimuli-responsive hydrogel. Details on the synthesis can be found in the Experimental Section; see also Figure S1 in the Supporting Information. This polymeric functional stack with a total thickness of less than 1 μm is processed onto 80 × 80 mm2 glass substrates using optical lithography to realize the predefined locations that will host the electronics (Figure 1b and Figure 3a). The IGZO-based individual thin-film transistors (Figure S2, Supporting Information), the key digital logic elements, i.e., inverters (NOT gates) and universal (NAND) gates, as well as the analog amplifiers with a total thickness of the stack of about 80 nm are prepared onto the lithographically predefined locations with a typical lateral size of 800 × 500 μm2 and 2000 × 1000 μm2. The circuitry is locally protected by the 40 nm thick photopatternable polychloroprene-based capping layer. By placing the substrate into a neutral aqueous solution of sodium diethylenetriaminepentaacetic acid (DTPA), the sacrificial layer is selectively removed leading to the free standing polymeric membrane with embedded IGZO electronics. The entire membrane is anchored at one edge to the handling glass substrate. The stiff polyimide reinforcement layer allows fixing the lateral dimensions of the devices at the top surface of the hydrogel. In the water solution, the hydrogel swells from below and generates thus the differential mechanical stress in its thickness that results in the upward bending of the entire architecture.30-32 Ultimately, the initially planar device will be transformed into a compact Swiss roll like architecture composed of one or several windings and with a lithographically defined length in the range of some millimeters (Figure 1c). The fabrication platform allows the realization of arrays of mechanically active microchannels with integrated high-performance IGZO microelectronics over the entire 80 × 80 mm2 glass substrates with a yield of more than 90% derived from 500 devices. Elastic moduli of the device: The mechanical properties of the polymers influence the self-assembly process and determine the final geometry of the device, e.g., diameter and number of windings. The elastic modulus of each polymeric layer with the thicknesses as in the functional stack was determined by measuring the force–distance curve with an atomic force microscope (AFM) tip indenting the layer (Figure 1f), accordingly to the routine proposed by Rosso et al.33 The data was fitted to the Hertzian model34 (see the Experimental Section for details) and the Young's modulus was determined by analyzing the slope of the linear part of the force–distance curve. We achieve a Young's modulus of the materials of 16.8 MPa for the capping polychloroprene layer, 4.5 MPa for the hydrogel layer, and 3.2 GPa for the stiff polyimide layer. The Young's modulus of the outer hydrogel layer and the inner polychloroprene layer is in the same range as the modulus of the protective tissues of the central and peripheral nervous system, namely dura and pia matter (20 MPa and 2.3 MPa, respectively), as well as the nerves itself (elastic modulus of the sciatic nerve is about 40 MPa).35-37 This renders our microchannels mechanically biocompatible for in vivo applications. Tailoring diameters of the microchannels: The diameter of the tubular structures can be tuned over a wide range, by adjusting the thicknesses of the individual polyimide, hP, and hydrogel, hH, layers. The chosen thicknesses determine the differential strain generated in the hydrogel layer upon immersing it into a solvent. Furthermore, the lithographically defined dimensions of the initial planar layout and the resulting diameter of the microtube determine the number of windings in the Swiss roll architecture. For instance, the self-assembly process of the initial planar device with an area of 800 × 400 μm2 consisting of the stack of 500 nm thick polyimide and 200 nm thick hydrogel rolls up into a tube with a diameter of 50 μm, leading to a Swiss roll architecture with 2.5 windings (Figure 1c, top inset). To realize a tube of 500 μm diameter, the thickness of the polyimide layer should be increased to 1400 nm, while the hydrogel layer should become thinner with hH = 150 nm. The possibility to tune the inner diameter of the tubes from 50 μm to ≈1 mm allows using these architectures as regenerative cuff implants to guide the growth of nerves and nervous fasciculus. Here, we use neural stem cells from the cortex region of E14 mouse embryonic brains to demonstrate their guided growth in the self-assembled tubular channels. 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The small thickness of the stack prevents the electronics from degradation upon severe mechanical deformations, e.g., bending to a curvature radius of about 25 μm. The elasticity of the inner polymeric layer is 17 MPa, which matches the elasticity of the protective tissues of the central and peripheral nervous We prove biocompatibility of the polymeric platform and demonstrate differentiation and guided growth of the neural stem cells in the channels formed by the The integrated microelectronics allows monitoring the of of ionic in the channel, the of processes in neural Furthermore, the demonstrated possibility to integrate high-performance amplifiers at the location where the signals of interest are acquired allows increasing the signal-to-noise ratio, which is of strong advantage for biomimetic microtubes a toward microelectronics for neural and The ability to polymers into upon external stimulation and mechanical properties should be of great reducing the of biological tissue and and of the polymeric layer including and sacrificial as well as is in the Supporting Information. Fabrication of IGZO In a single process on the same we prepared individual transistors and more devices including logic and The circuit is based on technology using IGZO thin-film have been using a Details on the fabrication are in the Supporting Information. We more than individual devices and after the self-assembly devices were and the the threshold over devices and after was V and The was to be and for planar and self-assembled the self-assembly active of the structures were protected with photopatternable Details on the synthesis of the photopatternable polychloroprene layer are in the Supporting Information. 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