Symposium SB08—Advanced Neural Materials and Devices

As our understanding of the brain’s physiology and pathology progresses, increasingly sophisticated technologies are required to advance discoveries in neuroscience and develop more effective approaches to treating brain disease. There is a tremendous need for advanced materials solutions at the biotic/abiotic interface to improve the spatiotemporal resolution of neuronal recording. Organic electronic devices offer a unique approach to these challenges, due to their mixed ionic/electronic conduction, mechanical flexibility, enhanced biocompatibility, and capability for drug delivery. We designed, developed, and characterized conformable organic electronic devices in the form of transistors and electrodes to efficiently interface with the brain and acquire neurophysiological activity not previously accessible with recordings from the brain surface. These devices have facilitated large-scale rodent neurophysiology experiments and uncovered a novelhippocampal-cortical oscillatory interaction. The biocompatibility of the devices allowed intra-operative recording from patients undergoing epilepsy surgery, highlighting the translational capacity of this class of neural interface devices. In parallel, we are developing the high-speed electronics and embedded acquisition and storage systems required to make high channel count, chronic neurophysiological recording from animals and human subjects possible.
This multidisciplinary approach will enable the development of new devices based on organic electronics, with broad applicability to the understanding of physiologic and pathologic network activity, control of brain-machine interfaces, and therapeutic closed-loop devices.

Designing materials using concepts inspired by and/or mimicking biology represents an attractive strategy for a variety of fields, including neural probes being developed to better understand the brain. In neural tissue it has been now well-documented that structural and mechanical differences between neural probes and neuron targets in the brain can lead to disruption of the native tissue that negatively impacts the capability to stably interrogate and modulate natural physiological activity. In this presentation, we will overview a new paradigm for seamlessly merging electronic arrays with the brain in three-dimensions (3D) using neuron-like electronics. First, considerations of matching size, mechanical and topological characteristics of neural probes and brain tissue will be discussed, thus leading to the paradigm of bio-inspired neuron-like mesh electronics. Second, extensive 3D subcellular resolution imaging of tissue containing implanted neuron-like electronics will be described to illuminate the immune-privileged nature of this material together with the unique seamless and nearly indistinguishable three-dimensional interpenetrating nanoelectronic and neural networks. Third, we will also describe studies showing how neuron-like electronics and biochemically-functionalized neuron-like electronics allows for controlled and selective manipulation of endogenous cells in the brains of live mice. Last, future opportunities and challenges will be discussed, including our goal of and potential for realizing precision electronic medicine in the brain.
Selected References
(1) http://meshelectronics.org/
(2) X. Yang, et al., Nat. Mater. 18, 510–517 (2019).
(3) G. Hong & C.M. Lieber, Nat. Rev. Neurosci. 20, 330–345 (2019).

Technological leaps to improve brain-machine interface devices require sophisticated integrated circuits based on ion driven transistors. These devices should be capable of forming complementary logic and analog circuits to allow acquisition, processing and manipulation of a biological environment. However, the lack of readily available p-type polymers for use in enhancement mode transistors and their complicated synthesis significantly limits the investigation and application of such devices in bioelectronics. To address this challenge, we propose a simple route for creation of enhancement mode Ion Gated Transistors (IGT) by combining a highly conductive poly(3, 4- ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) and a reducing agent polyethylenimine (PEI) to enable doping of the polymer in an aqueous environment. The protonated PEI bonds with the PSS- and de-dopes the PEDOT:PSS chain resulting in an initial off-state of the transistor. A subsequent application of negative gate voltage dopes the PEDOT:PSS switching the channel on. Furthermore, we introduce a novel operation mode of these organic transistors by utilizing contained mobile ions within the conducting polymer channel to permit shortened ionic transit time and high transconductance. The embedded ions within the channel are obtained through the addition of D-sorbitol, a biocompatible hydrophilic sugar alcohol, which uptakes water molecules keeping the channel hydrated through the generation of ion reservoirs. The distance ions have to travel to modulate the transistor is shorter compared to the one of electrolyte-based transistor resulting in substantially faster devices (time constant of 2.6 μs). To determine an optimal transistor configuration and material composition, we microfabricated transistor arrays of varying geometrical parameters. In doing so, we were able to extract conductivity, contact resistance, and electrochemical impedance values for all the critical interfaces of various composites. The resulting high-transconductance, high-speed enhancement-mode transistors were realized in the form of conformable depth probes fabricated on thin film plastics to enable in vivo electrophysiological recording of cortical activity from neonatal rats and conformable surface devices to be utilized for electrocardiography, electroencephalography and electromyography from human subjects. The ultra-high speed, improved transconductance and stability of these arrays were demonstrated by recording biosignals covering a full biological frequency band varying from single unit activity to local field potentials, cardiac and muscular activity, (0-1000 Hz) paving the way for use as a reliable neural interface device.

Implantable neurostimulation devices have been attracted considerable attention recently since neural disordered disease can be treated by electrostimulation. Microneedle electrode arrays have high aspect ratio that can sufficiently penetrate to the depth of target without causing any damage. Additionally, electrically controlled drug release has been particularly attractive for bioelectronics because the electrical signal is portable and controllable on-demand, without the requirement of large or special equipment. However, protein-based bioactives such as growth factors or antibodies are easily denatured to lose their bioactivity in response to external stimulation. It is challenging to develop a bio-electrode system that permits the electrically responsive drug release without damage.
In this study, we designed and fabricated an IrOx-based microneedle electrode array on a flexible Parylene C substrate. Then, the microneedle electrode array has been treated by a facile method to form a hybrid film of iridium oxide and bovine serum albumin (BSA). We carried out a cyclic voltammetry approach to co-electrodeposit iridium oxide and plasma protein on the microneedle electrode array. We characterized and evaluated the hybrid electrolytes and deposited films for bio-electrode applications. We also demonstrated the electrically controlled release triggered by pulse current stimulation. In addition, the biocompatibility of the microneedle electrode array was also investigated by testing the cell viability.

One of the most important scientific and technological frontiers of our time is the interfacing of electronics with the human brain. This endeavour promises to help understand how the brain works and deliver new tools for diagnosis and treatment of pathologies including epilepsy and Parkinson’s disease. Current solutions, however, are limited by the materials that are brought in contact with the tissue and transduce signals across the biotic/abiotic interface. Recent advances in organic electronics have made available materials with a unique combination of attractive properties, including mechanical flexibility, mixed ionic/electronic conduction, enhanced biocompatibility, and capability for drug delivery. I will present examples of novel devices for recording and stimulation of neurons and show that organic electronic materials offer tremendous opportunities to study the brain and treat its pathologies.

Release and reuptake of neuromodulator serotonin is central to mood regulation and neuropsychiatric disorders, whereby imaging serotonin is of fundamental importance to study the serotonin signaling system. Herein, we present a reversible near-infrared optical probe for serotonin that reports physiologically-relevant serotonin concentrations on relevant spatiotemporal scales, and is compatible with pharmacological tests. The probe responds with ΔF/F₀ of up to 194% in the near-infrared fluorescence emission window of 1000-1300 nm, and is constructed from semiconducting single-walled carbon nanotubes (SWNT), which have shown utility for non-invasive through-skull imaging in rodents. Synthetic molecular recognition for serotonin was conferred by evolving molecular recognition between single stranded DNA (ssDNA) and SWNT. To do so, we developed a high-throughput screening platform for evolution of serotonin molecular selectivity, in which systematic evolution of ligands by exponential enrichment is implemented on carbon nanotube surfaces, a process we’ve termed SELEC. 10¹¹ unique SWNT-ssDNA constructs can be screened for their ability to bind a target analyte and provide a selective near-infrared fluorescence signal. Iterative selection of analyte-binding polymers that form a SWNT-surface-adsorbed phase for serotonin recognition are identified through ionic desorption of sub-optimal polymers, and exponential amplification of nucleotides that recognize serotonin. The best-responding serotonin nanosensor is shown to bind serotonin with a K_d of 6.3 µM, is shown to be reversible, and exhibits unaltered performance in artificial cerebrospinal fluid. We show that our serotonin nanosensor does not respond to serotonin metabolites 5-hydroxyindoleacetic acid (HIAA), 5-hydroxytryptophan (HTP), and 5-methoxytryptophan (MTP) and of importance to understanding pharmacology in the context of serotonin signaling, we additionally show serotonin receptor-targeting drugs fluoxetine, MDMA, 25I-NMOMe, and quetiapine do not interfere with nanosensor performance. Lastly, nIRHT can be introduced into the brain extracellular space in acute brain slices, and can be used to image exogenous serotonin reversibly. Our results suggest evolution of nanosensors could be generically implemented to rapidly develop other neuromodulator probes, and that these probes can image neuromodulator dynamics at spatiotemporal scales compatible with endogenous neuromodulation. While we’ve implemented SELEC to develop an optical probe for serotonin herein, the platform is fundamentally generic for generating other optical probes of neurological relevance.

Flexible materials are important for the development of insertable neural probes for recording stable signals (spikes) in vivo. However, conventional neural probes such as silicon probes have several drawbacks, including high rigidity compared with soft brain tissues and poor mechanical properties during manipulation. In addition, the operator must handle the probe carefully because its tip is extremely small, sharp, and brittle, so it can be easily damaged if touched by a finger or dropped on the floor. To overcome these problems, here, we show inkjet-printed, flexible neural probes for spike recording by using polymeric ultra-thin films (nanofilms). The neural probes were constructed from 400 nm-thick poly(D,L-lactic acid) nanofilms, inkjet-printed lines consisting of Au and poly(3,4-ethylenedioxythiophene):polystyrenesulfonate nanoinks, and insulating fluoropolymer layers. The neural probe was prepared as following steps: i) 50-µm-wide conductive lines were inkjet-printed on the nanofilm, which was dip-coated with a fluoropolymer solution to make insulating layers. ii) The tip of the insulated nanofilm was trimmed with a razor, exposing the microelectrodes. iii) The resulting 6-µm-thick flexible electrode was twisted from a 2D sheet into a 3D needle, allowing it to be inserted into brain tissues. The electrode was also integrated into an optical fiber (i.e., optrode) to enable optogenetic recording. iv) To connect the electrode to the external amplifiers, the stiffness was gradually increased from the flexible nanofilm to the rigid device and fabricated pre-assembled connection. The nanofilm-based probe with a needle shape recorded not only in vivo neuron spikes from mouse thalamus, but also spontaneously and optogenetically evoked individual spikes from rat hippocampus with the optrode. The flexible and robust structure of the present neural probes will allow for tailor-made customization for various research requirements, such as changing the complex geometry, increasing the number of electrodes for spike signal sensing, and segmenting the electrode tips to precisely record in vivo neuronal activity.

Thermal drawing has been applied to produce flexible, microstructured polymer fibers integrating diverse functionalities. Because of their small size, flexibility, and biocompatibility of the component materials, these multifunctional fibers are attractive for applications in neuroscience to probe and modulate neural activity. Despite the advantages of thermal drawing, including simplicity of materials integration and high device yield, the functionality of thermally drawn fibers is constrained by the symmetry of the fiber along its axial direction. In the present work, we suggest two hybrid fabrication methods combining thermal drawing and photolithographic process to break this symmetry and enable interactions of fiber-based probes with multiple neural populations along their shaft following implantation into the brain.
We employed thiol-epoxy/thiol-ene click chemistry to produce a novel photoresist with low polymerization shrinkage stress and low oxygen inhibition during polymerization. The system is polymerized via two steps - thermal and photo curing. Thermal polymerization yields formation of linear chains through a reaction between thiol and epoxy functional groups resulting in a thermoplastic compatible with fiber drawing. In contrast, photo curing occurs only at areas exposed to ultraviolet light, generating an insoluble network structure through the thiol-ene reaction, allowing for the development of desired patterns. The reported photoresist can be applied either prior to or following fiber drawing allowing for the design of functional features along the fiber shaft.
In addition to offering greater utility of fibers for neuroscience and neural engineering, our technique will likely find broader applications in fields of optoelectronics, sensors and smart textiles.

We fabricated a transparent organic electrochemical transistor (OECT) using ultrathin Au (14 nm), and successfully recorded extracellar potential of induced pluripotent stem cell-derived cardiomyocytes.

OECT has attracted interests in both material engineering and biological engineering fields because its high transconductance and simple structures enable versatile applications by combined with other flexible materials. Monitoring electrophysiological activity of cells is one of those applications[1]. By using OECT instead of conventional electrodes, we could expect better data quality and high density measurement without lack of flexibility, which is usually realized on gel and provides bio-tissues with free movement and better maturity.
Although transparency is also an advantage of OECT, cell monitoring with transparent OECT have never been reported as far as we know. OECT usually consists of three layers other than substrates: metal source and drain electrode, organic semiconductor as channel, and passivation. Unlike other organic transistors, the channel of OECT can be made from poly(3,4-ethylenedioxy- thiophene):poly(styrenesulfonate) (PEDOT:PSS), which is a transparent semiconductor. Therefore, by replacing metal source and drain electrode for transparent conductors, you can make the whole device transparent. It enables the support of imaging technologies including fluorescence imaging or image based force mapping.
Transparent OECT for in vivo optogenetic experiment was previously reported[2], but its grid structured metal electrode may bother imaging in micro-meter scale cell monitoring. Thus in our previous report, we used Indium-tin-oxide/Au/Indium-tin-oxide (ITO/Au/ITO) structure as highly conductive transparent electrode instead[3].

Here we further optimized its structure. ITO/Au/ITO structure was replaced by simple ultrathin Au supported by polyimide coated parylene substrate. The photoresist used as passivation layer was also replaced for parylene to ensure its biocompatibility. Owing to the small total thickness, which is less than 5 µm, it can be used as highly flexible, fordable device. The mechanical robustness was limited so far due to the ITO layer. The ultrathin Au showed stable operation under bending test which is equivalent to 1.25 % tensile strain whereas ITO/Au/ITO started failing at 0.9 %. The sheet resistance was 8.6 ohms/sq. so that you can achieve sufficiently low parasitic resistance against channel resistance (~500 ohms). Owing to the simplified structure and low sheet resistance, transparent active matrix fabrication and extracellar potential recording were successfully demonstrated.

[1] Chunlei Yao, Qianqian Li, Jing Guo, Feng Yan, and I-Ming Hsing Adv. Healthcare Mater. 2015, 4, 528–533.
[2] Wonryung Lee, Dongmin Kim, Naoji Matsuhisa, Masae Nagase, Masaki Sekino, George G. Malliaras, Tomoyuki Yokota,and Takao Someya PNAS, 2017, 114,10554-10559.
[3] 2018 MRS Spring Meeting & Exhibit Yasutoshi Jimbo, et al., 2018.

Understanding how a network of interconnected neurons receives, stores and processes information in the human brain is one of the greatest scientific challenges of our time. Neurons encode information by electrical signals. Optical detection and imaging of electric activities provide unprecedented flexibility and parallelization. In the last decade, the advancement of voltage-sensitive fluorescent proteins, and small potentiometric dyes, has drastically enhanced the capability optical recording of electrical activity in cells. However, the use of fluorescence proteins requires genetic modification of the cells and the membrane insertion of voltage-sensitive probes can lead to membrane capacitance loading. The fluorescence-based imaging approach also suffers from phototoxicity, limited signal-to-noise, limited recording duration due to photobleaching, and time-dependent probe internalization and protein expression. We develop a novel technique to detect electric activities of a neuronal network in a label-free, parallel and non-invasive manner. Our detection technique exploits unique physical properties of electrochromic materials, whose optical properties can be modulated by the applied electric potential. Combined with sensitive optical detection, our method is able to achieve label-free detection of action potentials in neurons, cardiomyocytes, engineered spiking-HEK cells and brain slices over long periods of time.

Our ability to treat neurological disease is severely limited by the complexity of the nervous system and the quality of the information derived from recording devices. A device that holds great potential for recording high quality electrophysiology signals is the organic electrochemical transistor (OECT). OECTs are transistors in which the output current is regulated by the injection of ions from an electrolyte. They are fabricated from biocompatible materials and have been shown to provide higher signal-to-noise ratio compared to electrodes. Their unique properties pave the way for enhanced performance neural interfaces while minimising the invasiveness of the recording method. We report on how different parameters such as device geometry and bias conditions affect the noise characteristics of OECTs. These results show how to minimise noise and boost signal-to-noise ratio. We further show how these new design rules are applied to reduce the footprint of OECTs and drastically improve their recording capability when used in neural interface applications.

Introduction: Neural pathology is characterized by electrical and chemical dysregulation in distinct brain circuits. Chemical dysregulation can be monitored by measuring the local composition of interstitial fluid (ISF). The current state-of-the-art in neurochemical sampling, microdialysis, enables the collection of small, highly concentrated neurochemicals from ISF via diffusion across a semipermeable membrane. Large probe sizes (> 150 μm) limit spatial resolution, which can lead to tissue scarring and limit chronic recording. Moreover, relying on diffusion limits spatiotemporal resolution and increases detection limits. Membranes particularly limit measuring neuropeptides, which are prone to non-specific absorption (average recoveries < 20%) and are present at very low concentrations in ISF, and preclude measuring dense core extracellular vesicles (EVs), which play a critical role in cell-cell signaling. We have built a novel micro-invasive membrane-free sampling platform that enables direct sampling of ISF. This is a fundamental shift from microdialysis, which tracks small and abundantly present neurotransmitters, to a device that samples the complete biochemical milieu with pinpoint spatiotemporal resolution. We anticipate this will enable a deeper understanding of the onset and progression of neural pathologies.
Methods: We have manufactured a sampling platform composed of micro-invasive probes (80 μm outer diameter, 50 μm inner diameter) coupled to a custom-made nanofluidic pump (nanopump). Fluid flow within custom tubing (100 μm inner diameter) in the pump is driven by the sequential contraction of nitinol wires (shape memory alloy), and back flow is prevented by a nitinol powered valve. The nanopump is capable of bidirectional flow control with single nanoliter precision and negligent dead volume, a capability not demonstrated by the current state-of-the-art, and is fully portable for in vivo use. Flow is controlled via the electrically-triggered contraction of nitinol wires (Fig. 1A) and flow rates (1 nL/s) and sampling volumes (100-1000 nL) safe for in vivo operation in the brain were used. Sampled fluid is analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect neuropeptides at physiological concentrations (1-20 femtomolar).
Results & Discussion: We have shown that our micro-invasive probes minimize gliosis when chronically implanted in rodent brains, and retain fluidic functionality for 12 mos. post-implantation. Extensive characterization of nanopump-driven infusion into and sampling from agarose brain phantoms in vitro has enabled optimization of platform performance in response to a variety of parameters including wire number and pre-tension, tube diameter and length, capillary size, and flow control algorithm. Ex vivo infusions into rodent striatum have been characterized using both histology and 3D fluorescence imaging, showing bolus volume control as a function of infusion time. Nanopump-driven sampling from ex vivo and in vivo brains has also been performed, with confirmed presence of proteins specific to neural extracellular space, including brain acid soluble protein 1, myelin basic protein, and gamma enolase. Sample stability in different cryo-storage/processing conditions has been thoroughly characterized. Ongoing in vivo sampling studies will enable chronic tracking of covariant neuropeptides and EVs in physiological and pathological states. A first step will be long-term tracking of the spatiotemporal dynamics of dynorphin, a neuropeptide associated with stress, anxiety, and substance use disorders, a capability that is not possible with current tools.
Conclusions: We have developed a novel tool for biomedical engineers and neuroscientists to investigate the biochemical basis for neural pathology. Our micro-invasive ISF sampling platform has the potential to generate new fundamental knowledge and enable more accurate diagnosis and treatment of neural disorders in the future.

Recent advances in the field of neural interfaces have been developed to improve the precision with high resolution and specificity of stimulation to the level of individual cells. To activate neurons with enhanced selectivity, the efficient coupling between the cell membrane and electrodes is required. Here, we demonstrate a vertical nanowire multi-electrode array (VNMEA) with feature sizes and densities comparable to neural circuits which enable the individual stimulation by forming tight junctions with cell membranes. This study demonstrated stimulation-induced Ca²⁺ elevations in individual primary hippocampal neurons by direct intra-neuronal stimulation with a vertical nanowire multi-electrode array (VNMEA). VNMEA-mediated stimulation showed superior properties of peak amplitude of Ca²⁺ elevation and kinetics of recovery than those through field stimulus. Moreover, tight physical/electrical coupling with the individual neuron enables targeted stimulation of a specific neuron without activation of nearby neurons, and localization of spiking neurons in response to depolarization due to intra-neuronal stimulation, which is considered the connection and activation pathway of the neurons. From the results of simulation models, we demonstrate the spatial and temporal confinement of intracellular stimulation by 6.9-fold higher current density built in intracellular space compared to extracellular space. Considering the activation modalities of separate neurons are essential for a complete understanding of neural circuit dynamics, the VNMEA platform could further provide a versatile tool for both manipulating single neurons and probing the functional connectivity of specific neural networks.

Conducting polymer actuators are promising materials for biomedical applications ranging from artificial muscles to drug delivery devices. These devices rely on bulk volume changes of conducting polymers (CPs) which arise from electrochemical redox processes. The changes in polymer chains conformation combined with the transportation of ions and solvent in and out of the polymer matrix are responsible for the micro and macro-scale expansion/contraction of the CPs. Construction of bilayer/trilayer bending devices is a very common strategy to convert the reaction-driven volume changes into macroscopic motions, where electrical energy is transduced into mechanical energy via electrochemical redox reactions in the active CP layer. In such devices, the CP layer is adhered to a passive thin layer to convert the in-plane actuation strain generated in the CP layer into macroscopic bending movements. Prior works have focused on the actuation of bilayer/trilayer actuators based on polypyrrole (PPy) films. We have previously proposed a novel structure for PPy:polystyrene sulfonate (PSS) in the form of randomly-oriented nanotubes and studied their ion transport behavior during cyclic voltammetry (CV) via electrochemical quartz crystal microbalance. Here, we report a bilayer actuator based on PPy nanofibers doped with PSS and constructed on a passive layer of Au-coated polypropylene (PP) film (length= 20 mm, width= 1 mm, PP thickness= 30 µm, Au thickness= 180 nm, PPy thickness= 12 µm). The PPy nanofibers were fabricated using electrochemical deposition of PPy (charge density 3.6 C/cm²) around electrospun poly-L-lactide nanofibers with the average diameter of 140±4 nm. The average diameter of the resultant PPy nanofibers was 626±16 nm. The bending behavior of the PPy nanofibers was investigated by measuring the tip deflection of actuator in both liquid and agarose gel (0.2%) electrolytes containing 0.1 M NaPSS. The PPy nanofibers were subjected to CV in the potential range of –0.8 V to +0.4 V at various scan rates of 10, 50, 100, and 200 mV/s for 20 cycles. The actuator showed a reversible bending movement during each potential cycle. The maximum deflection of actuator decreased in both liquid and gel electrolytes by increasing the scan rate. The maximum tip deflection in liquid was 7.89±0.08 mm, 5.38±0.04 mm, 3.81±0.01 mm, and 2.52±0.01 mm, respectively at the scan rates of 10, 50, 100, and 200 mV/s. The maximum tip deflection in gel was 432±11 µm, 301±2 µm, 222±1 µm, and 148±1 µm, respectively at the scan rates of 10, 50, 100, and 200 mV/s. Ultimately, the actuation moment generated during cycling at various scan rates was calculated using linear bending beam theory and Bernoulli's equation for fluid drag force. The findings in this study may have a great impact on the utilization of CP nanofibers for development of bioactuators.

Peripheral nervous system injuries are a common accessory to trauma, constituting tens of thousands of cases per year. Without intervention, these injuries can compound into a permanent reduction or loss of limb function. Clinical treatments are limited to performing an autograft to close the gap. While effective, this method has some drawbacks, primarily nerve-tract mismatch in the host area and dysesthesias in the donor region. Artificial conduits have been widely studied as an additional intervention, but outcomes are not yet on par with autografts. Conducting polymers (CPs) have garnered much interest in neuroprosthetic applications due to their combination of unique electrical, physical, and chemical properties. Due to their biocompatibility, CPs can guide growing or regenerating axons, but the quantitative relationships between CP surface properties and axonal outgrowth are not yet clear. Characteristic feature size is known to be a crucial element in biomaterial substrates, enhancing, hindering, or entirely blocking the growth of cells cultured upon the biomaterial. Studies have demonstrated the effect of various substrate morphologies on neuron behavior and morphology, but the effect of spatially-variant surface features remains an unstudied realm.
Here we have investigated poly(pyrrole) (PPy) films fabricated using galvanostatic (GSTAT) electrodeposition at 0.5mA/cm² current density and poly(styrene-sulfonate) (PSS) dopant. An agarose gel was incorporated into the electrodeposition circuit to selectively apply Py:PSS monomer solution to controlled areas of an Au substrate. By positioning the gel on a computer-controlled motion stage, different regions of the substrate could be sequentially polymerized upon. Additional control software permits precise and variable control of the stage velocity; by changing the movement speed, residence time of the monomer liquid bridge may be adjusted. Increases in residence time caused increases in layer thickness and surface roughness. For example, by sequentially decreasing the stage velocity from 10.42µm/s to 3.21µm/s over 25 minutes, the PPy surface roughness (Rq) increased linearly from 2.75±0.69nm to 7.52±1.73nm, and the thickness increased from 318.5±75.9nm to 1,199.0±129.3nm. Various stage-velocity profiles allowed fabrication of multiple surface morphologies; constant-thickness, linear-increase (wedge-shaped), exponential-increase, and increase-decrease (hill) velocity profiles were successfully applied to 10 millimeters of Au substrate. Materials confocal microscopy was used to characterize film thickness and surface roughness as a function of substrate position.

Implantable neural electrodes are important tools for recording and manipulating functions of the nervous system. Neural network interfaces, such as electrodes, have been used to better understand neural network plasticity through recording brain signals, and stimulating neural electrodes have been used clinically for therapeutic and assistive purposes in people with disease and injury. The challenges facing neural interface engineering is to develop materials that can seamlessly interface with the biological environment of the brain over long time periods, consistently provide the desired therapeutic results, and mitigate health risks associated with chronic implantation. Coating electrodes with conductive polymers such as poly (3,4-ethylenedioxythiophene) (PEDOT). PEDOT has shown to enhance the performance of metal electrodes by decreasing the impedance and increasing the charge storage capacitance. PEDOT is an excellent candidate for interfacing with the brainbecause of its mixed ionic-electronic conductivity, biocompatibility, and electrochemical stability.

Here, stimulating platinum-iridium (PtIr) neural microelectrodes were coated with PEDOT:tetrafluoroborate through electrodeposition in the solvent propylene carbonate. Coated and uncoated stimulating electrodes along with tungsten recording electrodes were implanted in the hippocampus of mice. The coated/uncoated PtIr electrodes were stimulated daily, the recording electrodes measured the local field potentials generated by the stimulation, and the impedance before and after stimulation was measured at each electrode. The coated electrodes were able to effectively stimulate neuronal activity in the brain, thus demonstrating that PEDOT-coated electrodes are a viable alternative to bare PtIr recording electrodes.

In this presentation we focus on fabrication pathways established for development of a system for dual recording of neuroelectrical and electrochemical signals based on porous boron doped diamond (pBDD) microelectrode arrays (MEAs) and microprobes, enabling alternate, or even simultaneous, recording of two types of information from one biological sample during one measurement session, i.e. detection of action potentials and neurotransmitters. pBDD based MEAs were fabricated via a novel combination of ink-jet printing and microwave linear antenna plasma enhanced chemical vapour deposition (MW-LA-PECVD) techniques. Nanodiamond seeded SiO₂ nano-spheres in a dispersion of ethylene glycol were ink-jet printed onto a Ti/quartz MEA with 20 electrode pads. Following seeding the MEA substrate was loaded into a MW-LA-PECVD system for layer deposition. The selected growth conditions enabled co-deposition of conductive pBDD on seeded areas and insulating silicon carbide (SiC) on unseeded areas, and thus matching the functionality requirements of layers for in-vitro MEAs. This novel combination of techniques negates the need for complicated and time consuming lithography/masking/etching steps. Fabricated pBDD/SiC MEAs were then used to culture hippocampal neurons and record their activity using a MEA amplifier USB-MEA64. Regular measurements revealed neuronal activity on day 10 -12 in-vitro. Activity was maximal 19-22 days in vitro, later declined and after 40 days disappeared. Comparison of electric activity between neurons grown on commercial TiN MEAs and pBDD MEAs showed a 2x lower standard deviation of voltage noise recorded for pBDD MEAs.
For electrochemical recording, we aim to employ pBDD based microprobes positioned in close proximity to a firing neuron on a pBDD MEA electrode in order to record neurotransmitter (e.g. dopamine) activity. pBDD µprobes were fabricated following anisotropic chemical etching of metallic wires (W or Ti) and then coated with BDD using the same MW-LA-PECVD system. Electrochemical characterization of model planar and porous BDD electrodes has been performed by recording cyclic voltammograms (CVs) of the inner-sphere [Fe(CN)6]4−/3− and the outer-sphere [Ru(NH3)6]3+/2+ redox markers. The acquired ΔEp values approach 59 mV (a value for reversible one-electron system), thus confirming fast electron transfer kinetics as well as the high quality of the fabricated electrode materials. In addition, the electrochemical behaviour of 0.1 mmol L−1 dopamine was investigated by CV. Results indicate that both BDD materials can be applied for dopamine detection in media used for neuron culturing, e.g. HEPES.
In conclusion, we have demonstrated a simplified fabrication regime for the preparation of BDD based MEAs and microprobes with enhanced surface area. Functionality of the developed electrodes is shown by electrical recordings from firing neurons along with detection of neurotransmitters.

Acknowledgements: This work was supported by the Czech Science Foundation (contract 17-15319S)

We study synchronization patterns in microfluidic networks containing Belousov-Zhabotinsky (BZ) chemical oscillators. In our experiments, the auto-catalytic, light-sensitive, BZ reaction is confined to micro-fabricated wells constructed from the elastomer PDMS. Using soft lithography, PDMS networks are arranged into wells with controlled topology. Each well can be regarded as a single network node that sends and receives inhibitory signals. Here we present the dynamics of a 3-node ring network. This network has two equivalent circular traveling waves of excitation. Control over the chirality of the wave can be achieved by exploiting the light sensitivity of the BZ catalyst, which can modulate the frequency of an individual node. In experiment, we perturb the network by changing the light intensity and duration of each of the three BZ wells. This network provides a model of gait switching in central pattern generators and a dynamic method of information storage.
*We acknowledge financial support from the U. S. Army Research Laboratory and the U. S. Army Research Office under contract/ grant number W911NF-16-1-0094, and the microfluidics facility of the NSF MRSEC DMR-1420382.

Ion-sensitive transistors with nano- or micro-scale dimensions are promising for high-resolution electrophysiological recording and synaptic transistors. Technologies that are capable of patterning polymer functional materials directly from solution can effectively avoid any chemical damage induced by conventional lithography techniques. We report a method and process to pattern PEDOT:PSS-based transistors directly from their water-based suspension with high resolution. Gold electrodes with nanoscale channel width were also fabricated by firstly creating high-resolution polymer lines with mould-guided drying and subsequent pattern transferring, and PEDOT:PSS lines were then created through mould-guided drying on the predefined electrodes. The small devices with both nanoscale channel length and width exhibited good performance in electrical amplifying, high-frequency and multiple-frequency response. In addition, the assembled PEDOT:PSS lines showed anisotropy in electrical conductivity due to modified polymer chain alignment during drying.

Neural regeneration treatment after spinal cord injury (SCI) is still unsatisfactory despite the advances made in the field. One of the main challenges in neural tissue engineering is axonal growth and directionality. Cell and molecular therapies can enhance the axonal attachment and growth, however, axons may be unsuccessful to maintain their native organization and may grow in a disorganized fashion. Nanofiber scaffolds represent a potential solution for the problem of neural regeneration and axon guidance, as they can mimic the neural tissue extra cellular matrix (ECM) and combine the advantages of the combinatorial therapy for nerve injury in SCI cases. In this work, we aimed to fabricate a nanostructured scaffold that can be used as a physical support for maintaining axonal growth and regeneration in the lesion site. In addition to providing a suitable environment for the axonal extension to reconnect with their target neural tissues and restore their functional recovery. Anodized TiO₂ nanotube powder incorporated into Chitosan and Poly Vinyl Alcohol (PVA) nanofibers with different percentages as 0.5, 1 and 3%. The composite scaffold was fabricated using rapid break down anodization and electrospinning techniques for TiO₂ nanotubes and polymeric composite respectively. Characterization techniques such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) in addition to the viability assessment test using MTT assay with neural cell line were conducted. The results revealed that the scaffold with 0.5 and 1 % showed high biocompatibility material with neural cells which mimic the properties of the neural tissues in terms of biological and architectural properties and can be considered a regenerative treatment for axonal neural guidance of SCI.

Recently, the field of neuromorphic electronic system for mimicking diverse functionalities of biological synapse and massively parallel neural network found in human brain has been emerging as a promising approach toward energy-efficient computing technology¹. For wearable electronic technology, an ultra-flexible neuromorphic device platform could be envisioned as an on-body intelligent integrated circuits to instantly and proactively interact with a wearer for general or specific information and sensing technology purposes. Here, we introduce a new class of artificial synapse as a basic unit for flexible and wearable intelligent device applications². We fabricated a large scale of ferroelectric organic field-effect transistor memory (~ 500 nm total thickness) in a free-standing form using a pentacene and a ferroelectric copolymer, PVDF-TrFE, and utilized it as a free-standing artificial synapse. The device exhibits the reliable switching properties even in free-standing form, and it can be also properly operated on various corrugated surfaces such as a thermal-shrink plastic film, a jelly, a textile, a candy, a teeth brush, and a brain mold. By applying diverse electrical pulses with modulated relative time correlation between pre-synaptic (gate electrode) and post-synaptic neuron (drain electrode), diverse synaptic activities such as STP, LTP, LTD, and STDP have been implemented. Furthermore, it features sustainable synaptic functions for more than 6,000 times of input signals under extreme conditions such as transferred on the corrugated brain-like mold and completely folded with very small banding radius (R = 50 µm). Our demonstration suggests that the ultrathin conformable organic artificial synapse platforms are considered as one of key technologies for realization of wearable intelligent electronics in the future.

[1] Jo, S. H.; Chang, T.; Ebong, I.; Bhadviya, B. B.; Mazumder, P.; Lu, W. Nanoscale Memristor Device as Synapse in Neuromorphic Systems. Nano Lett. 2010, 10, 1297-1301
[2] Jang, S.; Jang, S.; Lee, E.-H.; Kang, M.; Wang. G.; Kim. T.-W. Ultrathin Conformable Organic Synapse for Wearable Intelligent device Applications. ACS Appl. Mater. Interfaces 2019, 11, 1091-1080

Reliable operation of neural probes over time scales ranging from minutes to years is essential to longitudinal studies of development, aging, and chronic neurological diseases. Recently developed fiber-based neural probes offer a promising platform for integrating multiple functions in a miniature, flexible form factor that is also biocompatible with the soft neural tissue.

In this presentation, I will describe strategies for further expanding the array of functions delivered by fiber-based neural probes by outfitting them with programmable, wireless optogenetics capability. The traditional approach to light delivery into the brain for optogenetics studies relies on tethering implanted fibers to external sources via optical cables, which limits the range of possible experiments, particularly those involving complex motor functions or social interactions among multiple animals. Here we present multifunctional fiber-based neural probes capable of complete wireless and programmed optical neuromodulation using integrated microscale LEDs for control of complex behaviors in freely moving untethered animals. The same fibers incorporate conducting electrodes that enable chronic extracellular electrophysiology and microfluidic channels for delivery of drugs and genes to the target neural tissue. The devices still maintain a miniature footprint (~270×200 µm²). A detachable wireless transponder with an on-board battery for power and low-energy Bluetooth for data transmission permit straightforward application of these probes in a multitude of behavioral assays.

Ever since stem cell technologies were developed several decades ago, researchers have produced a variety of cells without ethical issues which arise in obtaining cells from animal. Since embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can proliferate rapidly and infinitely, and possess multipotency, they are promising cell sources for bioengineering and regenerative medicine. Recently, efficient sensory neurons (SNs) differentiation methods from ESCs and iPSCs have been developed by using small chemicals and growth factors. The engineered SNs could be used to develop sensible neuroprosthetic devices and regenerated skin tissues for treating severe traumatic injuries or congenital malformations. However, there are still many issues remaining for current stem cell technologies regarding differentiation efficiency and controlling the structures and alignment of SNs. For example, matured SNs interconnected via dendrites were not amenable to enzymatic dissociation for reseeding due to the inevitable cell damage during dissociation process. Although immature SN progenitor cells (SNPCs) can be reseeded, they still possess stemness, resulting in differentiation into non-neuronal cells.
To solve the issue, at first, we investigated the optimal seeding method of SNPCs on a two-dimensional (2D) substrate. SNPCs reseeded onto a crude laminin-coated dish survived and elongated with neurites but a small amount of non-neuronal cells selectively proliferated and dominated to the culture space over the time. DAPT[l1] was used to efficiently inhibit the non-neuronal differentiation and the proliferation. Besides, subtypes of recombinant laminin were screened, revealing that specific subtypes of recombinant laminins retained SNPC as a neuronal progenitor cell and selectively induced matured SNs.
Second, we compared the biocompatibility of several hydrogels for SNs such as gelatin methacryloyl (GelMA), collagen type I, and Matrigel. Although GelMA is well-known as an appropriate biomaterial for creating various 2D and 3D tissue constructs, SNPCs showed less viability and no-elongated neurites in the GelMA hydrogel. On the other hand, SNPCs were able to elongate neurites in both collagen type I hydrogel and Matrigel and then they matured and connected to the adjacent neurons forming dense neuronal networks in 3D hydrogels.
Next, we performed 2D patterning of SNs using bioprinting techniques. Laminin solutions were printed onto conventional plastic tissue culture plates, SNPCs were then seeded, and specifically attached and elongated with neurites on the printed lines. However, the weakly attached neurons on the printed line were easily destroyed by mechanical stress such as medium replacement, and the patterned SNs couldn’t be sustained for a long time. [l2] In order to overcome this situation, SNPC patterns were embedded into collagen type I hydrogel. To do this, a sacrificial bioprinting technique was used, so SNPCs suspended in gelatin bioink were printed on the laminin-coated tissue culture plate and then physically crosslinked on ice. The printed SNPCs-laden gelatin lines were covered by collagen type I solution and solidified at 37°C. The printed gelatin was then gradually dissolved into the culture medium and hollow microchannels were left. The SNPCs originally suspended in gelatin bioink were attached on the laminin-coated plate. SNPCs were elongated with neurites following the hollow microchannel structures and connected to each other, forming straightforward lines at the centimeter level.
Now, we are developing and improving this method to form 3D neuronal wiring with printing SNPCs-embedded gelatin bioink in hydrogel bath constructs and connecting them to electrical devices like CMOS sensors to record the action potential of SNs later. These techniques above are expected to be applicable in generating 3D sensible prosthetics or skin tissues with complex wired sensory neuron networks in the future.

Recently, flexible electronic devices have shown their ability as tools for in vivo electrophysiological or electrochemical recordings of organs due to their conformability and biocompatibility [1]. However, flexible devices only can make contact on the surface of biological tissue. In general, the ability to insert sensors into the precise location of biological tissue also yields important capability to get signals from origins. Accessing the origins of the complex signal landscape over the organs of larger mammals will require conformal substrates, insert sensors, and biocompatibility simultaneously.
Here we present electrochemical and physiological needle based sensors on integrated substrates between rigid microneedle and soft flexible substrates. The microneedle (300-μm- caliber and 700-μm-height) is fabricated by epoxy hybrid materials on the 40-μm-thick PDMS substrate and passivation. The electrode channel is formed by Au/Ti which has a 35nm/5nm thickness, respectively. The electrode on the microneedle is coated by polyaniline through the electrochemical deposition. Polyaniline is formed well on the microneedle, which can be confirmed by scanning electron microscope (SEM) image. And their electrode shows low site impedance as 800Ω (1kHz) due to their porous structure, when the surface area is 0.5 mm². Additionally, the ability of pH detecting of flexible microneedle electrode is demonstrated by open circuit potential (OCP). As an analyte, phosphate-buffered saline (PBS) which has different pH controlled by hydrochloric acid, is used. The OCP measurement is performed between silver chloride and polyaniline microneedle, and it shows the 30 mV/pH. Finally, we successfully made 5×5 large areas flexible electrochemical needle array with 4.5 mm spacing. To enhance the biocompatibility of the microneedle, poly(3-methoxypropyl acrylate) (PMC3A) which has ion-permeability [1,2] is deposited by dip-coating.
Such integration of rigid and soft materials as one substrate could make the device to have the flexibility and insert sensors at the same time. This electrochemical and physiological sensor arrays hold great promise for medical applications including local field potential (LFP) measurement on the complicated organs of mammals.

[1] Wonryung Lee et al., Science Advances, eaau2426 (2018).
[2] Shingo Kobayashi et al., Biomacromolecules 18, 12, 4214-4223 (2017).

Most animals have the nervous system consisting of the central nervous system and the peripheral nervous system. Damage or malfunction of the nervous system causes serious diseases such as spinal cord injury, Alzheimer’s disease, and neurodegenerative disorders. Current in vitro nervous systems such as microarray have a limited ability to mimic a single cell-cell connection of neural networking and do not enable control over diverse aspects of the neural microenvironment.
In this study, we developed collagen-based various nanofibrous patterns (uniaxial, perpendicular, diagonal patterns) to mimic the extracellular matrix (ECM) and investigated neurite outgrowth and axon guidance depending on the patterns and the density of nanofibers. In general, neurons displayed the accelerated outgrowth and synaptic connection on fibrous patterns and showed nanofiber-guided growth of cell body. However, in this system, axon growth showed various directions and the interaction between nanotopography and neurons. Calcium imaging also showed different rate and intracellular calcium signals released from neuronal activity depending on arrangement and density of nanofibrous patterns.
This paper will discuss how nanotopological cues of collagen-based fibrous patterns to regulate neurite outgrowth and axon guidance, and optimize the neural activity within this system. The patterned nanofibrous system developed by our group could be extended to serve as a model for pathophysiological study of the nervous system and artificial 3D neural tissue engineering. Furthermore, our findings offer new insights into the design of nanofiber-based scaffolds for nerve injury repair and will provide new guidelines for the construction of neuronal network architecture.

ITO coated mica has been a promising flexible substrate for implanted electrochemical sensor of in situ sensing, monitoring of biological conditions (i.e. pH) and detect multiple species simultaneously. (i.e. glucose, dopamine and uric acid). Minimizing the pH sensor for implanting and long-term record could minimize the local tissue damages and improve the special resolution. Study the sheet resistance and curvature of substract also discussed in this study.
Iridium oxide has been an attractive material due to its advantages of electrochemical applications and electrophysiological sensing due to its excellent chemical stability, sensitivity, electrochemical catalytic activity and good biocompatibility. We developed a flexible iridium oxide based on ITO coated mica electrode for application in electrophysiological sensing by chemical bath deposited iridium oxide film. The characterization of thin film was investigated by SEM, XRD, XPS and XAS. Three-electrode system was selected as the sensing stimulation with pH varying from 1 to 11. The sensitivity was up to 80 mV/pH with an accuracy of 0.1 mV/pH. Moreover, the as-deposited sensor had an attractive properties of detecting muliple species of uric (AA) and dopamine (DA). The stability test was demonstrated by 1000 cycles of CV scanning. After finishing stability test, the charge storage capacity (CSC) and the charge injection capacity (CIC) maintained 96.3 % and 92 % of efficiency, respectively. Great electrochemical properties, high sensitivities, wide pH ranges, fast response time and great reversibility presented that the chemical bath deposited iridium oxide film presented well in electrophysiology electrodes.

The mission of the NINDS Division of Translational Research (DTR) is to accelerate basic research findings towards patient use for neurological disorders and stroke by providing funding, expertise, and resources to the research community. DTR provides funding and resources through grants, cooperative agreements, and contracts to academic and industry researchers to advance early-stage neurological technologies, devices, and therapeutic programs to industry adoption (i.e. investor funding and corporate partnerships). We have created a variety of programs that support the design, implementation, and management of research activities critical to translational challenges in the treatment of neurological disease. In addition, DTR plays an active role in the NIH BRAIN Initiative, SPARC Program, and HEAL Initiative. NINDS DTR is actively managing programs that support small molecule, biologic, and neural device therapeutics, biomarkers, and training through grants, contracts, and consultants. These programs cover all stages of translational research from early assay/biomaterial/device development and optimization to preclinical development and early clinical development. Funding opportunities and resources are actively supporting translational research in preclinical discovery and development of new therapeutic interventions for neurological disorders and stroke, as well as neuropsychiatric disorders and neurotraumatic injuries (BRAIN Initiative) and pain (HEAL Initiative). An overview of NIH/NINDS translational programs and resources will be presented.

Photo-sensitive conjugated polymers provide highly promising platform for visual prosthesis offering novel retinal biocompatible devices. Their high absorption coefficients, chemically tuneable bandgaps, the ability to be processed at room temperatures on flexible substrates, and signal transduction mechanisms that do not require external power sources make them ideal materials for retinal prosthetic applications. Despite ongoing research efforts in artificial retina field with organic semiconductor materials, full-colour sight restoration remains very challenging.
We demonstrate tri-colour optoelectronic devices based on three different band-gap conjugated polymers with absorption in red, green and blue spectral regions, mimicking absorption of human retinal cones responsible for full colour vision, as well as high-sensitivity broadband absorption polymer/small molecule blend imitating rods absorption. Photo-response of these devices, interfaced with biological electrolyte solution, is demonstrated with long-pulsed excitation from monochromator-filtered light source, and spectral response is shown to deviate from optical absorption of dry polymeric films. We compare polymers’ films response with retinal photoreceptors colour sensitivity and discuss the role of polymer-electrolyte interface for photo-transduction mechanisms, identifying the role of capacitive charging, and ions interactions with the polymer layers during light excitation process. Ink-jet printed devices are fabricated in the form of an array of semiconducting 50 to 90 micron diameter polymeric round pixels-photoreceptors with specific red, green and blue colour sensitivity. The devices do not require wiring or external bias to operate, and are stable in aqueous physiological conditions. Due to biological compatibility of organic semiconductors, high absorption and wide spectral tuneability, this device technology is expected to find medical applications as retinal bio-engineered prosthesis towards the restoration of human vision lost due to common eye diseases, including Age Related Macular Degeneration and Retinitis Pigmentosa.

Current scaffolds for soft tissue regeneration and rehabilitation are limited in that they are commonly fabricated from non-conductive polymers or have low-conductivity due to poor percolation of conductive components within an insulating polymer. When electroactive particles such as carbon nanotubes or metal nanoparticles are suspended within a polymer matrix, the conductive path is inadequate as the particles must be in close proximity to maintain conduction. This necessitates high concentrations of particles to facilitate sufficient conductivity, which in turn can be detrimental to the mechanical properties of the polymer system.

Conductive polymers (CPs) can have high conductivity, but are often stiff and friable, with typical moduli of 20 to 100 MPa. These poor mechanical characteristics have driven the need for new electroactive materials with softer, more robust mechanics. In addition, thermal processing of CPs, including MEW has not been achieved due to the thermal breakdown of CPs prior to reaching a melt phase. To achieve flexibility, conduction and thermal processability in a CP system, it is necessary to develop new CP based building blocks that can retain the CP chains in close apposition, while enabling movement and fabrication into devices.

New macromers of melt processable poly(2-oxazine) (POx) functionalized with thiophene have been developed. Using the synthesis strategy of Seeliger and Wittig et al. several thiophene-oxazoline/oxazine monomers were produced with a range of properties. The first isolated crystal structures of 2-oxazoline were observed. Using ring-opening polymerization (ROP) it was possible to polymerize homopolymers, copolymers and block copolymers. This broad variation is expected to enable control over material properties such as glass transition temperature (Tg) and fiber flexibility.

These new polymer building blocks were shown to retain the processability of the POx and enable the printing of a CP monomer using MEW for the first time. Specifically, poly(2-thiophene-2-oxazine) can be printed in continuous fibres with ~200 µm diameter. The MEW printing temperature was 160 °C, with voltages ranging up to 4kV. This molecule can polymerized to obtain a product with conductive properties reflective of a CP chain formation. Free PEDOT chains can be added to the co-polymer to improve conductivity, however, future work will focus on developing co-polymers where CP chain alignment can be facilitated during the printing process.

Fabrication of 3D conductive microstructures is a great of interest in the field of bioelectronics and biosensors. However, the main challenges are (1) inability of the commercially available 3D-printers to precisely control the spatial architecture and failure to maintain a high resolution and (2) incompatibility of the printing inks to fabricate functionalized and biocompatible structures. To overcome these challenges, we introduced a novel electronically conductive and biocompatible ink for fabrication of sub-micron resolution 3D-structures using two photon polymerization technique. The ink is consist of poly(ethylene glycol) diacrylate (Mn=700) as a crosslinker, ethyl (2,4,6-trimethylbenzoyl) phenylphosphinate as a photoinitiator, and high conductive grade conductive poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) dispersion in water. The 3D conductive microstructures were fabricated using a Laser uFAB Microfabrication Workstation (Newport, USA) at wavelength 800 nm with laser power 3.5 mW. Electrical conductivity of bar-shaped (800 µm length, 20 µm width and 1 µm height) structures with PEDOT:PSS concentration of 0 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt% and 0.5 wt% were 3.1 ± 0.6 S/m, 137.8 ± 11.9 S/m, 5502.9 ± 586.2 S/m, 13857 ± 927.9 S/m, 19042 ± 1302.1 S/m, and 25034 ± 886.3 S/m, respectively. These results showed the significant improvement of conductivity of 3D-structres up to 4 orders of magnitude (P<0.001). Furthermore, laminin protein was incorporated within conductive microstructure during fabrication process to enhance the cell attachment. Atomic Force Microscopy results revealed that the young modulus conductive microstructures was in the range of 200-500kPa. The development of conductive and bioactive 3D microstructures can be utilized in the field of bioelectronics and can be potentially utilized for neural interfaces. The future work will be focused on 3D-printing of organic neural microelectrode for recording brain signals.

We continue to investigate the design, synthesis, and characterization of electrically and ionically active conjugated polythiophene copolymers for integrating a variety of biomedical devices with living tissue. This talk will focus on our most recent results, including the development of several new monomers that be used can tailor the surface chemistry, adhesion, and biointegration of these materials with neural cells. Our recent efforts have focused on copolymers of 3,4-ethylenedioxythiophene (EDOT), functionalized variants of EDOT (including EDOT-acid and the trifunctional EPh), and dopamine (DOPA). The resulting PEDOT-based copolymers have electrical, optical, mechanical, and adhesive properties that can be precisely tailored by fine tuning the chemical composition and structure. We have also been investigating the ability of these materials to be deposited directly around living tissue, particularly peripheral nerves. We will present results on EDOT-dopamine bifunctional monomers, including EDOT-dopamide and EDOT-dopimine and their corresponding polymers. We will also introduce an EDOT-aldehyde monomer and show how it can be used as the starting point for a wide variety of functionalized monomers and polymers.

Memory devices based on organic materials are a promising alternative towards the next generation of nonvolatile nanoelectronics. The memristor is defined as any two-terminal electronic device that alters its conductivity based on its electronic history. In this effort, a methacrylate polymer is derivatized with an electronically active carbazole. Under an applied electric field, the carbazole groups rearrange their alignment, thus facilitating easier electron carrier delocalization and charge transport through molecular conformational changes. By tuning the structure of the polymer, the flexibility of the chain and the number of conductivity states can be further modified. The ability to have a multitude of conductivity states according to applied voltage makes the memristor a major candidate for emulating synapses in artificial neural networks – offering both the high connectivity and the high density required for efficient computing. Memristors, with a simple sandwich device architecture of ITO/poly(n-(9H-carbazol-9-yl)n-alkyl methacrylate)/Al, exhibit essential synaptic plasticity and learning behaviors, including short-term plasticity and spike-timing-dependent plasticity, through programmed waveforms inspired by neuronal action potential. Variations to the length of side chains that attach the carbazole moiety to the polymer backbone were studied for their effects on the optical and electrical properties of these materials.

Deep brain stimulation (DBS) is a well-established tool that is widely used in the clinical treatment of neurological disorders such as Parkinson's disease and depression. However, treatment advances can be limited by not fully understanding how the brain is affected, and thus animal studies in vivo are needed to investigate the mechanisms involved. In vivo studies using DBS require one or more electrodes be implanted in the brain so that an electric field can be applied across the targeted area. As these electrodes may stay implanted for several days to months, it is crucial to the success of the trial that the electrode materials are fully compatible with the brain. However, the electrodes currently available to scientists are rigid, causing significant inflammation and damage to the contrastingly soft brain tissue. While research is ongoing to develop soft electrodes capable of replacing their rigid counterparts, meeting the many requirements of stimulating electrodes, including biocompatibility, high electrical conductivity, low Young’s modulus, and a functional insertion mechanism, has posed a significant challenge to researchers.

To address this challenge, we have developed novel electrodes that are biocompatible, conductive, stiff during insertion, but soft once placed in the brain. The polyvinyl alcohol (PVA), cellulose nanocrystal (CNC), and carbon nanotube (CNT) composite is repeatedly frozen and thawed in fiber form to produce a soft, highly elastic, conductive cryogel material, which is then coated in polypyrrole to further increase conductivity and reduce excess swelling of the electrode that could damage surrounding tissue. When dry, the fiber can be made as thin as 90 microns diameter, and with a compressive modulus of 1.8 MPa, can apply enough force without bending to penetrate tissue (>1 mN). Once saturated, the Young’s modulus reduces to 3 kPa, approximately matching that of the brain (1-2 kPa). With wet conductivity as high as 150 S/m, we demonstrate that these electrodes can effectively stimulate mice in vivo. Furthermore, this material is shown to be 3D printable, with potential to print more complex electrode designs for advanced stimulation experiments.

Biopolymers have been utilized as a matrix for filamentary resistive switching memory due to its abundant, inexpensive, and biodegradable properties. Resistive switching characteristics can be governed by diverse factors, and ionic conductivity of the matrix is one of the most critical factors in filament formation. Here, we report biocompatible memory and synaptic devices based on the biopolymer matrix [1-2]. Filament formation dynamics are controlled by modulating the ionic conductivity of biopolymer matrix. In biopolymer matrix with low ionic conductivity, the volatile resistive switching behavior is observed. Synaptic characteristics such as short-term plasticity, paired-pulse facilitation and transition from short-term to long-term plasticity are emulated by exploiting the similarities between the volatile filament formation dynamics in the biopolymer matrix and Ca²⁺ dynamics in biological synapse. The devices also exhibit good non-volatile memory characteristics such as fast switching speed, low operation voltage, and high on/off ratio by improving ionic conductivity via chemical modification. This study may provide a new possibility for biomaterials to be used as biocompatible memory and artificial synapse devices. In this presentation, resistive switching behavior in biopolymer for memory and neuromorphic applications will be presented in detail.

[1] M.-K Kim and J.-S. Lee, ACS Applied material & interfaces 10, 10280-10286 (2018)
[2] M.-K Kim and J.-S. Lee, ACS Nano 9, 419-426 (2018)

Classically electrophysiological recordings have been performed with electrodes. From an engineering standpoint, this is an interesting situation as most biological events occur at lower frequencies where the impedance of standard metal electrodes considerably increases. Active devices, such as the organic electrochemical transistor (OECT), are promising transducers for biointerfacing due to their high transconductance, biocompatibility, compatibility with various form factors and low impedance at biologically relevant frequencies. To date, however, OECT performance has typically been limited near the 1 kHz range, a crucial frequency to resolve action potentials of electrogenic cells. Here we report on the fabrication and characterization of OECTs with vertically-stacked contacts. The resulting transistors exhibit a reduced footprint, increased intrinsic transconductance of up to 57 mS, and a geometry-normalized transconductance of 814 S/m. This 3D process allows for exploitation of the amplification properties while achieving a spatial resolution suitable for interfacing with small populations of cells or single cells. The vertical OECT has been fabricated on flexible implantable probes and interfaced with a wireless recording system, which utilizes an amplification circuit that avoids the previous pitfalls of layouts requiring a resistance in series with the transistor channel. Wireless recordings in vivo are utilized to demonstrate improvement in the signal detection capability vOECTs on organic implantable probes.

Graphene has been shown to exhibit an extraordinary range of tunable properties. High electrical and thermal conductivity coupled with exceptional mechanical properties and tunable chemical/biological activity has the materials science community enthralled. This combination of properties has led to the use of graphene to form electrodes for use in energy conversion and storage as well as in medical bionics.

We have been particularly interested in the processing of graphene from graphite in such a way that the above properties are retained but also the materials produced are amenable to a range of fabrication protocols. One approach to fabrication that has had a marked impact on the bionics area is our ability to produce long lengths of micron dimensional fibers with high electrical conductivity and exceptional mechanical properties. Our latest version of this is a fiber that can be sutured around individual nerves to enable stimulation and recording with unprecedented spatial resolution and sensitivity (1).

Here we will present the impact of these developments on the emerging field of electroceuticals - the use of electrical stimulation to treat disease.

Reference

(1) Wang, K., Frewin, C.L., Esrafilzadeh, D., Yu, C., Wang, C., Pancrazio, J.J., Romero-Ortega, M., Jalili, R., Wallace, G.High-Performance Graphene-Fiber-Based Neural Recording Microelectrodes Advanced Materials 2019, 31, 1805867.

High-speed, low-power, and biocompatibility are the main requirements of neural implantable devices at can acquire electrophysiological signal at high spatiotemporal resolution. However, the high ionic conductivity of biological tissue limits effective transmission of conventional electromagnetic based waves from inside of the body. In addition, such approaches often require complex implantable electronic components and circuits that increase the size and power consumption of the implanted device.

Here, we introduce a novel ion-based high-speed, low power consumption communication.

Three forms of ionic communication including capacitive, galvanic, and RF coupling were evaluated and characterized for their bandwidth, cross talk and scalability. Physical and geometrical factors of ion transport and movement including the distance between electrode pairs, implantation depth, electrode size, electrode material, ion concentration were determined to establish optimal high-speed ionic communication. To further increase the communication bandwidth, we investigated the feasibility of multiple parallel communication lines through manipulation of the spatial extent of ionic waves in the medium. Conducting polymer-based hexagonal electrodes were fabricated on conformable substrates to improve flexibility and maximize the electrode density. We evaluated the error rate of the ionic communication at different speeds and established four ion-based communication lines for validating the efficacy of this transmission in animal experiments.

We have performed in vivoelectrophysiological recordings of heart cells and neurons in anesthetized and freely moving rats. High-resolution electrophysiological signals were transmitted via ionic communication across the animal’s body without any extruding element, creating an imperceptible communication path between inside and outside the body. The proposed ionic communication scheme has broad applicability to implantable bioelectronics that require data exchange with external elements such as deep brain stimulators, pacemakers, implantable pumps, and brain machine interfaces. It will also enable development of novel implantable bioelectronic devices, with the potential to improve the care of patients who rely on medical devices to diagnose and treat disorders.

Calcium ions (Ca²⁺) plays an important role in neuronalsignaling, challenges to analyze various neuronal calcium sources is that they are not active one at a time, overlapping activity with strong interactions. An advance in this field would come from the development of selective, reversible fluorescent chemosensors, capable of repeated measurements. To this end, the rational design and fluorescence-based photophysical characterization of calmodulin calcium binding motif spiropyran-based chemosensors for Ca²⁺ are presented. Incorporation of spiropyran-based sensors into a peptide conjugation system in a crown shape has a promising results to yield significant signal to background changes with minimal sample volumes, a real advance in biological sensing that enables measurement on subcellular scale samples. In order to demonstrate chemo-peptide sensor compatibility within the light intense microenvironment photoswitching and photostability, revealing reversible Ca²⁺ binding with improved photostability compared to the non-photoswitchablecalmodulin calcium binding motifs. The spiropyran-based peptide chemosensor reported here highlight untapped opportunities for a new class of photoswitchable Ca²⁺ probe and present a first step in the development of a light-controlled, reversible sensor for Ca²⁺ .

Today Brain Computer Interface (BCI) offers a way to restore neuronal dysfunction due to degenerative diseases or accidents. Thus it becomes possible to restore vision with retinal implant or to offer tetraplegic a way to control a robotic tool by thought with electrodes implanted in the cortex. One major limitation of these systems is their insufficient stability. After several months of implantation, some modification can appear such as swelling of the passivation polymers thus inducing current leakage, or degradation of the electrode material. Moreover neuronal prostheses are poorly accepted by tissues and a glial reaction may appear at the vicinity of the implant. Hence for future generation of implants it will be crucial to limit glial reactions and fabricate a full hermetic implant. Several research teams proposed to protect the metallic tracks by encapsulating them in multilayers of in-organic materials like AL203 or TiO2 obtained by ALD. But in case of pin holes or adhesion problems between each layer, metallic parts will not be well protected. To overcome these issues we propose to fabricate a full hermetic diamond implant.

Diamond properties are very attractive for medical applications. Indeed diamond is a biocompatible material and it has no native oxide, so there is no degradation when the structure is placed in water or harsh environment. Being also a high density material, there are no species that can migrate inside diamond. Conductive diamond obtained by introduction of Boron dopants, called boron doped diamond (BBD) has a wide electrochemistry window compare to classical metallic electrodes. In parallel CVD growth diamond reactor offers a way to achieve a thin diamond film on silicon or glass wafer. This technology is compatible with classical micro fabrication process and offers an elegant way to achieve a full diamond implant. Using the same material for passivation and electrodes offers a way to achieve a full hermetic structure compatible with long term in vivo usage.

To validate this technology we started with the fabrication of a full diamond strip composed of Boron doped diamond for the electrodes and intrinsic diamond for the encapsulation. We have also produced several strips with more conventional encapsulating materials used for implants such as parylene, AL₂O₃ or Si₃N₄ and with several electrode materials like platinum, gold, PEDOT or black Pt. To compare both electrode and encapsulation materials, we designed strips with 3 electrodes (of diameter 60 µm or 30 µm) covered with same protective material. Two electrodes were continually stimulated during all the experiments while one was used as a reference (no electrical stimulation). To follow the evolution of the electrode and encapsulating materials, impedance spectroscopy was used periodically in buffer media (PBS)over several weeks, and first results clearly showed the advantage of diamond over the classical materials.

In parallel of these developments we have also achieved a preliminary version of a soft full diamond implant which will be used to test the surgical implantation procedure and to optimize the total thickness.

Axons navigate along specific pathways based on the gradients of guidance cues. Following substantial neural injury, axons may fail to fully regenerate without external intervention due to limited intrinsic axonal growth capacity and/or interference from scar tissue. Hence, development of platforms capable of providing gradients of guidance cues to facilitate axonal regeneration is crucial in the field of neural tissue engineering. Even though numerous technologies have already been established, effective axonal guidance over long distances is still challenging.
Here we report a novel method for creation of gradient of laminin on conducting polymer film. Conducting polymers (CPs) such as Poly(3,4-ethylenedioxythiophene) (PEDOT) have been widely used for neural interfaces, owing to their excellent biocompatibility, soft mechanical properties, relatively high conductivity and outstanding chemical stability. Laminin is a major substrate-bound molecule for axonal growth, known as a chemoattractant.
In an effort to mimic the extra-cellular environment, this study aims to provide different gradient shapes of laminin such as linear, hill, and exponential to modulate axonal regeneration in the nervous system. First, using micro-scale motorized X-Y-Z stages and nano-syringe pump, lines of laminin have been printed with various concentrations (based on the gradient profile) ranging from 20-100 ug/ml on the surface of a 2% agarose hydrogel slab (hydrogel thickness was 10 mm). The laminin concentrations can be accordingly adjusted to create different gradient shapes. Processing parameters including injection flow rate and stage velocity have been optimized to create minimum line widths (i.e. 200 um) which leads to generation of high-resolution 10mm-long laminin gradients on 10 mm length of agarose gel. The whole pattern is then transferred onto the surface of a Poly (L, Lysine)-treated PEDOT film. PEDOT was previously electropolymerized on the surface of gold coated silicon substrate using galvanostatic mode with charge density of 0.18 C/cm2. Immunohistochemistry was used to quantify various immobilized laminin gradients on the PEDOT surface.
We aim to culture dorsal root ganglion explants and cortical neurons on top of CP substrates printed with laminin gradient patterns. This will allow us to assess the neurite response to various gradients and compare these responses to find the optimum gradient type and concentration range for effective axonal regeneration. The outcome of this project will pave the way towards development of more effective engineered conduits for nerve regeneration and will help us unravel fundamental questions of axonal regeneration in the nervous system.

Photoconductive organic polymers are attracting considerable interest in tissue engineering and bioelectronics due to their remarkable light absorption along with tunable optical and mechanical properties.^[1,2] Moreover, these materials can be easily processed into precisely defined topographical patterns with large area coverage and low fabrication costs. We present a novel photoconductive biointerface which combines a multi-electrode array (MEA) functionalized with a semiconductive conjugated polymer patterned into defined microscale topographical features. In previous studies, photoexcitation of a light-sensitive semiconductive polymer, poly(3-hexylthiophene-2,5-diyl) (P3HT), was used to induce variations in the membrane potential of HEK-293 cells ^[3] and the occurrence of capacitive charging was observed at the P3HT/electrolyte interface that could potentially lead to photo-capacitive stimulation of the cell membrane.^[4] Our aim is to further this approach by developing a functional non-invasive tool for the optical and topographical modulation of primary cortical neurons in vitro.
Poly(3-hexylthiophene-2,5-diyl) was deposited on MEAs and patterned into conical micropillars to improve the cell-electrode coupling. Since P3HT is excited by visible light, the presented device can be easily implemented in any electrophysiological set-up without requiring complex optical systems. We observed that polymer photoexcitation leads to a significant decrease in the electrode impedance. Furthermore, we cultured primary cortical neurons and observed that microscale pillars significantly promote neurite growth and their alignment to the underlying topography in comparison to flat substrates commonly used in cell culture. Since light treatment did not result in significant detrimental effects on cell viability, we aim to optically stimulate primary cortical neurons using the presented device and further investigate whether polymer photoexcitation influences neuronal development on flat and patterned P3HT substrates.
In conclusion, the presented system is a step towards light-controlled manipulation of neuronal development and network activity which could have considerable implications for neural regeneration and the design of neuro-prosthetic devices.

[1] N. Martino, D. Ghezzi, V. Benfenati, G. Lanzani, M. R. Antognazza, J. Mater. Chem. B 2013, DOI 10.1039/c3tb20213e.
[2] V. Benfenati, S. Toffanin, S. Bonetti, G. Turatti, A. Pistone, M. Chiappalone, A. Sagnella, A. Stefani, G. Generali, G. Ruani, et al., Nat. Mater. 2013, 12, 1.
[3] N. Martino, P. Feyen, M. Porro, C. Bossio, E. Zucchetti, D. Ghezzi, F. Benfenati, G. Lanzani, M. R. Antognazza, Sci. Rep. 2015, 5, 1.
[4] G. Tullii, A. Desii, C. Bossio, S. Bellani, M. Colombo, N. Martino, M. R. Antognazza, G. Lanzani, Org. Electron. 2017, 46, 88.

Electrical stimulation is known to be beneficial for neuronal function, particularly on neurite growth. Electroactive materials as conductors and piezoelectrics are known to provide extra stimulatory signals for neural repair and have shown promising results. Poly (L-lactic) acid (PLLA) is a biodegradable and piezoelectric polymer studied as a scaffolding material and drug delivery system. It demonstrates and FDA-approved biocompatibility and biodegradability for particular applications, as screws for fixing fractures in orthopaedics [20] or in injectable filling for lipoatrophy.
In our work, we are exploiting the feasibility of combining electrical polarization in PLLA films and aligned nanofibers and investigate the biological outcome of these electrically-induced poled PLLA platforms on neuronal relevant in vitro models. Here we report on the production of electrically polarized PLLA aligned nanofibers along with a precise quantitative analysis of their polarization and stability. We advocate that electrical poling causes C=O dipoles orientation, which brings tensile piezoelectricity, and results in charged surfaces. Our results show the neuritogenic potentiating effects of this controllable electrically-induced polarization on human SH-SY5Y neuroblastoma cells and rat embryonic cortical neuronal differentiation.

Accurately mapping neuronal activity across brain networks is critical to understand cortical phenomena, yet it is challenging due to the need of tools with both high spatial and temporal resolutions. Here, penetrating arrays of flexible microelectrodes made of low-impedance nanomeshes are presented, which can record single-unit electrophysiological neuronal activity and at the same time, are transparent, allowing to bridge electrical and optical brain mapping modalities. These 32 transparent penetrating electrodes each with a small site area, 225 μm², have a low impedance of ~ 149 kΩ at 1 kHz, an adequate charge injection limit of ~ 0.76 mC cm^-2, and 100% yield. Mechanical bending tests reveal that the arrays are robust up to 1,000 bending cycles, and their high transmittance of 67% at 550 nm makes them suitable to combine with various optical methods. A temporary stiffening using polyethylene glycol allows the penetrating nanomesh arrays to be inserted into the brain minimally invasively, with in vivo validation of recordings of spontaneous and evoked single-unit activity of neurons across layers of the mouse visual cortex. Together, these results establish a novel neurotechnology—transparent, flexible, penetrating microelectrode arrays—which possesses great potential for brain research.

Flexible, polymeric-based electronics have the to potential to fundamentally change the design, fabrication and performance of bioelectronic devices. Contemporary bioelectronic devices are restricted to metals for charge transduction and injection in the body. The use of metallic conductors hinders the design and fabrication of truly stretchable and flexible electronics. The mechanical mismatch between metals and insulative polymers such as polydimethylsiloxane (PDMS) used in bioelectronic devices can lead to device failure due to issues such as mechanical failure and fluid ingress. Furthermore, the mechanical mismatch between metallic conductors and soft tissues contributes to chronic inflammatory responses upon implantation of bioelectronic devices.

Carbon-based conductors such as carbon nanotubes and conducting polymers provide a potential avenue for the synthesis of fully organic electrical conductors. In this study several approaches for the fabrication of soft, flexible organic conductors have been investigated and the resultant materials have been used in the fabrication of fully polymeric electrode arrays. Two key approaches for fabrication of conductive elastomers were explored. The first approach is the fabrication of a poly(ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) hydrogel which is impregnated with PDMS resulting in a bulk composite of an embedded PEDOT network within a PDMS matrix. The second approach entails dispersion of PEDOT fibres and nanowires within a polyurethane (PU) matrix to produce films via solvent casting. The effect of PEDOT loading on electrochemical and mechanical characteristics were investigated. Elastomers with 15 wt% PEDOT loading were identified as ideal for device fabrication, having conductivity of 4.15 S.cm^-1, young’s modulus of 20.5 MPa, an ultimate tensile strength of 7.4 MPa and strain at failure of approximately 265%. Cytocompatibility of conductive elastomers was assessed in vitro using ReNcell VM human neural precursors. Cultures on conductive elastomers were found to have an increased neural cell population compared to neat PU (33% compared to 53% of the cell population) due to increased surface roughness. However this was accompanied with a decrease in average neurite length due to the accompanied increase in Young’s modulus (298.4 ± 16.9 µm for PU versus 45.2 ± 1.5 µm for CE).

Flexible, fully polymeric electrode arrays were fabricated from conductive elastomers using conventional laser micromachining techniques. Methods to connect the polymeric arrays to traditional electronics were also developed. Arrays were comprised of nine, 900µm electrode sites in a hexagonal array. Charge injection limits (CIL) of the CE array were determined using biphasic stimulation with a phase length of 0.2 ms. CE electrodes were found to have a CIL of 0.020 ± 0.002 mC.cm^-2 compared to 0.013 ± 0.001 mC.cm^-2 for platinum electrodes of the same geometry. CE arrays were also found to have high charge storage capacity, 205 ± 55 mC.cm^-2 but also a high track impedance of 0.41 ± 0.16 Ω. Electrode arrays were demonstrated to maintain charge conduction and injection under strain and flexural deformation.

The formation of PEDOT networks within elastomeric polymer matrices has been demonstrated as a promising approach for the fabrication of soft, flexible bioelectronic materials. CEs facilitated the fabrication of a fully polymeric, flexible electrode array using conventional laser fabrication techniques. CE arrays were found to have favourable charge injection properties compared to platinum electrodes, however track conductivity is a current limitation which needs to be addressed in order to open up the application of CEs in the next generation of flexible bioelectronics.

Traumatic brain injuries (TBI) affect more than 2 million people in the US annually. Major causes include falls, motor-vehicle crashes, and assault/self-harm. Sports-related injuries suffered by athletes and blast injuries affecting soldiers are also notable contributors to the TBI patient population. Native brain microenvironment has low intrinsic regenerative capability and such injuries may lead to long-term effects such as dementia.

We describe a class of injectable materials that can manage and possibly reverse the harmful biological responses to TBI. These hydrogels, with material properties similar to native matrix in the brain, can liquefy during injections and be reconstituted in the brain after injection. We apply lessons learned from self-assembly of short peptides to create materials that can promote blood vessel formation, modulate the immune microenvironment, and facilitate survival and reconstitution of the functional neuronal circuit in and around the injury site. Our multi-disciplinary approach bridges chemical principles with neurobiological insights to set up a modular neuroprotective therapeutic platform with both stationary and diffusive biologic signals. This integrative approach will increase our understanding of the response of injured brain microenvironment to implanted biomaterials and solve a major problem in clinical management of injured brain tissue without complicated surgical intervention. The biomaterial scaffolds are based on functional self-assembling peptide hydrogels.

There are three distinct post-TBI responses that we can control with such implantable biomaterials: (a) enhanced angiogenesis, (b) immunomodulation, and (c) improved neuronal survival, recruitment of neural progenitor cells, neural sprouting, and synaptic restoration. Following implantation in the injured site in the brain, the peptides disassociate from the bolus, inducing angiogenesis, immunomodulation, and neuronal survival. The biocompatibility, tunable biodegradability, and facile material properties of the biomaterial platform complement the therapeutic efficacy of the peptide formulations. We compare the in vivo efficacy of the hydrogels in the injury site in a rodent model of TBI (fluid percussion injury) and determine the biodistribution of the hydrogels away from the injection site in a time-dependent fashion.

Self-assembled peptide hydrogels with covalently attached bioactive epitopes and sequestered immunomodulatory agents are promising materials for promoting neuro-regeneration after debilitating brain injury. Such injectable scaffolds that can create a neuroprotective niche after being injected in vivo have great potential for treatment of TBI, non-TBI stroke, and other ischemic conditions of the central nervous system.

As an important application of functional biomaterials, neural probes have contributed substantially to studying the brain. Bioinspired and biomimetic strategies have begun to be applied to the development of neural probes, although these and previous generations of probes have had structural and mechanical dissimilarities from their neuron targets that lead to neuronal loss, neuroinflammatory responses and measurement instabilities. Here, we present a bioinspired design for neural probes—neuron-like electronics (NeuE)—where the key building blocks mimic the subcellular structural features and mechanical properties of neurons. Full three-dimensional mapping of implanted NeuE–brain interfaces highlights the structural indistinguishability and intimate interpenetration of NeuE and neurons. Time-dependent histology and electrophysiology studies further reveal a structurally and functionally stable interface with the neuronal and glial networks shortly following implantation, thus opening opportunities for next-generation brain–machine interfaces. The NeuE subcellular structural features are shown to facilitate migration of endogenous neural progenitor cells, thus holding promise as an electrically active platform for transplantation-free regenerative medicine. Finally, I will discuss the potential application of NeuE to facilitate the recovery of brain injury.

Organic electrochemical devices, which use conjugated polymers in contact with an electrolyte, have applications in bioelectronics, energy storage, electrocatalysis, and sensors. Their operation relies on the oxidation (electron loss) or reduction (electron gain) of the polymer, which are traditionally described as Faradaic processes that transfer charge. However, recent evidence from various devices based on poly(3,4-ethylenedioxythiophene) chemically doped with poly(styrene sulfonate) (PEDOT:PSS) is consistent with a purely capacitive process that stores charge. To clarify whether PEDOT:PSS is an exception or the rule and determine which processes are capacitive and which are Faradaic, solid-state physics methodology developed to understand the operation of organic light-emitting diodes can be used. Such studies can pave the way for device optimization.

Within 1.3L of human brain billions of neurons connected by trillions of synapses are continuously exchanging signals. These signals govern the inner workings of the nervous system, and the aberrations in communication between neurons and other cells within the brain are manifested as the neurological and mental conditions that increasingly affect our aging society. While clinically available devices lack the finesse of the neural circuits and drugs often come with unwanted side effects, our team leverages the principles of optoelectronics and magnetism to develop tools matching the signaling complexity of the nervous system and cause minimal disruption to natural physiological function.
My talk will discuss how multimaterial fiber technology can be leveraged to produce compliant and miniature devices capable of electrical and optical recording and stimulation of neural activity, and delivery and sensing of neurochemicals. I will further highlight how multimaterial fibers can be employed for neural repair following traumatic injury as well as the potential candidates for artificial muscles with future applications in prosthetics. The second half of my talk will focus on using iron-containing nanomaterials as transducers of magnetic fields to biologically relevant stimuli such as heat and force. By tuning magnetic nanomaterials properties in conjunction with magnetic field conditions, it is possible to independently control multiple heat- or force-dependent biological processes. Finally, iron-containing nanomaterials can be used to aid minimally-invasive delivery of neurochemicals via magnetothermal and electrocatalytic means.

The auditory brainstem implant (ABI) is a neuroprosthesis that provides sound awareness to deaf individuals who are not candidates for the cochlear implant. The ABI electrically stimulates the surface of the cochlear nucleus (CN) in the brainstem. The complex anatomy and physiology of the CN together with the poor spatial selectivity of electrical stimulation and inherent stiffness of contemporary implants lead to only modest auditory outcomes in ABI users.
We propose a soft conformable ABI electrode array to improve the biomechanical match of the man-made implant to the curved CN surface. The conformable implant provides stable positioning of the implant in vivo and enables higher selectivity of electrical stimulation. The soft neurotechnology leverages miniaturization, high performance thin-film materials, and engineered mechanical compliance to produce implants compatible with the demanding ABI surgical insertion and conformability to the curvature of the brainstem. We developed elastic micro-structured multilayers, a soft electrode coating, and transient surgical features that allowed the fabrication of a scalable ABI from miniaturized mouse implants to human-size arrays.
This talk will report on the microfabrication and scaling process, mechanical and electrochemical characterization of the compliant ABIs. The soft electrode specifications are very similar to those measured in clinical ABIs with average impedance at 1 kHz of 5.78 ± 0.62 kΩ (electrodes of 0.385 mm² surface area). In a mouse model, we show that soft neurotechnology can be implemented to reliably activate auditory neurons in vivo for up to 4 weeks, a mandatory step before evaluating longer, chronic use of the technology. In a human cadaveric model, we demonstrate that the soft ABI is robust to surgical manipulation and insertion into the lateral recess of the IVth ventricle, and displays improved electrochemical performance compared to current clinical devices.

Aim: This study aimed to compare the chronic in vivo charge transfer properties of conductive hydrogel (CH) coated cochlear implant (CI) electrodes to traditional platinum (Pt) CI electrodes.

Background: CIs can improve quality of life in profoundly deaf people by restoring sound perception. Ideally, bionic devices should safely operate for a lifetime; however, host responses such as protein adsorption, inflammation and fibrosis can impact electrode impedance. These host responses can lead to inefficient stimulation and formation of toxic byproducts. CH coatings, based on poly(vinyl alcohol) (PVA) anti-fouling polymers modified with conductive polymers (CPs), can provide enhanced electrical properties, superior to those of traditional Pt electrodes. This technology can be directly coated onto metallic substrate of state-of-the-art bionic devices¹. This study tested the hypothesis that PVA conductive hydrogels can enhance safe charge injection and improve the quality of neural recordings.

Methods: CIs with 4 electrodes specifically designed for the rat cochlea were implanted for up to 5 weeks. The CH coating was fabricated using PVA hydrogel modified with 20 taurine groups for electrical doping and 5 methacrylate groups for crosslinking. The CP poly(ethylene dioxythiophene) (PEDOT) was galvanostatically grown through the hydrogel and arrays were ethylene oxide-sterilized (ETO) prior to implantation. Electrochemical impedance (EI), charge injection limits (CIL) and charge storage capacity (CSC) of electrodes was compared before and after implantation using a 3-electrode cell (vs Ag/AgCl). Sprague Dawley rats were unilaterally implanted with CH coated or uncoated Pt CIs. The animals were chronically stimulated using charge balanced biphasic pulses for ~20 hrs/week over the implantation period. To evaluate electrode performance in vivo, voltage transient impedance was measured 5 days/week, and EI, CIL and CSC was also recorded over the implantation period. Explanted electrodes were imaged using scanning electron microscopy (SEM) for evaluation of the CH attachment and for signs of Pt oxidation.

Results: Before implantation, EI of CH coated electrodes over a frequency range of 10Hz-50kHz was more than 3 times lower than uncoated Pt implants. CSC and CIL of CH-coated electrodes was over one and two orders of magnitude higher than Pt electrodes, respectively. During implantation periods, common ground impedance of CH-coated electrodes increased from ~4kW in the first week, and stabilized thereafter at ~6kW. In contrast, the impedance of Pt electrodes was approximately double that of CH immediately after surgery at ~8kW and after 2 weeks at ~13kW. Following implantation electrochemical impedance was maintained at close to pre-implantation levels in CH coated electrodes, which was significantly lower than in Pt. CSC and CIL decreased by ~40% and ~60% in CH coated electrodes following implantation, but both were still ~10 times higher than the Pt CSC and CIL. Preliminary observations of SEM images (n=2) showed that the CH coat remained attached to the cochlear implant throughout studies. However, further histological analysis will assess potential CH residues in cochleae tissue. Following removal of CH no signs of Pt oxidation were observed.

Conclusion: CH-coated cochlear arrays maintained enhanced electrical properties during and after implantation. These results support the potential of CH as an advanced electrode coating for cochlear implants with superior electrochemical performance than Pt electrodes.

1. Hassarati, R. T. et al. Improving Cochlear Implant Properties Through Conductive Hydrogel Coatings. IEEE Trans. Neural Syst. Rehabil. Eng. 22, 411–418 (2014).

Microelectronic devices placed in the nervous system present tremendous potentials for mapping neural circuits and treating neurological disorders. Currently, these devices often experience failures in part due to the electrical, mechanical, and biochemical, mismatch between the artificial device and neural tissue. Quantitative histology and 2 photon imaging have revealed neuronal damage and degeneration, inflammatory gliosis, blood brain barrier leakage and oxidative stress at the site of implants which may compromise the intended recording/stimulation/neurochemical sensing function. We use several biomaterial strategies to minimize these mismatches in order to achieve seamless and stable device-tissue interface. First, various conducting polymer based nanocomposites have been investigated as electrode coatings and facilitate the signal transduction/charge transfer between the ionically conductive tissue and the electrical device. Nanostructuring is employed to improve the adhesion, stability and charge injection and drug delivery capability of the conducting polymers to meet the material challenges in chronic interface. Secondly, to minimize the mechanical mismatch at the device-brain tissue interface, novel soft and elastomeric electrode materials have been developed with Young’s modulus approaching that of neural tissue (less than 1 MPa). Soft implants demonstrated reduced inflammatory tissue response in both CNS and PNS compared to stiff implants of similar geometry and surface chemistry. Thirdly, bioactive approaches are being developed to modulate the biological responses. One approach is to modify the implant surface with biomolecules or biomimics. Surface immobilization with these bioactive molecules significantly improved neuronal health and inhibited the inflammatory tissue response around the implants. Alternatively, therapeutics that control inflammation, neurodegeneration and oxidative stress have been delivered systemically or locally. These bioactive approaches have demonstrated significant benefit in neural recording quality and longevity. The ultimate solution to a seamless device/tissue interface may be a combinatorial approach that takes advantage of multiple biomimetic strategies discussed above and beyond.

In recent years, implantable bioelectronics has raised huge interest, especially for neural diseases. Among them, devices with a nerve guidance to guide neuron growth can enhance neural regeneration for traumatic peripheral nerve injury. However, the commonly used nerve conduit cannot provide a spatiotemporal regulation and in-situ monitoring for localized neurons. Accordingly, in this study, an implantable neural device that is built with microelectrode arrays on transparent and flexible substrate was developed to enhance cell growth through applying electrical stimulation and neural signals recording in a tissue-mimicked microenvironment. A new type of flexible substrate, nanoporous Parylene C, was designed and fabricated to exhibit tissue-mimic structural and mechanical properties. The microelectrode array, iridium oxide electrode for both neural stimulation and recording, was pre-deposited on a water-soluble sacrificial layer of poly acrylic acid (PAA) prior to the transfer print process. Such transfer printed tissue-mimic neural microelectrode array enabled conformable adhesion on a curvilinear nerve surface and electrical signals transduction from the microelectrode site, which is expected to provide higher performance of peripheral nerve regeneration. In addition, we characterized and evaluated the microelectrode array of iridium oxide film, including charge storage capacity, charge injection capability, and electrochemical impedance.

Lipid vesicles, also known as liposomes, present simple yet highly versatile nanocarriers for delivery of therapeutic and diagnostic agents. Composed of one of the most abundant biological molecules, lipids, these carriers have similarities to biological membranes and are thus, biocompatible, biodegradable, and easy to decorate on the surface. The lipid membrane in a liposome encloses an aqueous volume and therefore, liposomes are soft and highly deformable. Recent studies on delivery nanomaterials have suggested a prominent role for the mechanical aspects of these materials in their delivery performance (e.g. biodistribution). To enable variation of mechanical properties in liposomes, here we incorporate hydrogel materials in the lumen of liposomes. In this study, we present a straightforward and reliable technique for fabrication of nano-scale liposomes filled with photo-crosslinkable hydrogel, poly(ethylene glycol diacrylate) (PEG-Da). This technique relies on the lipid film hydration, extrusion, and proper incorporation of the photoinitiator into the liposomes followed by UV exposure to form a cross-linked hydrogel within the lumen of liposomes. We assess the resultant gel-filled liposomes for size and morphology using dynamic light scattering and scanning electron microscopy and confirm the presence of PEG hydrogel in the lumen of liposomes. We demonstrate that the present approach can successfully produce gel-filled liposomes with size range of 150-300 nm size. These gel-filled liposomes show good colloidal stability under physiological condition for at least 6 days. We demonstrate that this method allows for (i) changing the lipid composition of membrane and (ii) changing the hydrogel volume percentage. Mechanical testing of bulk hydrogels with various PEG-Da volume percentages reveled that increasing the PEG-DA volume percentage (10-40%) results in a significant increase in the elastic modulus of the hydrogel (0.1-6 MPa). The resultant gel-filled liposomes can, therefore, offer various levels of mechanical elasticity. Such membrane nanomaterials provide exciting and controllable platforms for delivery of therapeutic and/or diagnostic agents for different medical or biomedical applications.

Dental pulp derived cells are pluripotent stem cells which can be differentiated along odontogenic, osteogenic, adipogenic, or neurogenic lineages. Odontogenic and osteogenic differentiation, in the absence of dexamethasone, have been shown to be highly dependent on substrate morphology and mechanics. Here we focus on neurogenic differentiation, using the protocol described in [1], and its dependence on substrate nature. DPSCs were cultured on PLA, a biodegradable polymer approved for internal use. The upregulation of genetic markers was compared with that of cells plated on standard TCP. The role of substrate morphology was investigated by plating on electrospun fibers approximately 2.0 ± 1.0 μm in diameter and on spin-cast thin films . The influence of electrical conductivity was investigated through the addition of 3% and 10% graphene nanoparticles to the films and fibers respectively.
The aspect ratio of the cells was measured using confocal microscopy. Cells grown on graphene containing substrates had larger aspect ratios than their non-graphene counterparts , and cells grown on microfibers were longer than their counterparts on the flat films. But the cell aspect ratio did not necessarily correlate with genetic differentiation. The results after 21 days of incubation indicated that early markers (TBP, β-III tubulin), decreased uniformly on all substrates relative to day 0, with the largest decrease occurring on the PLA flat film with graphene. The late stage marker, NEFM, which indicates differentiation, was upregulated to a significantly larger extent on all PLA substrates. No difference was observed between the fibers and the flat film in the absence of graphene, thus morphology did not play a significant role on this polymer. Addition of graphene did not affect the outcome on the fibers, but significantly suppressed the gene expression on the flat films. These results indicate that PLA is a promising scaffold material for neurogenic differentiation.
[1] Arthur, Agnes, et al. “Adult Human Dental Pulp Stem Cells Differentiate Toward Functionally Active Neurons Under Appropriate Environmental Cues.” Stem Cells, vol. 26, no. 7, 2008, pp. 1787–1795., doi:10.1634/stemcells.2007-0979.

Intracranial aneurysms are a serious condition, affecting approximately 6 million people annually. The current method of treatment, endovascular coiling, utilizes a catheter to release platinum coils directly into the aneurysm which promotes diversion of blood flow. However, coiling is reported to be thrombogenic, and induces recanalization[1]. In order to circumvent these problems, we propose an alternative approach, where polymer gels are directly injected into the aneurysm. In this manner, the polymer can be engineered to occlude blood flow, while promoting endothelialization and minimizing thrombogenesis. A silicone model was 3-D printed from a cone CAT scan of a goat aneurysm. The model was connected to a peristaltic pump which simulated the pulsatile blood flow in the carotid artery. The polymer gels were then injected directly into the model, maintained at 37°C, via a Stryer Catheter, under radiological imaging. Two types of gels were tested. A physical gel composed of F127 Pluronic micelles was injected at 4°C, but warmed above the gelation temperature immediately after reaching the aneurysm. The retention time of the gels was then probed by x-ray imaging with induced contrast. Mixtures of F127 copolymer with multipblock (n=6), PF127 were tested in order to optimize retention time and injection viscosity. The F127 group of polymers are non cell-adhesive, and may need further modification to promote endothelial migration. Gelatin, was mixed with matri-gel or fibrinogen, injected, and gelled 37°C, using MTG. Endothelization was found to be successful on this surface. In this case, retention times in excess of 76 hours were determined. Even though both gelatin and Pluronic copolymers are presumed to be minimally thrombogenic, thrombus formation in both cases will be probed directly by circulation of platelet rich plasma across the aneurysm.

[1] W. Brinjikji, D. F. Kallmes and R. Kadirvel, Am J Neuroradiol. 36, 1216 (2015)