Symposium SM03—Advanced Neural Materials and Devices

The multimodal neural probe for simultaneous monitoring and manipulation of neuronal circuits in small animal models during behavioral tasks has advanced the understanding of complex brain functions. Although sophisticated and powerful in the manipulation and analysis of neural circuits, these multimodal neural probes are constrained by their inability to monitor changes in neurochemicals, which leaves many of the molecular mechanisms underlying neurological and psychological disorders unresolved. This limitation can be overcome by using systems that combine independently controlled neuromodulation with neurochemical sampling within a single platform. Conventional in vivo extracellular fluid sampling approaches such as microdialysis technology rely on rigid probes and semipermeable membranes, and therefore suffer from poor spatial resolution (~ 1 mm²), low temporal resolution (several minutes), and limited capacity to collect large molecules, such as neuropeptides and neuroproteins. Here, we demonstrate a battery-free, wireless, “all-in-one” neural probe for simultaneous optogenetics, neuropharmacology and neurochemical sampling in awake, freely moving animals. In vitro studies demonstrate the programmable operation for efficiently sampling various neurochemicals (K⁺, dopamine, and neuropeptide Y) with a small sampling area (10 × 10 µm²) and fast dynamics (< 1 min). Open field test reveals that the device implantation does not impact the movement and spatial preferences of mice. The probe is implanted in the brain of freely moving mice and could successfully sample the pharmacologically evoked release of the large molecule neuropeptide Y. The soft mechanics and lightweight construction allow for applications in freely moving small animal models to uncover the basis of brain disorders.

New technologies that can provide intracellular electrophysical access with high spatiotemporal resolution and minimal invasiveness could assist neurophysiological undertakings that aim to gain an in-depth understanding of how neurons regulate and orchestrate large network activity to coordinate cognition, disease, and function. Numerous nanoscale devices were invented in the last decade for probing intracellular potentials but such methods exhibit a compromise between stability and scalability. Of these, vertical nanowire arrays generally employ electroporation techniques which lead to a compromised stability of the nanowire-neuron interface and additionally suffer from low sensitivity. We address these challenges by innovating large scale vertical ultra-sharp nanowire arrays that are individually addressable and exhibit sub-10 nm tips. This silicon-based nanowire array interface system enables native recording of graded potentials proceeding high-amplitude intracellular action potentials without resorting to electroporation methods. Passive intracellular recording is performed stably with in vitro rat cortical neurons without attenuation of the amplitude for the duration of the experiment (several minutes). Systematic studies show chronically stable nanowire-neuron interfaces with somas naturally permeated by the nanowire, validated by FIB-SEM image of the cross-section at the nanowire-neuron interface. Pharmacological stimulation and inhibition of electrophysiological activities confirmed our results. By constructing a comprehensive small signal model used for modeling potential transients at nanowire-neuron interfaces, we demonstrate by simulation and experimental investigations the significant reduction of signal amplitude as we deliberately increased the number of nanowires per recording channel. Overall, our ultra-sharp nanowire array platform may pave the way to high-throughput and low-cost drug screening for neurological diseases and can enable future investigations of neuronal dynamics that coordinate function and of relevance to brain-machine interfaces.

Biological neurons process signals and compute by integrating incoming stimuli and initiating action potentials to convey information within the nervous system without loss. The process from generation to propagation is regulated by membrane potential and voltage-activated sodium and potassium channels. These key features inspire the design of neuron-like materials and neuristors for applications in signal processing, computing, and lossless propagation of information. To date, despite success in emulating neural functionalities, the state-of-the-art neuristors consisting of nanoscale memristors fall short in energy efficiency compared to biological neurons due to their electronic nature. Further, while suitable for neuromorphic computing applications, current neuristors lack biocompatibility and operate at non-biological timescales, which makes them incompatible with biological applications. In contrast, neuristors assembled from synthetic biomolecular elements are biocompatible, bear similarities in structure and dynamics to their biological counterpart, and can operate at biological timescales.

Here, we demonstrate a dynamic neuristor consisting of two biomolecular memristors in parallel operating according to the same principles as Hodgkin-Huxley. The biomolecular memristors consist of insulating biomembrane doped with voltage-driven, pore-forming peptide, alamethicin. To capture the switching dynamics of the sodium and potassium channels, we used DPhPC- and BTLE-based memristors, which can offer fast and slow-switching dynamics, respectively. The neuristor exhibits a firing behavior similar to a biological neuron as well as power efficiency that far exceeds metal oxide-based memristors. Further, we confirm that the addition of a lipid-based memcapacitor into the circuit embeds short-term synaptic plasticity dynamics within the neuristor—adding another layer of nonlinearity and complexity in signal processing. Our results demonstrate that the addition of a symmetrical memcapacitor resulted in a decreased firing rate (inhibition), while an asymmetrical memcapacitor resulted in an increased firing rate (facilitation). This novel neuristor composition can be implemented in an adaptive nonlinear control system, a brain-machine interface (BMI) device, as well as a wide range of applications in signal processing. Further, this neuristor can serve as a model to aid in fathoming the neuronal structure and functions.

With the rise of Artificial Intelligence and the Internet of Things in biological domains, it is imperative to construct computing devices that are biocompatible, low-power, and preferably ionic. We believe an approach that takes inspiration from the brain can yield a computational system that mimics synaptic and neuronal functionalities with the scalability of semiconductor processors, all while maintaining the biocompatibility and mechanical flexibility of biological materials. To this end, we have recently demonstrated that lipid bilayer membranes formed between two lipid-encased water droplets in oil (i.e., droplet interface bilayers or DIBs) can exhibit memcapacitive and memristive properties. To leverage all the diverse functionalities exhibited by a single membrane, large networks of functional membrane-based elements need to be assembled to achieve vast interconnectivity and flexibility—similar to how individual synapses/neurons operate together to perform complex tasks and functions in the brain. However, previous efforts to incorporate such components at a scale that could be usefully comparable to modern solid-state processors have been blocked by the difficulty of organizing liquid state DIBs into a dense, digitally addressable material. To address these shortcomings, we propose a pressure-driven PDMS microfluidic device to form lipid-encased water droplets in the initially liquid oil phase, organize them into DIBs, and connect them to electrodes forming a crossbar array. Crossbar arrays have been used in solid-state computers as a method to densely pack two-terminal devices while maintaining individual addressability to each device. The microfluidic system operates on the principle of a shift register where droplets pumped into the device fill the first available empty register and subsequent droplets move past onto the next register. This allows for the exact positioning of droplets within a 2D geometry which, in this case, is an n-by-n arbitrary-sized array with equal spacing between each DIB. Electrodes run the length of the top and bottom of the device such that the top electrodes electrically connect each row of the array, and the bottom electrodes connect each column of the array. Each DIB inside the device can be addressed by one combination of a single top and bottom electrode pair. Once all DIBs have been sorted inside the device, the oil phase is solidified, locking all the droplets in place. The solidified oil phase inside the PDMS microfluidic makes for a durable, yet flexible material that is capable of energy-efficient computation.

Development of soft and conductive microstructures has become a key research topic in organic electronics. Among various 3D printing techniques, Two-Photon Polymerization (TPP) based on direct laser writing technology stands out due to its unique capability to fabricate 3D complex architectures in sub-micron resolution. Herein, we have formulated a novel resin that can be fabricated into conductive microstructures via TPP lithography. We have also developed a strategy to fabricate multi-material (combination of conductive / non-conductive resin) microelectrodes with low impedance and high charge storage capacity.

Conductive resin consisted of poly(ethylene glycol) diacrylate (PEGDA), poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), dimethyl sulfoxide, and ethyl (2,4,6-trimethylbenzoyl) phenylphosphinate. Microstructures were constructed on a glass slide through 3D movement of XYZ stages and irradiation of 130 femtosecond pulses, which solidified the resin at the laser focal point. I-V measurements revealed that conductivity of microstructures fabricated by non-conductive resin (PEGDA) significantly improved from 3.4 ± 0.8 S m^-1 to 24248 ± 929.7 S m^-1 (n=5) by incorporation of PEDOT:PSS in the resin.

Michigan-style neural microelectrodes were fabricated in two steps via TPP: 1. Construction of conductive microstructures including cylindrical electrode sites with diameters of 1 µm (site 1), 5 µm (site 2), 10 µm (site 3), 20 µm (site 4), 40 µm (site 5), and 80 µm (site 6) with height of 7 µm, which were individually connected to cubical contact pads with dimensions of 20 µm (width) × 20 µm (length) × 7 µm (height) by wire connectors with height of 2 µm, using conductive resin (PEGDA/PEDOT:PSS), 2. Fabrication of electrode shank with height of 5 µm using non-conductive resin (PEGDA), which isolated the wiring and partially exposed the cylindrical sites and cubic pads. Electrochemical characterization of microelectrodes indicated that the impedance at 1 kHz was 63.13 ± 4.56 kΩ, 49.33 ± 2.98 kΩ, 42.82 ± 2.95 kΩ, 33.21 ± 3.69 kΩ, 26.66 ± 2.07 kΩ, and 19.28 ± 3.08 kΩ, and charge storage capacity was 2.38 ± 0.18 nC µm^-2, 8.61 ± 1.03 nC µm^-2, 16.36 ± 1.67 nC µm^-2, 28.43 ± 4.28 nC µm^-2, 61.07 ± 5.96 nC µm^-2, and 89.73 ± 15.14 nC µm^-2 for site 1, 2, 3, 4, 5, and 6, respectively (n=3). Development of these soft and electrically functional microelectrodes paves the way towards minimally invasive neural recording and stimulation.

Our current understanding of the spatiotemporal dynamics underlying neural function and disease is limited by (i) our inability to record brain dynamics at sufficient spatial and temporal resolutions and (ii) a lack of established frameworks for applying novel data analysis techniques to high-dimensional neuronal datasets.
Light-based approaches to manipulate and monitor neuronal activity have established neuroimaging as the principle technique for mapping the brain at cell-specific resolution and at large scale (>10⁶ cells). Optical imaging techniques, like calcium and voltage imaging, however, typically suffer from the slow kinetics, suboptimal intensity, and low photostability of the fluorescent reporter molecules, as well as from the limited speed of currently available imaging systems. Electrophysiological recordings with implantable microelectrode arrays can capture spikes from single neurons, but they can only resolve few tens of cells at a time.
Combining electrophysiological and optical modalities presents a unique opportunity to harness the spatial resolution of optical imaging along with the temporal resolution of electrophysiology. However, such a combination requires imaging and recording simultaneously in the same location, which is not possible with conventional microelectrode arrays composed of opaque metallic materials, as these block optical access and suffer from photoelectric artifacts. Furthermore, there is no established framework to combine these multimodal datasets operating at different spatiotemporal scales to elucidate the dynamics of brain networks during normal function and disease.
Here, we show how to address this essential challenge by leveraging flexible, transparent graphene microelectrode arrays to map the dynamics of onset and evolution of epileptic seizures. Through the transparent arrays, we acquired simultaneous electrophysiology and calcium imaging in vivo in mouse models during the transition from baseline to chemically-induced epileptiform activity. To integrate data features from both modalities, we established an analytical framework based on non-negative matrix factorization to identify transitions in brain state that occur during an epileptic seizure. Our analysis demonstrates that it is possible to capture complementary and otherwise hidden aspects of ictal dynamics operating on different spatial and temporal scales by leveraging data features from multiple modalities. Furthermore, we show how microscale brain dynamics are linked to clinically measurable markers of seizure states.

The number of people using Illicit drug aged 12 years and older reached 30 million in 2016, among which 2 million are cocaine users based on the national data. In order to fully understand the mechanism of action and develop effective therapy for addiction, a tool that can reliably and consistently measure in vivo cocaine concentration and neural activity with high spatial and temporal resolution is needed. Integrated biochemical sensing devices based upon microelectrode arrays (MEA) have emerged as a powerful tool for such purpose. The implanted MEA devices often face challenges in the in vivo environment where biological reactions could have detrimental effects on the sensing and recording capability of the device. To protect implanted MEAs from biofouling and harmful tissue reaction, zwitterionic polymer coatings have recently emerged as a promising candidate. In current work, cocaine electrochemical sensors made from synthetic DNA aptamers were functionalized on flexible MEAs and protected with antifouling zwitterionic poly (sulfobetaine methacrylate) (PSB) coating which prevents sensors from being degraded by host tissue. In vitro experiments showed that biofouling of plasma protein doesn’t play a significant role in sensor performance decline, while exposure to DNase have detrimental effects on sensor performance, causing a 57.2% signal reduction. The PSB coating successfully protected sensors from DNase enzyme digestion over a 24hrs period. Also, the sensor is stable after 12hrs of continuously applied square waver voltammetry (SWV). This novel integrated cocaine sensor can serve as a valuable tool for study of cocaine addiction mechanism, while the sensing and coating technology can be generalized to detect many other biochemicals of interest.

Intracortical microstimulation is not only a useful tool in neuroscience research for dissecting circuitry and modulating brain activity, it has also shown promise in restoring sensory functions in humans with neural deficits. Current electrode materials like platinum do not meet charge injection requirements for chronic microstimulation, which motivates the development of new stimulating electrode materials. One such material is the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) doped with acid functionalized multi-wall carbon nanotubes (CNT). Although promising, the in vivo stimulation performance of PEDOT/CNT(PC) remains to be fully characterized. Traditionally, characterizations of stimulation performance have been based on electrophysiological, behavioral or endpoint histology. Using advanced imaging technologies, we directly measured neuronal activity evoked by microstimulation in transgenic animals in real-time. Herein, we implanted microelectrode arrays coated with PEDOT/CNT (PC) and iridium oxide (IrOx) (a commonly used high charge injection stimulation material) in GCAMP6s mice and applied electrical stimulation while imaging neuronal calcium responses. We observed that PC coated electrodes produce more intense and broader GCaMP responses than IrOx. Using TPM we examined the effect of stimulation modality and pulse-width modulation on neuronal activation. We found that stimulating via PC coated electrodes activates significantly more neuronal soma and neuropil than via IrOx electrodes in constant-voltage stimulation and significantly more neuronal soma in constant-current stimulation. Furthermore, for both materials and the same injected charge, stimulating with a shorter pulse activated neural elements that are more spatially confined than longer pulses, providing an accessible means to tune stimulation selectivity. Using finite element analysis, we observed that the non-directional and rough edges of PC surfaces result in a more non-uniform and dense electric field, which increases the likelihood of activating nearby neural elements. Our results show that PC coated electrodes provide essential improvements in electrical stimulation applications in terms of energy efficiency for activating local excitable tissue.

Organic Electrochemical Transistors (OECTs) belong to a class of electrolyte-gated transistors with tremendous applications in bioelectronics due to their unique device architecture. The absence of an insulating layer between gate and channel and its replacement by an electrolyte creates an ideal environment for biological driven measurements as it allows for a direct integration of cell tissue on the device. For efficient gating, a non-polarizable Ag/AgCl electrode is used, which however limits the option for integrating more gates on a chip. Patterned polarizable Au gates on the other hand show strongly reduced gating due to a large voltage drop at the gate/electrolyte interface. Here, we demonstrate a novel method to induce efficient gating by patterned in-plane gate electrodes. For this, we make use of the fact that poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) exhibits a volumetric capacitance in an electrolyte. As a result, the capacity of PEDOT:PSS based gates can be strongly enhanced by increasing their thickness, thereby reducing the voltage loss at the gate/electrolyte interface. By combining two different fabrication approaches (spin coating and electrodeposition), we create gate electrodes of different thicknesses on the same chip. These electrodes gate the same channel revealing the effect of the gate film thickness on the gating efficiency. The results are compared to the ones of the typically used Ag/AgCl gate electrode, showing that the gating performed by a PEDOT:PSS electrode can be tuned to be comparable to the one from a Ag/AgCl electrode. Using these in-plane gates we study the device physics of multi-gated Organic Electrochemical Transistors and disentangle the part that the gate capacitance plays in gating. Our findings offer an extra degree of freedom in the design of OECT-based biosensors. Most importantly, they pave the way for an elegant integration of in-plane gate electrodes into the latter, leading towards the realization of “organ-on-a-chip” platforms.

Neural devices are important for understanding how the brain works and for treating neurological disorders. The commercially available conducting polymer PEDOT:PSS has emerged as a state-of-the-art material for neural devices due to its mixed conductivity, biocompatibility, and convenient deposition. I will present examples of implantable and cutaneous neural devices for bidirectional neural interfaces, explaining the advantages of this material over commonly used metal electrodes. I will discuss the relationship between materials properties and device performance, elaborate on device fabrication techniques, and discuss lessons learned in the design of competitive materials.

Two-way communication between electronics and neural tissue is key for advancing diagnosis and therapies for neurological diseases and disorders in both the central and peripheral nervous systems. Establishing such neural interfaces is a major challenge, as the tissue response to implants can have a detrimental effect on the signal quality and functionality of the implant. Also, electrical stimulation is inherently limited in its stimulation of neural tissue, in comparison to the sophisticated chemical signaling processes within biological tissue. Here, I present our efforts in addressing two critical material and device aspects for the creation of versatile neural interfaces: the matching of mechanical properties of tissues and electronics, and the development of fast and low-leakage chemical interfaces. Soft electronic biomedical implants have a demanding set of requirements, including biocompatibility, desirable mechanical and electromechanical properties, long-term stability and good electrode performance. To meet these requirements, we have developed a high-performance, long-term stable soft and stretchable conducting nanowire composite. Several fabrication strategies have been developed around this composite to enable the fabrication of soft neural electrodes of different dimensions and geometries. Based on this technology, we have developed devices for both the central and peripheral nervous systems. Next, I will outline our strategy for creating high-speed low-leakage chemical neural interfaces by first discussing the relationship between delivery delay and leakage, and then present an iontronic approach to achieve low leakage and small delivery delay simultaneously.
http://www.liu.se/soft-electronics

To better understand neuronal signalling behind brain activity, a proper detection of both electrical and chemical signals is essential. Indeed, action potentials (APs) generation and synaptic quantal release of neurotransmitters play a fundamental role in the cellular mechanisms underlying brain functions, being at the basis of information transmission and signal communication in neuronal microcircuits.
In the present work, we present the employment of diamond-based micro-patterned graphitic biosensors to extracellularly record the activity of excitable cells, to resolve APs waveforms and neurotransmitter release [1-2]. The multi-electrode-array sensors were fabricated using a three-dimensional patterning process by means of MeV He ion-beam-based lithography on an artificial-type-IIa single-crystal monocrystalline diamond sample (4.5×4.5×0.5 mm³), thus creating 16 independent graphitic electrodes embedded in its matrix. Being electrically conductive, the micro-channels of the sensors were used to investigate the in-vitro neuron activity both in terms of electrical signals generation (APs firing) and neurotransmitter secretion (quantal exocytic events).
The diamond biocompatibility allowed neuronal cells to be plated directly over the diamond device. Potentiometric measurements of APs generation were recorded from cultured hippocampal neurons, while quantal secretory events were amperometrically recorded from plated mouse dopaminergic neurons [3]. The electrical activity of an intact mouse sinoatrial node directly placed on the sensor was also recorded.
These results demonstrated the usability of diamond-based biosensors as promising devices for the simultaneous multi-parametric in-vitro detection of both electrical and chemical signals, representing in perspective a further step for a better understanding of brain functioning.

[1] Picollo, F. et al. Anal. Chem. 88, 7493–7499 (2016).
[2] Tomagra, G. et al. Front. Neurosci. 13, 288 (2019).
[3] Tomagra, G. et al. Carbon 152, 424-433 (2019).

Electrochemical and electrolyte-gated transistor architectures have emerged as powerful components for bioelectronic sensors and biopotential recording devices. For amplification of weak electrophysiological signals, maximum transconductance, high cutoff frequencies, and large on/off ratios are key desired parameters. Organic conducting polymer devices have recently dominated the field, especially where flexible and conformable in vivo electronics are necessary. Herein we report ultrathin, flexible indium tin oxide (ITO) electrolyte-gated field-effect transistors (EGFETs). These accumulation-mode devices combine high transconductance (g_m > 30 mS), excellent on/off ratio (10⁵), and fast modulation with excellent stability and the possibility of optically transparent layouts. While oxides are normally considered brittle, we obtain mechanically flexible and robust ITO layers by room temperature deposition of ultrathin (30 nm) amorphous layers onto parylene substrates. This approach results in low strain and the devices survive bending and deformation tests over hundreds of cycles. In addition to favorable material properties, these transistors offer low power consumption, as a result of the normally off state and low off currents. Furthermore, a wide variety of biosensor applications can be envisioned since the material is an oxide with active oxygen surface chemistry (hydroxyl groups) and facile chemical functionalization approaches may be employed. Based on the demonstrated stability and performance, as well as the future possibilities using these transistors, indium tin oxide EGFETs represent a promising avenue for bioelectronic devices.

Electrochemical actuators that transform electrical energy to mechanical energy through electrochemical reactions have numerous applications for biomedical devices, soft robotics, and bioelectronics. However, the design of high performance and durable electrochemical actuators that efficiently operate in biological environment remains a challenge yet. To address this challenge, here we developed and characterized a flexible organic electronic actuator based on poly(pyrrole) nanofibers (PPy NFs) that reversibly operates in liquid and gel polymer electrolytes under electrical stimulation. PPy is a versatile biocompatible CP which has been widely employed for bioactuators due to its low weight, fracture tolerance, and relatively large actuation strain. To fabricate the PPy NFs actuator, we electrodeposited an electroactive layer of PPy doped with polystyrene sulfonate (PSS) around template poly-L-lactide (PLLA) nanofibers that were previously electrospun onto a thin layer of gold coated poly(propylene) (PP) film (20 mm × 1 mm × 30 µm). The average diameter of template nanofibers and the resultant PPy NFs were 140 ± 4 nm and 626 ± 16 nm, respectively. The PPy NFs were subjected to cyclic voltammetry (CV) in an aqueous solution and 0.2 wt.% agarose hydrogel both containing 0.1 M poly(sodium 4-styrenesulfonate) (NaPSS) at various scan rates of 10, 50, 100, and 200 mV/s with the potential window of -0.8 V to +0.4 V. We demonstrated that the actuator kinematics (i.e. displacement and speed) can be tuned by adjusting the CV parameters (voltage and scan rate). Remarkably, the PPy NFs actuator demonstrated excellent properties, including low power consumption/strain (as low as 1 mW/cm²/%), relatively high actuation strain (up to 1.85%) a controlled cycling response, and excellent cycling stability (>96.5 % bending stability over 15000 actuations equivalent to 25 hr continuous operation). Overall, this study presents a new strategy for development of flexible, mechanically durable and dynamically tunable organic electronic actuators for variety of applications ranging from soft robotics to biomedical devices.

Conjugated polymers such as PEDOT:PSS provide soft interfaces with cells and tissue. Their large surface area and innate ability to transport ions make them attractive, high-performance materials that work in ion-rich, wet conditions.¹

We have developed a method of patterning PEDOT:PSS microgels into functional devices with heterogeneous structures. Using an out-of-shelf wax printer, we patterned filtrations membranes and have PEDOT:PSS microgels filtered through the membrane. PEDOT:PSS was retained on the hydrophilic region that was not covered by wax. We reached a resolution of ~100 um both at the patterned thickness and the gap distances between materials. Utilizing its microgel nature, we show that the patterns can be healed by water treatment. In its hydrated state, it can also be stretched by over 30% to 70%, depending on the stretching directions and the geometry of PEDOT.

By transferring PEDOT:PSS patterns onto adhesive substrates, followed by consecutive transferring of other colloidal materials, such as carbon nanotube (CNT), we demonstrate functional PEDOT:PSS electrodes and electrochemical transistors. We also developed methods to tune the water stability of the devices. This allowed us to perform ON/OFF switching cycles on the transistors in water for over 500 cycles.

Finally, we demonstrated biocompatibility of the materials by cultivating human tumor-derived U87 glioma cells as well as Lund Human Mesencephalic (LUHMES) neuronal cells on patterned PEDOT:PSS microgels electrodes, thus showing the possibility to use this method in both tumor-biology studies and more demanding neural models.

(1) Zeglio, Erica, et al. "Conjugated Polymers for Assessing and Controlling Biological Functions." Advanced Materials 31.22 (2019): 1806712.

Microelectrode arrays (MEAs) have been widely used in recent years for in-vitro investigation of neuronal cells and neural networks, as they enable long-term bi-directional interfacing with networks of living cells [1, 2]. Modern CMOS-based active MEA devices, especially high-density MEAs, provide the capability to simultaneously perform electrophysiological recordings from thousands of electrodes at cellular/subcellular spatial resolution [3-6]. The high spatio-temporal resolution enables better separation and assignment of neural activities for closely spaced neurons as well as localized and specific stimulation of single individual neurons [7].
An exemplary CMOS high-density microelectrode system includes a large sensing area of 4.48 × 2.43 mm² comprising 59’760 (332 × 180) electrodes of 3 × 7.5 µm² size at a pitch of 13.5 µm [6]. The system incorporates several types of sensing units with the aim of extracting a wide spectrum of information from the same neuronal culture or brain slice: 16 dual-mode stimulation buffers, 2048 action-potential recording channels, 32 local-field-potential recording channels, 32 current recording units, 32 impedance measurement units and 28 neurotransmitter detection units [6].
By using such a system it was possible to record subcellular-resolution data in various preparations, ranging from organotypic and acute slices to cultures of dissociated neurons and stem-cell derived neurons. It was also possible to detect low-amplitude signals of action potentials traveling along thin axons (~100 nm diameter). Moreover, the stimulation features of CMOS microtransducer arrays and integrated microsystems offer the capability to bi-directionally interact, also in closed loop and real time, with potentially every single neuron in a given neuronal network. Applications include research in neural diseases and pharmacology.

Acknowledgements
The work was supported by the European Community through the ERC Advanced Grant “neuroXscales” under contract number AdG 694829.

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Organic Bioelectronics operating in the electrochemical mode has been widely explored to record and stimulate signaling of neuronal systems. To make this technology successful in therapy and prosthesis applications as a future med-tech platform, included devices and materials should possess minimal invasiveness and must operate at a proximity, addressability and signal specificity to capture as much as possible of the signaling spectrum of the nervous system. Here, recent progress of the development of Organic Bioelectronics is presented that address these challenges, specifically targeting the development of devices integrated into capillaries and also self-organized neuro-bioelectronics.

Three-dimensional (3D), sub-millimeter-scale constructs of neural cells, known as cortical spheroids, are of rapidly growing importance in biological research because these systems reproduce complex features of brain architecture and organization in vitro, at levels qualitatively more sophisticated than those found in traditional two-dimensional (2D) cell cultures. Despite their great potential for studies of neurodevelopment, neurological disease modeling and evolution, detailed investigations of these miniature, 3D living objects cannot be accomplished easily using conventional approaches to neuromodulation, sensing and manipulation. This talk describes classes of microfabricated 3D frameworks as mechanically compliant, multifunctional neural interfaces to individual spheroids and to engineered, interconnected collections of them, known as assembloids. Electrical, optical, chemical and thermal interfaces to cortical spheroids derived from human induced pluripotent stem cells demonstrate some of the capabilities. Complex architectures and high-resolution features, in isolated or arrayed configurations, and in layouts that enable full surface coverage highlight the design versatility. Detailed studies of the spreading of coordinated bursting events across the 3D surface of an isolated cortical spheroid and of the cascade of processes associated with formation and regrowth of bridging tissues across a pair of such spheroids represent two of the many opportunities in basic neuroscience research and regenerative medicine uniquely enabled by these platforms.

Microelectronic devices placed in the nervous system present tremendous potentials for mapping neural circuits and treating neurological disorders. Currently, the performance of these devices is sub-optimum due to electrode material limitations and undesired host tissue responses. 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 responses in order to achieve seamless and stable device-tissue interface. 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 at the neural interface. As we continue to improve our understanding of the implant induced tissue response, bioactive approaches are being developed to modulate the cellular responses for seamless integration. Surface modification with bioactive molecules or anti-fouling materials have been found to significantly improve neuronal health and inhibit the inflammatory tissue response around the implants. Alternatively, therapeutics that control inflammation, neurodegeneration and oxidative stress can be delivered systemically or locally. These bioactive approaches 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 biomaterial strategies discussed above and beyond.

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, the Blueprint for Neuroscience Research, the HEAL Initiative, and the SPARC Program. 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). An overview of NIH/NINDS translational programs and resources will be presented.

As a cancer therapy, hypothermia has been used at sub-zero temperatures to cryosurgically ablate tumors. However, these temperatures indiscriminately damage both tumorous and healthy cells. Additionally, therapies targeting single molecules or pathways are highly susceptible to evolutionary escape. To address these limitations, we studied the use of hypothermia as a broad cytostatic tool against cancer and deployed it in rodent models of glioblastoma (GBM). To identify the minimal dosage of ‘cytostatic hypothermia’, we cultured at least 4 GBM lines at 4 continuous or intermittent degrees of hypothermia and evaluated their growth rates through a custom imaging-based assay. This revealed cell-specific sensitivities to hypothermia. Subsequently, we examined the effects of cytostatic hypothermia on these cells by a study of their cell-cycle, energy metabolism, and protein synthesis. To assess the efficacy of cytostatic hypothermia in vivo, we report the design of an implantable device to focally administer cytostatic hypothermia in rats bearing F98 and U87-MG GBMs. Hypothermia at supra-cytostatic temperatures significantly doubled the median survival of rats bearing F98. Cytostatic hypothermia delivered in rats bearing U87-MG resulted in no deaths during the study period. An absence of gross behavioral alterations was also observed and in concurrence with literature suggesting the brain is naturally resilient to focal hypothermia. We also investigated the use of cytostatic hypothermia as an adjuvant to chemotherapy and CAR T immunotherapy. Our studies demonstrated that cytostatic hypothermia did not interfere with Temozolomide in vitro and may have been synergistic against at least 1 GBM line. Interestingly, we also demonstrated that CAR T immunotherapy can function under cytostatic hypothermia. Based on these findings, we anticipate that focally administered cytostatic hypothermia alone has the potential to delay tumor recurrence or increase progression-free survival in patients. Additionally, it could also provide more time to evaluate concomitant, curative cytotoxic treatments.

We continue to investigate the design, synthesis, and characterization of functionalized variants of poly(3,4-ethylenedioxythiophene) (PEDOT). PEDOT and its derivatives have proven to be of considerable interest for a variety of applications, particularly bioelectronic neural interfaces. Substituted variants of the 3,4-ethylenedioxthiophene (EDOT) monomer, including EDOT-carboxylic acid (EDOT-acid) and EDOT-maleimide (EDOT-MA) make it possible for us to create a variety of functionalized PEDOT copolymers. This allows us to optimize electronic and ionic transport, adhesion to surfaces and biological tissue, and mechanical properties. Here, we will present results from our recent studies of PEDOT functionalized with cholesterol side groups, attached either via the EDOT-acid (PEDOT-cholesterol) or EDOT-MA (PEDOT-MA-cholesterol) chemistries. Cholesterol is the most abundant steroid in the animal kingdom, and provides flexibility and resilience to cell membranes. It is also known to promote the formation of chiral liquid crystalline mesophases. We will discuss the structure and properties of PEDOT-cholesterol using a variety of characterization techniques, and compare these results with previous studies of PEDOT and PEDOT copolymers.

Ongoing interest in the interface between the manmade device and live neural system is at the heart of many researchers that develop new materials to facilitate the neural interface. The ultimate goal in the neural materials is to seamlessly integrate the neural system with the device so as to monitor and modulate signals from neurons. In this regard, there are two major technical hurdles in connecting electronic devices and neural systems. First, the neural signal transmits across synaptic cleft through elaborate secretion and recognition of neurotransmitters, which are tricky to mimic using artificial gadgets. Second, it is difficult to secure stable and intimate contact between the neural system and the electronic device because of dissimilar material properties such as flexibility and softness. In this talk, we suggest our two approaches to get over the challenges.
One is the gel-based iontronics to the neural interface. Iontronics is the field of ion-based information processing inspired by the neural system and various biological structures such as ion channels. Materials that allow an electric field to manage gradients of ions, such as polyelectrolyte gels (charged hydrogel), can be used as components of iontronics. Taking hints from it, we constructed microfluidic chips such as a bipolar ionic diode, transistor, and logic circuits through polyelectrolyte gels. Even a fully aqueous and ionic circuit can be created on a chip powered by reverse electrodialysis (RED), which is reminiscent of neurons that transduce signals just by the ion transport. PDMS, a transparent and flexible material, can be employed as a chip substrate to expand the libraries of materials available for iontronics. Recently, we invented a new form of iontronic tools, i.e. an ion current rectifying micropipette, to address the neural system for chemical stimulation with high spatial and temporal resolutions.
The other is a biologically spontaneous and steady conjugation between the neural systems and electrode through synaptic proteins. One of the key issues in functional neural materials is persistent stability to minimize inflammation and neuron cell loss. We materialized the neural interface harnessing the synaptic proteins, paying attention to the fact that neuron stably transmits signals through neurotransmitter secretion in the synapse. Engineered the Neuroligin1 (Nlg1) as known to induce the presynaptic differentiation is immobilized onto the substrate to call for its counterpart protein, Neurexin (Nrx), in the live neuron that contact with the modified electrode. Heterogeneous association of the two proteins belonging to natural and artificial surfaces facing each other comes to form a unique junction that is mechanically as well as biologically stable enough to be persistent for several days.

Optogenetics is a powerful neuromodulation technique that enables dissection of neural circuitry and treatment of neurodegenerative diseases based on its cell-type specificity and high temporal resolution in control. Implementing optogenetics traditionally relies on fiber optics or microfabricated optical probes. Although highly reliable and effective, however, these approaches do not support facile and rapid design adjustment to meet various needs for in vivo neuroscience. Specifically, optical fibers are hard to be transformed to 2D or 3D configurations to enable multisite neural interfacing. Microfabricated probes can address this limitation by providing design versatility. However, the fact that microfabrication requires special cleanroom facilities and complex processes makes this approach expensive and time-consuming, hampering easy optimization and rapid prototyping for targeted in vivo applications.
To address these issues, we have developed a 3D printing-based manufacturing technique for rapid and customizable fabrication of optogenetic probes. The 3D printed optogenetic probes (3D-POPs) consist of microscale inorganic light-emitting diodes, electrical interconnects made of silver paste, and thin 3D printed substrates (60 μm thick). The 3-D printed patterned substrate determines the overall framework of the probe and facilitates electrode formation using silver paste in its groove patterns. The design can be easily altered using a 3D computer-aided design (CAD) program, thus providing facile design customizability. This straightforward 3D printing scheme can not only enable low-cost ($0.54 per 5 mm-long probe) and mass production of probes (fabrication of 50 probes (printing area of 1800 mm²) in 3 minutes), but also significantly reduce the time and cost needed for developing new devices optimized for specific applications. Successful experiments with live mice show biocompatibility as well as wireless functionality of 3D-POPs, verifying their practicality for in vivo optogenetics. We foresee that this 3D printing strategy can be applied to cost-effective production of diverse bioelectronics beyond neural probes.

Background: Low charge transfer, mechanical mismatch and host response to the implant, can ultimately compromise the long-term performance of deep brain stimulation (DBS) devices. Conductive hydrogel (CH) electrode coatings are hybrid systems that address these limitations by combining the high electroactivity of conductive polymers with the soft mechanical properties of hydrogels. Successful use of CHs on DBS electrode arrays relies on their ability to withstand mechanical stress during electrode placement. A key challenge in use of CH electrode coatings is dimensional change due to swelling of the hydrogel component. This work aimed to control CH swelling via modifying hydrogel chemistry, and assessing the effect of insertion on electrode coating stability in vitro and in vivo.
Methods: Two CH formulations based on poly(vinyl alcohol) were fabricated with low or high crosslinking densities (CD). DBS arrays modified for use in rats were dip-coated in polymer solution, polymerised, electrodeposited with poly(ethylenedioxytiophene) (PEDOT), and ethylene oxide (ETO) sterilised. Electrode coating stability was assessed in vitro via insertion in 0.6% agarose gels before and after ETO sterilisation. Stability was evaluated by impedance spectroscopy (EIS), charge storage capacity (CSC), and charge injection limit (CIL). The higher-crosslinked, lower swelling formulation was tested in a rat animal model and compared to Pt DBS arrays over 8-weeks implantation.
Results: CH coated DBS arrays maintained superior electrochemical performance when compared to Pt model electrodes in vitro. Following insertion in agarose gels, there was no significant impact on electrical properties of DBS coated arrays. ETO sterilisation significantly affected CSC and CIL as well as the coat adhesion on Pt electrode substrates. Nonetheless CH coated electrodes electrically outperformed Pt counterparts in vivo allowing for high fidelity neuronal recordings following chronic implantation.
Conclusions: CH coat dimensional stability can be improved by tailoring hydrogel crosslinking density without affecting electrical performance. Further research is required to understand the effect of sterilisation techniques on coating stability.

Neural regeneration is important for repairing nerve injuries and treating neurological disease. Despite with improved understanding of pathophysiology, a critical problem of slow nerve regeneration remains unsolved and often leads to a delayed and incomplete functionality recovery. Current stimulation technologies, such as electrical, ultrasound stimulation and optogenetics were found to improve nerve regeneration often due to an enhanced production of neurotrophic factors. However, electrical stimulation is invasive. Ultrasound lacks spatial specificity. Optogenetics, as a genetics-based technique, has limitation in translation from bench to patients.
Photoacoustic (PA) neural modulation is an emerging light-mediated technique allows temporally and spatially precise control of neural activity. The photoacoustic process is initiated by shining a pulsed laser on the absorber, and then broadband acoustic waves will be generated by the transient heating and thermal expansion. In this study, we developed a composite film of silk fibroin and carbon nanotubes (CNT) as a novel neural interface with the function of PA stimulation upon near infrared (NIR) second window excitation. PA signals can be controlled by varying the laser power and the concentration of CNT. To investigate the neural stimulation function, we examined the response of cultured cortical neurons to PA stimulation under different stimulation conditions. By time-resolved calcium imaging, we demonstrated this PA stimulation can robustly generate neural activations. Furthermore, as a key messenger in nerve system, we found that the calcium influx triggered by the PA stimulation can also modulate the neurite outgrowth of cultured rat dorsal root ganglion (DRG).

Accurate control of cell differentiation has received lots of attention because of due to its high applicability in personalized medicine. For this reason, numerous researches for control of cell differentiation have been reported such as introduction of functionalized nanoparticles or surface modified electrodes. However, these methods are difficult to apply directly to cell-based therapy because it is hard to simultaneously control the time and region in which cells are induced to differentiate into desired lineages. In this study, nanostructured biohybrid material composed of metalloprotein, DNA, gold nanoparticle (GNP), and electrically controllable complex was developed to control the neural differentiation electrically. The electrically controllable complex composed of 5,5-Bis(mercaptomethyl)-2,2-bipyridine, cucurbit [8] uril, and retinoic acid modified WGG tripeptide (RA-WGG). The result indicates that the neural differentiation was successfully conducted due to the release of the RA-WGG from electrically controllable complex by applied electrical stimulation. Furthermore, by adjusting the time and location of applying electrical stimulation to nanostructured biohybrid material, it was possible to increase the efficiency of neural differentiation over a long period of time and various regions on substrate. Proposed nanostructured biohybrid material should be used for differentiation of various stem cells as an innovative method in cell therapy. Acknowledgments: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1A2C3002300) and by the Ministry of Education (No. 2016R1A6A1A03012845).

Implanted neural prosthetics are electrical interfaces enabling neural recording and stimulation for the treatment of multiple neurological diseases. The typical neural prosthetic devices are designed for neuromodulation, while they cannot permit neuroreplacement or neuroregeneration which is a critical goal for rehabilitation in nervous injury. Herein, we developed tissuelike microelectrode arrays (MEAs) composed of bioresorbable materials to show the functions including neural recording, neural stimulation, and stem cell therapy. The electrode, PDGA, was synthesized with copolymerized poly(3,4-ethylenedioxythiophene) (PEDOT) and gelatin methacrylate (GelMA) to exhibit both nerve tissue-mimicked properties and hydroresponsive conductivity. Based on the thermal-responsive and photo-responsive sol-gel transition properties, PDGA can be directly micropatterned into a circuit in a resolution down to 30 μm, followed which an adhesive hydrogel substrate synthesized by gelatin and Transglutaminase (mTG) can directly transfer print the PDGA micropatterns. Applied with PLGA as the insulation layer, the integrated final MEAs demonstrated tissue-like mechanical and structural properties, adipose tissue-derived stem cell (ADSC) -laden capability, and electrical monitoring/neuromodulation. With the examination of physical, chemical, and biological properties, this device was expected as a revolutionary platform to promote nerve regeneration.

Treatment of brain tumours, particularly maglinant one such as glioblastoma, is extremely challenging because the efficacy of the conventional chemotherapy is quite limited due to the blood-brain barrier. Meanwhile, the wireless integration of wearable devices with implantable drug delivery devices can present a new opportunity in the development of unconventional devices for the treatment of fatal seizures. In this talk, recent progresses in the wirelessly-controlled implantable drug delivery devices for brain diseases, such as brain tumour and fatal sezure, will be presented. In the first part, an implantable drug delivery device using a flexible, sticky, and bioresorbable drug reservoir integrated with the biodegradable wireless electronics for the treatmet of glioblastoma will be presented. In the second part, a device that consists of a soft implantable drug delivery device integrated wirelessly with a wearable electrophysiology sensor for the treatment of status epilepticus will be presented. Such novel wirelessly-controlled drug delivery devices are expected to present new opportunities for the treatment of brain diseases.