This paper reports on the characterization of a microfabricated wafer-scale, graphene-based, soft implant for spinal cord applications. Graphene is used because of its high transparency and good conductivity, making it suitable for optogenetic applications. Moreover it has a high mechanical strength and is potentially biocompatible. The implant consists of multi-layered chemical vapor deposited graphene, in the form of electrodes and tracks, encapsulated between 2 layers of silicone. Methods such as Raman spectroscopy, optical transmittance, and electrical measurements combined with bending tests and in-vitro experiments, using phosphate-buffered saline (PBS) solution, were employed to characterize the device. The results have shown high bendability and no critical damage of the graphene after immersing the device in PBS solution up to 7 days. To the authors' best knowledge, this is the first work that presents a soft and fully scalable optogenetics-compatible graphene-based spinal cord electrode array.@en

This work aims to develop a smart neural interface with transparent electrodes to allow for electrical monitoring of the site of interest during optogenetic stimulation of the spinal cord. In this paper, a microfabrication process for the wafer-level development of such a compact, active, transparent and flexible implant is presented. Graphene has been employed to form the transparent array of electrodes and tracks, on top of which chips have been bonded using flip-chip bonding techniques. To provide high flexibility, soft encapsulation, using polydimethylsiloxane (PDMS) has been used. Making use of the "Flex-to-Rigid" (F2R) technique, cm-size graphene-on-PDMS structures have been suspended and characterized using Raman spectroscopy to qualitatively evaluate the graphene layer, together with 2-point measurements to ensure the conductivity of the structure. In parallel, flip-chip bonding processes of chips on graphene structures were employed and the 2-point electrical measurement results have shown resistance values in the range of kΩ for the combined tracks and ball-bonds.@en

In the emerging research field of bioelectronic medicine, it has been indicated that neuromodulation of the Vagus Nerve (VN) has the potential to treat various conditions such as epilepsy, depression, and autoimmune diseases. In order to reduce side effects, as well as to increase the effectiveness of the delivered therapy, sub-fascicle stimulation specificity is required. In the electrical domain, increasing spatial selectivity can only be achieved using invasive and potentially damaging approaches like compressive forces or nerve penetration. To avoid these invasive methods while obtaining a high spatial selectivity, a 2 mm diameter extraneural cuff-shaped proof-of-concept design with integrated Lead Zirconate Titanate (PZT) based ultrasound (US) transducers is proposed in this paper. For the development of the proposed concept, wafer-level microfabrication techniques are employed. Moreover, acoustic measurements are performed on the device, in order to characterize the ultrasonic beam profiles of the integrated PZT-based US transducers. A focal spot size of around 200 ?m by 200 ?m is measured for the proposed cuff. Moreover, the curvature of the device leads to constructive interference of the US waves originating from multiple PZT-based US transducers, which in turn leads to an increase of 45% in focal pressure compared to the focal pressure of a single PZT-based US transducer. Integrating PZT-based US transducers in an extraneural cuff-shaped design has the potential to achieve high-precision US neuromodulation of the Vagus Nerve without requiring intraneural implantation.@en

In the emerging research field of bioelectronic medicine, it has been indicated that neuromodulation of the Vagus Nerve (VN) has the potential to treat various conditions such as epilepsy, depression, and autoimmune diseases. In order to reduce side effects, as well as to increase the effectiveness of the delivered therapy, sub-fascicle stimulation specificity is required. In the electrical domain, increasing spatial selectivity can only be achieved using invasive and potentially damaging approaches like compressive forces or nerve penetration. To avoid these invasive methods while obtaining a high spatial selectivity, a 2 mm diameter extraneural cuff-shaped proof-of-concept design with integrated Lead Zirconate Titanate (PZT) based ultrasound (US) transducers is proposed in this paper. For the development of the proposed concept, wafer-level microfabrication techniques are employed. Moreover, acoustic measurements are performed on the device, in order to characterize the ultrasonic beam profiles of the integrated PZT-based US transducers. A focal spot size of around 200 ?m by 200 ?m is measured for the proposed cuff. Moreover, the curvature of the device leads to constructive interference of the US waves originating from multiple PZT-based US transducers, which in turn leads to an increase of 45% in focal pressure compared to the focal pressure of a single PZT-based US transducer. Integrating PZT-based US transducers in an extraneural cuff-shaped design has the potential to achieve high-precision US neuromodulation of the Vagus Nerve without requiring intraneural implantation.@en

Multimodal platforms combining electrical neural recording and stimulation, optogenetics, optical imaging, and magnetic resonance (MRI) imaging are emerging as a promising platform to enhance the depth of characterization in neuroscientific research. Electrically conductive, optically transparent, and MRI-compatible electrodes can optimally combine all modalities. Graphene as a suitable electrode candidate material can be grown via chemical vapor deposition (CVD) processes and sandwiched between transparent biocompatible polymers. However, due to the high graphene growth temperature (≥ 900 °C) and the presence of polymers, fabrication is commonly based on a manual transfer process of pre-grown graphene sheets, which causes reliability issues. In this paper, we present CVD-based multilayer graphene electrodes fabricated using a wafer-scale transfer-free process for use in optically transparent and MRI-compatible neural interfaces. Our fabricated electrodes feature very low impedances which are comparable to those of noble metal electrodes of the same size and geometry. They also exhibit the highest charge storage capacity (CSC) reported to date among all previously fabricated CVD graphene electrodes. Our graphene electrodes did not reveal any photo-induced artifact during 10-Hz light pulse illumination. Additionally, we show here, for the first time, that CVD graphene electrodes do not cause any image artifact in a 3T MRI scanner. These results demonstrate that multilayer graphene electrodes are excellent candidates for the next generation of neural interfaces and can substitute the standard conventional metal electrodes. Our fabricated graphene electrodes enable multimodal neural recording, electrical and optogenetic stimulation, while allowing for optical imaging, as well as, artifact-free MRI studies. [Figure not available: see fulltext.].@en

Patients affected by spinal cord injuries (SCI) are usually unable to perform trivial motor activities and thus, for therapeutic purposes, epidural spinal cord stimulation (ESCS) is currently used. Moreover, more exploratory research, using optogenetics, is being conducted in rodents for a better understanding of the mechanisms that occur while delivering specific therapies. However, the availability of tailored neurotechnologies for such experiments is limited. This work reports the development and characterization of flexible, active spinal cord implants with optogenetic compatibility1,2 (Fig.1). A scalable and reproducible microfabrication process has been developed, using graphene3, a transparent, flexible and conductive material, to form the electrodes and interconnects of the implant. Small and thin4 electronic chips were assembled via flip-chip bonding processes either on graphene or on metal-on-graphene layers. Soft, polymeric encapsulation was employed to sustain the high flexibility and transparency of the implant. The result is an active prototype consisting of a multi-layered graphene structure between two polymeric-based encapsulation layers, with thin chips integrated on the implant and test pads for interconnection to the outside world. Raman spectroscopy and optical transmittance were employed for the characterization of the graphene layer while cyclic voltammetry and electrochemical impedance spectroscopy were performed to benchmark the electrical properties of the device. The assembly process of the chips was evaluated using four-point electrical measurements. In this work, the first transparent, graphene-based active implants have been developed (Fig. 2 and Fig. 3). The prototypes were extensively characterized and the results showed a transparency of ~80 % as well as no deterioration over time when soaked in saline solution or when bent under various angles. The graphene electrodes showed an impedance of ~8 kΩ at 1 kHz frequencies and the resistance after the bonding process ranged from 10 mΩ up to 16 Ω for individual connections, depending on the substrate used @en

The most important symptoms of spinal cord injuries (SCIs) are partial or complete loss of sensory and/or motor functions caused by the disruption of the neural pathway between the brain and the extremities of the body. Recent studies have shown that epidural spinal cord stimulation (ESCS) can promote locomotor recovery in patients affected by SCIs, thus becoming one of the most promising means of treatment for the lesion. Devices currently available on the market, consist of active components, enclosed in a hard case and connected via leads to the electrodes that form the interface between the stimulator and the biological tissue. The presence of leads along the spine, may be an important cause of failure for the device. Moreover, the overall stiffness of the stimulator does not resemble best the anatomical structure of the human body. Flexibility and optical monitoring of the biological tissue during implantation and stimulation are very important aspects and both can be improved with a proper choice of materials. The goal of this work is to develop a compact, active, transparent and flexible spinal cord stimulator that could be implanted at the site of stimulation. To provide high flexibility, soft encapsulation, using polydimethylsiloxane (PDMS) has been used. To ensure transparency but also mechanical stability of the electrodes and tracks, graphene has been chosen as a replacement for the conventional metals. Integrating active components, in the form of application specific integrated circuits (ASICs), on a graphenebased substrate, constitutes the biggest challenge. To this end, flip chip bonding techniques using a metal layer as an interface between graphene and the chip’s stud-bumped pads, are being investigated. Preliminary measurements after bonding have shown resistance values in the range of kΩ, thus taking the project one step closer to achieving the desired goal. @en

In an attempt to reduce the side effects caused by the chemically-based drugs used to treat neurological disorders, the field of bioelectronics has been focusing on the development of smart and reliable solutions that could, ideally, interact with the tissue at a resolution of individual cells.1 Conventionally, electrically-based systems have been used.2 However, increasing the resolution at which they interact with the body leads to the development of invasive electrode arrays, which can cause long-term side effects.3 Another approach, based on acoustic waves, has recently emerged. Ultrasound (US) neuromodulation has been proven to be effective in modulating the response of peripheral nerves, in an in-vivo setup4 and has the potential to achieve higher spatial selectivity.5 In this work, we aim to fabricate an implantable cuff for US neuromodulation, which would employ an array of US transducers to deliver focused US to specific nerve areas in a non-invasive manner. To this end, the potential of different US transducer arrays for peripheral nerve applications is evaluated, assessing the acoustic performances as well as ease of assembly and integration. More specifically, two of the most important parameters that affect neural excitation are the frequency and output pressure generated by the US transducers.4 Conventional bulk PZT transducers can generate a wide range of output pressures but these are not small enough for this application. PZT-based arrays, integrated on CMOS have recently emerged, and will be part of this evaluation6. However, these have not yet been integrated on flexible substrates. On the other hand, micromachined US transducers (MUTs) have been gaining a lot of interest, particularly capacitive MUTs (CMUTs) which can operate at high frequencies, thus reducing the focal point significantly.5 CMUTs can be fabricated on flexible substrates, using biocompatible materials, rendering them a very attractive candidate for the envisioned cuff. However, CMUTs usually feature lower Q factors compared to PZTs, hence the output pressure still has to be evaluated for neuromodulation. In addition, this work will also discuss important characteristics of the materials used for encapsulation, as these should ensure the required flexibility of the cuff without negatively affecting the acoustic performance of the transducers.@en

Ultrasound (US)-based neuromodulation has recently emerged as a spatially selective yet non-invasive alternative to conventional electrically-based neural interfaces. However, the fundamental mechanisms of US neuromodulation are not yet clarified. Thus, there is a need for in-vitro bimodal investigation tools that allow us to compare the effect of US versus electrically-induced neural activity in the vicinity of the transducing element. To this end, we propose a MicroElectrode-MicroTransducer Array (MEMTA), where a dense array of electrodes is co-fabricated on top of a similarly dense array of US transducers.In this paper, we test the proof of concept for such co-fabrication using a non-monolithic approach, where, at its most challenging scenario, desired topologies require electrodes to be formed directly on top of fragile piezoelectric micromachined ultrasound transducer (PMUTs) membranes. On top of the PMUTs, a thin-film microelectrode array was developed utilizing microfabrication processes, including metal sputtering, lithography, etching and soft encapsulation. The samples were analysed through focused ion beam–scanning electron microscopy (FIB-SEM), and the results have shown that damage to the membranes does not occur during any of the process steps. This paper proves that the non-monolithic development of a miniaturised bimodal neuroscientific investigation tool can be achieved, thus, opening up a series of possibilities for further understanding and investigation of the nervous system.@en

Ultrasound (US)-based neuromodulation has recently emerged as a spatially selective yet non-invasive alternative to conventional electrically-based neural interfaces. However, the fundamental mechanisms of US neuromodulation are not yet clarified. Thus, there is a need for in-vitro bimodal investigation tools that allow us to compare the effect of US versus electrically-induced neural activity in the vicinity of the transducing element. To this end, we propose a MicroElectrode-MicroTransducer Array (MEMTA), where a dense array of electrodes is co-fabricated on top of a similarly dense array of US transducers.In this paper, we test the proof of concept for such co-fabrication using a non-monolithic approach, where, at its most challenging scenario, desired topologies require electrodes to be formed directly on top of fragile piezoelectric micromachined ultrasound transducer (PMUTs) membranes. On top of the PMUTs, a thin-film microelectrode array was developed utilizing microfabrication processes, including metal sputtering, lithography, etching and soft encapsulation. The samples were analysed through focused ion beam–scanning electron microscopy (FIB-SEM), and the results have shown that damage to the membranes does not occur during any of the process steps. This paper proves that the non-monolithic development of a miniaturised bimodal neuroscientific investigation tool can be achieved, thus, opening up a series of possibilities for further understanding and investigation of the nervous system.@en