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Neuronal NanoCarbon Interfacing Structures

Final Report Summary - NEUROCARE (Neuronal NanoCarbon Interfacing Structures)

Executive Summary:
Medical implants can repair the nervous system following an accident or disease, notably to correct the loss or impairment of eyesight (through retinal degeneration) or hearing (through damaged cochlea). Traumatic spinal injuries, drug-resistant epilepsies, psychiatric disorders and chronic neurodegenerative pathologies can also be treated (in the cortex) with such reconstructive approaches. NeuroCare aims to create better retinal, cortical and cochlear implantable devices through the use of improved interfacing between the electronic implants and living cells. The roadmap for market deployment of these implants predicts their market penetration within 10 years following clinical trials.

The NeuroCare concept involves high-quality, low-cost, carbon-based materials. These materials are well-adapted for use in medical implants, because they (i) offer a wide range of electronic properties (metal, semiconductor and insulator), (ii) are bio-inert and (iii) are physically robust. Nanocrystalline diamond (NCD) thin films and graphene layers were studied as novel biocompatible interfacing materials. These carbon implants considerably reduced the formation of glial scar tissues and enabled long lasting operation with reduced biofouling.

The Neurocare project has led to a tremendous number of breakthroughs with respect to the adaptation of carbon technologies towards their use as biointerfacing devices. For example, flexible graphene microelectrode arrays were fabricated and their biocompatibility assessed in-vivo. This sole development has gone beyond the hope of the European graphene flagship, and was recently included as a novel workpackage to this very ambitious project, and conducted by the Neurocare partners of the activity. Similarly, diamond coatings were made available towards applications for microelectrodes arrays for electrophysiology, cochlear implantable electrodes for the stimulation of the cochlea, for cuff electrodes for the stimulation of peripheral nerves, for cortical electrodes for epi-cortical recording and stimulating brain research, as well as for two distinctive forms of retinal implants, namely rigid epiretinal or soft subretinal ones. Although at the beginning of Neurocare, the project tended to demonstrate that diamond, although offering remarkable biocompatibility was not providing performances beyond its competitor in terms of electrochemical performances, novel concepts of nanostructured 3D materials were proposed, enabling to promote by typically two orders of magnitude the performances of the material, opening the route to carbon materials for implant materials.

Project Context and Objectives:
Living cells generate an imbalance of charged molecules and ions between their interiors and the external environment. This results in a potential difference across the cell membrane. In certain cell types, such as neurons and muscles, the cells have harnessed these electrical properties to process information or do work. However, these properties also mean that the living cell can be thought of as an electronic element. Numerous devices have been developed to connect living tissue to man-made electronic systems. Among the most prominent of these are heart-pacemakers, cochlear implants, and devices for deep brain stimulation. These initial implants for electronically interfacing with cells utilize millimetre sized electrodes to stimulate or record from large regions of tissue in their target organs. Within the Neurocare project, one WP (WP4) aimed to evaluate the range of cell signal detection and action potential (AP) stimulation using much smaller extracellular devices. These devices, such as field effect transistor (FET) arrays and micro-electrode arrays (MEAs) are manufactured so that each element in the array is similar in size to a single cell. These devices non-invasively detect the activity of neurons and muscle cells to allow continuous monitoring of various bodily systems. Furthermore, applying an electric pulse to an MEA electrode can be used to direct activity of the cell network. An increase in the resolution of these devices is desirable to improve the specificity with which the implant can interact with the network of cells in a tissue. However, there is usually a trade-off between resolution and effectivity of the devices. As these devices are staged to form the basis of future active implants in the body to restore diseased or damaged neural function, such as hearing, sight, or misfiring brain pathways, their resolution and the interpretation of the signals recorded is essential. As such, within the Neurocare project, we aimed at implementing optimised forms of these MEAs in terms of size, shape, flexibility or pixel sizes to fabricate implants aiming at cochlear electrode arrays for the stimulation of the cochlea, cuff electrodes for the stimulation of peripheral nerves, cortical electrodes for epi-cortical recording and stimulating brain research, as well as for two distinctive forms of retinal implants, namely rigid epiretinal or soft subretinal ones.

The project was constructed around 5 major technical WPs. In WP2, NeuroCare aimed to take benefit of the newly developed materials of the project to create better retinal, cortical and cochlear implantable devices through the use of improved interfacing between the electronic implants and living cells. Then WP3 concentrated on the technological developments to implement for the fabrication of all devices, namely rigid MEA substrates for cell and tissue recordings, as detailed in WP4, and soft or implantable devices for tissue stimulation in vivo, in WP5. All the dissemination and impact are the concern of WP6.

The idea of WP2 aimed to start from experienced state of the art devices in different existing applications (retinal, cochlear, peripheral nerve stimulation, cortical recording) provided by their “product owner” and try to find from road map up to physical concretization the way to demonstrate major functional enhancements that could be provided by introduction of NCD coating. On the other hand, while technical feasibility and clinical exploration of new functional performances are the main scientific objectives of this task, a closed monitoring of both economic and industrial aspects are maintained all along the project. Transferring such a new technology in industrial process remains the principal target of the Neurocare project. The objectives of WP2 were then to define rigid MEA specifications for in vitro tests and implantable MEA specifications for in vivo applications, and to provide the consortium with a roadmap to be shared with all partners to ensure that the results of all WPs lead to implantable devices meeting real-world needs and objectives.
The answers to WP2 specifications were the objectives of WP3, where NCD and graphene had to be implemented in similar approaches as current versions of these devices when made on metals like gold, platinum, or black platinum that utilize process technology from the electronics industry, to allow the microfabrication of todays state-of-the-art MEAs. FET sensors are similarly implemented using semiconductor technology in silicon materials. Within WP3, Various techniques were implemented according to the types of substrates used, namely cochlear implantable electrodes consisted of 3D volumic cylinders, whether cortical electrodes were metal chips, cuff electrodes metallic rods, and retinal implants were either solid substrates or flexible foils, according to the location where they aimed at stimulating the retina, namely subcortical or epiretinal, respectively.

The WP3 thus aimed at investigating how carbon materials may further improve these devices. From the outset, the goal of the project was to benchmark boron doped nanocrystalline diamond (BNCD) as a MEA material, and graphene as a FET gate material. We expected BNCD MEAs to be superior to metal MEAs because less glial scarring is observed on BNCD materials than on metal devices. We further expected graphene to act as a superior FET gate for two reasons. First, because it is a flexible material, GFET arrays could be made in a way that conforms to tissue convolutions to improve cell-device contact. Second, since graphene is a two dimensional material, the surface effects of cell behaviour on the gate are not significantly diluted by bulk effects in the material. The device developments from WP3 also aimed to expand the design of devices from two dimensional structures to three dimensional structures using nanoscale 3D electrodes. Nano- and micro- structuring of the device allowed us to increase the effectivity of stimulation and decrease the level of noise without compromising the lateral resolution of the detection or stimulation area. This was achieved successfully as will be described below.

All data concerning properties and functionality of implantable devices were assessed within WP4 and WP5, which targeted the consolidation of the observed performances and their assessment by end-users. Medical teams and electrophysiologists had to determine to what extent their needs and specifications were met. They aimed at evaluating the degree of improvement of devices and results. Finally, the end-users had to evaluate which device is the best compromise in terms of material, design, functionality, cost, etc.

For this, devices were evaluated in laboratory animals for example towards retinal stimulation to demonstrate their robustness. These implants aimed at considerably reducing the formation of glial scar tissues, enabled stimulation currents to be raised by more than one order of magnitude before causing visible chemical alteration, and enabled long lasting operation with reduced biofouling. The project also aimed at evaluating long term use of these implants. For example, rats where implanted for 8 weeks with retinal implants, and mini pigs for 3 months with cortical implants. This will be detailed in WP5 below.

Project Results:
WorkPackage 2: Retinal, Cochlear, Cortical and Peripheral nerve Implantable devices – Specifications and Assessment

Within this WP, the tasks were split according to the different applications: Retinal, Cochlear, Cortical and Peripheral nerve Implantable devices. The expectations and results are so differentiated according to applications but also to initial existing “state of the art” of the application.
Retinal implants were considered as the application where the most significant breakthroughs were to be gained. This topic was be highly prioritised in the project. The cochlear implant application is a field already well-established in our modern societies and it was very challenging for NeuroCare implants to pretend to become a new standard. As such the cochlear implant application has remained in the project a practical evaluation of the benefits our implants may offer. If the benefits would reveal high, then industrial partners of NeuroCare may have to push this technology to a future market.
For the cuff electrodes dedicated to peripheral nerves stimulation, major increase of the recruitment performances should be demonstrated. Other points, like robustness of the coating during manufacturing process, biocompatibility/invasiveness of the coating while implanting and implanted will decide the future of the envisaged process including diamond/graphene coating. Economic and cost assessments will be also conducted.
Finally cortical implants are seen as very novel. To date they are only used in animals or in laboratory experiments, and are very far from becoming a standard for medical applications. Years of medical research will still be required after the end of NeuroCare before such implants become clinical devices. Within NeuroCare, the cortical implant research will thus focus on evaluating the benefit of our technology and how it may be beneficial in terms of stability and signal to noise.

Retinal implants
NR aimed to lower the power for retinal stimulation and gaining better neuron-device interaction; the NCD coating to be developed had to provide a localized spatial response with biocompatible properties. The development of the diamond coating for the NR 3D MEAs was extremely challenging as structured electrodes had to be efficiently coated at the end of their tips. The processing route implemented will be presented with WP3. NR obtained 3D electrodes MEAs uniformly coated with respect to the 3D MEAs structure and with a good adhesion
The test achievements of this project included electrode threshold measurement of electrical activation of retina neurons was established in different stimulation patterns. Calcium imaging was established with electrical stimulation of dissociated chick retina. This imaging demonstrated local activation along with transferring the stimulation toward the optic nerve. These parameters were fully correlated to the threshold level measurements. The technical challenges of 3D NCD coated electrode that created significant delays in the project required cancelation of the initially agreed testing method. In agreement with the consortium, NR performed electrochemistry tests to the NCD NR 3D MEAs in order to demonstrate its electrochemical properties. However, the prototypes used were test samples where all electrodes provided by NR were shortcircuited together. It was thus impossible to characterise each individual electrode performance. Nevertheless, test results showed capacitive behavior within narrow potential boundaries, with consistent behavior. Strong faradic currents are present beyond this narrow potential range (typ. ±0.5V). The average capacity found was ~900nF.
In conclusion, those developments were very successful towards NCD coated NR 3D MEAs with respect to NR requirements. Grinding stability and surface coverage of the NCD coating appeared feasible, and the uniformity of the NCD coating with respect to NR 3D electrodes structure was remarkable.

Cochlear implants
For the cochlear implant, major reduction of liminar thresholds was to be demonstrated. Other points, like robustness of the coating during manufacturing process, biocompatibility -invasiveness of the coating while implanting and implanted could decide the future of the envisaged process including diamond/graphene coating.
While coating of cochlear arrays was successfully obtained, cochlear in vivo tests were withdrawn from the project, following modification of MXM corporate structures after the sale of its Neurelec subsidiary.
However processes up to coating of cochlear electrode arrays were achieved and briefly summarized.
• NCD was grown on cochlear implant contacts and it appears that, thanks to MXM proprietary EDM manufacturing processes, the initial roughness of the material led to avoid interlayer (Tungsten…) before NCD coating. Where material does not exhibit such property, delamination occurs.
• MXM production team did not report special difficulty in the process of integration, which is excellent news for an industrial use of this kind of diamond, except some handling scratches in some points.
• The behaviour of these probes was tested test in saline buffer (PBS). It appears in the EIS/CV of representative contacts that the impedance on the diamond coated electrodes is higher than on bare PtIr contacts due to a smaller active surface which is disadvantageous.
• It finally appears that the NCD deposition process is responsible for this smoothening of the surface so that the surfaces originally obtained in EDM manufacturing were greater than those covered, and so “integrated”, by the NCD coating.
As such the cochlear implant application will remain a practical evaluation of the benefits our implants may offer. If the benefits would reveal high, then industrial partners of NeuroCare will have to push this technology to a future market.

Cuff electrodes
MXM’s efforts were then turned to produce other demonstrator vehicles like cuff electrodes in which MXM is also specialized in. Adaptation of existing design for in vivo trials on rat sciatic nerves of NCD coated and non-coated electrode has been undertaken. Parts including NCD coated contacts and non-coated electrode were produced (D2.3 D3.8). These demonstrators were then transferred to other partners for further tests and preclinical evaluation in WP5. The cuff electrodes were characterized by means of impedance spectroscopy and cyclic voltammetry (EIS/CV). On some parts, the diamond electrode has a lower impedance than the platinum electrodes and the cut-off frequency is either lower. The reproducibility of the integration is limited; the active area is not defined accurately on account of the mesh and the adhesive rubber.
Based on the biocompatibility tests performed by University of Mainz for diamond in cortical contact in D5.1 there was no further investigation as the results appeared very convincing (see WP5 description or the second mid-term report). For these acute tests, there was no problem, nor issue of toxicity.
In terms of stimulation performances, the study is also reported with WP5. In brief, all the animals recovered after surgery within a night; the electrodes and the connector were adjusted to meet to the surgery constraints.
Concerning the stimulation performances, the cuff electrodes were used to trigger leg contraction. The experimental set-up is able to check the efficacy of the stimulation but the major conclusions from such acute experiment is that diamond can match platinum performances.

Cortical implants
Within NeuroCare, the team has assessed the benefit of the boron doped diamond technology compared to platinum iridium disk. How it may be beneficial in terms of stability and signal to noise. The expectations in terms of enhancement provided by NCD coating were biocompatibility, long term stability, and electrical charge transfer improvement leading to possible reduction of active surface, increasing number of addressable poles, selectivity. The diamond coating appears to be sufficiently robust to comply with surgery and in-vivo experiments In terms of biocompatibility, the results confirmed the high biocompatibility of diamond. Long term stability was only assessed during three weeks. We could not assess long term stability due to several connector issues.
To ensure the adhesion of the diamond coating we had to increase the roughness of the electrodes. Following the experiments in cochlear contacts showing excellent results in terms of adherence, the consortium decided to switch to flat disks diced with a specific electroerosion process (EDM) which increased drastically the roughness and the diamond coating adhesion. Process parameters implies high temperature not suitable with polymers, the need of mask for the catalyst deposition is also an issue because this generates a constraint of alignment. In conclusion, in terms of impedance for the cortical electrodes, the introduction of the nanostructuration improves the behavior of the electrodes. Concerning the Cathodic charge storage, the values are comparable when considering the whole electrochemical window. Taken all these parameters into account, we conclude that the project managed from nano structuration to improves the electrodes behavior by two orders of magnitude to match those of the best performance black Pt or PtIr electrodes. The main advantage for electrical stimulation of diamond remains therefore its biocompatibility and large electrochemical windows.

Work package 3: Technology developments: from MEAs to implantable prototypes

This WP aims at the technological development of novel prototypes for the improvement of the performance of neural interfaces for the various applications targeted within the project. The WP achievements will be the development of new technologies for the fabrication of diamond based microelectrode arrays and implants, as well as of arrays of graphene FETs for a new generation of biocompatible, low noise, and highly sensitive carbon devices on flexible substrates for in-vivo cell interfacing.

Diamond Microelectrode arrays
Several specific designs for novel MEA developments have been proposed to the MEAs end-users of the project (UPMC, Juelich and CNRS). Two end users are using a standard MCS recording and stimulation system while Juelich has developed its own system. Specific designs were implemented according to the needs of the medical partners of the project; they consisted of arrays of 8x8 electrodes, or of linearly aligned electrodes, or also of varying size and configuration arrays. A specific process to allow selective growth on 4 inch substrates was assembled (Figure 1).
To optimise the performances of electrodes and particularly with respect to planar ones, a new approach has been developed to reduce noise level and increase the capacitance. This approach consists of achieving a textured surface of diamond in order to increase the active electrochemical surface of the electrodes while their intrinsic diameter remains constant. All technological partners contributed to improve the feasibility of these tasks. The structure was obtained using a vertically aligned CNT forest as a template layer, on which boron doped diamond was grown. The surface of the CNT is at the end of the process completely buried by diamond to avoid any possible contact between tissues and CNTs. To implement this technology on the MEA, a new process has been developed, starting from a substrate on which a thin layer of boron doped diamond is prepared. With the pattern of the required electrode design, a thin layer of nickel was prepared at the future location of electrodes, and then treated thermally in order to create small drops of nickel at the surface. The nickel drops were used as a catalyst for the growth of the CNT.
After CNT growth, the CNTs were then covered by a layer of diamond nanoparticles using a conformal technique that enabled the growth of diamond uniformly over the 3D structured surface of the vertically aligned CNTs. Photolithography was then used to define the electrodes and tracks in diamond. To reduce the resistance of the tracks over long distances a metal is used and particularly around the electrodes. After those steps, a passivation layer is used over the entire device surface, and then openings are prepared facing the 3D diamond electrodes for their contact with the tissues. The Figure 2 below summarizes the process.

In parallel, all fabricated MEA substrates were mounted by QWANE in order to be used for in-vitro assessments with conventional biology equipment. QWANE assembled several batched received mostly from ESIEE of MEA devices for local stimulation. These new MEAs incorporate a counter electrode around all 60 recording electrodes. These MEAs have been assembled onto a printed circuit board, sealed and a glass ring defining the culture chamber has been added, as illustrated in the Figure 3 below.
The finished MEAs have been shipped to medical partners for testing purposes. Also further reference MEAs with platinum and platinum black electrodes, as designed at beginning of the project have been manufactured and shipped at same time to INSERM as reference samples.

Graphene FET arrays
In parallel, The TUM team has designed 8x8 pixel arrays of graphene field effect transistors. Large area graphene layers prepared on Cu foils were transferred to sapphire substrates pre-patterned with TiAu metal layers as drain and source electrodes. Oxygen-plasma processing was used to define the active area of the 8x8 (64) transistors. Finally, SU8 resist was patterned to prevent the contact between the metal layers and the electrolyte. Figure 4 below shows an optical micrograph of the 8x8 transistor array before the SU8 layer, and a graph with the in-electrolyte characterization of the solution-gated field effect transistors.
In a joint task carried out by the ESIEE and TUM teams, a first generation of devices with an implant design has already been produced. The fabrication yield for this particular sample is already relatively high, although it has to be further improved. Figure 5 (left) shows tens of implants during the fabrication on a 4-inch wafer; the right photo in Figure 5 shows a picture of a single device after the release process.

Transistor curves of the devices with the implant design are shown in Figure 6, revealing the expected “V-type” shape. However, a fast saturation of the drain-source current afar from the Dirac point can be observed. This is attributed to a large access resistance, caused by e.g. the larger access areas (region between source and drain contacts and the graphene as shown in the insert of Figure 6 that is not in contact with the electrolyte. The fabrication process has later been optimized to lower the access resistance by decreasing the access areas, which is expected to improve the device performance in terms of transconductance.

Cochlear and cortical implants

Several strategies have been studied in order to coat homogenously cochlear and cortical implants with nanocrystalline boron doped diamond. Initially, the investigations have been conducted on planar PtIr disks as reference, provided by MXM, before moving on so-called PtIr “bowl hats”’ also provided by MXM.
In all cases, the diamond growth is performed at low temperatures (<550°C), through a specific CVD process previously developed at CEA-LIST, ensuring a high boron doping level in the layer ([B]~1021 atoms.cm-3). The challenge lies in the adhesion, the homogeneity and the crystalline quality of the deposited diamond layer, which mostly depends on the quality of the interface between the substrate and the coating. Therefore, different nucleation techniques and interlayers have been tested, as well as pre-treatments on the substrates (Figure 7).
The characterisation of fully assembled cochlear implant prototypes validated the approach. Efforts on these developments were however stopped with that of our partner activities on cochlear research. The result was a shift of research activities towards the successful development of cuff electrodes aiming at the stimulation of peripheral nerves (Figure 8).
A similar approach was initiated to fabricate cortical implants, based on over-growth of diamond on PtIr chips (Figure 9).
In order to benefit from the more recent developments where 3D nanostructured electrodes exhibit a much higher capacitance, with expected improved performances for stimulation, it was planned to add those developments to PtIr chips for cortical implants. In this respect, a process was optimised between CEA-List and CEA-Clinatec where (i) after PtIr surface preparation, (ii) a first NCD diamond layer is deposited on the chip surface, to allow (iii) Ni seeds to be prepared to enable SW-carbon nanotubes growth, then (iv) nanodiamond seeding and (v) diamond growth was performed on MWCNT bundles as described earlier. The technology developed demonstrated the feasibility of this approach for cortical implant preparation. Prototype series of chips were prepared for CEA-Clinatec to enable the preparation of those novel 3D diamond cortical implants (Figure 10).
The nanotubes have a good density and remain relatively vertical, in the end due to Van der Waals forces as shown on Figure 11 (left) one can identify the formation of CNT bundles, “teepees” like. Figure 11 (right) also displays the 3D nanostructured diamond coating on the PtIr chips. The frontier between diamond coating and the 3D nanostructured diamond coating can be easily distinguished.
This work led to the first nanostructured 3D diamond based cortical implants. They were implanted in minipigs for several weeks as will be detailed in WP5 (Figure 12).

Retinal rigid implants for epi-retinal stimulation

Another approach for retinal implants aimed at fully processed solid state devices, where arrays of tips are used as epi-retinal implants. A new technological route developed enabled NCD diamond growth at low temperatures, solely on the extremity of the electrode tips. Figure 13 display such a structure, where the white top end of tips reveals the high diamond conductivity. The technology to fabricate those devices was particularly challenging. It consisted of applying a selective coating of diamond solely at the end of tips.


Retinal soft implants for subretinal stimulation
Neurocare aimed at soft implants design for subretinal stimulation. The process flow here was adapting the MEA process flow on flexible polyimide, using a liftoff process. The process was well documented in the mid-term reports. Those implants were provided to UPMC for in vivo implantation in rats over periods of several weeks while electrochemical measurements were regularly performed as will be presented in WP5.

WorkPackage 4 : Cell-device interfacing in vitro: bidirectional recording and stimulation

Micro-Electrode array performances

The first task in evaluating neural stimulation with MEAs was to test the spatial extent of stimulation in vitro. Networks of dissociated cortical neurons, retina explants, and hindbrain-spinal cord explants were tested in tissue specific setups to determine the extent of cell activation in response to chip based stimulation using state-of-the-art metal MEAs. Custom chips produced with tissue specific layouts were provided by one of the company beneficiaries. These MEAs all utilized platinum electrodes to establish the basic conditions for chip-based stimulation in each model system and to define our understanding of the extent to which an electrode can stimulate an area of the cell network. In the case of hindbrain-spinal cord explants and dissociated cortical neurons, single cells within the network were recorded one at a time with the well-established method of whole cell patch clamp recordings. This method does not allow long term recordings because it punctures the cell in order to record the intracellular potential. However, because the patch clamp electrode is completely separate from the chip, the response of the cell can be continuously recorded, even when the nearest MEA electrode is being used to apply a stimulation pulse. Electrodes at varying distances to the cell were then used to apply a stimulation pulse. The patch clamp recording was used to evaluate if the electrode stimulation was successful in triggering an AP at that distance. Different electrode sizes were also tested to determine what the smallest electrode capable of injecting enough current to trigger an AP is. Two important findings were made. First, the current injection threshold necessary to trigger an AP does not increase with distance as quickly as previously thought. Measurements on hindbrain-spinal cord explants were able to trigger APs farther away and with lower currents than predicted, as seen in Figure 14.

Secondly, the response of dissociated cortical neurons suggests that when a voltage controlled stimulation pulse is applied to a very small electrode, the high impedance of that electrode can cause the signal to be coupled to nearby electrodes whose connection lines lie close to those of the stimulation electrode for long distances over the chip. This was observed when cells very close to these ‘neighbor’ electrodes could be stimulated, despite the small electrode’s failure to trigger APs in cells directly next to itself, see Figure 15.

The stimulation range in retina was measured by an optical method that allowed evaluation of large areas at once. A setup was established where a fluorescent calcium indicator, which gets brighter when the cell fires an AP, was used to monitor a track of retinal neurons while stimulation was applied via an electrode under the retina. Using this setup it could be seen that the AP propagated along the neuron track, but higher current stimulation pulses triggered APs in neurons of the track farther from the electrode, see Figure 16.

In all cases, the stimulation of these different neuronal cell types showed that even when using small electrodes, large regions of tissue may be activated. Special design considerations are thus necessary to target regions of the cell network that participate in similar parts of the information processing task, and to reduce the effect of on-chip crosstalk between electrodes. One way to focus the stimulation field was shown to be the use of a surrounding ground material. A further approach is to introduce guard-lines of conductive material to prevent coupling of one electrode to another. Increasing the surface area of the electrode should improve its ability to transfer charge to the cell and stimulate an AP. However, expanding in the lateral dimension would reduce the resolution of stimulation.

The Consortium therefore pursued the use of three dimensional nano-structured diamond electrodes. The development and production of the three dimensional boron doped diamond (3D-BDD) material as an electrode is described in WP3. The improvement in impedance and reduction in noise can be seen in Figure 17. In terms of noise reduction, the 3D-BDD outperforms not only planar diamond electrodes, but also planar platinum electrodes, which are the standard material for these devices. The 3D-BDD noise level is on par with that of platinum black, an extremely high performing material that suffers from low stability in most bioelectronics applications. The structure and how neurons interact with 3D-BDD is further discussed in section WP4.3. Spinal cord explants could be stimulated to fire bursts of APs using 3D-BDD electrodes with only a 20 µm diameter lateral dimension on chip. The coated devices were tested electrochemically and indicated that once handling difficulties are overcome, BNCD coatings could allow a larger safe stimulation window on these devices.

Values reaching a reduction of the impedance of electrodes by two orders of magnitudes were obtained, as visualised in figure XX. It leads to the reduction of cell recording noise by about the same range, a very significant improvement for diamond that could here therefore match its best competitors, while improving the biocompatibility

The Neurocare project enabled to characterize how embryonic mouse hindbrains as well as neuron cells interact with these three dimensional structures. The quality and shape of recorded signals were compared to traditional flat devices. The mode of coupling between the device and cell were investigated and determined how existing models of the cell-device interface are appropriate for the new three dimensional system. Furthermore, the range of stimulation for these three dimensional structures was evaluated. These results were described in details in the second mid-term report.

Once the devices and cell-device interfaces were characterized, we aimed to use recordings from small to moderate sized neural networks to gain an increased understanding of how biological systems process information. We then developed very novel research aiming at the probing of controlled networks on these devices that will allow research into basic neuroscience and pharmaceutical developments.

Graphene FET array performances

To make such devices flexible, graphene FET arrays appeared as a preferred approach for the Neurocare partners, and TUM in collaboration with ESIEE developed the technique enabling such devices to be available for medical experiments. A very innovative approach has been proposed to enable the fabrication of soft flexible graphene FET arrays. The technology developed has given rise to intellectual property application. Novel devices including soft implants were realised, and provided to electrophysiologists and biologists partner teams for in-vitro as well as in-vivo evaluation. Medical recording with those devices however has demonstrated the necessity to locate the ability to preamplify the signal in the near proximity of the implant. This has implied discussions between several partners in order to propose an easy solution (Figure 18).

Implants

In parallel to these developments with respect to the development of rigid microelectrode arrays, the use of diamond and graphene was adapted to the needs of implantable devices, including cochlear, cuff, and cortical electrodes enabling the measurement of in-vivo action potentials.

Finally, one of the most convincing use of carbon materials remained the development of retina implants.

An additional concern is the safe charge injection capabilities of the electrode. Using a pH sensitive fluorescent dye, we were able to show the safe charge injection limits of diamond electrodes. This safe window of operation is determined by the point at which water breaks down at the surface of the electrode due to the applied potential. This is of particular concern in systems when the current applied is controlled using whatever voltage is necessary to achieve it. We have shown that currents sufficient for stimulating neurons with diamond electrodes can be safely generated. This further validates the use of diamond electrodes in bioelectronic applications due to the material’s larger safe water window.

Recording neuronal activity with carbon based devices.
APs may recorded with two fundamentally different types of extracellular sensors; microelectrode arrays (MEAs) and solution gated field effect transistors (FETs). MEA measurements rely on a coupling of changes in the cell potential to changes in potential at the surface of the electrode. In contrast, FETs use interactions between the cell and the FET gate to change the current flowing from source to drain through the gate.
Current state of the art FET sensors for APs use silicon based technology. However, we investigated the use of FETs that use graphene as the gate material (GFETs). As a two dimensional material, graphene’s conductivity is expected to be more severely influenced by events at its surface. The GFETs were shown not only to detect changes in intracellular potential, but also to exhibit a signal component that is dependent on the ion fluxes into and out of the cell during depolarization. A model was developed to correctly predict this combined voltage and ion sensitivity signal, which allows a back-calculation of underlying ion channel events in the cell from extracellular electrical recordings (see Figure 19). We also cultured cortical neurons on the GFET arrays and were able to detect spikes consistent with spontaneous activity of the neural network.

The alternative method of AP detection, using MEAs, normally utilizes microelectrodes of gold, platinum, or platinum black – with platinum black yielding the best sensitivity but the lowest stability. In the course of the project, one of the company beneficiaries developed the fabrication of MEAs with PEDOT:PSS coatings. These electrodes showed a 1kHz impedance module of 25 kOhm and charge storage capacity of -3.5 mC/cm2. The beneficiary is pursuing this as a product development in addition to the technologies originally intended in the project. 3D-BDD MEA electrodes were shown to have similar characteristics to this new state of the art material. 3D-BDD electrodes showed more variability, most likely due to their complex structure. However, 75% of electrodes exhibit an impedance module less than 100 kOhm, with values typically about 50 kOhm. In physiological buffer solution the charge storage capacity of 3D-BDD electrodes ws 6.8 mC/cm2.
The 20 µm diameter 3D-BDD MEA electrodes were successfully used to detect APs in the spinal cord. Spikes of approximately 75 µV were recorded with characteristic fast onset and slower recovery, reflecting the time-course of an action potential in a single cell in the network. 3D-BDD MEAs were able to record low frequency spontaneous activity in the spinal cord, as well as high frequency AP bursts triggered by electrical stimulation.

The cell-device interface.
The 3D-BDD material was tested for its compatibility with neuronal cultures in vitro. Hipocampal, cortical, and spinal neurons were shown to grow as effectively on 3D-BDD as on planar BNCD material. The metabolic output of neurons grown on 3D-BDD was also tested and found to be normal. Closer investigation of the 3D-BDD material indicated that cells avoided areas of the substrate that were scratched or displayed broken nanostructures. Cell bodies preferred to rest on top of the nanostructure tips. Using a focused ion beam, the cell grown on the 3D-BDD could be cut and viewed in cross-section (see Figure 20). This showed that while the cell bodies do not engulf the nanostructures, the neurites can traverse the spaces between the structures. An analysis of the fraction of the cell membrane in close contact with the nanostructures suggests that a similar fraction of cell is tightly adhered to the substrate as in flat substrates, such as protein coated glass. Different aspect ratios of the nanostructures did not affect cell viability or cell wrapping of the 3D-BDD. However, further investigation is necessary to quantify if the fraction of membrane contacting the structure is different when different nanostructure spacing is employed.

Network-level activity recorded with carbon based devices.
In order to evaluate the information processing in networks of neurons on chip, it is desirable to control the position and connectivity of neuron growth. One way to achieve this is the use of micro-fluidic channels to guide neurite outgrowth and the connectivity between sub-populations of cells. In order to produce durable and re-usable chips with such guiding structures, QWANE developed a technique to pattern channels into the SU-8 epoxy that also serves as a chip’s passivation layer. These devices allowed the separation of two populations of neurons with electrodes under each population. Specially designed electrodes were then aligned under the microchannels that linked the chambers, where only axons could pass from one population to the other. This system allows investigation of the intrinsic cell signals that pattern connectivity in the brain, as well as testing the effect of disease factors or drugs on the downstream targets of the network – elucidating knock-on effects to damage in one part of the neural network. 3D-BDD devices were able to record bursting activity in neurons of the spinal cord downstream of those stimulated by 3D-BDD electrodes on the same chip. In addition to stimulating activity in spinal cord, networks of hippocampal neurons could be induced to fire bursts of APs using the 3D-BDD electrodes. The stimulated bursting activity lasted for hundreds of milliseconds and could be detected by the stimulating electrode once the stimulation pulse was completed.

WP4 Summary
The consortium has determined the necessary charge injection threshold to extracellularly stimulate various types of neurons using MEAs. The extent of such stimulation was spatially mapped and found to be larger than previously predicted. We developed new models which better reflect the stimulation range observed experimentally to predict the impact of MEA stimulation protocols. Two new types of devices were validated for recording cell signals; solution gated field effect transistors with graphene gates, and micro-electrode arrays with nanostructured boron-doped diamond electrodes. GFETs were shown to not only detect changes in intracellular potential, but to simultaneously detect changes in ion concentration at the gate. Low noise recordings were achieved with spontaneous neural activity registering as high signal-to-noise ratio spikes. 3D-BDD MEAs were also shown to be able to record neural APs from spinal cord explants. These APs could be spontaneously recorded, and could also be triggered in bursts in response to stimulation pulses applied via the chip. MEA recording was possible at distant positions of the array concurrently with stimulation from single electrode points to characterize network responses to the stimulation. To further advance investigation of network activity, a new design of MEA that allows separation of different neural populations was developed to the pre-market phase. These MEAs utilize an epoxy process to incorporate microfluidic structures directly on the chip. Two culture chambers separate cell bodies, while micro-channels allow the neurites to form connections between the two populations. Specially designed electrode patterns allow monitoring of the individual populations, as well as the information flow between them.

WorkPackage 5: Histology, toxicity and performance - preclinical evaluation

Majors concerns in this WP were the testing in vivo of the prototypes developed within the previous WPs. In-vivo evaluation concerned histology assessment, but also optimising the surgery for cortical and retinal implants, as well as characterising in vivo the performances of the devices.

Histology and general biocompatibility assessments.

Using several tens of prototype electrode chips were provide to UM for in-vivo implantation. They consisted of diamond on PtIr electrodes, graphene on polyimide, diamond and polyimide, as well as reference samples of PtIr, polyimide, and carrier samples. They were bilaterally implanted subdurally in a total of 65 rats. For comparison polyimide or parylene without electrode material were used. Tissue reactions were assessed for up to 83 days. Tissues were immuno-stained for GFAP, Iba-1, and Interleukin1. DAPI was used to highlight cell nuclei. In some animals studied 4-14 days after implantation there was a mild glial reaction, which, however this was also seen below in implants lacking diamond or platinum and, therefore are due to surgery. Day 83 animals showed little reactions. Then test samples Microglial activation as sign of inflammatory processes were quantified by evaluation of morphology changes from the resting state with many branches to an activated state shown by loss of branches and rounding of the cell body. This was done by measuring cell perimeters and areas using ImageJ software. Cell perimeter decreases with activation. From these two parameters, an index can be calculated: 4*pi*area/perimeter. This index is called ‘circularity’. It increases with activation (1=maximum). Another index is ‘solidity’ (cell area/convex area), which also increases with activation to a maximum of 1. None of the time points 14-84 days after implantation of any of the electrode materials tested yielded significant changes as compared to naive animals or compared with each other, Sham-operated animals were sacrificed after 4 days to show surgical trauma effects. When compared to naive rats without surgical trauma all three indices showed statistically significant inflammatory reactions, which, however were minor, e.g. circularity increased from 0.039 to 0.063 (when considering the possible maximum close to 1.0). Similar signs of mild surgical trauma were seen in rats with parylene+platinum and polyimide+diamond electrodes sacrificed after 4 and 14 days. We refer to the second interim report for a more detailed observation of the trends, that were overall extremely positive for all carbon materials assessed.

Similarly, with respect to retinal implants, surgeries were developed by NR to introduce retinal implants in the subretinal space of blind rats. The tissue of animals were examined after 6 weeks of implantation, and no particular inflammation was observed in the epiretinal sections.
Finally, in order to evaluate the biocompatibility of nanostructure 3D materials, samples consisting of PtIr chips coated with CNT diamond were assembled as electrodes and mounted on the minipig cortex. All the animal surgery was developed for this particular experiments, where 3 minipigs were implanted for 3 months. A craniotomy sized around 3.5 cm x 1.5 cm was performed bilaterally exposing the Dura mater. The Dura was opened longitudinally (Figure 21 A). The multielectrode arrays (MEA), one with nanostructure 3D diamond and one without, were then placed over the brain cortex (Figure 21 B) according to the implantation map shown in Figure 21D. Then the Dura was put over the MEA and sutured (Figure 21 C). The bone removed during the craniotomy was replaced and fixed with screws and plates (Depuy Synthes).
According to the animal, recordings were performed on the electrodes to survey somatosensory evoqued potential. The electrical stimulation was done to the median nerve anterior member and tibial nerve of posterior member, using isolated electrical stimulator (Energy light) until eliciting threshold responses. Recorded Cortical responses were processed on line in Micromed and averaged using an external trigger coming from the isolated electrical stimulator. Electrodes: 1 to 6 in BDD and electrodes 7 to 12 in Pt/Ir. The recordings further completed using electrochemical impedance spectroscopy in vivo.

Histological examinations were carried out post-mortem, 13 weeks after implantation, for the mini-pig. After euthanasia of the animal and removing skin and muscles covering the implant, observation of the implantation sites showed a thickening of the sutured dura mater in front of the craniotomy frame in comparison with the constitutive dura mater. After separation of cerebral hemispheres, we observed integration of Diamond and iridium/Platinum MEAs into a translucent and thin (around 40 µm) neoformed tissue embedding the MEA (Figure 22 A, B). The adhesion of the neoformed tissue with the cortical surface below and the dura mater above was also tested and we observed focal adhesions between the tissue capsule and the Dura mater and the brain cortex (Figure 22C, D) without macroscopic sign of tissue defect. As shown in Figure 22D, the cortical surface was modified by the pressure of implant and the MEA imprint is delimited by a dashed line.

In conclusion, during this task, the Neurocare project enabled to design a complete experimental set-up to chronically record ECoG, monitor impedance spectroscopy and evoked potential. The behaviour of the boron doped electrodes was comparable to the behaviour of the nanostructured platinum iridium electrodes. In terms of biocompatibility, the boron doped diamond was chronically implanted on large animal. Obviously the statistic shall be improved but we can report that the methods from the surgery to the use of specific antibodies satisfy the requirements of the project.

Potential Impact:
A high impact from the different results of the project is expected in the following technical areas, as highlighted at the inception of the project.

Using diamond and graphene nanobioelectronic devices for neuron interfacing has applications in (i) in vitro pharmacological research and in (ii) the medical treatment of vision, hearing and cognitive/motor impaired patients.
Concerning the first point, Neurocare enabled the development of Micro-Electrode Arrays based on diamond that exhibit the electrical performances of the commercial ones in terms of impedance and noise levels further to offer a better biocompatibility, stability, and to be “reactivated”, namely cleanable. In fact, porous metal electrodes used in electrophysiology often result in progressive degradation of the electrode surface properties, whether diamond electrodes do not. The developments led to neuronal culture preparations in vitro that preserve a variety of native neuronal network properties under appropriate conditions and are in many aspects advantageous for physiological studies and pharmacological characterization when combined with extracellular recording systems. Such systems can be used for neuropharmacological studies by measuring spontaneous activities of neuronal networks and study how the activity pattern changes related to specific substances applied. This will help identifying new drugs to treat disease and to predict drug toxicities or adverse reactions. NeuroCare has increased the signal to noise ratio by a factor of 100 using 3D nanostructured diamond, and stimulation capabilities of MEAs with respect to the “conventional” electrode arrays, and this will give a significant commercial advantage over other MEAs available on the market. In parallel, the first graphene FET arrays were fabricated during the project, using innovative technological protocols optimised with Neurocare, and exhibiting recording level that out-take all other materials. Their performances are at the highest state of the art with respect to other electrode systems. Marketing such modified electrode and FET arrays to the growing research and pharmaceutical market could be a next future priority for QWANE, for example, although other electrophysiological apparatus have also expressed their interests to the consortium.
Concerning the second point, Neurocare has paved the way for novel implantable arrays of electrodes, addressing the ability to stimulate neural tissues in vivo. New fabrication protocols were here also proposed and optimised in order to provide biologists with electrically active arrays of conductive electrodes, aiming at transmitting electrical action potentials. Neurocare enabled the fabrication of novel prototypes aiming at the stimulation of the cochlear, the cortex, and the retinal, as well as cuff electrodes for peripheral electrodes. In most cases, their performances reached those of conventional materials, although they often have tens of years of background research, and while Neurocare Carbon materials also demonstrated remarkable biocompatibility and a wider electrochemical window. Although it always take time to get normative approvals for the use of such devices in clinical uses, several tests were performed in vivo on rat retinas, mini-pig cortex, and peripheral nerves. The idea is to propose palliative systems that enable to alleviate disabilities of patients affected by neurodegenerative pathologies. In fact, the average increasing life expectancy has led to as-yet unmet medical challenges (Alzheimer’s, Parkinson’s, dementia, spinal cord lesions, retinal degeneration, deafness, etc). By 2050, people older than 60 will represent 50% of the French population, for example. Recent advances in nanotechnology have opened new opportunities for novel implants through MEAs, an increasingly common part of treatment strategies. These implants are used to electrically stimulate neuronal networks. These prostheses relay signals within an altered neural network and can restore lost functions, or record and inactivate the network when it generates false information. For example, commercial neuronal implants emit high-frequency stimulation to the thalamus for treating Parkinson’s disease or to the inner ears of deaf patients. Other applications could include treatments of epilepsy, dementia, headaches, or visual disabilities.
In addition, retinal degeneration affects almost 12 million people in the EU+US, and studies predict a growth of yearly sales of retinal drugs and prostheses to 1 billion USD (Figure 23). By 2012, retinal degeneration is expected to affect over 50 Million people, afflicting 1% of the population at age 55, 10% at age 65, 25% at age 75, and up to 60% at age 90. One of the most frequent pathologies, age-related macular degeneration causes blindness through photoreceptor degeneration. Although some degeneration also affects inner retinal neurons, the remaining neuronal circuit can still convey information to the higher visual centres. Retinal prostheses propose to restore vision by targeting electrical stimulation of this remaining retinal circuit. The concept has been validated in several clinical trials showing that patients were able to follow moving light targets and were able to identify known contrasted objects.

A clinical trial on this subject is ongoing under the supervision of Prof. Sahel (UPMC) in which 6 patients were implanted with a 60 pixel epi-retinal implants from various supplier. Their clinical evaluation has already shown that patients have a functional gain in vision when the implant is switched on. However, the success yield of improvement has remained very poor, and reasons are still being evaluated. They involve the risk of gliosis around the implant as a foreign body,that therefore limits the injectable charge capability, thus the lifetime of the implants. We have validated Neurocare implants in several configurations (e.g. the retina and the epi-cortex), and we have disseminated those results to our partners as well as to other industrial partners. For example, our partner NanoRetina aims at proposing a full implant solution within the next few years. However, so far no company is really adopting a similar approach where the specificity relies on the use of extremely innovative implant materials such as diamond and carbon. In fact, although the biocompatibility of diamond is a very convincing motivation, as well as the performances of graphene electronic devices, very few groups have reached the ability to adapt nanotechnology processing routes to these levels enabling the fabrication of implants. Thanks to Neurocare the association of partners like CEA-LIST, ESIEE, and UPMC, led to several related patents that have been recently either approved or at least filed, so far mostly covering retinal implant applications, but that could be extended to other types of implants.
Regarding cortical implants as a functional help for blindness , the stimulation of the primary visual cortex in higher visual regions of the brain may also be an interesting strategy for restoring vision for patients struck with specific diseases. In fact, it is expected that visual and hearing impairments could also potentially be treated by direct cortical stimulation (see e.g. the RETINE project, FRENCH National project running from 2009-2011). Partners CEA-LIST, CEA-Clinatec, UPMC, and ESIEE have recently approached Pixium-Vision with respect to the proposal of a project named VISOR that targets this approach, towards French institutional funding. If accepted, the Visor project will directly benefit from the Neurocare developments for these applications.
Also, the ability to stimulate the cortex constitutes one of the most promising therapeutic approaches to alleviate disabilities for tetraplegic patients. Early research in US (2005) has for example enabled one tetraplegic patient paralysed from the neck to “think his TV on and off, to change the channel and alter the volume” Many etiologies of tetraplegia or locked-in syndrome (for example, brainstem stroke and ALS) can leave cognitive and cerebral motor control structures largely intact. Since these structures contain motor signals directly related to lost functions, they are a promising source of relevant command signals. Thus, some types of brain computer interfaces (BCIs) aim to decode neural activity related to intended movements in order to provide control signals for external devices. The field of application is broad, and covers several pathologies. However, this research remains very exploratory and has to make subsequent progress before it can reach the clinic to alleviate disabilities. So far this field of research only belongs to few specific institutes in the world, and namely CEA CLINATEC, where the topic constitutes one of the major R&D motivations for the fabrication of innovative brain computer interfaces.
Regarding cochlear implants, more than 188,000 people worldwide have attained some degree of sound perception thanks to these devices. A retrospective study of patients who have multiple-channel cochlear implants, showed a mean gain in health utility of 0.24 which corresponds to a favourable cost-utility of $9530 per quality-adjusted life-year thanks to declined depression and increase in job prospects to name a few. Most recent implants use MEAs to give better speech recognition. NeuroCare advances may enable users to perceive music and discern specific voices in noisy rooms.

New medical sensing devices providing less invasive, fast and accurate, and selectively sensitive diagnostic functions
NeuroCare has generated a substantial basic knowledge for the next development in the biosensing and neuron interfacing area with outputs in several applied fields. In a mid-term it brings to the fore emerging potentials in biology, food industry, biotechnology, neurosciences and medicine for fabrication of specific bio and chemical sensors. In the long-term, it can allow coupling of cells or neural networks for medical physics and disabilities treatments using new multifunctional devices, combining solid state physics with biological systems in hybrid electronic architectures. Eventually this could lead to a whole generation of intelligent, implantable devices for repair of the nervous system after damage through disease or accident. This is a truly adventurous concept to which Neurocare has brought its contribution.
The NeuroCare concepts for implants have been developed through the active participation of 5 material technologists, 1 laboratory specialised in implant-cell interfacing, 3 SME end-users and 3 clinical partners. These partners have used their extensive experience in exploiting research results and translating them to the clinic and to industry. For example, the start-up company PIXIUM-Vision has been co-funded by 3 of the Neurocare partners, namely UPMC, ESIEE, and CEA, and although the Neurocare implants are not industrially ready in terms of approval authorities and labelled biocompatibility, discussions have occurred were diamond technologies were identified as future potentially market opportunities. Such a start from Pixil would certainly boost the impact of R&D results obtained during our project, that aims at new medical sensing devices and implants that are less invasive, fast and accurate, with selectively sensitive diagnostic functions.

Potential for spin-off applications
NeuroCare is in line with the European strategy to move from resource-based to knowledge-based industries. For example, QWANE, one of the smallest SME of the consortium, has developed a new device within Neurocare, that enables cell guiding through a patterned device. Truly, their optimised micro-electrode array systems developed through Neurocare and enabling neuron guiding has now become a novel product in their catalog. It can provide applications to material, pharmaceutical, medical device and biotechnology fields. Many new markets and breaking innovations are at this interface, we are confident that partners will continue their histories of finding new spin-off applications for their discoveries. The two major applications at which are aiming are (i) in vitro cell interfacing for pharmacological research and (ii) medical treatment of patients with various diseases/injuries.
The NeuroCare concept has involved the development of implants made through microelectronics mass-production processes. Following NeuroCare, the production chain, costing or upscaling will not require extensive development. For the case of retinal chips tested in rodents by consortium partners, approximately 200 chips were built simultaneously on a 100mm silicon wafer. In R&D-scale fabrication, the approximate cost is less than 10€ per chip. Once the chips are released from the wafer (which can be recycled), there is only one packaging step to connect them to an Implantable Pulse generator. Therefore, results would be immediately transferable for direct or spin-off applications.
The developed nanostructured electrode or NCD/graphene coated MEA and FET devices can be used for a variety of applications in health care, patient compliance monitoring and detection of diseases. By specific simple modification of the diamond/graphene surface chemistry, sensors for many applications can be developed including the detection of drugs (patient compliance), sensors for cardiac disorders, diagnosis of metabolites or biological markers of diseases (uric acid, ketones, lactose, glucose, histamine, narcotics testing, DNA sensing etc). The universal concept of cell sensors can be applied to numerous medical applications with the advantage of unmatchable sensitivities. Cell sensors have also numerous applications including the food industry as well as drug, explosive and toxic substance detection. There are other advanced applications such as HIV detection, electrodes for patches, surface probes, electrocardiograms, cancer detection, and skin-penetrating electrodes.

New opportunities for European biomedical industries and advanced SMEs fabricating implantable devices.
Nano-diamond and graphene research are important field in Europe, as demonstrated most prominently by the fact that two European researchers recently won the Nobel prize for their discovery of and work in graphene, that consecutively led to the EU Graphene Flagship. In addition, Europe hosts several priority conferences of CVD diamond growth applications. The recent progress in Europe on NCD and graphene films offers a European lead over the current state-of-the-art in Japan and the USA. Concerning diamond, significant attention is paid to CVD diamond research for applications in electronics and bioelectronics using mainly single crystal diamond which is small size and expensive or passive UNCD films. Nevertheless, competition from outside Europe in these fields is being reinforced. Concerning graphene, a new leading “Graphene Research Center centre for biological studies” has been set up at ICN2 institute is opening a biological institute in the forthcoming months, as part of the EU Flagship project. Neurocare opened up the route for the emergence of a new WorkPackage in the FS aiming at neurointerfacing applications, and where UPMC and TUM are already identified as leading partners, to whom Pixium-Vision has agreed to contribute. For instance, this activity has already offered position to Neurocare participants and postdocs.
To the longer vision, the medical, scientific, economic and industrial impacts of NeuroCare are expected to be great, based on the results and the IP generated by the project. Neurocare has paved the way for the development and commercialisation chain of new products from R&D of MEA systems, to packaging and implants towards clinical studies. This ensures that the inertia built-up by the partners will lead NeuroCare innovations in the medium term.
Furthermore, the medical impact of implants is growing exponentially, which is expected to create increasing demand, especially given the low material cost of the NeuroCare solutions. In addition, these new devices will be more robust and less invasive because they are miniaturised, biocompatible, durable and easier to produce industrially. In fact, the wider electrical window of diamond should also enable the use of much less energy to run.
High technology, research-intensive SMEs are the ideal players for benefitting from these newly developed technologies. The thorough involvement of 3 successful and growing SMEs that are end-users of the project’s research results will hopefully enable the uptake and commercialisation of these technologies.

Dissemination activities and exploitation of results
Several results of NeuroCare have become publicly available, that contains over 100 items including conferences, scientific publications, or media disseminations actions (see the full list available below). This consists of a consistent proof that Neurocare has successfully convinced its scientific communities from biology to nanotechnology. Further, the intellectual property acheivements have been numerous, with a long list of patents, although some were only partially or marginally the result of Neurocare. For example, they cover the fabrication of the electrodes, the 3D nano structuration of the diamond, the technological fabrication of graphene flexible implants, the brain connectors for animal testing of visual or cortical implants, etc. Nevertheless, the rules to share this IP are described in the Consortium Agreement, including the right of access to the "proprietary technologies" of the project members by other project members.

Project Identity
The first step of the dissemination plan was to define the project identity. It led to an attractive project logo and slogan. The NeuroCare website then played and will keep playing a major role in informing public awareness and opinion. In addition, templates for dissemination activities were provided to all partners for PowerPoint presentations about the project’s results. Finally, regular editions of the project brochure and regular project flyers were provided to partners for distribution at trade fairs, conferences and other meetings.

Target Audiences and adapted dissemination channels
Target audiences, during and after the project lifetime, includes society-at-large, the scientific community, medical implant and related industries, patient groups, medical charities and the clinical community.
NeuroCare disseminated to the scientific, clinical and industrial communities though traditional channels which include peer-reviewed publications, specialist websites and scientific conferences.
To ensure that the NeuroCare project is appealing to stake-holders in the medical implants and related sectors, including materialists, clinicians, fabricators of implants, and patient groups, the NeuroCare consortium has constructed its objectives in line with clinical and industrial needs.
To ensure the involvement of communication and media professionals in the Dissemination, all partners’ public relations and communications departments were provided with dissemination materials about NeuroCare, including up-to-date brochures and flyers for public-private and business-to-business communication via commercial, technical, financial and industrial publications; broadcasts; and trade fairs and seminars. Emails were sent to all public relations and communications departments before major trade fairs and conferences, requesting that NeuroCare communication tools be presented along with other partner information. Finally, targeted customer visits were encouraged in order to raise technology profile awareness.

Results and Exploitation
• Plan for the use and dissemination of knowledge
All foreground and all dissemination activities are accurately reported in the plan for the use and the dissemination of foreground, which is described in detail in Section 1.2.

• NeuroCare Exploitation and clinical roadmap
It is well known that introducing a new technology in the medical sector often faces numerous bottlenecks such as very high investment level and long testing and development phases. The NeuroCare project has been designed to minimise these bottlenecks. Indeed, it was set-up in line with a longer term vision, summarised in the roadmap below.
As shown in in Figure 24: NeuroCare Clinical Roadmap, the NeuroCare project lasted for 3 years. The remaining innovation chain is expected to take about 3 more years. During this 3-year period, partners expect to benefit from internal company revenues, venture capital, business angels, acquisitions and public subsidies in order to fund the subsequent steps expected to lead to the market penetration of NeuroCare technologies (technology transfer, upscaling to larger animals, clinical trials, etc.).

List of Websites:

The address of the project public website is given on the front page of this report:
http://neurocare-project.eu
The main contact is the project coordinator: Prof Philippe Bergonzo, CEA.

Please note that the list of scientific publications (Part A1) and the list of disseminations activities (Part A2) has been uploaded as an attachment under the pdf file name: "Final Report Neurocare - Part A1 and A2".