SiNAPS: An implantable active pixel sensor CMOS-probe for simultaneous large-scale neural recordings

Abstract Large-scale neural recordings with high spatial and temporal accuracy are instrumental to understand how the brain works. To this end, it is of key importance to develop probes that can be conveniently scaled up to a high number of recording channels. Despite recent achievements in complementary metal-oxide semiconductor (CMOS) multi-electrode arrays probes, in current circuit architectures an increase in the number of simultaneously recording channels would significantly increase the total chip area. A promising approach for overcoming this scaling issue consists in the use of the modular Active Pixel Sensor (APS) concept, in which a small front-end circuit is located beneath each electrode. However, this approach imposes challenging constraints on the area of the in-pixel circuit, power consumption and noise. Here, we present an APS CMOS-probe technology for Simultaneous Neural recording that successfully addresses all these issues for whole-array read-outs at 25 kHz/channel from up to 1024 electrode-pixels. To assess the circuit performances, we realized in a 0.18  μ m CMOS technology an implantable single-shaft probe with a regular array of 512 electrode-pixels with a pitch of 28  μ m. Extensive bench tests showed an in-pixel gain of 45.4 ± 0.4 dB (low pass, F-3 dB = 4 kHz), an input referred noise of 7.5 ± 0.67 μVRMS (300 Hz to 7.5 kHz) and a power consumptio

Conception et caractérisation de microélectrodes flexibles pour le développement de neuroprothèses implantables

Brain-Computer Interfaces ensure a bidirectional connection between a patient and his environment. Using an implant in his brain, a patient suffering from severe motor deficiency can control external devices through the sole action of his cerebral activity. One of the major requirements for such application is to conceive an implant, also called neuroprosthesis, that is able to be implanted for long periods of time.Micro-nano-technology enable the fabrication of a neuroprosthesis that gives in the demands of such item: efficient, reliable and limiting body rejection mechanisms. To that aim, the implant is designed on a flexible substrate provided by a biocompatible polymer. Implant flexibility allows for better compliance with the brain tissues and insures a more intimate contact with the neurons while maintaining minimal inflammation. This implant is inserted in the brain using a bioresorbable support made of silk fibroin. First tests in vitro on culture cells, and in vivo on mice showed promising results in terms of biocompatibility and biostability in the short and medium term.Les Interfaces Cerveau-Machine assurent une connexion bidirectionnelle entre un patient et son environnement. Un patient atteint de déficience motrice lourde peut, au moyen d'un dispositif implanté dans son cerveau, commander des objets connectés par la seule action de son activité cérébrale. Une des premières exigences que cela requiert est de concevoir un implant, dit neuroprothèse, susceptible de rester implanté de façon chronique. L’utilisation des micro-nano-technologies permet de fabriquer une neuroprothèse qui réponde aux exigences d’un tel dispositif : performant, fiable et limitant la réaction de rejet par l’organisme. Pour cela, l’implant est conçu sur un substrat flexible à base de polymère biocompatible. La souplesse de l’implant lui permet de mieux s’adapter aux tissus cérébraux et d’assurer un contact intime avec les neurones en diminuant la réaction inflammatoire. Cet implant est inséré au moyen d’un support rigide biodégradable issu de la fibroïne de soie. Des premiers tests sur culture in vitro et sur petit animal (souris) ont montré des résultats prometteurs en termes de biocompatibilité et biostabilité sur le court et moyen terme

In vitro and in vivo biostability assessment of chronically-implanted Parylene C neural sensors

International audienceParylene C has rapidly gained attention as a exible biomaterial for a new generation of chronic neural probes. However, polymeric material failure in the form of delamination, swelling or tearing, often compromises device biostability in the long term. This work constitutes a rst step towards lifetime assessment of Parylene C implanted devices. We have conceived a Parylene C-based neural probe with PEDOT-nanostructured gold electrodes for the recording of brain activity. The material response to its biological environment was studied through in vitro soaking tests and in vivo wireless recordings in mice brain, both carried out for up to 6 months. Impedance monitoring and SEM images indicate that over the length of this trial, none of the implants presented with apparent signs of material degradation. Packaging reliability was a predominant factor in device failure, with a certain number of faulty connection appearing over time. This parameter aside, all soaked devices were stable in Articial Cerebro-Spinal Fluid, with impedances within 10% of their initial value after 6 months at 37°C. Besides, at least 70% of the implanted device were able to accurately record wirelessly high amplitude hippocampal Local Field Potentials from freely-moving mice, with steady Signal-to-Noise Ratio. In other terms, Parylene C implantable sensors responded minimally to articial and actual physiological conditions during a period of 6 months, which makes them promising candidates for reliable, chronically implanted sensors in the biomedical eld

Biostability Assessment of Flexible Parylene C-based Implantable Sensor in Wireless Chronic Neural Recording

International audienceThe stability of polymer-based sensors in a biological environment remains a challenge, as delamination and swelling often compromise mechanical and electrical capability. We have developed a neural implant based on Parylene C, a biocompatible flexible polymer, with PEDOT-nanostructured gold patterns to record the brain electrical activity. Here, we show first evidence of device biostability through in vitro soaking tests in artificial brain environment and in vivo recording in mice. Our results indicate that after over the six months trial, more than 75% of the in vitro electrodes have stable impedance, and the implanted sensors in mice were able to accurately record signals from mice hippocampi. None of the implants presented with signs of Parylene degradation or metal corrosion. Overall, the devices are promising candidates for reliable, chronically implanted sensors in the biomedical field

Plasma etching of thick Parylene C for fabrication of biocompatible electrodes

International audienceHere we report on the plasma etching of thick parylene C in order to define flexible implantable probes for neural applications. To reduce the foreign body response and to ensure a good stability for chronic in vivo recordings, the chosen substrate for the neural probes is Parylene C, a polymer known for its high biocompatibility, flexibility and chemical inertness. In the manufacturing process, highly defined structuration steps of Parylene C are essential. Techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures, i.e. with good dimensions control and without residues. As an alternative, plasma etching has shown great promise in this area. However, because of the highly crystalline structure of this polymer, fast etching rate, lack of residues and high aspect ratios remain hard to achieve, especially for deep etching of thick layers

Plasma etching of thick Parylene C for fabrication of biocompatible electrodes

International audienceHere we report on the plasma etching of thick parylene C in order to define flexible implantable probes for neural applications. To reduce the foreign body response and to ensure a good stability for chronic in vivo recordings, the chosen substrate for the neural probes is Parylene C, a polymer known for its high biocompatibility, flexibility and chemical inertness. In the manufacturing process, highly defined structuration steps of Parylene C are essential. Techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures, i.e. with good dimensions control and without residues. As an alternative, plasma etching has shown great promise in this area. However, because of the highly crystalline structure of this polymer, fast etching rate, lack of residues and high aspect ratios remain hard to achieve, especially for deep etching of thick layers

Deep plasma etching of Parylene C patterns for biomedical applications

We report on the plasma etching of thick Parylene C (~25 µm) in order to define flexible implantable probes for neural applications. Parylene C is a transparent polymer that presents with high biocompatibility, flexibility and chemical inertness, and has gained increased attention over the years in the biomedical field. In the manufacturing process, highly defined structuration steps of Parylene C are essential, but techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures, i.e. with good dimensions control and without residues. Here, for the first time, negative resist (BPN) coating followed by RIE-ICP are used in order to pattern Parylene C-based structures, with a clean cut, vertical profile, fast etching rate (~0.8 µm/min) and conservation of device biocompatibility

NeuroNets: Advancing SU-8-Based Flexible Self-Standing Microdevices for 3D Brain Model Interface

National audienceNeural spheroids and organoids stand as the next-generation in vitro models of the brain, offering a more precise replication of human-specific physiopathologies. To validate the functionality of these emerging 3D biomodels, capturing the maturation of neuronal activity through electrophysiology is imperative. So far, this aspect has been significantly underexplored, frequently constrained to sporadic, invasive and/or destructive testing methodologies. Organoids transferred onto Microelectrode Arrays (MEA) tend to flatten and spread onto the MEA surface, while optical methods such as calcium imaging, are mainly implemented in slices/dissociated organoids due to scattering effect in a 3D sample [1-2]. Self-rolled biosensors and other flexible electronics offer improved contact compared to MEA, but still face challenges accessing the interior of the 3D model [1]. In this sense, recent advancements in mesh electronics are gaining interest in the field to address the limitations imposed by the three-dimensional nature of brain organoids [3]. In this work, we have developped a thin, flexible electronic mesh to be integrated in 3D brain models during early-stage development for prolonged, in-depth monitoring of electrical activity. This microdevice, called NeuroNet, is fabricated through standard process lithography of SU-8 (Fig 1A ), a negative epoxy-based photoresist used in a wide range of biomedical device [4]. It comprises of two layers of 1µm SU-8 patterned in a serpentine shape, with layers of Ti/Au and Pt as contact lines and electrodes/pads, respectively (Fig 1B ). A final thick SU-8 layer on the device periphery enables device handling with tweezers (Fig 1C). To release the devices, we have studied different sacrificial layers before chosing TiW, on the grounds of i) biocompatibility (H2O2 can be washed easily), ii) compatibility with process steps (solvent-compatible, stable up to 125°C), iii) release time (<1 day) and iv) yield (~95% success rate). In this process, a special attention has been brought upon the issue of SU-8 cross-linking over metallic surfaces. Indeed, on the platinum electrodes, a residual layer of unexposed SU-8 has shown to prevent proper electrode opening. To circumvent this issue, a small study was conducted to optimize ICP-RIE plasma etching process of SU-8 residues as well as stripping techniques, to obtain a smooth opening on the metallic electrodes, with limited residues (Fig 2 ). Following process optimization, we proceeded with biological pre-validation. While SU-8 has been reportedly used in diverse biomedical applications, SU-8 formulations contain radical monomers and antimony (Sb) salts as photocatalysts, both of which are cytotoxic [5]. When processed as recommended by the manufacturer, SU-8 devices soaked 24h in cell culture medium were highly harmful for SH-SY5Y cell line (Fig 3A), resulting in massive cell death. We developped a biocompatibility protocol as a final step for our SU-8-based devices, comprising of extra UV-exposure, followed by extensive development, thorough washes and a 3-days bake @125°C. As a result, cell viability was ensured, as established by our MTT cytotoxicity assay (Fig 3A + data not shown). Finally, we show some preliminary results on the transfer of primary cortical spheroids from rodents into our NeuroNets. All spheroids (N=16) remained viable throughout the 18-day culture period, displaying promising indications of neuronal branching onto the device (Fig 3B). Overall, our research establishes the technical groundwork necessary for advancing a novel generation of flexible electronics seamlessly integrated into early-stage 3D neural models, facilitating continuous electrophysiology recordings over extended periods. Our next phase will now prioritize the optimization of mesh internalization inside 3D neural models to perform valuable in-depth electrical recordings

Deep plasma etching of Parylene C patterns for biomedical applications

International audienceWe report on the plasma etching of thick (~23µm) Parylene C structures. Parylene C is a transparent polymer that benefits from high biocompatibility, flexibility and chemical inertness, and has gained increased attention over the years in the biomedical field. In the manufacturing process, highly defined structuration steps of Parylene C are essential, but techniques based on laser, scalpel and wet etching have shown to be unsuitable for properly cut structures. Plasma etching remains nowadays the most widespread option, though fast etching rate, lack of residues and high aspect ratios are still hard to achieve. To overcome these issues, the selection of both mask material and plasma conditions is crucial. Here, three masks-metal, positive and negative photoresists-are tested as stencils, and several plasma parameters are briefly studied in order to obtain the highest etching rate while maintaining good coverage. We showed that increasing the RF power up to a considerable 2800W while maintaining a moderate physical contribution (bias power, pressure, temperature), is optimal in the achievement of fast PaC etching without inducing thermal stress. Besides, the addition of a short fluorinated plasma in the midst of the process is shown to alleviate residues. For the first time, negative photoresist Intervia Bump Plating (BPN) coating followed by ICP 1-RIE 2 are used in order to pattern Parylene C-based structures, with a clean cut, vertical profile and fast etching rate (~0.87±0.06 µm/min) and a selectivity of 0.5. This solution was carried out to release unitary Parylene-based neural probes from a silicon wafer. Finally, cytotoxicity assays on these neural implants were performed to make sure that no trace of mask or stripper residues would jeopardize device biocompatibility

Nanowires vs Neurons : engineering network scale guidance and promoting biointerface through nanotopographical cues

International audienceHigh-aspect-ratio surface nanostructuration have gathered increased attention in many biology fields. Indeed, nanostructures features are of similar length scales to cellular systems, and enable a wide toolbox to sense and perturbe at the single cell level. Depending on the nanostructure geometry, spacing, sharpness, height and mechanical properties, they have shown to strongly influence cell behaviour such as morphology, differentiation, mobility, adhesion and division [1]. Particularly, these altered behaviours originate from membrane-material interactions, as the cultured cell undergoes deformation of its membrane around the highaspect-ratio structure (engulfment or endocytosis), sometimes leading to actual penetration. In the field of neurology, this tight seal between nanostructures and a neuron holds the potential to perform intracellular electrical measurements with the help of electroporation or optoporation, or even intracellular-like potentials when a very tight seal is achieved [2]. On the other side, surface nanostructuration over large scales have gathered a great interest in understanding and altering neuronal dynamics and network formation. For instance, neuronal cells cultured on densely-nanostructured surfaces have served as physical cues to guide neurite growth [3] or to reduce glial adhesion [4]. In this work, we performed a systematic assay to study the influence of nanotopography on axonal growth and network formation of embryonic cortical neurons from rodents. We demonstrated a controlled, top-down process to obtain 3µm-high silicon nanowires (NW) over a wide surface of several mm (Fig 1-A). The process, using projection lithography and DRIE, enables high throughput fabrication at wafer scale (4 to 6") of NW arrays with controlled dimensions (minimum 250nm and 5µm in height) and spacing. The axonal guidance of neuronal cultures was investigated on top of NW compared to flat surfaces (Fig 1 -BC). More particularly, through immunostaining and SEM observations, we studied the impact of both nanowire diameter (400nm to 2µm) and pitch (500nm to 5µm) on the privileged directions of neurites (axons and dendrites, Fig 2). While 70% of all neurites were aligned axially and diagonally on NW up to 2µm in diameter, our work highlights a strong impact of NW spacing in the formation of an aligned network (~98-100% alignment for a 500nm spacing to 43-47% for a 5µm spacing). This observation aligns with previous research findings, indicating that neurons cultured on nanotopographical surfaces follow a distinct developmental pathway compared to those on flat surfaces. This pathway is marked by the early emergence of elongated major neurites, ultimately resulting in accelerated polarization [5]. In this sense, we captured unprecedented images of neurite sprouting, commonly referred to as axonal growth cones, directing neuritogenesis along a preferred trajectory (Fig 2C). Finally, we examined nanowire engulfment/bed-of-nail behaviour tuning depending on nanowire dimension and density (Fig 3). Based on this study, we are able to propose an optimal design of 400nm diameter, 2µmpitch nanowires, exhibiting axonal guiding in privileged directions with close to 100% accuracy, while maintaining an engulfment state optimal for high-resolution electrophysiology [5]. Overall, this work elucidates cues on brain development and circuit formation and paves the way to more efficient biosensors and neuroprosthetic scaffolds