Drug and toxicity assays are necessary to develop advanced treatments, as a way of improving medical diagnostics and testing the potential toxicity of new chemicals. In vitro testing is a powerful pre-clinical method used not only to initiate the drug development but also to iterate on the molecule to improve its safety and efficiency before the clinical trial. The actual in vitro models are currently not on par regarding the in vivo system they attempt to mimic. A great deal of information is missing from the basic in vitro assays, because the in vitro models are overly simplified and mainly monitored via visual criteria. Two research direction can be invested in to overcome this issue: a) elaboration of more complex in vitro biological model directed towards complete autonomous organ-on-chip systems and b) improvement of the monitoring of these complex biological models. To do so, we propose a multi parametric system approach, by integrating bioelectronic devices. Electrophysiological monitoring offers a complementary approach to optical techniques. It allows label-free detection of multiple, simultaneous biomarkers occurring in the in vitro model. To merge the electronic and biological assays protocols, we integrate Organic Microelectrode Arrays (OMEA) into standard biological experiment protocols. This approach, from the petri dish to a multi-parametric device, is the core of this work, presented in this thesis.

A review on Organic Microelectrodes introduces the field of organic material, their fabrication, and applications in microelectrode arrays devices. Specific properties of conjugated polymers such as PEDOT:PSS are of interest to interface with biology. . This organic material can be transparent, conformable, easy to fabricate, and mechanically matched to soft biological tissues. More importantly, the conducting polymers are excellent transducers of biological signals: they can be ion-to-electron transducers or vice-versa. Bioelectronic devices create a bridge between the electronic and the biological worlds. For instance, electrophysiology performed using organic microelectrodes arrays delivers extremely valuable data for drug or toxicity assays. Different methods are implemented to process the organic materials into microelectronic fabrication protocols such as patterning and encapsulation steps. In the introduction section, a number of applications are detailed for in vitro studies.

à [ɛl] s,

To include electrophysiology monitoring steps into usual biological experiments, the planar bioelectronic chips are assembled in the form of a standard cell biology tool: the 12well plate. The OMEAs are arranged in a multiparametric format enabling simultaneous visual and electrical monitoring. Furthermore, this "smart well plate" fits most of the biological protocols for in vitro assays. A specific design adapted from the 12-well plate done using computer-aided design software. In addition, a supplementary supported holder and wire system is integrated into the design concept. It enables the smart well-plate to fit inside generic biological equipment such as microscopes, hoods and incubator and simultaneously performs continuous electrophysiological monitoring.

In electronic setup and architecture, one of the main restrictions is the number of channels that are available for both input and output data. A way to reduce the channel number and to maintain the same number of electronic components is studied, namely matrix design architecture. Organic electrochemical transistor (OECT) design has an arranged matrix structure. First, OECTs fabrication steps were optimized. From metal deposition to encapsulation technique, the chapter 3 describes the methodology of the OECT matrix microfabrication protocols. Then, a second process is developed to perform multi-stacking of conductive tracks over encapsulation layers to design a matrix architecture. Electrical and structural characterizations are then performed on the matrix device. Furthermore, the bioelectronic device's ability to monitor biological experiments in vitro is tested. First, an OMEA is used to monitor the viability of lipid bilayer viability in Chapter 4.

Then, they are used to measure the formation of the epithelial barrier, and finally to detect electrogenic cell activity in Chapter 5. In the first experiment, transmembrane proteins (TMP) embedded in a lipid bilayer are deposited on an OMEA using PEDOT:PSS. The organic material created a soft interface with the lipids and provided a negatively hydrated charged cushion to support the proteins. By doing so, the organic polymer mimics the inner cell polarity and softness and enables the correct positioning of the TMPs inside the lipid bilayer.

Electrophysiological assays were performed to study the behavior of proteins using the organic electrode. Then, two in vitro experiments are achieved using the OECTs matrix devices. The first experiment aims to monitor the epithelial barrier formation of MDCK cells seeded directly on the device's surface. The measurement of the cut-off frequencies of the transistors gives information on the barrier integrity and density area upon the channel. The second experiment integrates the OECTs matrix devices in a 3D in vitro model of electrophysiological cells. Motor neurons and Schwann cells are mixed into a spheroid, then seeded into a specific PDMS mold to develop into a peripheral nerve. The OECT matrix device used as the biological model substrate is used to detect action potential from the nerve fibers.

In conclusion, we have shown that including bioelectronic devices into generic tools from the biological experiment is possible. The matrix multi-stack devices were successfully performing electrophysiological monitoring in vitro. The etching fabrication method to pattern organic material is a promising technique to industrialize OMEAs This study opens new avenues for further in vivo testing, especially for long-term experiment.

Résumé

Les études pharmacologiques sont primordiales dans le développement des traitements de pointe. Elles permettent d'améliorer les diagnostics médicaux et de tester la toxicité potentielle de nouveaux produits chimiques. Les tests in vitro font partie intégrante des méthodes précliniques utilisées pour initier le développement d'un médicament, et pour itérer sur la formulation moléculaire afin de s'assurer de son innocuité et de son efficacité avant les essais cliniques. Malheureusement, les modèles biologiques actuels in vitro font défaut et ne sont pas prédictifs du système in vivo qu'ils tentent d'imiter. Beaucoup d'éléments ne sont pas inclus dans les tests in vitro basiques. En effet, les modèles biologiques sont ultra simplifiés, et de façon générale, contrôlés sur des critères visuels. Deux axes de recherche sont investis pour répondre à cette problématique : 1) Élaborer un modèle biologique in vitro plus complexe, tels que des systèmes complets et autonomes appelés organ-on-chip. 2) Améliorer les méthodes de contrôle de ces modèles biologiques complexes.

Aussi, nous proposons un système de suivi multiparamétrique in vitro intégrant des dispositifs bioélectroniques. La surveillance électrophysiologique offre une approche complémentaire aux techniques de contrôles optiques. Elle permet la détection simultanée de plusieurs biomarqueurs se produisant aléatoirement dans le modèle in vitro. Pour fusionner les protocoles d'analyses électroniques et biologiques, nous intégrons des réseaux de microélectrodes organiques (OMEA) dans des protocoles d'expérimentations biologiques usuels. Cette approche, allant de la boîte de Petri à un dispositif multiparamétrique, est au coeur du travail présenté dans ce manuscrit.

En premier lieu, une revue portant sur les microélectrodes organiques introduit le domaine des matériaux organiques, de leur fabrication et leurs applications. Les propriétés spécifiques des polymères conjugués tels que PEDOT:PSS sont adaptées pour interfacer avec la biologie. Ces derniers peuvent être transparents, conformables, faciles à fabriquer et s'accordent mécaniquement avec les tissus biologiques mous. Plus important encore, les polymères conducteurs sont d'excellents transducteurs de signaux biologiques : ils peuvent être des transducteurs ion-électron ou vice-versa. Les dispositifs bioélectroniques créent un pont entre le monde de l'électronique et celui de la biologie. Par exemple, l'électrophysiologie réalisée à l'aide de OMEA fournit des précieuses données pour la pharmacologie. Différentes méthodes sont évaluées pour intégrer les matériaux organiques dans des protocoles de fabrication microélectroniques tels que les étapes de modelage et d'encapsulation. Pour terminer, quelques applications in vitro des dispositifs sont détaillées.

Pour intégrer le suivi électrophysiologique dans des expériences biologiques usuelles, les puces bioélectroniques sont assemblées sous la forme d'un outil standard utilisé en biologie cellulaire : la plaque 12-puits. Les OMEA sont disposées dans un format multiparamétrique permettant une observation visuelle et électrique simultanée. De plus, cette « plaque intelligente » s'intègre dans la plupart des protocoles biologiques de test in vitro. Une conception spécifique adaptée de la plaque 12-puits est réalisée à l'aide d'un logiciel de conception assistée par ordinateur. Un ensemble connectique permettant à la plaque de s'intégrer dans des équipements biologiques (microscopes, hottes, incubateurs…) permet d'effectuer une surveillance électrophysiologique continue. Dans l'architectures des systèmes électroniques, l'une des principales restrictions est le nombre de canaux adressables pour les données d'entrée et de sortie. À cet égard, un moyen de réduire le nombre de canaux et de conserver le même nombre de composants électroniques est élaborer : l'agencement matriciel. La conception du transistor électrochimique organique (OECT) est adaptée pour une lecture matricielle. Tout d'abord, les étapes de fabrication des OECT ont été optimisées. Cette section décrit la méthodologie des protocoles de micro-fabrication de matrice OECT : du dépôt de métal à la technique d'encapsulation. Puis, un second processus est développé pour créer un réseau de pistes conductrices sur des couches d'encapsulation afin de concevoir une architecture matricielle.

Des caractérisations électriques et structurelles sont par la suite effectuées sur le dispositif matriciel.

Enfin, la capacité du dispositif bioélectronique à monitorer des expériences biologiques in vitro est évaluée. Premièrement, un OMEA est utilisé pour contrôler la viabilité d'une bicouche lipidique. Ensuite, ils sont utilisés pour mesurer la formation d'une barrière cellulaire épithéliale, et pour détecter l'activité des cellules électrogéniques. Dans la première expérience, les protéines transmembranaires (TMP) intégrées dans une bicouche lipidique sont déposées sur un OMEA. Le matériau organique crée une interface souple pour les lipides, et forme un coussin hydraté chargé négativement soutenant les protéines. Ce faisant, le polymère organique imite la polarité et la souplesse interne du corps cellulaire, et permet le positionnement correct des TMP à l'intérieur de la bicouche lipidique. Des mesures électrophysiologiques sont réalisées pour étudier le comportement des protéines à l'aide d'électrode organique. Par la suite, deux expériences in vitro sont réalisées en utilisant les dispositifs matriciels d'OECT. La première expérience vise à surveiller la formation de la barrière épithéliale des cellules ensemencées directement à la surface du dispositif. La mesure des fréquences de coupure des transistors donne des informations sur l'intégrité et la densité de la barrière cellulaire formée sur le canal. La deuxième expérience intègre les dispositifs matriciels OECT dans un modèle 3D in vitro de cellules électrophysiologiques. Des neurones moteurs et des cellules de Schwann sont mélangés pour former un sphéroïde, qui est ensemencés dans un moule PDMS. Sa forme spécifique permet aux cellules de se développer en un nerf périphérique myélinisé. Le dispositif est utilisé comme substrat sur lequel repose le modèle biologique, et est utilisé pour détecter le potentiel d'action des fibres nerveuses.

En conclusion, nous avons montré qu'il était possible d'intégrer des dispositifs bioélectroniques dans des équipements standards utilisé en biologie cellulaire. Les dispositifs matriciels multicouche ont effectué avec succès un monitoring électrophysiologique in vitro.

La méthode de fabrication par gravure pour modeler la matière organique est une technique prometteuse pour l'industrialisation des OMEA. Cependant, des tests in vitro supplémentaires sont nécessaires pour valider leur utilisation dans une expérience à long terme.

Acknowledgement

I would like to first to my thesis Directors Pr Roisin Owens and Pr Therry Djenizian for advising this PhD, their support and interest, availability, and guidance. I am grateful to my mentors that gave me the opportunity to join their department, Flexible electronics (FEL) and Bioelectronic (BEL). I am especially thankful to Roisin Owens, that kept pushing me forward from internship to PhD, no matter how difficult the distance made this PhD organization. I felt privilege to have been able to join her new Bioelectronic system laboratory (BEST) periodically, meet and learn from new colleagues. You taught me to be independent and confident, and I will look up to you as a model of Professor.

I would also express my infinite gratitude to Dr Bradley Schmidt and Dr Pierre Leleux, that have trusted me from the very beginning of this journey and offered me a place in their company PANAXIUM. Not only have they been supportive in many aspects, but they also opened doors of great science, project, and perspective that expended the scope this PhD. I would like to thank Dr Loïg Kergoat, that also supervised my thesis and helped me during the hard time of redaction and defense, providing wise advice and guidance. To Mrs Carolyn Aubry, that has been a pillar throughout these years in the company, thank you for your patience, and carrying support. And a big thanks to my Panaxium colleagues that I look up to, and helped me growth within the company: Jolien, Isabel, Marcel, Aaron, Olivier, Karima, SiMing, Marie, Bahar Alwin and Jean. I felt grateful to Pr Rodney Oconnor, that, more than allowing me to stay and work in the BEL department, included me in his wonderful team/family that I have been honored to be a part of. Thank you Rod for your support. David Moreau, Martin Baca, thank you for helping me daily, you are essential to this lab and support student to overcome their struggles.

Rémy, you are an amazing PhD student. Be more confident of your work and capabilities. You have already become an excellent microfabrication expert in a few months, and the devices you nicely gave me worked perfectly and saved my last experiment. Thank you for your kindness and generosity. Gerwin, special thanks to my office buddy. It has been a pleasure to be able to exchange not only scientifically, but also chit chat on more general ideas and values. I value our discussions and will follow your new journey starting after your PhD. Lionel, thank you for working with me in the clean room, you have been a real support during those harsh time when I was struggling on technical issues. Also thank you for being this straight honest colleague, that do not hesitate to speak for others when we needed it. Mary, you've been a mentor I've tried to follow the tracks. You are a reference in science to me and I will always remember how you made science easy and accessible to beginners. Magali, it has been a chance to meet you when I arrived at the CMP. I felt lucky to have started my PhD with you, and I would like to thank you for all the knowledge you shared with me about biology. We became friends and I saw you overcoming the struggles of PhD with harsh work, and it showed me what was strong and powerful dedication to research. To my BEL colleagues, Ian, Hajar, Marina, Attila, Charles, Esma, Romanos, and Rita, thank you for your presence, support and scientific knowledge that you kindly shared with me.

Many people from different teams and groups also greatly contributed to this PhD. I would like to thank Chris Martin, and the whole Cornell Nanofacility team for introducing me to microfabrication, and for their exceptional warm welcome to Ithaca. Also, a huge thanks to the clean room team of the CMP, Gaëlle, Jean and Frederic, as well as the CMP IT team: Michelle, Anaïs, Veronique, Gracien and Thierry. You've been life savior many times during these three years.

To my lovely "special team", that saw me at my best and worst for four years, without whom this thesis would have been a much more difficult exercise: Marie, in all objectivity, you are one of the best scientist/researchers I had the privilege to work with. You make miracles in the biological field and are skilled in many other fields. As a friend, I wish you the best for your future project. Thank you for teaming up with me, even if it did not fit into your own PhD projects. You gave me your time and energy, and I could not thank you enough to have enlighten my last and favourite PhD project. Severine, thank you for your kindness and patience. Having gone through similar struggles during your PhD, I valued a lot your wise advice that helped me overcome many difficulties. I am looking forward to your future path as a strong and inspiring scientific researcher. Bastien, you are way more thoughtful than I would ever be by moving a step backwards and see the totality of the aspects of challenges and difficulties. More than once, you were able to take me out of stuck situations and find a way out / solutions to my PhD struggles. I am excited to join Panaxium and work with you more as a colleague. SBAM, thank you for your daily support. It was a relief and pleasure to wake up and get to work with you every day. I hope to be able to return the favor to you somehow someday. Many thanks and you have my forever gratitude.

To my dear parents, you gave me the most valuable gifts a child could hope for: the strength to challenge any obstacles in life, as harsh and unexpected they could be. The opportunity to growth and freely support my own convictions, despite being different from yours. And your unconditional love and support, specifically during this PhD adventure. Thank you Quentin, last but not least, my foremost thanks for keeping up with me in these particular years of PhD. You've provided me support, love, and confidence I needed in the harsh moments. Thank you for keeping me company in my sleepless night of insomnia, wiping away tears of stress, discouragement, and frustrations. Thanks, my love, for overcoming this endeavour with me, and giving me bright and joyful days.

ABBREVATION LISTE

Organic microelectrode arrays for biomedical applications

This chapter is an ongoing works and will be part of a future publication in collaboration with

Zixuan lu and Róisín M. Owens

A MEA is a platform gathering multiple microscopic electrodes in a small area. They are made with biocompatible materials interfacing biology to record electrophysiological signals. Made upon rigid or flexible substrates, they allow extracellular recordings of biological events alongside optical monitorings, if the substrate is transparent. They are used both for in vitro and in vivo applications.

In industry, rigid MEAs used for in-vitro experimentation are built on two types of substrates: silicon and glass/quartz. The latter enables applications where high optical transmission, high thermal conductivity and strong chemical stability are required. They are typically preferred for optical monitoring using microscopes. As will be described in section 3 below, the micro electrodes, their conducting lines and connecting pads are mostly patterned using lithography, which is the most common technique used in the microelectronics industry.

In the last decade, industrial processes have begun to implement organic materials into MEAs, where gold, platinum, and silicon nitride where the main material used in their composition.

In this review, we will focus on the Organic Micro Electrodes Arrays (OMEAs). First, we discuss why organic materials are used and what their useful properties are. Then, we explain the device physics of OMEAs, and highlight the properties of different organic conducting materials. We describe OMEA fabrication protocols, and finally list the advantages of OMEAs in different in vitro / in vivo applications.

Why Conducting Polymers?

Inorganic materials used as active sensing materials limits the signal detection and amplification between biotic/abiotic interfaces 1 . Intercellular activity and interactions usually rely on ionic transport, thus traditional electronics based on metal, metal oxide, and other semiconducting materials have advantages for processing electronic signals but not ionic ones. Single crystal semiconductors (such as Si, Figure 1 (b)), the most common materials for electronics, consist of a rigid lattice of covalent bonds. In order to have mobile charges in the silicon lattice, elements are often added into the lattice as dopants by either direct addition during single-crystal-formation or post addition during device fabrication 2,3 . Group III and group V elements can accept holes (positive mobile charge) and electrons contributing to the conduction in the lattice, respectively 1 . Often, the charge carriers in the silicon lattice have very high mobility (around 10 3 cm 2 /V•s electron mobility with 10 22 m -3 dopant concentration at 300 K) 4 , but they hardly have any ionic conductivity. Since the ionic current is one of the major signals existing in biological systems, the inorganic semiconducting material cannot support the communication at biological-electronic interfaces. Also, dangling bonds on the inorganic crystal surface can easily form a layer of oxide, which acts as an insulator blocking electrons and ions, further hindering signal transduction. Conducting polymers (CP), a new class of organic electronic materials with both electrical and ionic conductivity, have emerged showing great promise for biological applications. The most commonly used conducting polymer in the field of bioelectronics, poly(3,4ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) is shown in Figure 1(a). The p-type PEDOT has π-conjugation throughout the backbone, through which the mobile charge can transport freely 5,6 . With the presence of the negative dopant poly(styrene sulfonate) (PSS), the holes are stabilised on the backbone of poly(3,4ethylenedioxythiophene) (PEDOT), so that holes become mobile with an applied-voltage potential. In addition, since the mixture of PEDOT and PSS has a loose and disordered structure, the hydrated metal ions in the aqueous environment can move into the PEDOT:PSS bulk and change its conductivity 5,6 . This type of polymeric material, also known as organic mixed conductors (OMCs) 7,8 , that can support mixed ionic and electronic conduction, exhibits many advantages for interfacing with biological systems. First of all, the modification of πconjugation and side-chain groups can be used to engineer the properties of the polymer 6 .

For example, for a bithiophene-thienothiophene backbone (2T-TT), when the side chain is switched from alkoxy side chain to glycol side chain, the ionic injection into the film is facilitated without decreasing electric motilities 9 . Nielsen et al. have shown that the molecular design of the backbone of a series of conducting polymers can be tuned in many aspects, such as ion and hole transport, electrochromic properties, operational voltage, and material stability 10 . Second, the organic conducting polymer used as the active material for bioelectronics can be deposited with wide-range of methods avoiding high-temperature processing 6 . The polymer can be positioned at the desired area with a solvent process, such as spin-coating 11 , inkjet-printing 12,13 , screen-printing 14 , and in-situ electrochemical polymerization 15,16 . Third, the OMC provides an oxide-free interface between a biological system and the devices 1,5 . Unlike silicon, OMC has no dangling bonds, so it has no active sites to form oxides 6 . Fourth, the conducting polymers provide great ionic transport at room temperature 6 . Relatively large distances and weak van der Waals forces between the organic molecule chains allow water molecules and hydrated ions to move into the material 6,8,17 .

Lastly, the injection of the ions into the organic materials can modulate the doping states, and directly influence the electric carrier population 5 . Because of this, the OMC is able to transduce ionic signals from biological systems into electronic signals.

Alongside these key features of common OMCs, PEDOT:PSS, as the all-around champion material also has shown many more great properties. Biocompatibility 5 , tissue-like mechanical properties 18 , transparency 19 , long-term stability in cell culture 20 , resistance to physical deformation 21 , autoclavabilty 22 , and commercially availability are highlighted advantages that have been proved and confirmed in the literature. Thanks to these features, PEDOT:PSS based bioelectronic devices can be introduced to diverse applications, such as electroactive cell recording 23 , barrier-tissue integrity monitoring 24 , metabolite sensing 25 , drug delivery 26 , bioimaging 19 , and many other fields of applications.

Organic Microelectrodes

Device physics and Characteristics of OMEs

Organic microelectrodes (OME) are one of the common devices applied across a wide range of biological sensing applications and studies. Since the invension of conducting polymer, researchers started to develop organic microelectrodes (OMEs) by coating traditional metal microelectrode with conducting polymers as the active-functional material 27,[START_REF] Cui | Surface modification of neural recording electrodes with conducting polymer/biomolecule blends[END_REF] .

Working Principle of OMEs

To describe their working principle, the energetic diagram of an organic electrode is demonstrated in Figure 2(a) to illustrate the charge transport in 5 phases. From the working electrode, a hole is injected into the highest occupied molecular orbitals (HOMO) (Phase 1) [START_REF] Berggren | How conducting polymer electrodes operate[END_REF] .

The holes can be transported through the delocalized HOMO throughout the polymer chain (Phase 2) [START_REF] Berggren | How conducting polymer electrodes operate[END_REF] . Meanwhile, the anions are drifted by the potential applied by the counter electrode toward the polymer film (Phase 3) [START_REF] Berggren | How conducting polymer electrodes operate[END_REF] . Then, the anions can travel within the free volume between the polymer crystals and chains (Phase 4) [START_REF] Berggren | How conducting polymer electrodes operate[END_REF] . Lastly, the hole and the anion electrostatically compensate each other at a combination site (Phase 5) [START_REF] Berggren | How conducting polymer electrodes operate[END_REF] .

For the traditional metal (such as Au) electrode, the anions directly compensate with the accumulated positive charges at the metal-electrolyte interface forming electric-double layer, which can be considered as a capacitor. Likewise, the OMC film possesses an ion-storage feature also behaving like a capacitor. Also, the OMEs have much free volume within the OMC film, so they have more surface area to electrostatically stabilise the injected anions. Thus, in the aspect of capacitance, the OMEs have much higher capacitance than the metal electrodes.

As a result of this, the organic electrode with a larger effective area for ionic-electronic transduction lowers impedance compared to bare gold electrodes with the same size (Figure 11,20 . This relates to the charge-storage capacity of OMC, and this property can be depicted with its volumetric capacitance (C * ) [START_REF] Rivnay | High-performance transistors for bioelectronics through tuning of channel thickness[END_REF][START_REF] Proctor | Understanding volumetric capacitance in conducting polymers[END_REF] . Moreover, the impedance of a the overall capacitances can be expressed by equation 1:

(b))

Zc= 1/(j2πfC) (1)
where f is the frequency; C is the capacitance; j is the imaginary number unit. Therefore, the impedance of OMC is inversely proportional to its capacity. The OMC is a highly-capacitive (high C * ) material [START_REF] Rivnay | High-performance transistors for bioelectronics through tuning of channel thickness[END_REF][START_REF] Proctor | Understanding volumetric capacitance in conducting polymers[END_REF] , so the OMC coating causes the decrease of the impedance when measured via electrochemical impedance spectroscopy 16 .

Steady-State Characteristics Explored by Cyclic Voltammetry

The ion injection/ejection and ionic impedance behaviors are the intrinsic device properties of OMEs, which can be categorized with steady-state and transient characteristics, respectively. The ion injection and ejection into the OMC film can be explored by voltage sweep with a constant scan rate, whereas the ionic impedance can be investigated by sinusoidal voltage wave with frequency variations. To analyze these properties, the threeelectrode configuration is usually established. As Figure 2(a) demonstrated, OME is the working electrode (WE) carrying out the measurement of interest. The current is applied by the counter electrode (CE) to sustain the ionic interaction within the conducting polymer of the WE. The reference electrode (RE) essential to control the potential and avoid potential fluctuation causing by large current changes [START_REF] Kissinger | Cyclic voltammetry[END_REF] . The RE is usually made out of Ag/AgCl because it is a material operating under reduction-oxidation reaction. This eliminates the double-layercapacitance effect to maintain applied voltage throughout the electrolyte before reaching the WE surface. Ag/AgCl RE is usually kept in KCl solution, and in order not to contaminate the measured biological solution, a salt bridge is used to avoid diffusion of KCl solution.

For the steady-state characteristics, the cyclic voltammetry (CV) is the common analysis technique. CV is a traditional method to observe rapid redox reaction over a large range of voltage potential [START_REF] Kissinger | Cyclic voltammetry[END_REF] . Since the nature of redox reaction is gaining and losing of electrons, the characteristic curve describes the current response induced by reduction and oxidation reaction over a range of voltage. With the present of redox molecule (X), when the forward voltage scan induces a oxidation reaction, the anodic current rises and reaches a peak as the oxidation species X start to deplete while X + accumulate at the surface of the electrode [START_REF] Kissinger | Cyclic voltammetry[END_REF] .

Soon, the anodic current decline to the switching point (SP) where the species X + is zero at the electrode surface [START_REF] Kissinger | Cyclic voltammetry[END_REF] . Then, the reverse scan initiates the oxidation reaction converts X + back to X and the cathodic (reduction) current behave with the same fashion to the anodic (oxidation) current curve 32 . Due to its unique shape, the current-voltage curve is denoted as "duck-shape curve", and this redox reaction dominate behavior is called a Faradaic process (Figure 2(d)). Also, without redox molecules, some conducting polymer, such as Polyaniline (PANI) undergoes this Faradaic process as well. Its Faradaic process is related the conversion between its two redox-couples.

For the case of PEDOT:PSS and non-faradaic OMC coated microelectrodes, their operation involves ion injection and ejection instead of a redox reaction, and CV is used to monitor the electrostatic interaction ion charge within the OMC. When the forward voltage is applied, a large amount of previously diffused cations (ex. Na + ) are extracted and a great number of anions (e.g. Cl -) are injected 17 . Then, this doping process comes to saturation with a small increase of cathodic current 17,[START_REF] Dijk | Influence of PEDOT:PSS Coating Thickness on the Performance of Stimulation Electrodes[END_REF] . When, the reverse scan start, the de-doping process quickly begins with cation injection and anion extraction 17 . Because of the shape of this CV curve, the PEDOT:PSS (usually scan voltage range from 0 -0.5V) represent a common behavior called "box-shape curve". These OMC coated electrode operation involves charge compensation, so this phenomenon is also named as the capacitive process (Figure 2 (b)) [START_REF] Berggren | How conducting polymer electrodes operate[END_REF] .

Researchers recently obtained a deeper understanding of the properties of OMEs based on their steady-state measurements. Ganji. et al proved that PEDOT:PSS coated microelectrodes enhance up to 9.5 times of charge injection capacity compared to bare metal (Au) electrodes, and the maximum injected current increases with scaling up the OME size [START_REF] Ganji | Scaling Effects on the Electrochemical Stimulation Performance of Au, Pt, and PEDOT:PSS Electrocorticography Arrays[END_REF] . Also, PEDOT:PSS/Au OMEs demonstrated the excellent capability of convert available stored charge into injected electrical pulse (high charge injection capacity/charge storage capacity ratio) [START_REF] Ganji | Scaling Effects on the Electrochemical Stimulation Performance of Au, Pt, and PEDOT:PSS Electrocorticography Arrays[END_REF] .

By combining the measurements of CV and quartz crystal microbalance (QCM), Savva. et al.

showed that the thickness of the film contributes to the de-doping efficiency 17 . The thicker PEDOT:PSS film (~300 nm) can receive much more cations than the compensation sites and trap more cation and water molecules within the film even when the driving potential is paused 17 . If a sensitive ionic current detection or delivery is needed, the thickness of the film is a direct factor to be considered.

Frequency-dependent Characteristics Explored by Electrochemical Impedance

Spectroscopy

For analyzing the transient characteristics of OMEs, electrochemical impedance spectroscopy (EIS) is a typically used technique. It also adopts a three-electrode configuration (Figure 2 (a)). Besides the working (WE) and reference electrode (RE) which control and measure the potential difference, a sinusoidal current is applied across the WE [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] . With this setup, the impedance spectra can be recorded for the analysis and study of the impedance characteristics of the OMEs with/without the biological system. According to Ohm's law, the impedance under an applied alternating voltage is determined by the division of the applied alternating voltage with the recorded alternating current signal [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] . The Impedance magnitude and phase can be plotted against the corresponding voltage frequency, which is usually called the Bode plot (Figure 3 (a)). In the ideal case, because the impedance of a resistor is independent of the frequency, it is shown almost like a horizontal straight line [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] . The capacitive component can be expressed by equation 1, so when both the y-axis (impedance) and x-axis (frequency) is plotted on a logarithmic scale, a capacitor's behaviors is shown as a straight line with a slope of -1 [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] . The OMC-coated microelectrode has both resistive (electrolyte resistance (Re)) and capacitive (OMC capacitance (Cp)) behaviors and the equivalent circuit is shown in Figure 3 (b) 16 . Therefore, at higher frequencies (10 2 -10 5 Hz), the electrode is in the resistive domain, which behaves almost like a horizontal segment (Figure 2 (d)) 16 . At low frequencies (10 -1 -10 2 Hz), the same electrode operates in the capacitive domain 16 . Furthermore, the voltage is often applied as a sinusoidal waveform, so the current response could have a time shift caused by characteristics of the circuit elements. This time shift is often expressed as an angular phase difference between the voltage and current waves [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] . This phase shift is plotted against each frequency on a phase diagram (Figure 3 (a)). A resistor's current response is immediate, so the phase is located around 0°. As a result of capacitance inducing current lagging 90° from the voltage input, a horizontal flat line appears along the 90° mark. Therefore, for an OME, at the resistive domain, the phase remains around 0°, and once reaching the capacitive domain, the phase stays at 90° [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] .

Independent of whether the electrodes are bare metal or coated with any conducting polymers, the impedance spectra always demonstrate the "universal curve" in electrochemistry with a 45°-slope-plateau shape. Owning to the impedance of the capacitive component being very high at high frequencies and the resistive component dominating the whole circuit at low frequencies, the pure resistance and capacitance behaviors are always isolated in an impedance spectrum. Even if the equivalent circuit is complicated by integrating biological systems, the resistance and capacitance of both electrode and bio-substances can be discerned. This components impedance spectrum is tightly related to equivalent circuit modelling, which will be discussed in a later section. Furthermore, a Nyquist plot in EIS is another way to illustrate impedance characteristics, which plots the imaginary part versus the real part of the impedance. It displays the real and imaginary parts, so it is easy to note the impedance contribution due to each equivalent circuit component. For example, since a biological barrier has both resistive and capacitive features, they can be expressed separately as the real part and imaginary part of the impedance, respectively. Hence, it is a complementary graph to the impedance information for the Bode plot. However, the Nyquist plot does not explicitly plot the frequency, so the frequency of each point has to be traced back to the raw data [START_REF] Loveday | Evaluation of organic coatings with electrochemical impedance spectroscopy: Part 1: Fundamentals of electrochemical impedance spectroscopy[END_REF] .

Material Choice

Recently, many organic mixed conductors have been heavily developed to promote the performance of organic electrochemical transistor (OECT), which consists of OMC layer as the channel linking the source and drain electrodes, and a gate electrode injecting ions into the OMC to dope or dedope the channel. Some of these novels and highly performing materials have not much been evaluated on MEAs for certain biological applications. However, the electronic and ionic conductivity properties of these materials from OECTs can also be the figures of merit to direct OMEA applications. For example, Electronic mobility (µ) and volumetric capacitance (*C) are the crucial properties for OECT performance, and these two properties combining with other OMEA properties, such as redox activities (from CV) of some promising and popular OMCs from OECT characterization are summarized in Table 1.

Besides PEDOT:PSS, some other conducting polymer materials have been coated onto microelectrodes and OECTs. These conducting polymers have reported conductivity ranges from a single digits to hundreds of S cm -1 , and band-gaps are around 1-3 eV. First of all, polyaniline (PANI), p-type semiconductor, shows conductivity when present in acidic environments (pH < 3) [START_REF] Dhand | Recent advances in polyaniline based biosensors[END_REF] . When the nitrogen atoms on the PANI backbone are protonated, bipolarons are formed, and the cation radicals work as holes. The hole continuous can form on the backbond by successive transforming of bipolaron into polarons. The attractive property of PANI is its two redox-couple: leucoemeraldine (LE) can be oxidised to emeraidine (ED) form, and then ED is oxidised to Pernigraniline (PG) as the fully oxidized state [START_REF] Dhand | Recent advances in polyaniline based biosensors[END_REF] . The tworedox couple of PANI can facilitate the electron transfer between polymer and enzyme without extra charge-transfer mediators, so it provides a simplified metabolite sensing system [START_REF] Karyakin | Processible polyaniline as an advanced potentiometric pH transducer. Application to biosensors[END_REF] . However, the presence of oxygen induce ageing problem of PANI when the potential is around 0.5 mV, and the PANI has a dramatic loss of conductivity after a few CV cycles [START_REF] Dhand | Recent advances in polyaniline based biosensors[END_REF] . Moreover, polypyrrole (PPy) as one of the earliest CP used in the field of organic bioelectronics, It intrinsically has very high volumetric capacitance (400-500 F cm -3 ) 38 .

However, the dense arrangement within the bulk block the dopant ion access to the interior sites, so a thicker PPy does not have a high capacitance per mass [START_REF] Snook | Achieving high electrode specific capacitance with materials of low mass specific capacitance: Potentiostatically grown thick micro-nanoporous PEDOT films[END_REF] . PPy also shows conductivity degradation once the voltage potential is applied above 0.3-0.4 mV [START_REF] Kros | Poly(pyrrole) versus poly(3,4ethylenedioxythiophene): Implications for biosensor applications[END_REF][START_REF] Yamato | Stability of polypyrrole and poly(3,4ethylenedioxythiophene) for biosensor application[END_REF] .

In the case of polythiophene (PT), it has very high conductivity around 300 -400 S cm -1 , and it is very stable in air and moisture at its p-doped and undoped forms [START_REF] Snook | Conducting-polymer-based supercapacitor devices and electrodes[END_REF] . However, It has very low n-doped potential (lower than -0.2 V) and loses conductivity due to oxidation at n-doping state, so the Pt-based devices do not have the desired life-time [START_REF] Snook | Conducting-polymer-based supercapacitor devices and electrodes[END_REF] . To overcome these problems, researchers have found that once phenyl, ethyl, or alkoxy groups substitute into the position 3 carbon on the thiophene ring (Table 1), the electric properties are greatly preserved for longer term [START_REF] Snook | Conducting-polymer-based supercapacitor devices and electrodes[END_REF][START_REF] Villers | The Influence of the Range of Electroactivity and Capacitance of Conducting Polymers on the Performance of Carbon Conducting Polymer Hybrid Supercapacitor[END_REF] . The well-known PEDOT-based materials with excellent stability at n-and p-doping conditions are typical examples of this modification [START_REF] Villers | The Influence of the Range of Electroactivity and Capacitance of Conducting Polymers on the Performance of Carbon Conducting Polymer Hybrid Supercapacitor[END_REF] . However, if just by PEDOT itself, it has low specific capacitance (92 F g -1 ) [START_REF] Snook | The measurement of specific capacitances of conducting polymers using the quartz crystal microbalance[END_REF] , because its highly-packed interchain structure caused by high molecular weight [START_REF] Ouyang | Imaging the Phase Separation between PEDOT and Polyelectrolytes during Processing of Highly Conductive PEDOT:PSS Films[END_REF] . When PEDOT is doped with poly(styrene sulfonate) (PSS), PSS can function as an ion conductor, which can greatly improve the ion transport and capacitance within the film [START_REF] Yamamoto | Controlling the Neuromorphic Behavior of Organic Electrochemical Transistors by Blending Mixed and Ion Conductors[END_REF] . Nevertheless, one of the drawbacks of PSS addition is that bulky PSS chains can lower the PEDOT volume faction, so the sulfonate anion/hole pair within the material is limited and hence the volumetric capacitance [START_REF] Proctor | Understanding volumetric capacitance in conducting polymers[END_REF] . One way to increase volumetric capacitance is to increase the anion/hole sites by doping PEDOT with smaller anionic molecules [START_REF] Proctor | Understanding volumetric capacitance in conducting polymers[END_REF] . Once PEDOT is doped with small molecules, such as tosylate (TOS, the pendant group in PSS, Figure 5 (f)), much anion/hole sites are created and the C* is improved with a neglected change of electronic mobility 5,[START_REF] Inal | Benchmarking organic mixed conductors for transistors[END_REF] . A large range of PEDOT-based materials are created by mixing with different additives and polymerizing with diverse methods to achieve versatile properties, and these materials have been summarized in a recent review [START_REF] Donahue | Tailoring PEDOT properties for applications in bioelectronics[END_REF] . Among these polymers, p(g2T-TT) is currently holding the record of highest µ and *C values since 2016 9,10 . Researchers have shown that the strategy of increasing the glycol side chain on p(gNDI-g2T) increase the ionic capacitance [START_REF] Paterson | Water stable molecular n-doping produces organic electrochemical transistors with high transconductance and record stability[END_REF] . When NDI-T2 copolymer with alkyl chains demonstrate high electric mobility, but it has a great loss of mobility when 25% of the alkyl chains are replaced by glycol chains [START_REF] Giovannitti | The Role of the Side Chain on the Performance of N-type Conjugated Polymers in Aqueous Electrolytes[END_REF] . Therefore, side-chain engineering is a new route to modify the electric and ionic properties of OMCs. Furthermore, to the best of our knowledge, different biological conditions have not been systematically studied or tested for the for these new materials, so the characterization of them under various biological-relevant environments should be one of the future focuses of investigation, and the knowledge will help us to expand the library of OMC for bioelectronics. High µC* value (228 F cm -1

V -1 s - 1
) with high mobility (0.9 cm 2

V - 1 s -1
) and high volumetric capacity

(241 F cm -1
).

Performance can be modified with side-chain engineering.

Lack of more systematic studies for biological applications.

cm 2 V -1 s -1
) 54

Organic Microelectrode arrays: fabrication and micro manufacturing

In this section, we will describe conventional microfabrication of MEAs, and then discuss the adaptation and options available when integrating organic materials into standard microfabrication processes.

MEAs are dependent on fabrication within specialized laboratories. Microfabrication 55 takes place in the specific environment of a cleanroom. The cleanroom provides a "clean" environment that ensures the quality of the product fabrication steps. Submicron to micron sized particles are maintained at low levels during the multiple steps of the process thanks to High Efficiency Particles Air (HEPA) filters that prevent the particles from landing on the substrate and affecting the MEA fabrication. The cleanliness of each room is labelled "class °" (or "ISO °"), where ° indicates the number of particles per cubic meter. Humidity and temperature are also maintained constant for the chemical stability of different materials used during microfabrication. Specific lighting may also be required, for example no UV lights, to ensure integrity of photosensitive materials.

A microelectrode array is made of 4 distinctive parts:

1. Substrate: Platform on which every element of the MEAs is built during microfabrication steps. The material chosen, and its surface characteristics must guarantee a good thermal and chemical stability. Adequate surface topology should be optimized where adhesion of material layers is required. 2. Conductive tracks (CT): Lines, usually made of a material with an excellent electrical conductivity, carrying electrical signals obtained from the electrophysiological recording. They end at a contact pad connecting to the hardware systems. 3. Insulation layers: Coating made from electrical insulating material beneath and top to the CT. They prevent cross talking in between tracks and protect the MEA circuitry from the external environment (humidity, oxidation, mechanical stress etc...). 4. Interface: Areas in contact with the biological system studied. It is not covered by the insulation layers as they record electrophysiological activity from the model. Made of biocompatible materials, usually metals (Gold, platinum, ITO…), they form the electrode sensing/effective area.

Each of these four MEAs component influence the fabrication process, hence, the material used. As described in the previous sections, organic materials are quite often sensitive to temperature, harsh chemicals and sometimes light. In the following section, we describe typical processes for fabrication of inorganic MEAs, and go on to describe the adaptations required to adjust a fabrication protocol for use with organic materials.

1.4 What are the constraints and limitations when integrating organic materials in the devices?

Substrate:

The

Conductive tracks:

The second category of material answers the requirements of carrying the electrical information recorded, to the contact pads. Conductive tracks (CT) are usually made of material with low impedance and high signal to noise ratio properties, to ensure a good quality of the signal recorded. Furthermore, if encapsulation cannot be achieved at 100%, biocompatible materials are preferred during elaboration of the MEA to reduce toxicity risks. In that sense, gold, platinum, titanium and iridium titanium oxide are widely present as conductive track materials. Where gold is more ductile and conductive, ITO 56 offers transparency and platinum a higher Youngs modulus, providing a better resistance to mechanical deformation that could occur during the fabrication process or on flexible device. Any material chosen to convey the electrophysiological information from the biological model to the hardware is to be in contact with the interface material: in our case, the OCM. The first characteristic and limitation to consider is the contact resistivity at the interface between the organic material and CT material. The thickness of the materials over layers impacts the behavior and quality of the recording as well.

Insulation/encapsulation

The encapsulation layer has to perform its purpose while remaining innocuous to the sensitive OM. Some microfabrication techniques and materials described below have shown their ability to match OM properties.

In industry [START_REF] De La Oliva | On the use of Parylene C polymer as substrate for peripheral nerve electrodes[END_REF] . This material provides good chemical stability, excellent conformability over rough surfaces, transparency and hydrophobicity 11 . Finally, PDMS has proven its mechanical and thermal stability, as well as a relatively acceptable performance as an encapsulation layer 62 in aqueous environments.

Deposition techniques for OMC

Spin Coating: As depicted in Figure 4 (a), the substrate is centered on a plate above a rotation axis in a bowl dedicated to spin-coated processes. This technique is used for OCM in liquid form, which are drop-cast at the center of the substrate. A homogeneous coverage of the material is obtained thanks to the high rotation movement of the substrate, spreading the OMC all over the surface. Micrometer to millimeter scaled thickness can be obtained depending on the viscosity and the amount of material dispensed, and the rotation speed of the platform. The geometry of the substrate influences the edge effect appearing during spinning, making film thickness inhomogeneous on the extremities of the substrate or on nonplanar surfaces [START_REF] Tait | Spray coated high-conductivity PEDOT:PSS transparent electrodes for stretchable and mechanically-robust organic solar cells[END_REF] .

Spray coating: droplets of the OMC are sprayed on top of the device (Figure 4 (d)), forming a layer by accumulation. The micrometer-sized drops coat the surface homogeneously and it is theoretically uses less material than the spin-coating technique. This also offers a better homogeneity of coverage on edges and textured surfaces than achievable via spin coating.

The solvent evaporation rate during flight is the first parameter to consider, as is the viscosity of the resist chosen. Droplet propulsion speed, temperature and solvent concentration in the atmosphere are the three other parameters to consider when elaborating the fabrication protocol [START_REF] Tait | Spray coated high-conductivity PEDOT:PSS transparent electrodes for stretchable and mechanically-robust organic solar cells[END_REF] .

Thermal deposition: Deposition of organic polymers in their solid form can be achieved through thermal heating. The substrates are placed on a plateau facing the material source, in a thermal /electron beam metal evaporator vacuum chamber [START_REF] Ryynänen | Transparent microelectrode arrays fabricated by ion beam assisted deposition for neuronal cell in vitro recordings[END_REF] . The surfaces face the metal targets crucible at the bottom of the machine, while undergoing rotation to ensure homogeneous deposition. An electrical current, or a laser beam, is applied on the crucible containing the OMC performing sublimation phase. A crystal placed inside the machine measures the thickness and deposition rate. Depending on the material, a faster deposition will result in bigger aggregate of particles than a slow evaporation rate, slightly changing he mechanical and electric properties of the material.

Chemical vapor deposition (CVD):

The chemical vapor deposition technique allows formation of a thin film, and uniform coating over patterned surface. A thin film is formed via a thermal induced chemical reaction at the substrate surface. Reagents present in their gaseous form in the environment will dissociate or chemically react upon heating, plasma or light activation.

Some applications targeting PEDOT CVD deposition [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly(3,4-ethylenedioxythiophene) films[END_REF][START_REF] Dominguez-Alfaro | Tailored Methodology Based on Vapor Phase Polymerization to Manufacture PEDOT/CNT Scaffolds for Tissue Engineering[END_REF] have been elaborated.

Electrodeposition: This process schemed Figure 4 (c) revolves around deposition of materials on conductive substrates [START_REF] Murbach | In situ electrochemical polymerization of poly(3,4ethylenedioxythiophene) (PEDOT) for peripheral nerve interfaces[END_REF][START_REF] Carli | Electrodeposited PEDOT:Nafion Composite for Neural Recording and Stimulation[END_REF] . The electrodeposition technique can be divided in two different categories:

• Electroplating 69 : inducing a potential between a substrate (cathode) and an electrode made of the desired material (anode) to coat the substrate, dipped in an electrolyte containing the ions corresponding to the material. An oxide reduction reaction occurs in electrolyte, the cathode being the substrate to receive the coating, and the anode an electrode made of the same material.

• Electrochemical 70 : the CP coating onto the surface of a conductor substrate is done by electrolysis. Ions of the CP are in suspension in a solution in which the substrate is dipped and electrolysis is performed. Two subtypes are available : constant voltage and constant current deposition.

Electrospinning. This new process produces long fibers of polymer 7172 , hydrogels [START_REF] Creighton | In situs 3D-patterning of electrospun fibers using two-layer composite materials[END_REF] , metal [START_REF] Miyamoto | Inflammation-free, gas-permeable, lightweight, stretchable on-skin electronics with nanomeshes[END_REF] and even composites [START_REF] Creighton | In situs 3D-patterning of electrospun fibers using two-layer composite materials[END_REF] , with a diameter ranging from the micrometer to the nanometer scale [START_REF] Kim | Low-Cost Fabrication Method for Thin, Flexible, and Transparent Touch Screen Sensors[END_REF] . Easy to process, the electrospinning method requires a solvent in which nanoparticles of the OMC are dispersed [START_REF] Chronakis | Conductive polypyrrole nanofibers via electrospinning: Electrical and morphological properties[END_REF] . A simple set up detailed Figure 4(a) involves a syringe containing the mixture, and a voltage applied at its needle. The parameters defining the diameter size and density of the fibers are: the intensity of the electric field applied, the viscosity of the mixture and the concentration of nanoparticles, the humidity in atmosphere, distance between needle and target substrate.

Bar coating and doctor blading: Bar coating and doctor blading are similar techniques. Excess amount of material is deposited on the substrate. While the first one involves running a blade over the substrate, the second one uses a bar, and spreads the material on the surface. The gap between the tools and the substrate define the thickness of the material deposition.

These methods require a certain viscosity of the material, and thickness under 10 micrometers cannot be achieved [START_REF] Elschner | The spectral sensitivity of PEDOT:PSS films[END_REF] . Nevertheless, these methods are inexpensive and easy to process, and are often used in research and industry.

Additive materials for improving adhesion and biocompatibility

Conductive polymers are interfacing biology and its harsh environment: humidity, proteins, serums and cells themselves can be damaging to the sensitive material and its device. One common issue regarding fabrication and longevity of the bioelectronic structure is delamination and foreign body response. In this regard, studies have been made to improve adhesion of the CP to the device, and its contact to the cells. These can be categoriesed into three types: . Mechanical, chemical and biological.

For delamination of OMC, surface topology of the material withstanding the OMC can be tuned to improve adhesion. Creating roughness impacts the surface tension properties [START_REF] Cui | <Electrochemical-deposition-and-characterization-of-poly-3-4ethylenedioxythiophene-on-neural-microelectrode-arrays_2003_Sensors-and-Actuators-B-Chemic[END_REF]

Patterning techniques for organic materials

Lithographic based techniques

Photolithography is a fabrication technique widely used in microelectronics, allowing to transfer patterns onto a substrate via shadow mask and several electron or light exposures.

Those operations are based on chemical and physical properties of materials and resists, labelled electron sensitive and photosensitive respectively. Their main property changes the chemical structure when electrons or a certain UV wavelength is applied to the material. For photosensitive resists, it modifies the dissolution rate of its areas exposed. For OMC, chemical bonds structure are changed [START_REF] Rutledge | Etch-free patterning of poly(3,4ethylenedioxythiophene)-poly(styrenesulfonate) for optoelectronics[END_REF] . For photosensitive resists, the non-cross-linked parts are removed when the carrier is dipped into the appropriate developer, displaying the electronic circuit in a negative or positive tone, depending on the type of resist. Two step options are following in order to achieve the micro patterning by removing the excess material: etching or lift-off.Based upon photolithographic techniques, patterning of OMCs can be achieved directly or indirectly.

Etching techniques

Etching techniques require that the OMC has been deposited before the photosensitive resist. For the specific application of patterning resist over such sensitive materials, three requirements have to be fulfilled:

• The resist, solvent and developer used through photolithography steps should be chemically inert to the OMC.

• The UV range applied over the device during exposition should not interfere with the OM electrical and chemical properties.

• Thermal baking of the resist should be at a temperature that the OMC can withstand.

The etching step is divided in two categories ( (a)): Dry (plasma) and wet (chemical) etch. The first requires a plasma generator and gas delivering system inside a chamber under vacuum. The second is a quartz tank filled with liquid etchant that can be heated. In both cases, three main mechanisms are necessary to achieve material removal. The flow of etchant over the substrate, the surface treatment (chemical and physical reaction) and the material removal. Reactive Ion etching machine [START_REF] Laermer | Deep Reactive Ion Etching[END_REF] : Compared to wet etching, the material is not submerged in a liquid containing etchants. This technique revolves around chemically reactive plasma etching.

The material is eroded, combining two effects, mechanical and chemical reaction with the ionized gas in the chamber.

ION BEAM etching Milling machine:

A specific type of dry etch. Compared to other methods, the process doesn't require high pressures. A larger variety of materials can be etched.

Indeed, one key aspect of this tool is that it can remove material solely by physical bombarding, hence giving the tool a broad spectrum of material etch ability. The substrate is fixed on a rotating stage/platform, slightly tilted in front of the plasma source, at a certain distance, hence, the substrate is not surrounded by the plasma, and no radiation damage is received. Onto this plasma created inside the chamber, a direct beam is applied to control ion trajectory and energy toward the substrate.

Lift-off techniques

In opposition to the etching technique, where the material to pattern is deposited before the resist, the lift-off technique creates the patterning of the resist before the deposition of the material. When dipped in the appropriate solvent, the resist is removed alongside the OMC deposited over it. This process schemed Figure 5 (b) will mainly fit anisotropic types of material deposition, since the sidewalls of the resist pattern mustn't be covered in order to ensure a good lift of the material. Furthermore, from the thermal deposition, the resist used could undergo strong cross linking due to long exposure of the substrate to high temperatures in the chamber, hence making the lift-off difficult. This process technique is used if the resist employed for etching (dry or wet) have poor adhesion properties to the material that need to be patterned.

In the case of organic patterning via lift-off technique, two options are available 11,[START_REF] Defranco | Photolithographic patterning of organic electronic materials[END_REF] :

• Use of resist patterned via photolithography as a lift-off material. The OMC is deposited over the patterned layer, and once stabilized, the resist is removed in the appropriate solvent.

• Resist is patterned over third type of material as a hard mask for etching step. Once patterned, OMC is deposited over the layers. Then, the material is peeled-off manually leaving only the OMC on the surface.

UV / laser patterning

An alternative option to pattern an organic film sensitive to light is via UV patterning [START_REF] Rutledge | Etch-free patterning of poly(3,4ethylenedioxythiophene)-poly(styrenesulfonate) for optoelectronics[END_REF] . It has been shown that under proper wavelength exposure, PEDOT:PSS thin layers increase in resistivity [START_REF] Duffy | Synthetic bioresorbable poly-α-hydroxyesters as peripheral nerve guidance conduits; A review of material properties, design strategies and their efficacy to date[END_REF] . By doing so, the OM loses conductivity on the area exposed to UV (Figure 6 (b)).

This process allows patterning of the material conductivity, which is different from material removal patterning. It is free from any etching step and resist patterning, thus preserving the PEDOT:PSS film chemical integrity. Laser ablation of OM has been used to create porous organic membrane 86 .

.

Soft lithography patterning / Stamping

To pattern OM, soft lithography is a technique based on printing/stamping with a soft elastomer (usually PDMS) pre-patterned through photolithography [START_REF] Zhang | Conducting Polymer Nanowires and Nanodots Made with Soft Lithography[END_REF] . This low-cost technique (Figure 6 (a)) can be divided into 3 subsections: micro-contact, replica and solvent assisted micro-molding.

Hybrid deposition and direct patterning of organic materials Printing /additive processes

As additive manufacturing rises as a reliable and widely used process in many different industries, its translation to the microelectronic field is recent [START_REF] Hebner | Ink-jet printing of doped polymers for organic light emitting devices[END_REF][START_REF] Bharathan | Polymer electroluminescent devices processed by inkjet printing: I. Polymer light-emitting logo[END_REF] . Indeed, precision and critical dimension of resolution were restricting parameters for their used in microelectrode fabrication [START_REF] Bao | Materials and fabrication needs for low-cost organic transistor circuits[END_REF] . Technical limitations have been overcome and new opportunities and capabilities are available in fabrication processes for microelectrodes; stretchability, bioresorbability and conformability is now feasible through printing techniques for biosensors [START_REF] Didier | Capabilities and limitations of 3D printed microserpentines and integrated 3D electrodes for stretchable and conformable biosensor applications[END_REF] . Printing on fragile scaffold, such as bioresorbable materials, is conceivable [START_REF] Voss | SI -Additive manufacturing of hydrogel-based materials for nextgeneration implantable medical devices[END_REF][START_REF] Adly | Printed microelectrode arrays on soft materials: from PDMS to hydrogels[END_REF] .

Inkjet printing techniques are enlarging their formulations to enable new material deposition such as ITO 100 or organic inks 101 .

Dip-pen / Nano lithography and Scanning-probe-based lithography

Dip-pen nanolithography is a direct printing-based technique using AFM. Similar to nanoimprint, this patterning technique uses probes and tips of the AFM to deposit ink materials at a nanoscale precision [START_REF] Lim | Electrostatically Driven Dip-Pen Nanolithography[END_REF]102 Similarly, scanning probe lithography patterns materials using scanning probes, and can reach 10nm features precision. In contrast to the dip-pen technique, the scanning probe created an electrochemical reaction at the surface of the OM to change its structure: It locally crosslinks the polymer over the substrate.

Applications of OMEAs

OMEA have been widely used for the biological studies and medical research. Here, we generally categorise mainstream MEA related applications either as in vivo or in vitro, and within these two categories, the overview of each subcategorises are based on the device configurations, such as surface conformable OMEAs, deep-insertion OMEA probe, and rigid planar OMEAs, as illustrated in Figure 6. Generally, the OMEA probe-brain recording systems are modelled with the equivalent circuit in Figure 8 (a). Ve usually is the biological voltage source, and Rspread is the resistance of the extracellular space (media) between the active cell and electrode 105,106 . Re and Ce are electrode resistance and capacitance, which are related to the intrinsic properties of the polymer coating, and Rm is the resistance of metallic connections 105,106 . In the case of neuron stimulation, the same circuit applies. On one hand, a large electrode area can reduce the Rspread and increase Ce having advantages to detect local field potential (LFP) for a population of neurons 106 . On the other hand, a smaller electrode has larger Rspread and areal reduction of Ce. For smaller size electrode for tissue implatation, a the high capacitance and low resistance OMC coating can still sustain high injection limit and avoiding voltage drop at the interface, so such microelectrodes are excellent at recording a small number of cells 106 . However, the circuit of recording/stimulation (Figure 5 (b)) is an ideal situation of the recording circuit. Once the probe is inserted into the brain tissue, an inflammatory response can quick effect the recording sites by releasing proinflammatory factor and astrogliosis (forming scar around the probe) 110 . The microglia and astrocyts inflamation response and populate around the recording sites increasing recording and stimulation impedance. During this process, the resistance induced by these scar and dead/injured neurons forms an extra impedance layer frustrates the voltage propagation to the recording electrodes. Accompany with this, neuron population loses around the inserted probe, so the recording signal can also be weaken by the distance 107 . Martin's group have demonstrated that the probe with PEDOT coated electrodes showed a drastic decline of impedance at the recording site and increase the amplitude of the signal comparing to the uncoated iridium electrode [START_REF] Ludwig | Chronic neural recordings using silicon microelectrode arrays electrochemically deposited with a poly(3,4-ethylenedioxythiophene) (PEDOT) film * Related content Poly(3,4ethylenedioxythiophene)[END_REF] . The PEDOT electrodes sustained longer-term (40 days) through the whole immune response periods: initial trauma, early reactive response, and sustained immune response, especially after around 20 days, the PEDOT coated electrodes provide much high SNR compared to uncoated ones [START_REF] Ludwig | Chronic neural recordings using silicon microelectrode arrays electrochemically deposited with a poly(3,4-ethylenedioxythiophene) (PEDOT) film * Related content Poly(3,4ethylenedioxythiophene)[END_REF] .

From the device-design perspective, minimizing the probe size and matching Young's modulus of probe material with the tissue's are the two direct paths to reduce inflammatory responses 106 . Anikeeva's group applys thermal-drawn method produce filberic neuronal probe with diameter of crossesional area around a few hundred micrometer (Figure 8(d)). By selective etch of the cadding of the fiber, the fiber stiffness is greatly reduced to 5 N/m 108 . The brain implantation experiments show that the forgeign body responses, such as glial response and blood-brain-barrier (BBB) breach, is minimized, so two-month consistent recodings were achieved 108 . The thermal-drawn method can also be multifunctional by building in optical waveguides for optical stimulation and fluidic channels for drug delivery 108 . To bring the Young's modulus of probe close to tissues, Malliaras group frabricate PEDOT devices on 4 µm parylene flexible substrate and implant into rat cortex with SU-8 shuttle. The shuttle can be delaminated from parlene substrate after implantation and there is no noticeable gial scar even after 1 month implatation 109 . Based on this work, they recently also developed "NeuroRoots", which consists of multiple separated flexible electrodes 111 . The NeuroRoots are guided by microwire during implatation, and they can delaminate from the microwire and remain in the brain for long-term measurements with high desinty recording sites 111 . Besides reducing probe size and matching material flexibility, coating the electrode with bioactive molecules, such as extracellular matrix (ECM), is also applied to reduce inflammatory response 110 . ECM can greatly promote neuron attachment on to the recording site and enhance the signal from the source 110 . Even though OMC coated electrode probe can significantly reduce the forgein body response and measure the brain activity with high signal quality, but the demages from probing process can still be a unignoriable side effect. Then, Khodagholy and colleagues further reported another system of conformable OMEA, called "NeuroGird", to record LFPs and action potentials from surfaces of hippocampal and cortex 114 . The organic electrode dimensions and density (10 µm X 10 µm electrodes with 30 µm spacing) specifically matches the neural cell body size and density so that the action potentials from an individual neuron were stably recorded over 10 days 114 . Also, the LFP has monitored over a wide range of frequency implied to the mouse's daily movements and capsulation process even provides a 3D mapping of single field opential propagation across the spheroid 117 . Hence, recent various studies, such as long-term brain-health monitoring, point-of-care monitnoring, and even in vitro electrogenic tissue 3D mapping, not only have opened up our vision of conformable OMEAs , but also brought and guided our interests toward the further development their potentials in many more biomedical applications.

Planar OMEAs for In Vitro Applications

Planar OMEAs are the most common configuration found, and are used for many in vitro applications, such as metabolite detection, electroactive tissue and cell recording, and barrier cell and native cell membrane impedance sensing. Among these applications, OMC coatings show great versatility in sensing ion current, lowing signal impedance, and ease to functionalization, so the planar OMEAs catch increasingly amounts of attention in the field of bioelectronics.

Enzyme-Base Metabolite Sensing

Enzyme-based detection of metabolites and biomolecules from body fluid or gas is one of the most common applications of planar OMEAs. Generally, the detection mechanism is based on sensing the products of redox reaction or redox current generated by the metaboliteenzyme (redox) reaction. Usually, cathode or anode peaks of cyclic voltammograms are used to quantitatively measure the concentration of metabolite of interest. Glucose detection still dominates the market and research fields of metabolite biosensing 25,118 . Here, the development of glucose sensing with OMEA can be illustrated as a model. In many cases, glucose oxidase (GOx) is a specific recognition glucose enzyme, and it contains two molecules of cofactor, flavine adenine dinucleotide (FAD), which can oxidise glucose into gluconolactone meanwhile it accepts two electrons and yield hydrogen peroxide (Figure 9 (a)) 119 . Therefore, GOx-base sensors are designed by detecting either hydrogen peroxide or redox current.

Owing to the outstanding electric conductive of conducting polymer, OMEAs have been used to sense glucose concentrations through redox current (Figure 7 (a)-(c)). Furthermore, the layer formed from the enzyme might inhere an electron shielding effect, which frustrates the electron transport towards the electrode surface, 25 so a mediator is used to promote the electron transfer between enzyme and electrode surface 25 . Thanks to excellent electron transport properties of the conducting polymer, and once they can be intimately incorporated with the enzyme, they are qualified to be an excellent mediator to conduct redox current.

Electroactive Cell and Tissue Monitoring and Barrier Cell Impedance Recording

Similar to in vivo recording from brains, electroactive cell or tissue slides can be recorded by rigid planar OMEAs in a standard culture environment. Rigid OMEA as in vitro tool has been much investigated for pathology and medication to fight against the diseases related to electroactive cell/organ dysfunctions. Sessolo et al. fabricated OMEA with "parylene liftoff" method and successfully detected LFP and action potentials from slices of rat hippocampus with low noise (± 10 µV peak-to-peak) 11 . Taking advantage of the glass substrate, the recording site of each electrode was positioned with an optical microscope (Figure 8 (h)) 11 .This proved to be a powerful tool to spot the origin of the abnormal firing (i.e., epilepsy). However, there are two limitations of tissue slice recording. The first is that the presence of a layer of dead or injured cell acting as an insulator reduces the recording signals 11 . The other problem is that slice only can stay active for a short time period (several hours), so long-term study could not be carried out. Therefore, culturing electroactive cells on MEA is a more sustainable, simplified, low-cost, ethical-issue-free approach. Once cells are confluent on the filter, the TJ can form intercellularly, so the cell layer creates a barrier to inhibit the ionic flow and influence the impedance spectra 125,133,134 When AC voltage sweeps through mid-frequencies, the cell monolayer characteristics can be measured. Taking Caco-2 monolayers as an example, the frequency from 1 to 10 2 Hz explores the resistance effect of the paracellular pathway (between the cells), whereas at the higher frequency (10 2 -10 3 Hz), the current provides the capacitance characteristics of the transcellular pathway (through the cells) (Figure 10 (j)) 24,125,130 . Furthermore, the cell monolayer and transwell OMEAs are commonly modelled with the equivalent circuit shown in Figure 10(i). RMEM is the tight junction resistance between cells, and CMEM is the capacitance element contributed to the cell. Rs and CEL are electrolyte impedance and the electrode capacitance, respectively 125 . Through this equivalent circuit model, the impedance (capacitive and resistive) properties of the cell monolayer can be extracted through the fitting of the impedance spectra.

However, according to the work from Koursouras et al., the barrier cell-impedance characteristic can only be sensed by relatively large electrodes (5000 µm X 5000 µm and 10000 µm X 10000 µm). Essentially, the sensitivity is due to the ratio of impedance modulus of the cell barrier (|Zcell|) to the impedance modulus (|Zelectrode|) of the electrode ( Figure 10 (k)) 125 . On one hand, when the |Zelectrode| is greater than the |Zcell|, the system is in the non-sensing regime, so the electrode cannot pick up any impedance signals from the cells. On the other hand, when the |Zcell| is greater than |Zelectrode|, the electrode can sense the cell-barrier impedance, which is noticeable both in the Bode plot and phase diagram (Figure 10 (c)). Therefore, the size of the electrode is a very crucial parameter for designing sensitive microelectrodes for various biological applications.

Native Cell Membrane Impedance Monitoring

The cell membrane, as the outermost barrier of individual cells, functions as first defensive screen, nutrient selector, and supplier, which maintains a stable intracellular environment.

Cell-membrane-on-chip is recently becoming a popular topic on bioelectronics. Researchers have investigated Impedance properties of the native cell membrane with OMEAs. Pappa et al. demonstrated that human embryonic kidney (HEK) cell membranes were reconstructed on the PEDOT:PSS electrode which in turn sense the impedance increase compare to the bare electrodes, using EIS measurements 135 . The native membrane fused microelectrode can be simulated with an equivalent circuit shown in Figure 11 (a). The Cm and Rm in parallel represent the membrane resistance and capacitance, respectively, and the Rs is the electrolyte resistance above the microelectrode, and Cp is the capacitance of PEDOT:PSS coating 135 . With this equivalent circuit model, the membrane impedance characteristics Cm and Rm can be extracted 135,136 .

With this integration native cell membrane and OMEA, the ion channel activation and deactivation can be monitored with EIS measurements. On the HEK membrane, the TREK-1 potassium ion channel was overexpressed throughout the membrane, so when arachidonic acid (AA, a TREK-1 activation molecule) was added, it induced the ion flow through the membrane and the membrane impedance had a large drop (97% decrease) 135 . Spadin, a TREK-1 blocker, suppressed the ion current and caused the rising of membrane impedance 135 .

Besides the ligand-gated ion channel, Liu and colleagues also managed to read out the impedance reduction caused by open the ATP-gated PX2P-ion channel on baby hamster kidney (BHK) membrane with the same membrane-microelectrode system 136 . This innovative membrane impedance sensing with OMEA breakthroughs the limitation of both low throughput of ion-channel activity monitoring and cell viability maintaining from traditional ion channel monitoring methods (i.e., patch clamp), which is opening a new window for ion channel protein-related research and its drug screen studies.

Outlook

The recent rapid advances in the field of OMEAs truly demonstrates its capacity as implantable health monitoring devices, in vitro diagnostic sensors, tissue-on-chip models and many more. It will keep catching more attention in the field of medical care and lab research in the future. For further advancements in the field, there are few points of view worth considering. Firstly, to reduce the wiring resistance and fabrication difficulties, most of the wire design needs to keep a reasonable width and proper arrangement around the working electrode areas within the same plane. Thus, the wires occupancy may limit the packing density and deduction of the electrode sizes. The ideal separation of wiring and functional electrode into two (multi-) planes adapted from matrix technology might give more room to arrange a denser array-packing 137,138 . In the area of tissue or cell recordings, culture on the planar device is not representative of the native environment. Recently, researchers cocultured multiple cells within conducting polymer scaffolds and monitored cell growth with impedance recordings 139 . The conducting scaffold not only maintained the needs of cell growth but also create a 3D and more biologically relevant environment. The combination of the spacial recognition of an array and the scaffold culture could enhance the sensing resolution and improve cell growth environments. Furthermore, in the field of biosensing, "sponge-like" surface of conducting polymer is a great advantage of incorporation of the enzyme and conducting polymer, but the balance of enzyme coverage and charge transport is important to maintain proper sensitivities, which needs much investigation and experience in the field. Last but not least, the ongoing and increasing amount of collaborations among material chemists, device engineers, and biological/medical researchers all over the world should be highly encouraged to make OMEAs more powerful and promising tools to study biological events and fight against diseases.

12-well plate & Micro Electrodes Arrays

Introduction As shown in Figure 12, the in vitro models are evolving and getting more complex to get closer to the in vivo system 2 . Several parameters such as physical constraints, cell culture The transconductance is determined by the volume of the conducting polymer, hence the size of the channel 10 . An amplifier can be obtained by tuning the OECT geometries, and achieving high transconductance at zero hate bias 11 .They are able to detect ionic flux and its propagation occurring from an action potential 12 , measure cell barrier functionality and integrity during toxicology assay 13 , and can be functionalize to detect different metabolite in blood samples 14 . To achieve this concept, transparent Organic Micro Electrode Arrays (OMEAs) were integrated into the 12-well plate device structure. In this configuration, the biological models can be evaluated in any standard biological laboratory equipment.

I
Visual and electrophysiological studies can be performed simultaneously. Thus, this "smart" 12-well plate allows a multi parametric and simultaneous monitoring of the biological model studied.

In this section, I will describe the assembly of the "smart' 12-well plate detailing its different pieces: firstly, the plate/lid structure design and the material choices, then define the creation of the Printed Board Circuit (PCB). Afterwards, I will detail the design and fabrication of the OMEAs according to the restrictions and expectations from the in vitro experiment. Finally, in the last section, I will develop the assembly through material and electronic hardware used for both electrical connections and sealing, and the integration of the tool into standards biological plan and experiment.

Integration and advantages of Organic Micro Electrode Arrays in standard biological equipment

As described briefly in chapter 1, MEAs are adaptive tools and a solution that can be integrated in in vitro experiments and facilitate electrophysiological monitoring of more complex and elaborated structures such as co-cultures of biological models. As they are fabricated on glass or other transparent substrate, they allow optical and electronic characterizations simultaneously. Furthermore, the materials used for their fabrication process allows sterilization through ethanol, UV or autoclave 15 As seen in Figure 13, these setups can be bulky and troublesome for users, as they are time consuming, non-intuitive for biologists and might damage MEAs chips or the biological matter over repetitive measurements. The chips and their monitoring hardware must be redesigned and reshaped to facilitate in vitro experiments in laboratory.

Single MEA chip VS multiple MEAs chips on a well plate

Regarding user-friendly handling and ease of use, a parallel can be made between petridishes or multiple well plates, and single unit or multi-unit recording chips. Figure 14 represents the analogy of single VS multiple test experimentation in biological assays protocol.

Reproducibility is expected between the cell cultures, as well as for MEAs chips. Biological experiments should be conducted simultaneously and be repeatable. An additional asset derived from mimicking the design of this standard tool is that this "smart" well plate fits with biological equipment: incubator, hoods, microscope, autoclave etc. The concept of lab on chip is initiated in the development concept of the 12-well plate.

The assembly of biology, biochemistry and electronics are part of the exercise to develop these new diagnostic tools. Microfluidics, thermodynamic and mechanics are additional parameters added to create lab on chips. Here we are focusing on the electronic devices (MEA) layout and their integration in a biological structure (12-well plate), that can undergo thermal and chemical treatment.

Traditionally, MEAs are single unit chips, on which a well is attached, containing the biological element for the in vitro experiments. Various models have been investigated in the Bioelectronic laboratory through this set up: epithelial barrier formation over MEAs through cell culture insert (Transwells) 13 , action potential detection from neurons cultured directly on top of MEAs 16 , or wound healing assays 17 . They also were used to monitor 3D spheroids of coculture cells in microfluidic chip 18 .

As we are moving toward multi parametric and simultaneous recording system for several individual MEAs chip, their layout must be adapted to new requirements.

Choice of material

A transparent plastic, polyurethane, was chosen per the biological experiment requirements. The transparency of the material is mandatory as the visual control of the cell, volume and colors of media are important during experiments. Unfortunately, the cost of a biocompatible and transparent material was too high to be considered. To circumvent this issue, we decided to cover the overall 3D structure with PaC, which is biocompatible. The instruments has to undergo sterilization before going under any other laboratory tools, such as the incubator and the hood. Considering that the melting point of polyurethane is 98°C, it doesn't suit the autoclave sterilization process, which requires temperature of 120°C.

Deformations of the 3D printed circuit also occurred during the steam cycle for sterilization, rendering the tool inoperable. Therefore, the ethanol bath solution is the best approach to fulfill sterilization requirements. We did not consider UV sterilization because organic materials are known to be sensitive to UV 20 and the performance of the MEAs would be deteriorated. The "smart" well plate could be sterilized with ethylene oxide. Unfortunately, this option was not available in the laboratory.

PCB designs and fabrication

Well plates dedicated to biological experiments use a specific standardized design. To fit into standards equipment found in biological laboratory, a Printed Board Circuit (PCB) was sketched to dispose the MEAs correspondingly to a 12-well plate pattern. The printed board circuit assembling MEAs is designed using Dip trace software. On side A, the restrictive environment is attached with PDMS. On side B, MEAs are electrically connected to the PCB.

The conductive trace designed on this PCB gather all the connection pads of the 12 MEAs on a single edge, similar to a PCI slot. This enables easy access to the recorded data. The whole set up is maintained inside a microscope thanks to an aluminum holder. We have designed its structure to allow good optical focusing on the MEAs bottom part, and a solid holding of the tool to ensure security of the set up. By plugging the PCI slot into a flat cable extracting the data from the experimental area, it is possible to monitor optically & electronically biological events all together.

The first design draft integrated 12 MEAs in a 12-well plate design is shown in Figure 18.

The space available by the surface of the PCB was the limiting factor to draw the conductive lines between the chip and the external PCI connector. Consequently, the number of connective pads available at each of the 12 MEAs was reduced to a maximum of 8 pads. The lines conducting data to the PCI end were drawn on both the top and bottom side of the PCB.

Therefore, a total of 96 external PCI slots are spread equally on both ends.

The initial PCB design underwent modifications to achieve a higher number of sensing sites on the MEAs. As the external geometry itself cannot be expended due to standard dimensions, an extra conductive layer was added inside the PCB to allow more electric lines and contact pads to be drawn, hence increasing the number of sensing sites achievable. Inscription and numeration were directly printed on the PCB as illustrated in Figure 18. A second functional PCB was design for geometry and microscope lens focus purpose. In Figure 19 a), the PCI (peripheral component interconnect) slot needed to be elevated to reach the microscope plateau thanks to the PCB n°2 seen Figure 19 b). The PCB n°3 reorganized the electrical connection from the PCB n°2, allowing easy measurement connecting small banana slots and cables to harmonized with the NI system seen in Figure 19 d).

OMEAs design and fabrication protocol

The first OMEAs design integrated a choice of electrodes, OECTs or both at simultaneously in a single chip. They were made using glass slides as an easy and affordable substrate. The

Assembly

Electrical connection

The first attempt to create electrical connections between the PCB and the OMEAs was to use copper tape. It would be easily cut with laser but challenging to put in place precisely. The second attempt involved silver paste deposition by hand using a needle to drop the material on the PCB pads. This technique failed due to the gap existing on the PCB gold pads (500um), avoiding nanoparticles to form proper electrical contact.

The final solution adopted to create the electrical continuity between the PCB and OMEAs was obtained through a combination of adequate anisotropic conductive paste (ACP) and a semi-automated bonding machine. This technique allowed constant deposition of ACP material, reproducible and precise alignment of the OMEAs and PCB's connection pad

Sealing and biocompatibility (PDMS)

To prevent potential leakage of media or any other liquid from the wells, we have used PDMS as sealing material. The deposition was made manually on the bottom ring of the glass well and centered on the PCB holes. While hardening on top of a hot plate at 80°C for two hours, the polymer glides into the PCB hole, reaching the OMEAs close surface and performs adequate sealing. It also provides biocompatibility, and the joint prevents the biological matter from being in contact with the toxic material from the PCB. This material can also undergo autoclaving and ethanol sterilization mainly used in in vitro experiments. Its curing and thermal range of applications are also compatible with the PCB and OMEAs material and do not degrade any of the assembled components. PDMS is also perfectly compatible with biological experimental protocols, and widely used in microfluidic systems 21 , 3D structures for pressure sensors 22 , cell restrictive environment 23 and more generally for lab-on-chip devices [24][25][26][27] .

Conclusions

A smart 12 well plate integrating MEAs was developed. Electrical conductivity from the MEAs to the third "banana" PCB was shown to be effective as resistivity recorded from a multimer indicated approximatively 30 ohms of resistivity. The tool can be used both inside microscopes, incubators, and biological hoods as show in Figure 24. By maintaining transparency and multi-channels recording, this equipment could allow simultaneous electrical and optical monitoring.

a)

Figure 24: Smart well plate system in use for experiment inside a biological hood. a) The smart well plate, alongside its holder, are placed inside the cabinet. A flat wire connects the inside part of the system to the outer part of the hood to a plugging PCB. d) and e) shows media poured into a well. the transparency of the system allows to visually monitor the media colors.

Unfortunately, the plastic used to prototype the 12 wells would not offer sterilization in an autoclave with a temperature of 110°C. Autoclaving devices at this temperature are often mandatory when experimenting with extremely sensitive cells. Plastic deformation would occur during the sterilization program and deform the structure. As such, the device could not be used for most of the biological applications carried in the laboratory. The cost of a biocompatible, transparent and autoclavable material was too high to be considered. To finalize this part of the project, further modifications are investigated:

1. Use of a different polymer for the cell restrictive environment. Some polymers used as dental prosthetics are now available and more affordable. They are transparent and fully biocompatible. Only autoclaving sterilization would still not be an option as those polymers melting point are below 120°C.

2. Investigation of a different manufacturing processes for cell restrictive / biological container than additive manufacturing. Plastic injection could allow a wider choice of biocompatible and transparent material, with a higher melting temperature. By doing so, a completely new prototype design would have to be created, as this technique requires a mold for injections, and some angles and shape would not fit the fluidic mechanics of the injection 3. As PDMS can be removed from the different parts of the smart 12 well plate, a solution to remove MEAs and their electronic paste from the PCB should be reflected on. The idea of a reusable set up to reduce cost and environmental impact would greatly advantage the tool industrialization 4. To completely free the smart well plate from any bulky cable wiring, a wireless antenna could be implemented on the PCB or on the holder. This would further enhance the user-friendly use of the tool.

Microfabrication and design for Organic electrochemical transistors

Introduction

Merging electronics and biology

The transistor is a semi-conducting device used in electronic systems 1 . It enables modulation of signals when used as a switch, or can be used as an amplifier 2 . Developed in 1947 at Bell laboratories, the transistor has been widely used as an essential electronic component in the evolution of communication 3 and computing systems 4 . During the last decades, coupling electronic devices with medicine 5 paved the way for innovative diagnostics and therapies for medical applications. The field of bioelectronic based on electrophysiology provides new methods and monitoring for biological studies 6 . The Organic electrochemical transistor (OECT) is an alternative for electrodes, broadly used in medical studies, as described in chapter 1. Using the transistor function of amplification makes the OECT the perfect candidate to monitor biological events. Initially developed by Mark Wrightson's group in the 80's 7 , the OECT is a three-terminal system (drain, source, gate). A semi-conductor or a conductor made from a conjugated polymer is used as the channel and connects the source and drain. The whole system is immersed in an electrolyte, where the gate regulates the drain current by doping or de-doping the polymer. OECTs have been used in many broad applications for in vitro and in vivo experiment 8,9 . From detection of analytes 10,11 (glucose, lactate sensing), to neuron action potential recording 12 and epithelial cell membrane integrity 13 . As briefly described in Chapters 2, OECTs designs may vary in number, spacing, size, and sometimes shape 14 . Nevertheless, the recording method and hardware remain identical.

OECTs electronic structure & matrix configuration

In previous OECTs designs, each transistor was individually monitored, and their recording was performed one after the other. Some biological events can occur simultaneously in multiple areas. Data recorded with individually monitored OECTs, one at a time, aren't representative of the biological events recorded. The detection of biomarkers is often missed, or the signal spreads too fast to be tracked. For example, reporting epileptiform activities such as seizure 15 , or tissue barrier disruption 16 , requires real-time monitoring on several locations.

To achieve an optimum electrophysiological recording, the sensitive area should be dense and filled with multiple OECTs, all of them recording simultaneously. Unfortunately, the electrical connections and the hardware system required to perform such measurements are heavy and unhandy.

To record from a single OECT, three contactors and three cables are necessary. One for source, drain, and the gate. For n OECTs individually monitored, the number of contact and cables is n × 3. The numerous wires, pogo-pins, contactors, and NI system coupling create a messy and bulky environment. This limits the number of OECTs that can be recorded from.

To overcome those limitations, a passive matrix circuitry type is considered, as described in Figure 25.

In mathematics, a matrix refers to an arrangement in rows and columns of elements.

In electronic circuitry, it indicates a method to arrange and control different components.

Matrix circuitry, also referred to as gate arrays for transistors 17 , arose during the computer era as a mean to enhance Complementary Metal Oxide semiconductor (CMOS) architecture.

By organizing the electrical components, the commands efficiency and their energy consuming is optimized. The development of LED displays for screens has accelerated the integration of organic materials in electronic 18 (passive matrix display of organic LEDs). Its

Microfabrication protocols optimization

In undercut requires precise control in every section of the photolithography step: the thickness of the resist, the temperature and time of the pre and post-baking steps and the development bath. All of those parameters are evolving and changing according to features geometries and sizes. This is challenging to obtain a similar undercut over different feature sizes.

Metal deposition

Table 2: resist double stack for easier and fast lift off using Orthogonal SL1 Neg1

Step Tool Parameters

SL1 spincoating

Manual Spin coater 60 sec : 1000 rpm : acc 0

SL1 soft baking

Hot plate 90°C : 1 min

Neg1 spincoating

Manual spin coater 60 sec : 3500 rpm : acc 1

Neg1 soft baking

Hot plate 110°C : 1 min

Photolithography

Photo aligner UV line 365nm : 70mJ/cm²

Post exposure bake

Hot plate 110°C : 1 min

Development Neg1

Crystallizer bath 1 min in AZ 300 MIF bath then rinse 1 min in water

Development SL1

Wet etcher Puddle developed SL1 using Developer 100 for 30 seconds, Spray rinsed for 5 seconds during drying spin cycle Repeated Puddle for 10 more seconds with an additional 5 second spray rinse during drying spin cycle

E-beam metal deposition (Gold deposition over Alliance e-beam and BOC Edward)

In the standard fabrication protocol elaborated in the laboratory of BEL, chromium is used as an adhesive layer evaporated between Parylene C and gold.

The option of using titanium instead of chromium, which is less biocompatible, was evaluated. The alliance concept tool deposits metal by e-beam and is fully automated. As this machine allows a more directional deposition of materials, it has facilitated the lift-off of metal as resists side walls were not covered.

Unfortunately, gold deposition by e-beam was not effective. As depicted in 2 , further optimization has been made on the layer thickness to reach higher definition of features. SL1 thickness is 900nm, and the Neg1 is 1.2um thick. The undercut depth reaches 700nm in a U shape as highlighted in the orange disc.

Figure 27, holes and craters appeared on the surface of the metal and aged poorly. As such, this material type of deposition and tool were not considered any further. Furthermore, as isotropic deposition will be required later for the second layer of metal deposition, e-beam technique was left aside.

Figure 27. Titanium and gold deposition on PaC after lift-off. Using laser beam to heat the gold to perform sublimation for deposition appeared to be difficult in our equipment. Visible crater on metal appeared as soon as e-beam deposition is done. Aging of the devices deteriorate their appearance.

Etching organic material using OSCOR 5001

The development of organic patterning was based on previous development using Orthogonal Inc resist for etching technique. OSCOR 5001 was investigated in collaboration with the Panaxium team as a mask resist to etch away exceeding organic material. A protocol has been elaborated and results

were obtained through etching and patterning over a layer of PEDOT:PSS spincoated and cured. Table 3 details the fabrication protocol established. Unfortunately, lifting the resist after the RIE step appeared to be tricky and residues remained on the substrate as seen in Figure 28 c) and d). The stripping required mechanical strength through spin coating. Spraying vigorously over the device could not completely remove the OSCOR 5001 residues from the device.

DE1 encapsulation

In the BEL fabrication protocol, the encapsulation and organic patterning are performed in the same step. Two layers of PaC are deposited on the device, separated by a thin soap layer. The first layer being the encapsulation and the second the sacrificial layer used to pattern the organic material. Photolithography is performed on the stack, and openings are created with RIE etching. Once the organics are spin-coated and hard-baked on the device, the PaC sacrificial layer is peeled off. As such, encapsulation and organic patterning are done.

Less time-consuming steps in fabrication were considered to replace the PaC double stack and the peel-off technique. A new resist from Orthogonal Inc, the DE1, was developed as a dielectric layer compatible with organic materials. Its patterning protocol is detailed in Table 4.

Conclusion and transition to the final fabrication process

Device's designs, a wide range of feature sizes can be drafted: from micrometer to millimeter scale geometries. As such, the double-stack SL1 + Neg1 was replaced by a negative resist: AznLOF2070. Its undercut provides more homogeneity as it is controlled over baking temperature and exposition rate.

Because many issues with our e-beam machine appeared, and as a more isotropic deposition of metal will be preferred (described in this chapter part II), I decided to use the same thermal deposition tool as the BEL protocol: the BOC Edward. It has a smaller chamber and the distance between the devices and the source of metal is short, allowing a more isotropic deposition of the metal.

To overcome the difficulty of resist residues by removing the OSCOR5001, Panaxium fabrication team has integrated a protective layer made of Polyvinyl alcohol (PVA) above the PEDOT:PSS layer. OSCOR 5001 is deposited and patterned on top of the PVA, which is not soluble in the developer of OSCOR 5001. After etching the non-protected PVA/PEDOT:PSS stack, OSCOR 5001 is stripped and then PVA is stripped in water. This method leads to a much lower contamination of the PEDOT:PSS surface by residues of OSCOR 5001.

Finally, the development team decided to move from the DE1 resist as an insulator to PaC. The reason being that the resist isn't, yet, FDA approved for in vivo biocompatibility, where the PaC is. This decision relies on the industrial approach to facilitate the commercialization of devices with material already approved for biocompatibility.

Once

Double layering connections and etching features

The right area shown in Figure 33

Fabrication steps controls

In process characterization and alignment mark

The last parts, center + external are illustrated in Figure 34, and include specific test structures, allowing some characterization tests during the fabrication process. Thickness, conductivity of the different materials, and design / geometric tolerances of patterns.

Alignment marks are found on the external part in the form of two verniers. The Verniers are specific structures used to precisely align a mask during photolithography exposure step over previous structures patterned on the substrate. Characterization of each fabrication step is an essential key to obtain reproducibility between each single device and in between batches. As the machine used has inconstant behavior, modifications in the design were done: line resistivity and thickness are measured using the circle figure at the center of the design. For a bigger device than a glass slide, this type of figure should also be added at the extremity of the design to extract average values of characterization all over the device.

Step 1: Conductive Track metal deposition.

Thickness and critical dimension are measured with the MET Figure 35 Step 2: the insulating layer deposition and patterning.

Test design conclusion

As a result of the design test batch, three parameters were validated.

1. The Pac insulation layer should be as thin as possible. It must allow contact between the two metallic layers but also ensure good insulation over the metallic layer crossing of the matrix. In my design, approximatively 450 nm PaC thick was decided.

2. The profile of the etched PaC for opening must have a maximum angle of 70° for the metal overlay to create electrical contact

3.

The gold deposition could only be performed in one specific thermal evaporator in our cleanroom. The BOC Edward machine model used, having a small chamber and a short samples/crucibles distance, allows, to some extent, an isotropic deposition of metals shown in Figure 39. Following the batch production, we performed electrical characterization. A PDMS well is attached to the surface of the devices and filled with PBS. A silver/silver chloride (Ag/AgCl) pellet is dipped into the electrolyte and used as the gate for the OECTs.

To characterize an OECT, three main parameters are evaluated: its drain current modulation under variation of the collector voltage, providing its maximum transconductance, its cut-off frequency, and its time response. In the first experiment, only the transconductance and the cut-off frequency were of interest.

The matrix devices of 16 and 60 transistors were characterized using a NI system and a digital multimeter (DMM) with a built-in Labview software. The electrodes were characterized through electrochemical impedance spectroscopy (EIS) using an Autolab potentiostat (PGSTAT128N).

EIS impedance for electrodes and cut-off frequencies, IV curve and bandwidth for OECTs were extracted. All measurements were made with NI system and customized Labview software. Data were treated using a Matlab program (shown in supplementary figure).

-0.5 -0.4 -0.3 -0.2 -0.1 0

V DS [V] -1.5 -1 -0.5 0 I D [mA]
V GS = 0.0V to 0.6V result is extracted from one transistor of the matrix. The IV curve is obtained by fixing the gate voltage and changing the drain voltage from 0 to 6V. We the applied a drain bias VD = 0.6 V, and changed the gate voltage from 0 to 0.6 V to obtained to transconductance curve.

V GS = 100mV 0 0.2 0.4 0.6 V GS [V] -1.5 -1 -0.5 0 I D [mA] V DS = -0.5V to 0.0V V DS = 50mV -0.5 -0.4 -0.3 -0.2 -0.1 0 V DS [V]
-0.5 -0.4 -0.3 -0.2 -0.1 0 V DS [V] -1.5 -1 -0.5 0 I D [mA]
V GS = 0.0V to 0.6V

V GS = 100mV 0 0.2 0.4 0.6 V GS [V] -1.5 -1 -0.5 0 I D [mA]
V DS = -0.5V to 0.0V Unfortunately, as the machines used for the micro-fabrication are not performing constantly due to the PaC layer and the PEDOT:PSS etching step. Initially, the pad areas are fully etched. The idea is to remove any excess material in between pads to ensure good bonding with the connecting systems. By doing so, the CT were cut at the etched and nonetched junction of the design. As such, many devices were lost.

V DS = 50mV -0.5 -0.4 -0.3 -0.2 -0.1 0 V DS [V]
A second issue appeared through the PEDOT:PSS etch. Again, the same inhomogeneity in the RIE machine led to a PEDOT:PSS residue all over the device. Currently, this residue is much reduced but did not completely during the etching step.

Second designs: results and characterization

A second device was designed to increase the number and density of OECTs. 60 transistors are spread on a 1cm diameter disc surface. At the center, an electrode of 1mm diameter is used as a coplanar gate for the transistors. Cell culture will done upon those devices for further applications described in chapter 4. This design corrects the etching marge of error that was detected in the previous design. To overcome this potential issue during etching steps, the opening of the pads is now only restricted to a smaller diameter circle area inside the gold pads. By doing so, the full CT length is not going under the etching procedure.

The etching step is terminated on top of the gold pad. It is worth noticing specific RIE tools should be dedicated to the step etching landing on gold (or other metals) for regular and industrialized process lines, as it produces particles that aggregate in the chamber and pollute the machine.

A first electrical characterization is operated. Compared to the 16 electrodes matrix designs, those transistors show degraded electrical characteristics compared to the previous design, as seen in Figure 44. This might be due to the CT resistivity increasing because of their width being reduced from 200 to 60 µm. The cut-off frequency of the bandwidth is around 100 Hz, and the maximum transconductance is 4mS. The gain from the transconductance is not extremely high, but the cutoff frequency value enables detection of some biological markers, or cells tissues formation (cf. chapter 4). Cross talking between OECT was investigated as well. To do so, we connected one source and drain of one transistor only and measured its conductivity. We reiterate the same operation but connect all the other sources and drains to the ground. By obtaining the same conductivity, we made sure the transistor was not connected to the others by crosstalk. By keeping the same connections where all source and drain were connected, we performed a measurement on line 1-A, where no transistor should be found. The absence of conductivity proved that the transistors were not cross-talking.

To perform those measurements, the devices were previously soaked in deionized water overnight. It is worth mentioning that every characterization step was performed in phosphate-buffered saline solution (pH = 7.4). This parameter is of great importance since degradation of the transistors will be observed during their application for in vitro experiments, detailed in the next chapter.

Conclusions

The OECT fabrication protocol and configuration were successfully revisited. Two processes were developed and translated into applications detailed in Chapters 4 and 5. One to pattern the PEDOT:PSS using an etching technique, and the other to replace PaC as insulation layer by the photo patternable resist DE1. Patterning organic material via etching instead of peel-off makes the protocol more suitable for industrial processes, repeatable, and scalable. DE1 resist allows thin layer and smooth edges of the features. These properties are more compatible with cells than harsh geometries and surfaces, described in the next chapter.

The matrix OECTs configuration and protocols were demonstrated. Its in vitro application are detailed in chapter 5. By arranging drains and sources in rows and columns, OECTs density can be increased, and their monitoring hardware simplified.

Coupling the matrix devices with an Arduino system to record simultaneously from multiple OECTs should be investigated in future development.

Cell study

In biology, cell study, or cytology, revolves around understanding of the single cell's structure and function. A cell is described through three main parts draft in Figure 45:

• the nucleus, containing the DNA

• the cytoplasm. All the cellular contents present between the nucleus and the plasma membrane

• the plasma membrane that forms the outer flexible surface of the cell. It separates the internal environment of the cell from the external environment. able to open ionic channels from the membrane by applying pulses, creating an electric field.

To some extent, it creates holes in the lipid bilayer and porates the cell 7 , momentarily or permanently. Its main application target cancerogenic cells from skin or subcutaneous tissues. Cochlear implants are an example of therapy using stimulation at the cellular levels, transducing external sounds to electrical impulse via electrodes placed along the nerve in the cochlea.

As mentioned above, the cell is structured by its plasma membrane. Its basic framework is the lipid bilayer. Two types of proteins are present in the membrane: The peripheral ones, where proteins are attached to the polar heads of the lipids, and the transmembrane proteins that are firmly embedded in the bilayer, protruding inside and outside the cell membrane. In this chapter, we will focus on the latter type: Transmembrane proteins (TMPs). The transmembrane proteins serve different purposes and functions: receptor, enzyme, linkers, cell-identity markers, ion channels and carriers. It is worth noticing that the membrane is a fluid structure, meaning lipids and proteins can move and rotate in their own half of the bilayer. This specificity is extremely important as it allows multiple function of the cell, such as their interaction on the membrane, the TMPs assembly, the self-healing mechanism, and the movement of some components of the membrane for growth, division, cellular junction

In this chapter, we study TMPs as part membrane permeability actuators by creating electrical gradient across the lipid bilayer. Ions channels and carrier allow selective permeability of the membrane by helping a specific molecule or ions to pass through the bilayer. By doing so, the plasma membrane has an influence on the ions and molecule concentrations inside and outside the cell. Usually, the inner part is more negatively charged, and the outer part positively charged. This electrical gradient occurring at the membrane is called membrane potential. This phenomenon is monitored by electrophysiology, by detecting the electrical gradient near the cells as shown in Figure 46.

Bioelectronic devices technology

When interfacing biology, organic materials possess the particular propriety to conduct both ionic and electrical carriers 8 . Furthermore, they offer an improved mechanical matching with the organic tissues 9 . They are soft and compatible with flexible substrates 10 , reducing the inflammation and body response to the device 11 . As such, bioelectronic devices are the ideal tool to transduce biological event from living organism into electrical data 12 . In this chapter, I will describe the efficiency of OMEAs to monitor the lipids barrier and the TMPs. This work was published in Langmuir in 2020 13 . All figures reproduced with permission. In this study, innovative methods were developed to create SLBs 15 . Recreation in vitro of the lipid membrane has been achieved over transparent bioelectronic devices, thus enabling dual electrical and optical monitoring of TMP function in its native membrane environment. This bilayer is supported by a soft conductive organic material, PEDOT:PSS. Used as a negatively charged hydrated cushion under the lipid's bilayer, PEDOT:PSS acts as the inner part of the cell to the bilayer, and allows absorption of ions from the outer part, and helps positioning of the TMPs on the lipids membrane. As the recreation of SLBs on a softer substrate than glass is more complex, a protocol to extract native membranes from cells and transfer them to the PEDOT:PSS surface was developed 16 . Then, we investigated the proteins functional properties and orientation of our model using a protease assay.

To conclude, we used our model to study the ATP-gated P2X2 ion channel, a therapeutic target for multiple pain modalities including neuropathic, inflammatory, and chronic pain states.

Here we describe a platform adapted and tuned to a biologically complex model. Its surface has friendly properties to lipids bilayers such as smooth, hydrated, and soft structures.

Plus, by providing transparency with the OECT, the device allows simultaneous optical tracking of single protein motions to characterize protein diffusion; and sensing of TMP function using electrical impedance spectroscopy (EIS). This monitoring technique measures the impedance of the biological system over a range of frequencies. To do so, an equivalent circuit of the SLBs is elaborated (Figure 50 e). From there, The Nyquist plot is drowned, representing the real and imaginary part of the impedance. Each point of the curves is obtained from one frequency.

The Shape of the curve is characteristic of the different element present in the model measured.

The conducting polymer (PEDOT:PSS) is used to support the SLB. Our platform maintains the compositional complexity of the native plasma membrane, the membrane fluidity of the lipids and proteins, the native protein orientation in the membrane. It can directly monitor ion channel activity across it as drawn in Figure 48. Protocols for assembling complex biological SLBs over the topographic and highly negatively charged surface of the hydrated polymer were adjusted, as the surface of PEDOT:PSS makes the self-assembly process that is easy on glass, significantly more difficult.

Lipids bilayers and TMPs experiment

Cells and plasmids

BHK cells were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle medium (DMEM) (CellGro) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 10 μg/ml streptomycin (CellGro), 1% HEPES buffer (CellGro). The plasmids used in this work have been reported in previous literature 17 .

The pYFP-GPI-N1 or pINR3-Neon-THR-P2X2 were used for transfection of BHK cells to express a glycophosphatidylinositol (GPI) anchored yellow fluorescent (YFP) protein or neon green fused transmembrane P2X2 receptor. The pYFP-GPI-N1 plasmid and the pINR3-Neon-THR-P2X2 plasmid were gifts from the Baird/Holowka and the Kawate research groups at Cornell University, respectively. Preparation of plasma cell membrane vesicles (blebs): This procedure has been reported in previous literature 16 , but salient details are provided here. In brief, 9 × 105 BHK cells were seeded in 10 cm culture dishes (Corning) and grew for 24 hours in a 37 °C, 5% CO2 incubator. 18μL of Turbofect transfection reagent (ThermoScientific) and 6μg of DNA plasmid were used to transfect the BHK cells and incubated for 24 hours. Then, cells were washed with GPMV buffer (2 mM CaCl2, 10 mM HEPES, 150 mM NaCl at pH 7.4). 4 ml of GPMV buffer with 25 mM formaldehyde (FA) and 2 mM dithiothreitol (DTT) (0.075%FA) were incubated with the cells to induce the formation of plasma cell membrane vesicles (blebs) for 2 hours at 37 °C. The solution containing the cell membrane blebs were settled on ice for 15 min to separate cell debris from the more buoyant cell membrane blebs, which were then collected from the supernatant. Characterization of blebs were reported in previous literature.

Preparation of liposomes :

The process of forming the liposomes has been described in previous literature 16,17 .

Liposomes of desired compositions were made from the following components: 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), purchased from Avanti Polar Lipids. The desired ratio of lipids was mixed in chloroform and dried by nitrogen stream. Then, the lipid films were stored under the vacuum for three hours to evaporate chloroform. PBS buffer were added to the dried lipid film to concentration of 2 mg/ml. Once rehydrated, the solution was extruded through a 50 nm membrane (GE Health) 15 times. Characterization of these vesicles is provided in Supporting

Formation of SLBs

To assemble a planar cell membrane bilayer on a support, the 100 µl of cell membrane bleb solution at approximately 5 × 108 blebs/ml were added into the well and incubated for 15 mins. After incubation, the well was rinsed with PBS buffer. Then, 100 µl of liposomes at 2 mg/ml was added into the well and incubated for 30 mins. Next, the well was washed with PBS buffer. 100 µl Polyethylene glycol 8000 (PEG8k, 30% w/v) in PBS buffer was added into the well and incubated for 15 mins more. The well was rinsed with PBS buffer to remove PEG8k after bilayer formation. For supported bilayers made from lipids-only, the procedure is the same as above without the step that introduces the blebs.

Modelling of EIS measurement :

The measured impedance spectra were fitted to a model using the EC-lab software To form the native SLB, a multi-step process was carried out. First, cells were transfected with the membrane protein of interest and chemically induced to bleb off small vesicles from the plasma membrane, resulting in proteoliposomes containing the native lipids of the cell membrane and the expressed proteins. These proteoliposomes, commonly referred to as "blebs", were then adsorbed onto the PEDOT:PSS surface, followed by the addition of a small amount of positively charged liposomes. These liposomes (Figure S1), act as nucleation sites for planar bilayer formation, more easily rupturing to form an SLB on the negatively charged PEDOT:PSS surface and simultaneously inducing rupture of the neighboring absorbed blebs. Although only a small fraction of positively charged lipids are used in this step, it is essential to note that these lipids are not naturally present in cell membranes, but used here to facilitate the SLB assembly process. Eventually, the patches of SLBs heal together to form a continuous layer. To further ensure complete rupture of any remaining blebs or liposomes, we add a buffer spiked with polyethylene glycol (PEG) to create osmotic stress to drive the completion of this assembly process.

The membrane formation and the continuity of the healed patches were optically verified using a photobleaching experiment (Figure 50 b). A membrane intercalating fluorophore, R18, was used to label the blebs before rupture. A ~20-micron diameter spot was bleached in the plane of the fluorescently-labeled bilayer and its recovery was monitored over time.

Fluorescence of the bilayer approaches full recovery after 300 seconds, with a diffusion coefficient of 0.91 ± 0.03 µm 2 /s and mobile fraction 99.5 ± 0.05%. The mobility confirms that the lipids within the membrane plane are mobile and that the membrane probe, initially confined to the ~100 nm bleb membrane surface, is now free to move within the SLB over at least the 20 microns scale.

EIS was used to confirm the presence of the membrane and monitor each stage of the selfassembly process electrically (Figure 50 c). At first, we see the intrinsic features of the PEDOT:PSS surface alone in the bathing buffer solution. Next, the addition of the blebs results in a shift toward higher resistance. With the addition of liposomes, the shift is greater, until finally, after PEG8k solution was added and rinsed out, we see the highest complex impedance values for this system. Impedance values of the native membrane were compared against a liposome-only membrane (Figure 50 d) and resistance and capacitance values were calculated (after modeling the equivalent circuits for each of the bilayers using the model in Figure 50 e) Rb, native = 0.14 kΩ.cm2, Cb, native = 0.17μF/cm2 and Rb, lipid = 0.78 kΩ.cm2, Cb, lipid = 0.18μF/cm2. The substantially lower membrane resistance value of the native SLB, compared to the liposome only SLB, is attributed to the molecular complexity of the native membrane, that is the presence of ion channels or other "leaky" elements, or a poorer ability to pack as tightly as the pure lipids. Another possibility could be the difference in membrane selfassembly quality between native SLB and liposome only SLB. This comparison highlights the sensitivity of the device in detecting the difference in membrane composition or quality. We show the time-resolved evolution of the membrane formation using continuous EIS monitoring (Figure 50 f&g). In both cases, it was observed that the PEG-induced osmotic shock results in a marked increase in the resistance after further promoting the "healing" the bilayer. We verified that increase in resistance is not due to the presence of any remaining PEG in the solution after rinsing Finally, we investigate our ability to monitor disruption of the assembled cell membrane using ethanol, known to have a strong effect on the permeability of membranes. The EIS measurements show a decrease in membrane resistance with increasing concentration of ethanol, supporting our conclusion that the device is sensitive enough to quantitatively detect disruption of the membrane layer by ethanol.

Membrane protein mobility and correct orientation is crucial for TMP function. In particular, for ion channels, protein units move and undergo conformational changes necessary for opening and closing; and orientation is critical for the proper directionality of ion flux. To show the power of our platform in sensing TMP function, we studied the ATPgated P2X2 ion channel, a therapeutic target for multiple pain modalities including neuropathic, inflammatory, and chronic pain states. Protein ion channels in cell membranes are key elements in pain signaling with around 80 ion channels having strong links to pain sensation.34 However, only a few of these channels are understood well enough to be successfully drugged for pain management. The effective treatment of pain remains a challenge that has been exacerbated by the opioid crisis, stressing the urgent need to better understand the underlying causes of pain and find new targets to manage it with minimal side effects. In the case of P2X2, many questions persist about its function, including how lipid regulation may influence ion flux, the process by which monomer units may come together to yield an ATP-sensitive gating events, and more. Thus, having a platform that provides a native environment of lipids and proteins from the plasma membrane surface with the ability to study these interactions and factors would greatly enhance such studies.

As a proof of concept, we use our platform bearing baby hamster kidney (BHK) However, within a device, there is a clear distinction when ATP is added or not, especially when P2X2 is expressed in the membrane. The significant decrease in the membrane resistance observed for the first case, is likely due to P2X2 activation and opening, allowing small positive ions, like Na+ or Ca2+ present in our buffer, to pass across the membrane. Dual modality monitoring using our platform thus confirms that the P2X2 protein is mobile, correctly oriented, and functioning as an ion channel in response to native gating ligands. The bioelectronic chip enables direct electronic read-out of specific interactions taking place at the cell membrane related to key physiological events, while also allowing for conventional optical characterization. The ability to electrically discriminate between membrane compositions, the ability to readout small molecule interactions with these membranes, and, for the first time, the ability to readout the activity of mammalian ion channels expressed within those membranes when subjected to changes in stimuli that alters their response, was shown. As ion channels and other TMPs represent an important class of targets for drugs to combat disease, the ability to scale up these devices, through lithography for the electronic components and self-assembly for the biological components, will be greatly beneficial. Rapid screening for active drugs against ion channels is a critical need for pain management, neurological disorders, etc. More fundamentally, many basic questions remain unanswered regarding how lipid-protein or protein-protein interactions lead to biophysical changes in TMP function and activity. Here, we offer a platform that can begin to get at the heart of these questions by using molecular biology tools to tailor the membrane compositions examined with these devices, opening a technology that is unmatched by any current platform for these kinds of studies.

Epithelial VS electrogenic cell application

Introduction

Merging biology and electronics promises tremendous development of novel therapy and diagnostic through bioelectronic devices 1 . Organ-on-chip models are now becoming more common in drug and toxicology assays 2 . As an example, the study of tissues interface in vitro 3 can be performed by Electrical impedance sensing (EIS), in addition to the optical methods traditionally used. Electrodes on transparent substrates and microscopes combined allows simultaneous recording of the biological events. OECTs are bringing advantages to this study on multiple aspects: they are biocompatible and mechanically matching the living tissues, but they are also transparent and amplify the signals locally 4 .

What are electrogenic and non-electrogenic cells?

Electrogenic refers to the creation of a net flow of charge. In the biological field, this flow designates the ion flux inside and in between cells. Embedded in the cell membrane, ion channels, known as electrogenic pumps/channels, are creating this net flow of charges. We will define an electrogenic cell as a cell able to produce electrical activity via ion pumps. Nonelectrogenic cells doesn't create electrical activity (via action potentials). Epithelial cell and muscle cell are respectively examples of non-electrogenic and electrogenic cells. While the first acts as an impermeable barrier, the second reacts to and produces electrical signals.

In this work, I investigate the use of bioelectronic devices in vitro. On one hand, biocompatibility and long-term electrical stability were performed on control devices immersed in cell culture media. On the other hand, electrical monitoring was performed to evaluate the two categories of cells. Firstly, I have monitored the growth of an epithelial cell barrier using OECTs. Placed in between the gate and the channel of the transistor, the cells grow directly on the surface of the PEDOT:PSS. The barrier integrity is modeled as a resistance increasing as much as the cells close tight. Then, I have measured action potential from a nerve bundle placed over bioelectronic devices. Electrodes successfully detected the ion flux propagation from one axon of the bundle to the other.

Following characterization in PBS of the two matrix designs described in chapter 3, I have implemented, using the OECT matrix, a biological tissues model to validate the proper functioning of the device. The aim is to confirm the design and the fabrication protocol of the matrix structure. This experiment is based on previous work done by Ramuz et. al 5 : Madin-Darby canine kidney (MDCK) II cells were cultivated directly on the channels of transistors. An identical biological set up is applied on top of 2 nd generation matrix system, comparing two organic patterning protocols: the etching and peel off technique. In the first section of this chapter, I demonstrate the efficiency and robustness of the peel-off protocol over the etching protocol, as the last one couldn't survive during the long-term biological experiment.

Finally, a second experiment using electrogenic cells was performed on the same matrix design, with the etching protocols to pattern the organic material. As previously suspected, the devices performances failed, and no measurement could be obtained neither from the OECTs, or the electrode. A second device made with a peel off technique and electrodes successfully recorded action potentials from the nerve bundle.

Electrogenic cells: Nerve-on-chip

The brain gives, processes, and receives information from and to the body: sensory and motor signals. They need to travel a long distance and rapidly. This is possible thanks to the nerve. Their structures support a fast propagation of the signals and protect the neuron fibers 8 . Nevertheless, little is known on the mechanism of nerve disruptions diseases, or degeneration and reparation of the neurons, despite the many tragic outcomes that might come from it 9 . Electrophysiology on the peripheral nerve system (PNS) is a field developed since 1952 10 to study the propagation of action potentials (AP), and understand the functions and properties of the neurons and their structure. Recently, bioelectronic studies have spread the use of electrodes and MEAs to understand and treat PNS disorders 1 , in vitro 11 or in vivo 12 .

Organic materials newly investigated 12 in the fabrication of MEAs allow a better interface with the PNS.

Neurons can integrate and propagate electrical signals. In the peripheral nervous system (PNS), nerve structure gathers bundles of axon in the connective tissue named endoneurium.

Assembled in fascicles, they are wrapped in a protective sheath called perineurium.

Themselves can be grouped in the epineurium, the outermost layer of the nerve structure in the PNS. Nerve from the PNS serve two function:

1. sensory conduct from receptors to central nervous system (CNS), or in between organs called afferent nerve For in vitro assays, monitoring is generally optical and achieved through microscopes.

In this work, a nerve-on-a-chip platform was constructed as shown schematically in Figure 53, and a biological experiment to follow the propagation of an AP from a myelinated axon using an OMEA was developed. Similar to the in vitro and in vivo work previously published on OECTs for AP detection of epilepsy on the cortex 15,16 , this model was designed to prove the same property of OECT to detect and amplify the signal from a motor neuron of the peripheral nervous system.

MDCK II cells seeding

Madin-Darby canine kidney cells II were cultured in DMEM low glucose media supplemented with 10% of fetal bovine serum, 430 mg/mL glutamine, 50 U/mL of penicillin, 50 μg/mL of streptomycin and 50 μg/mL of gentamicin. These products were purchased from Invitrogen.

The cells were incubated in a humidified atmosphere at 37 °C and 5% CO2. MDCK II cells were seeded at 5 × 10 4 cell/cm 2 in each well and growing for three days to obtain an epithelial monolayer. The media was replaced every two days.

Nerve-on-chip

Human Schwann cell (hSCs) culture

Container Preparation

A solution of 0,01% poly-L-ornithine (A-004-M, Sigma) in sterile water was mixed and used to cover the surface of a cell culture flasks. After one hour of incubation at 37°C, the solution was removed, and the flask was washed four times with sterile water. Then, 7.5mL of 10ug/mL laminin solution in PBS was added into the flask and stored at 4°C overnight.

Specific medium was prepared by mixing 500 mL of Schwann cell basal medium, 25mL of Fetal bovine serum, 5mL of Schwann cell growth supplement and 5mL of penicillin/streptomycin solution (Kit P60123, Innoprot). The laminin was removed, and 15 mL of culture medium equilibrate at 37°C is added to the container before plating.

hSCs plating

The frozen hSCs (cat# 10HR-188, iXCells Biotechnologies) vial from Fujifilm was thrown in the water bath two minutes at 37°C to defrost and added to flask. The culture was left undisturbed in incubation at 37°C for minimum 16 hours. Then, medium media was changed every 24 hours until cell passage.

Passaging cell

When 80% of confluence was reached, media was removed, and cells were rinsed with 10mL of PBS. Then, 3mL of Trypsin EDTA were added and cells were incubated at 37°C for 3 minutes. When detached, 8mL of hSCs medium was added, and the total volume was transferred to a 50mL falcon tube. The tube was centrifuged at 200g 5 minutes at room temperature (22°C). The supernatant was discarded, and the pellet was resuspended in 1mL of hSCs culture medium. Cells are then added in a new flask coated with 10mL of fresh medium.

Human Motor Neuron (hMNs) culture

Medium preparation

The medium for hMNs was mixed from a kit sold by Fujifilm: 100mL of iCell Base medium (#C1050) + 2mL of iCell Neural supplement A (#M1032) + 1mL of Nervous supplement B (M1031). The medium was warmed at room temperature and 1mL was added in a 50mL falcon tube.

hMNs thawing

1mL of hMNs medium is added in a 50 mL Falcon tube. The hMNs frozen vial was warmed 2 minutes 30 seconds in water bath at 37°C, and the totality transferred to the 50mL falcon tube containing medium, dropwise with a swirling motion to minimize osmotic shock. The vial was rinsed with 1mL of hMNs medium and transferred to the falcon tube. The total volume was then adjusted to 10mL by slowly adding hMNs medium dropwise while swirling. The solution was transferred to a 15mL falcon tube and centrifuged at 200g for 5minutes at room temperature. The supernatant was removed, and the pellet resuspended in 1mL of hMNs medium.

Results

MDCK II barrier formation over OECTs

The characterization done in the biological hood confirms the pre-measurement carried out in the electrical lab. The 60 OECTs incorporated in the matrix design were performed identically as two weeks before. Then, media and cells were seeded, and a second measurement was done the same day. Three days into post seeding, OECTs from the etching process did not show any sign of conductivity in their channel, thus being unable to perform electrical characterization of the epithelial barrier formation. The conductivity between source and drain ids decreasing, and more noise appears during recording as seen in Figure 57 f). The OECTs made from peel off technique are still performing well as seen Figure 56 C2) and Figure 57 c). The decrease of cut-off frequencies during day 2 and 4 shows the monitoring of the MDCK II barrier development over the channels. No electrical recording was performed using the matrix device. The drain current dropped in a similar way than the MDCK II experiment. This issue questions the performance of the etched devices to monitor long-term biological experiment. To validate the biological model, we have decided to use external MEA to record and stimulate the nerve. These In the Figure 62, we used the following parameters: one electrode (B10) was used to stimulate the bundle with 5 "A for 100"s. The recording was performed using the 9 other electrodes.

Three stimulation were manually triggered from the electrode B10. Artefacts are seen on the 10 channels depicted in the Figure 62. A pattern is expressed sequentially with an offset on each electrode monitoring channel. This could be the expression of the signals input travelling from B10 electrode through the nerve bundle, to the other electrodes. shown and corrected during an isolated test. Secondly, PEDOT:PSS residues over the surface devices were popping irregularly depending on the batches fabricated in the cleanroom.

Conclusion

In vitro testing is essential to treatment development and innovation in pharmacology.

To improve their predictivity and accuracy, involved biological models are developed into more complex structures, thus becoming closer to the in vivo system they intend to reproduce. However, the foremost blocking parameters to the understanding of biology relies on the limited technique of observations and analysis available for in vitro experiments. Indeed, most monitoring protocols are optically based, which limits the survey coverage to the optical scope, camera sensitivity and frame rate. Biomarkers can occur simultaneously at different locations, for example during epileptic activity, or be too fast for the microscope's camera. Spatial temporality of the biological might even be lost during the monitoring of the Chapter 2 focused on the OMEAs chips insertion into a 12-well plate format. To fit easily into biological experiment protocols or standard equipment, a "smart well plate" based on standard biological tool was designed and prototyped. Using CAD software, the 12-well plate's skeleton structure was sketched, modified to include OMEAs. Twelve chips are inserted in the wells, all connected via a PCB gathering the electrophysiological data to an external port. The transparency and geometry of the system allow standard microscopy imaging. Plus, the holder and cables integrated in the tool conception grant continuous monitoring of the experiment inside hoods, incubators, microscope. Thus, the full system enables to perform multiparametric monitoring of the in vitro assay, including simultaneous and continuous recording of optical and electrophysiological data.

In a second experiment, the OECT-matrix device was used to monitor the bioelectrical activity of a developed 3D nerve-on-chip model. In this assay, only the etched-type matrix device was used. To create the 3D model, a spheroid of Schwann cell and motor neuron coculture was seeded in a PDMS well containing Matrigel. The Spheroids developed myelinated neurites, growing over OECTs channels. As the devices failed to record activity from the biological model, we used different OMEAs to validate the nerve biological integrity.

Spontaneous activity and induced activity from stimulation were recorded. This shown that the nerve structure biological model produced compound action potentials spontaneously or induced by stimulation.

Outlook

Organic electronic and conjugated polymers have shown promising results as a multiparametric device to monitor in vitro experiment. Integrated in a standard biological tool, they provide electrophysiological data that complete the optical monitoring results for drug and toxicity assessments. The pharmacology industry could greatly profit from the use of bioelectronic devices. Further development is required for the etch process to pattern organic when the devices are used in long-term in vitro experiment. Industrialization of the microfabrication protocols are necessary to increase the stability and repeatability of the bioelectronic devices. As briefly mentioned in the chapter 1, additive manufacturing is pushing boundaries of the electronic manufacturing field. Applications involving hybrid processing of organic material are bridging the gap with photolithographic technique and reach micrometer resolution of organic features printing.

As in vitro biological models evolve toward 3D structures and autonomous organ-onchips, planar and rigid bioelectronic devices will reach their limits. 2D OMEAs designs won't be sufficient anymore to monitor biological events from these models. New fabrications methods and structures are emerging such as the PEDOT:PSS scaffold, or conductive hydrogels.

Figure 1 :

 1 Figure 1: The structural differences between inorganic semiconductor materials and organic mixed conductors for bioelectronics: (a) scheme and structure of disordered PEDOT:PSS film in the biological environment; (b) scheme and structure of the rigid lattice of boron (p-type) doped single crystal silicon with silicon dioxide layer (modified from Rivany, et al, Chemistr of Materials, 2014) 1 .

Figure 2 :

 2 Figure 2 : (a) The scheme of organic microelectrode three-electrodes measurement set up with equivalent circuit (Re is electrolyte resistance, and Ce is the conducting polymer capacitance); (b) the energy-level diagram of a microelectrode operation in the electrolyte with five phases: (1) a hole is injected from the metal electrode into the HOMO level of the polymer (HOMO and LUMO refer to highest occupied and lowest unoccupied molecular orbitals, respectively); (2) the hole is transported in the delocalized HOMO level in the polymer; (3) the anion is driven into the polymer by the potential; (4) anion travel within the free volume between the polymer chains and crystals; (5) electrostatic compensation of holes and anions at the combination site in the bulk; (c) and (d) cyclic voltomary and I-V plots for capacitive and Faradic process of conducting polymer coated electrodes (modified from Berggren and Malliaras, Science, 2019) 29 .

Figure 3 :

 3 Figure 3: The electrochemical impedance spectroscopy of conducting polymer coated electrode: (a) the impedance (red) and phase (blue) versus frequency (Bode plot) plot of typical PEDOT:PSS coated electrode with resistive and capacitive regimes labelled by the electrode circuit; (c) The impedance lowering effect of PEDOT:PSS coated electrode comparing to gold electrode (adapted form Sessolo, et al. 2013) 11 ; (c) The semicircle characteristics of a biobarrier (lipid membrane, red dots) presented in Nyquist plot (bare electrode is presented in black dots).

  capacity, longterm stability, biocompatibility, and commercial-availability Bulky structure of PSS dilutes PEDOT content (lower volume fraction); higher Young' s modulus (few hundred MPa) than most of the biological tissues (

Figure 4 :

 4 Figure 4 : Organic material deposition techniques. a) Spin-coating b) electrodeposition c) electrospinning and spray deposition are widely used in the research field when OM are disperse in a liquid phase.

81

 81 , and can degrade or enhance adhesion, depending on the scale. Additives can be added on the substrate to answer the issue of delamination. For example, Polyurethane 78 allows strong adhesion of wet type of CPs such as PEDOT:PSS, PAni, or PPy on glass, PDMS, kapton, ITO or gold. Addition of Protein 79 , or polymers like 80 help reduce the foreign body response (FBR) and improve the cell adhesion to the surface of the device.Table 2: Summary of OMC deposition techniques: Picture of a drop of Ag nanowire ink on the top of the pristine PTB7-Th:PC71BM/ PMA:PEDOT:PSS • Scalable process • Easy and fast to elaborate • Less material loss • Doesn't lose uniformity of the deposition over large substrate

1 . 2 .

 12 Wet: Based on redox reactions, four steps are generated when materials are wet etched: Oxidation, dissolution, diffusion and convection. The parameters are: type of liquid and time of the bath and temperature. The etching rate is determined by either a slow chemical reaction, whether the bath time is the determining factor, or a fast reaction where the amount of etchant is the limiting factor. The disadvantages of the technique are under etching and lift off of small structures. Dry: This technique involves removing of material by ionic bombardment inside a reactive ion etcher. Patterned are achieved by using a hard mask leaving areas exposed to the ions. Different parameters can improve the etching: speed, power, directionality (anisotropic or isotropic) Parameters to be optimized include: temperature, RF power, pressure, and time of etching. It is possible to obtain isotropic and anisotropic effects by customizing the parameters. The dry etch technique applied to OMCs such as PEDOT:PSS has been developed firstly using a PaC protective layer 83 , then replacing it with specific resists 60 . The latest processing steps involve chemically inert solvent and developer to the OMC.

Figure 5 :

 5 Figure 5 : Etching OM VS Peeling off OM technique

Table 3 :

 3 Patterning techniques for OMCs

Figure 6 :

 6 Figure 6 : Patterning techniques for OM : a) stamping method using a PDMS patterned stamp. B) Laser direct writing used on UV sensitive technique. Applied on OM such as PEDOT:PSS, the laser pattern the conductivity of the material. c) Did-pen nanolithographic technique used on conducting polymers.

Figure 7 :

 7 Figure 7 : The OMEA applications are categorised based on their configurations: (a) in vivo applications: deep-insertion OMEA probe and surface conformable OMEAs; (b) in vitro applications based on rigid planar OMEAs.

  MEA probeNeurological disorders, such as Alzheimer's, Parkinson's, and epilepsy continuously burden the attention of the global health, and in vivo application with OMEA probes advance the neurosurgical community from the traditional brain probing systems (Figure7(a)). Michiganstyle probe 103 and Utah arrays 104 are commercially available silicon-based probes with iridium and platinum active recording sites. Silicon based insertion probe has inconsistent performance in long-term recordings because of foreign body responses, such as impedance increase by immunocells and neuron loss. Compared to these inorganic probes, OMEA probes are showing excellent properties in recording and stimulation, reduction of recording impedance, high signal-to-noise ratio (SNR), high stimulation capacity, and lower inflammatory responses are the top advances contributing to the conducting polymer coating.

Figure 8 :

 8 Figure 8: cellular immunoactivity between brain tissue and microelectrode: (a) astrocytes populating between neuron and electrode after probe insertion with an equivalent circuit model of between brain tissue the recording systems (Ve: biological voltage; Rspread: spreading resistance; Zscar: scar impedance; Re: electrode resistance; Ce: electrode capacitance; Rm: metal resistance) parallel to the physical structure 105,106 ; (b) inflammatory scar tissue labelled by cell-specific-markers: inflammatory cells (ED1), astrocyte (GFAP), and Neuron (NeuN) ( adapted from Biran R. et al., Exp. Neurol., 2005) 107 . (c) 16-site and 4 shank machigan style probe with PEDOT film coated electrodes (darken) and iridium (shiny) electrodes (Ludwig, K. A. et al., J. Neural Eng., 2006) 88 ; (d) and (e) thermal-drawn fiberic probe: (d) The diagmiter change during thermal draw process; (e) the crossesstional area of the multifunctional fibric probe with conductive polyethlene (CPE) electrodes and hollow core for chemical delivery (COC: cyclicolefin copolymer; CP: polycarbonate) (adapted from Canales, A. et al., Nat. Biotechnol., 2015) 108 ; (e)-(h) The layout and insertion process of substrate-delamiating probe: (e) layout of microelectrodes and OECTs; (f) The insertion step; (h) delamination and remove of shuttle part (Williamson, A. et al. Adv. Mater.,2015) 109 .

Figure 9 :

 9 Figure 9 : the conformable OMEA applications: (a)-(b) NeuroGrid: NeuroGrid structure conform on an orchid petal surface (scale bar, 100 mm); the insert shows the electrode array (scale bar, 100 µm); (b) NeuroGrid conforms on the surface of a rat somatosensory cortex (scale bar, 1mm) (adapted from Khodagholy, D. et al., Nat. Neurosci., 2015) 114 . (c)-(e) anisotropic PEDOT composite coated soft electrodes: (a) image of two arrays bonded by PEDOT composite material; (b) high-density array adhered on human wrist for recording; (d) The electromyography (EMG) and electrocardiography(ECG) signal recorded grom wrist and biceps (Jastrzebska-Perfect, P. et al. Sci. Adv., 2020) 115 ; (f)-(h) conformable brain probe prined by 3D printing techniques: (f) the schematic of printable PEDOT:PSS ink produced by lyoppilization and rediposion in water-dimethyl sulfoxide (DMSO); (g) the whole structure of printed PEDOT:PSS array with PDMS printed as insolation layer; (h) 3D printed neuron probe for rat brain recordings (Yuk, H. et al. Nat. Commun., 2020) 116 ; (i)-(j) self-roll 3D-recording OMEA array: (a) the self-roll mechanism; (b) confocal image of OMEA array wrapping around cardiac spheroid recording (scale bar: 50 um) (Kalmykov, A. et al., Sci. Adv., 2019) 117 .

  activities (ie. Non-rapid eye movement sleep) 114 . Despite clean room microfabrication of flexible OMEAs is still dominating in the field, researchers achieved high quality devices by 3D printing approach. Yuk et al. is able to 3D print PEDOT:PSS flexible electrodes for in vivo rate brain recording, and they successfully pick up LFP, continous action potential, and single unit potnetials 116 . This 3D printing technique is promising for mass production and commercalise these OMC based probes in the future. Beside in vivo recording, flexible property of OMEA array is also capable for wearable health monitoring and 3D recording from tissue surface. Recently, Jastrzebsk-pefect et al. developed flexible MEA from PEDOT:PSS particles and chitosan polymer composite with anisotropic electric resistance 115 . These devices can interface with human skin and conduct electrocadriogphray (ECG) and electromyography (EMG) with high spatiotemporal resolution 115 . Moreover, take the advantage of flexible substrate, the comformable OMEA can also used to monitor single cardiac spheoids. Kalmykov et al. fabriacated self-rolled MEAs by mismatch the residual stress bewtween the bottom and top layer of SU-8; these devices can capsulate a single human cardiac spheroid and maintian their viability 117 . The self-rolling-

  Kros and coworkers showed that once GOx is directly incorporated with PEDOT-based surface, the redox current can be directly transported to PEDOT layer 119 . Later, Wang et al. demonstrated that ferrocene bound to a mixture of polyethyleimine nanobead (BPEI-Fc) and GOx and coated on PEDOT:PSS-carbon electrode, had very high glucose sensitivity (66 µA mM - organic-electrode sensors are promising candidates as low-cost, disposable, and eco-friendly point-to-care devices. Many academic research demonstrate the sensing chemistry with a single electrode, but the capability of producing replicates and statistical analysis can be enhance by electrode arrays. Bihar et al. fabricated glucose-sensing electrodes with completely inkjet-printed method onto a paper substrate (Figure 9 (c)) 121 . This device provided a high sensitivity of glucose concentration for differentiating non-diabetic to diabetic saliva 121 . Besides glucose, other metabolites, such as creatinine 122 , ascorbic acid 123 , dopamine 123 , uric acid 123 , and many more can also be sensed based on their unique redox activities (sensed by CV or Differential pulse voltammogram), which attracting great interest within the field of enzyme-based OMEA sensing. Overall, the high-electronic transport, intimate incorporation with enzymes, and low fabrication cost are all crucial factors making enzyme-based OMEAs sensors powerful and promising tools for future point-to-care technologies.

Figure 7 :

 7 Figure 7: Biosensing with OMEA: (a) The redox reaction series of the sensing mechanism; (b) the structure of PEDOT:PSS-carbon electrode with mixture of polyethyleimine nanobead (BPEI-Fc) and GOx; (c) glucose-sensing CV curves with ink-jet-printed PEDOT:PSS electrode under four different measurement conditions (black line: before PEDOT:PSS deposition; red line: PEDOT:PSS electrode measured in PBS; blue and yellow lines are measuring with 1mM and 10mM glucose present; the insert is the electrode structure)(Bihar et al., npj Flex. Electron., 2018); (d) Differential pulse voltammogram of ascorbic acid (AA), dopamine (Dop), and uric acid (UA) shows high selective at -40, 150 and 280 mV with electropolymerized PEDOT microelectrodes.

Figure 10 :

 10 Figure 10 : Comparison of direct culture and transwell culture configuration of planar OMEAs: (a) Scheme of electroactive cells directly cultured on planar device for recording and stimulation; (b) scheme of seeding barrier cell layer onto transwell MEAs for impedance characterisation; (c)-(h) various type of electroactive cell or tissue system on chip: (c) neurosphere recording (PEGDA (blue dashed band) patterns with showing neuron (MAP2, green), astrocytes (GFAP, orange), cell nuclei (bisbenzimide, blue)); (d) cortico-hippocampal network recording (adapted from Blau, A. et al. Biomaterials, 2011) 124 ; (e) PEDOT encapsulation SH-SY5Y neuroblastoma; (f) glioblastoma stimulation; (g) neural stem cell 3D stimulation with pillar electrode array; (h) brain slice recording planar MEA; (i)-(h) the barrier effect of cell monolayer with EIS measurements: (i) the equivalent circuit model of cell monolayer on transwell membrane of MEAs; (j) the impedance regimes of each equivalent circuit element represented in Bode plot; (k) sensing regime and non-sensing regime separated with impedance spectra of specific electrode area (red curve) where |Zelectrode| is equal to the |Zcell|(adapted from Koutsouras et al. Adv. Healthc. Mater., 2019) 125 .

Figure 11 :

 11 Figure 11 : : The EIS measurement of ion channel activity on membrane integrated with OMEAs: (a) a scheme of HEK-TREK-1 membrane on PEDOT:PSS coated microelectrode with equivalent circuit model, and each circuit element representing the physical element in the system (separated with red dashed lines); (b) a scheme of opening and closing the TREK-1 ion channel with AA and spadin, respectively; (d) spadin (10 µM) addition suppressing the TREK1 activity inducing an increase of membrane resistance; (e) AA activating TREK1 causing a decrease of normalized membrane resistance (Pappa, A.-M. et al. ACS Nano acsnano., 2020) 135 : (d) schematic of PX2X ATP-gated ion channel; (e) Nyquist plot of bare electrode (black), P2X2-rich membrane, and membrane treated with ATP (blue); (f) Membrane impedance of baby hamster kidney (BHK) membrane expressing P2X2ion have more significate decrease when it is treated with ATP channel comparing with normal BHK membrane (Liu, H. Y. et al., Langmuir, 2020) 136 .

  Cell study and bioelectronic systemsCell culture is a major tool to study biology. It provides models to experiment on, study cells behavior to drugs or interactions among themselves, and perform toxicology assays for pharmacological development. It allows experiments on specific topics such as cancer, aging, or bacterial and viral infections. Nevertheless, there is the weak rate of success of drugs assays based on simple cell culture systems from in vitro to in vivo model. Often, cell culture would hardly be representative of the original biological model it tries to mimic. In vivo, cells interact with other cells, fluids, air or even evolve under mechanical constraints. These biological models lack of predictivity and might result in misleading conclusions 1 obtained from over simplified in vitro cell culture experiments.

heterogeneity 3

 3 can be added in the biological model. Mixing different cells cultures and integrating microfluidics systems also improve the similarity of the in vitro model to the in vivo one. Those enhanced cell cultures require a complementary monitoring tool. Optical screening through microscope could hardly monitor events occurring inside 3D structure 1 , or simultaneous biomarkers spreads in large areas or multiple cell culture. As an example, the confocal microscopes: they can perform visualization of 3D biological structure but might lose temporal resolution of a rapid and prompt biological event occurring in multi distanced areas. Whereas electrical recording can perform real time monitoring of the event occurring in different location. To overcome this limitation, bioelectronic devices offers a complementary monitoring to the visual control. It extracts electrophysiological information from the electrical signals of the biological model studied. As such, combining MEAs to optical tools offers a multiparametric monitoring. To meet the requirements and expectations of researchers and manufacturers from the bioelectronics field, a survey has been conducted 4-7 , and lead to the development of an innovative technology platform. With this instrument, adapted to technical demands and standards of traditional laboratories, different biological models can be more accurately validated. Multi parametric, dynamic and long-term monitoring are upgrading the

Figure 12 :

 12 Figure 12: scheme showing the evolution of biological set up and experiment towards in vivo relevance. From Mono-culture models in petri dishes to organ-on-chip multiparametric systems. In vitro optical monitoring evolution. Resolution of the microscopes allows sub cellular monitoring of the in vitro systems studied. Different technique and options emerged as the fluorescent microscopes. The integration of electrophysiology into the biological equipment panel adds complementary data recording and stimulation from optical results.

  have developed an in vitro instrument for biological assays based on the 12-well plate device. This tool is a standardized and easy to handle consumable, widely used in biological experiments for analytical research in in vitro model and diagnostic testing. Coupling the well plate device with bioelectronics technology is the key to improve and evolve the diagnostic and drug toxicity testing. Companies like Multichannel Systems have developed consumables and hardware such as their MEA2100-technology and Multi-well system. Applied biophysics have developed their ECIS, and Mimetas their OrganoPlate systems family.

  steam. MEAs translate ionic flux from the biological event to electrical impulses in its conductive tracks. Electrical data are extracted from the chip via a combination of hardware: a PCB gathering the chips connections, a National instrument system (NI system) performs the power transfer measurements, two Digital multimeter NI-PXI-4071 measured source and drain currents, and a NI-PXI 6289 for the drain gate voltage, wires, pogo pins.

Figure 13 :

 13 Figure 13: Electrical hardware and set up to extract electrophysiological & optical data. A)shows the amplifiers and the digital multimeter monitoring the biological set up in area B). In the latest, the in vitro culture on MEA is placed under a microscope surrounded by a faraday cage (not mandatory) and contact electrical hardware. C) is the software and hardware control set up from which the data are extracted.

  The multi-well plate tool answers those challenges by providing a single device on which multiple sets of experiments can be conducted simultaneously that are easy to use during experiments, ensuring reproducibility and stability of the trial. To maintain these advantages, and providing optical monitoring and electrical characterization simultaneously, I have designed MEAs in a 12-well plate format.

Figure 14 :

 14 Figure 14: Single unit VS multi-well experiments. Petri dishes are used to perform one protocol on a large sample. Multi well plates perform simultaneously several analysis protocols in wells containing the same biological model. MEA initially performed biological experiment with single unit chip. Combining the multi well plate and the MEA chips creates a dynamic, simultaneous and multiparametric tool for biological experiments.

Figure 15 :

 15 Figure 15: Comparison 12 well plate standard and our smart well plate. Dimensions and spacing of the wells are identical, as well as external geometry. Only the PCI connector add an extrusion to the prototype. The material from the prototype is becoming yellow as it ages. A different choice of material would be necessary

Figure 16 :

 16 Figure 16: Geometry and dimensions of the 12 well plate. Standard dimensions are applied to the smart well plate in blue. External geometry of the biological container part (blue) follows the same structure as the 12 well plate. The cap is identical in size and structure. To fit the PCB and the chips, a third section in added beneath the cell container shown in Figure 17 e).

Figure 17 :

 17 Figure 17: Solidwork rending of the smart well plate. Exploded view of the structures displays the different components. From top to bottom: the lid, cell restrictive environment top part. The PCB and its MEA supported by the bottom part of the smart well plate. And the holder with its additive PCB and cap as g) and h).

Figure 18 :

 18 Figure 18: From Solidworks rending to final design PCBs n°1. a) and b) display the First PCB layout and design using solid works software. The spacing between wells exactly fit the 12 well plate standards seen in Figure 16. MEAs chip are electrically bonded to the PCB upon the bottom face a). Each one have 8 connections pads maximum. On the top face b), single glass wells could have been added individually, stucked to the PCB with a PDMS creating. c) and d) are pictures of the final PCB prototype. Well and pads numbering have been added to visually facilitate the user handling. Chips are now connected via 8 pads dispatched in circle as seen in c). A third layer of conductive tracks have been added inside the PCB. It allows to increase the spacing between the connecting pads and eased the bonding between the MEA chips and the PCB.

Figure 19 :

 19 Figure 19: Connector and PCB card design for data extraction from biological equipment. a) is is showing the PCB n°1 PCI connectors chosen to gather all the conductive tracks from the MEA chips to the PCB n°2. b) displays PCB n°3, reorganizing the wells slots and pads from the chips in rows and column.

  fabrication protocol used is based on the organic etching technique, which is more suited for the microfabrication industrialization. The conductive tracks were made of gold deposited via thermal deposition and patterned through lift-off technique. PaC deposited via CVD encapsulates the device. Organic material was patterned through holes made in the PaC. To do so, a patterned photoresist was used as a hard mask for the etching technique. Then, the organic material was spin coated on the device and patterned using etching technics over specified photoresist. The fabrication protocol will be detailed in chapter 3. Different patterns were designed depending on the biological experiment to be studied: action potential detection, lipids bilayer formation and MDCK II barrier growth and disruption. Electrodes of 10, 25, 50 and 100 micrometers were designed. Figure20shows different designs of electrodes and OECTs chip for the smart well plate.

Figure 20 :

 20 Figure 20: Different design of electrodes and OECTs for the chips. Multiple spacing and sizes of electrodes and OECTs were elaborated in order to fit different types of biological experimentations.

Figure 21 :

 21 Figure 21: MEAs chips electrical connection to the PCB. a) Pictures of the different attempts to electrically bond the chips and the PCB pads together.Silver paste and copper scotch tape were tries as seen in a) but has poor electrical conductivity. Indeed, the topology of the connective pads of the PCB doesn't allow these technical solutions as the gap from the pads is too deep as seen on b). c) Final solution adopted using ACP and a bonding machine was successful. 14 Ohms resistance maximum was measured using a multimeter between an electrode on the MEA chips to the PCI slot corresponding

Figure 23 :

 23 Figure 23: Smart well plates in use. The Holder is fitting inside the microscope plates as shown in a). The smart well plate is then inserted inside the holder b) and the focus is made on the MEA chips beneath. Finally, the data are gathered in the PCB n°3 outside the microscope thanks to the flat cables.

  translation to the field of bioelectronics came alongside photovoltaic technology rising in the late 20's. Many approaches in the bioelectronic field have reached functional passive and active matrices devices19 , 20 . In the case of a passive matrix configuration, each transistor is controlled by an intersection of row and column in the matrix grid. An active-matrix display includes a capacitor prior to the component targeted. It allows to switch transistors much more rapidly.

Figure 25 :

 25 Figure 25: Design sketches for connection pads. The number of contactors required is shown for multiple electrodes and transistors individually monitored. The matrix design circuitry decreases the number of pads required.

  collaboration with Orthogonal Inc, I have investigated new photolithographic products and techniques to improve and industrialize the fabrication protocol. Optimization attempts based upon the BEL protocol (in supplementary figures) have been made for the double layer resist for lift-off techniques, the dielectric resist patterning for insulation, and the organic friendly resist for etching sensitive material. Indeed, their chemical products are fluorinated (containing fluorine). There are of interest since as fluorosolvents and fluoropolymers, they are non-reactive and non-contaminating for our organic materials. Their chemical stability is improved: they are inert to other material compound by integrating a fluor atom in their chemical structure. This is of interest when looking for industrial protocols to process PEDOT:PSS patterning and other sensitive materials.Going through the list of Orthogonal Inc photoresists, I will first explain the attempts to improve the metal lift-off step by studying SL1, Neg1 double layer, and the e-beam deposition tool. Then, I will develop our first protocol using OSCOR 5001 to pattern PEDOT:PSS via etching technique. Finally, I will describe the fabrication step involving the resist DE1 as our device surface insulator.

•

  photoresist

Figure 26 .

 26 Figure 26. SL1+Neg1 double stack SEM pictures. The resists are separated by the orange line on the left picture. The double layer is resting on a sliced silicon wafer, bounded by the red line on the right picture. From the parameters detailed in the Table2, further optimization has been made on the layer thickness to reach higher definition of features. SL1 thickness is 900nm, and the Neg1 is 1.2um thick. The undercut depth reaches 700nm in a U shape as highlighted in the orange disc.

Figure 28 :

 28 Figure 28: PEDOT:PSS patterning using etching protocol and the OSCOR 5001 resist. a) b) and c) are SEM pictures upon different magnitude and zoom of a same pattern of a 10 µm diam PEDOT:PSS discs. Some residues are remaining. c) and d) show the residues post etching and stripping step.

Figure 29 :

 29 Figure 29: Organic patterning and DE1 encapsulation. On the left, a microscope pictures displays OECTs patterned with the etching process and the insulate with DE1 resist.

  the material and steps were set by the fabrication team, I adapted their fabrication protocol to a new multi-layered device design. Extracting each block of the etch process, and putting them back together in a different order, I elaborated a new process. The aim is to obtain a matrix layout to organize OECTs and minimize the contact pads. I have first created a test design to elaborate and control each step of the multi-layer assembly, then moved to a simplified version of an OECT matrix. Test Design for transistors and double layering fabrication protocol To investigate the different structures of the matrix device and their layering, a specific set of masks was designed. The aim is three-fold: optimize the base, the inter insulating and the PaC encapsulating layer. Etching, patterning, and additive layering steps are paramount to the device fabrication protocol. Deposition and patterning of the organic material and the conductive tracks overlaying the PaC steps shown in Figure 30 are investigated in this first test design. As seen in Figure 31, the design is divided into four distinct parts: left, right, center, and external areas detailed below. These test features allow the characterization of the layers and process step by step. Mechanical profilometry, microscope and optical reflectometry are used to control material thickness and feature sizes. Using such test features allows non-destructive monitoring of the fabrication protocol, avoiding characterization directly in sensitive areas of the device.

Figure 31 :

 31 Figure 31: test design for multi layering fabrication protocol. External sections are used to place alignment mark for layer superposition during photolithography steps. The center part contains a circle with test figures to characterize each layers of material, the features definitions, and the material conductivity. Finally, the section. on the left contain a large OECT to test its electrical behavior on a multilayer structure. The right section contains rows and columns of gold layer, with PaC opening in between. It is used to test the etching limits and PaC wall angle minimum to perform a metal overlaying connection.

  consists of features to characterize the various etching steps and the feasibility of metal overlays depending on the PaC wall profile. Fifteen conductive lines, ranging from 10 to 1000 µm large from the top layer overstep 15 other similar metallic lines, separated by a patterned layer of PaC with holes to enable contacts. Depending on the size of the patterns and the thickness of the PaC insulating layer, the etching edges profile differs from another. The etching protocol recipe mainly impacts the profile according to those parameters. If the edges are too straight/vertical, the gold deposition from the second metal layer won't create a smooth bond with the first layer (sketch). A minimal angle of 70 degrees is required to perform a bond between the two layers of metals, crossing over the insulating PaC layer.

Figure 33 .

 33 Figure 33. metal overlaying PaC. a) SEM pictures of gold deposition overlaying a Parylene C step. b) test figure used to characterize conductivity of the metal overlay

Figure 34 .

 34 Figure 34.. External and center parts of the test design for matrix devices. The Serpentine for the organic conductivity measurement

  figures.

Figure 35 .

 35 Figure 35. first layer of target test figures

Figure 36 a

 36 Figure 36 a) shows the central target with opening features for the insulating PaC layer. It is measured using a mechanical profilometer. The green areas represent where the PaC is etched. Its thickness is approximately 500 nm.

Figure 36 .Figure 37 .

 3637 Figure 36. a) Central target for PaC insulation patterning. b) test figure for insulation patterning between two metal deposition and for overlaying metal conductivity

Figure 38 .

 38 Figure 38. a) Serpentine used to characterize the organic material conductivity. The conductivity of the material is estimated using a multimeter and the formula of line resistivity. b) central target with the Organic material feature step in navy blue. The rectangle represents the area where the PEDOT:PSS will remain on the device.

Figure 39 .

 39 Figure 39. metal deposition over resist structure patterned on a wafer in the BOC Edward thermal deposition machine. The SEM pictures shows the double-stack layer SL1+Neg1 upside down, covered with gold deposited using the BOC Edward. The edges and walls of the features are covered with gold, showing an isotropic deposition during the evaporation process.

Figure 40 .

 40 Figure 40. Fabrication protocols. Base process made from the etch fabrication protocol. Organic patterning is then split in two different lines: etching technic and BEL peel off step

Figure 42 .

 42 Figure 42. IV curve and transconductance curves extracted from the Matlab program written for data treatment. This result is extracted from one transistor of the matrix. The IV curve is obtained by fixing the gate voltage and changing the drain voltage from 0 to 6V. We the applied a drain bias VD = 0.6 V, and changed the gate voltage from 0 to 0.6 V to obtained to transconductance curve.

  From the IV curve extracted from OECT n°8 characterization seen in Figure42, we can confirm the gating effect and the transitive aspect of the transistors elaborated. From the bandwidth curve of the same transistor shown in Figure43, the maximum transconductance shown is at Vds0 V and is 5 mS, and the cut-off frequency reaches 10 3 Hz. Those values suggest an excellent behavior of the OECTs.

Figure 43 .

 43 Figure 43. Bandwidth, impedance, and phases data from OECT n°8 characterization. The drain current is fixed at 0 V. A sinusoidal voltage is applied at the gate at different frequencies ranging from 1 to 1 kHz.

Figure 44 .

 44 Figure 44. a) Matrix device made on glass slide. 60 transistors and 1 central electrode are displayed in this configuration. C) electrical characterization of a single OECT. Bandwidth and IV curve results. D) normalized mean transconductance of the 60 transistors over one matrix

Figure 45 :

 45 Figure 45: scheme of cell structure

  The electrodes interface in vitro this new model established to study the cell's membrane and some of its proteins embedded. This work was done in Collaboration between the group of Pr. R Owens in Cambridge, and the group of Pr Susan. Daniel from Cornell. I have provided one type of bioelectronic devices including electrodes covered by PEDOT:PSS and DE1 to perform the electrophysiological recording of the biological model.

Figure 46 :

 46 Figure 46: Electrophysiology: when electronics merge with biology. Example of ionic flux expressing by cells' membrane through transmembrane proteins. Detection of ion flux with a bioelectronic device

Figure 47 :

 47 Figure 47: SLBs on rigid and. insulating substrates. Ions cannot pass through TMPs and protruded proteins are poorly embedded in the lipid bilayer.

Figure 48 :

 48 Figure 48: Electrophysiological model studied from the Lipids Bilayer and its TMPs experiment.

  Device fabrication: The fabrication protocol relies on a 4-stack layer step. First, 3 µm thick Parylene C layer is added through chemical vapor deposition on top of the wafer. Then, Cr/Au 150nm thick electrodes are patterned using photolithography with AZnLof 2070 as the lift off resist. PEDOT:PSS solution, Clevios PH1000 containing 5 vol% ethylene glycol and 1 vol% (3glycidyloxypropyl) trimethyoxysilane, is spin-coated at 1500 rpm for 90 seconds, baked on a hotplate at 130 ºC for one hour and dry-etched using orthogonal resist 5011 as protective mask. Finally, a dielectric resist, DE1 from Orthogonal Inc, is spin-coated and patterned on top as the encapsulation layer via photolithography. The devices pictures and set up are shown in Figure 49.

  . The equivalent circuit used to extract the electrical properties of the membrane consist of a resistor and a capacitor in series, accounting for the electrolyte and the PEDOT:PSS film respectively, along with a parallel resistor-capacitor component also connected in series accounting for the membrane. (Figure 50 e) The PEDOT: PSS electrode is represented by a capacitive element due to its purely capacitive behavior as suggested in recent studies. A parallel resistor and capacitor component represent the membrane (SLB) system. The parallel resistor and capacitor circuit represents the cell membrane. It is typically used in literature to account for passive membrane or transmembrane ion transport in biological layers (i.e., cell membranes, cell monolayers).

4. 3

 3 Result & discussionLipids and TMP platformEIS was used to record the presence of the membrane and electrically monitor the assembly process of the SLB. The resistance of the film increases as every step is added on top of the devices: blebs, then liposomes. EIS monitored the for long term membrane formation, disruption, and healing. It shows the increase of resistance during the formation stage, and its decrease when ethanol is added as proof of disruption of the membrane

Figure 49 :

 49 Figure 49: Device used for the in vitro monitoring of the TMPs function. a) microscope image of the electrode used. b) SEM picture of the sensitive area of the device. c) experimental scheme of the lipid bilayer deposition over the device

Figure 50 :

 50 Figure 50: Combined optical and electrical monitoring of Baby Hamster Kidney cell SLB formation. (a) Cell bleb rupture and bilayer self-assembly on a PEDOT:PSS film after the addition of positively charged liposomes and soluble PEG (8k). (b) A photobleached spot in an R18-labeled SLB illustrates the fluidity of the membrane over the micron scale, with the corresponding recovery curve used to calculate the diffusion coefficient. (c) Nyquist plots showing the formation of the native planar cell membrane at each stage in (a); after addition of cell blebs (orange), after addition of the liposomes (purple); and after addition of PEG (red). (d) Comparative Nyquist plot of the native SLB (red) and a liposome-only SLB (grey). (e) The equivalent circuit used to model the EIS data. (f) Time resolved changes in resistance as the SLB forms on the polymer surface. (g) Resistance changes for the supported native membrane formation over time.

  membranes expressing P2X2 ion channels to measure the ion channel response under different conditions. P2X2 ion channels are ATP gated; open in the presence of ATP and closed in its absence (Figure 51 a).Figure 51 b shows the impedance profile changes in response to ATP treatment. The plots show the impedance value for the bare electrode in black, the value for the membrane prior to treatment with ATP in red, and then a shift to lower impedance values once ATP is added to the solution in blue. The relative difference in the resistances between +ATP and -ATP after fitting the impedance data with the model is captured in Figure 51 c in the first set of bars. In a control experiment, carried out on a different electrode with a membrane not expressing P2X2, the relative change in resistance +/-ATP is much less, as shown in the second set of bars. In this bar chart, we have normalized the resistances to the values without ATP on each device for comparison, noting that the electrodes themselves have some variation that makes it difficult to compare absolute values across the two devices.

Figure 51 :

 51 Figure 51 : Ion channel function study using the native membrane on an organic electronic transducer. (a) Schematic of PX2X ATP-gated ion channel. (b) Nyquist plot of the electrode prior to (black) and after (red) the P2X2-rich membrane formation as well as after treatment with ATP (blue). (c) Calculated membrane resistance of the P2X2-rich membrane before (red) and after (blue) ATP (10 µM) treatment showing a clear decrease in the resistance of the membrane in response to ATP and the corresponding values of the BHK membrane not expressing any P2X2 (patterned histograms) (n = 3, p = 0.009).

5. 1

 1 Non-electrogenic cells: epithelial barrier growth and disruption detection Epithelial cells form tissues that allows organs and the whole body to interact with both its internal and external environment. They are held together by cell junctions. I will focus on tight junctions and gap junctions in this chapter. Tight junctions are occurring when transmembrane proteins (TMPs) strands fusing together between two cell membranes. An epithelial layer is the combination of epithelial cell held together by cells junction arranged in layers, more or less dense and tightly packed, as shown in Figure52.MDCK II are cells that can form an epithelial barrier in vitro. They form a confluent and dense cell layer in a couple of days6 . The ionic permeability of the formed tissue decreases as the cell-junctions increase. To determine the barrier integrity, trans-epithelial electrical resistance (TEER) is characterized using electrochemical impedance measurements7 . Cells were seeded on top of OECTs devices and an Ag/AgCl pellet was used on the media surface as gate. As the cells grow and form a barrier, its TEER increases, and the ionic current gets limited between the gate and the OECT channel as depicted in Figure52. The drain current and resulting transconductance show a lower cut-off frequency as the epithelial barrier is forming over the transistor channel.Following the decision detailed in chapter 3 to dismiss the use of DE1 as insulating layer of the device, the new process involving PaC and organic etching were used. Two types of devices were then crafted following BEL and the etch technique processes, to compare efficiency of the new fabrication technique for long term in vitro experiment.

Figure 52 :

 52 Figure 52: Electrophysiological model to study non electrogenic cell barrier. MDCK II cells are directly seeded on OECTs. When a voltage is applied to the gate in the upper media of the model, an ion flux in created toward the transistors. If the epithelial barrier integrity is not compromise, the ions cannot reach the OECTs. If the barrier is damaged, then ions penetrate the OECTs and modulate its transconductance.

2 .

 2 motor function, called efferent nerves, guiding signals from the CNS to musclesThe electrical signals conveyed through the body are based on electrochemical impulses called action potentials (AP). Their speed can reach 120m/s alongside axons. To achieve such speeds, the peripheral nerve axons possess a myelin sheath, made from Schwann cells. Similar to oligodendrites in the brain, they provide support and protection to the axons. Additionally, they insulate the axon fibers by sections, resulting in the transmembrane current only occurs at the node of Ranvier, creating the saltatory conduction. Instead of travelling all along the unmyelinated axon, the action potentials jump from node to node, where sodium channels are grouped, accelerating the impulse propagation. To study these signals and diagnose disorders in the PNS, physical examination can be performed. Otherwise, more in-depth imaging technique such as magnetic resonance neurography or ultrasonography allows studies of structure of the nerve13 . Electrophysiology assays of the PNS recording the compound action potentials (CAP)14 , or a single AP propagation, from the nerve in vitro or in vivo, are a new method to study the function of the nerve. However, as described above, the structure of nerves of the PNS is extremely complex and difficult to reproduce in vitro. Coculture of dorsal root ganglion and Schwann cells are usually standard and efficient models.

Figure 53 :Figure 54 .

 5354 Figure 53: Electrophysiological simplified model to study action potential propagation. OECTs are placed beneath myelinated axons and a 1.5mm diameter electrode is placed under the bulb of the motor neurons. The OECT should be able to monitor the propagation of the AP alongside the axon, by detecting the ion flux gradient impacting the PEDOT:PSS channel conductivity.

Figure 54 :

 54 Figure 54: Scheme of the biological experiment. a) myelinated nerve growth over matrix device. b) section of myelinated fibers covering the OECTs and electrode from the device

  Two days after seeding, pictures b in Figure55, OECTs and matrix devices made using etching protocol for organics start to deteriorate. As shown in Figure56, some area where MDCK II are forming a dense epithelial barrier are detected by the OECTs located beneath, decreasing their cut-off frequency.Transistors perform almost identically, without noticeable changes in their electrical behavior as expressed by the bandwidth seen in Figure56 b1) and c1). It is worth noticing that the contact pads are being scratched (shown in supplementary figure). The metal is ripped off the surface as much as the pogo pings are placed and removed upon them alongside the multiple measurements.

Figure 55 :

 55 Figure 55 : MDCK II seeding over OECTs matrix device microscope pictures. a) device control before cell seeding. b) device using peel off technique day 5 post seeding c) device using etching technique day 3 post seeding. The epithelial barrier was fully developed and pack at day 5.

Figure 56 :

 56 Figure 56 : Comparison of two OECTs from the same matrix made using the peel off technique. a) shows the OECT cut off frequency characterized in media at day 0. b1) OECT A and c1) OECT B cut off frequencies at day 1 after cell seeding. At day 4, b2) displays the cut off frequency of the OECT A, that is not covered by cells, and C2) displays the decreased cut off frequency of the OECT B covered by epithelial cell. The bandwidths differences show that the presence or not of the cell barrier coverage is detected by the matrix of OECTs.

Figure 57 :

 57 Figure 57: Mean and Normalized transconductance curve from devices used. a) evolution of the mean transconductance of the 60 OECTs from a peel off device over time. The cut-off frequency decreasing is significant of the epithelial barrier forming over the matrix b) 2 days c) 4days. Results from characterization and comparison between processes. d) control devices from the experience compared to devices e) peel-off and f) etching bandwidths.

Figure 59 :

 59 Figure 59: Assembly of the Nerve-on-chip. a) PDMS mold aligned to the matrix for the electrode to be in the bulb section and the OECTs in the axon's growth axe. b) complete device. Two parts are used, one for the NoC and the other for the control. c) Microscope pictures of the spheroids seeded in the bulb section embedded in Matrigel in control sample cultivated in Transwell. d) Developped nerve bundle on top of the matrix devices after 3 weeks of culture in Matrigel and Media

Figure 60 :

 60 Figure 60 : Nerve-on-chip cells culture growth 20 days after the spheroid seeding. a) Control cell model. The PDMS mold and its spheroids are placed inside a Transwell. The bulb developed multiple neurites that spreads inside the tube section of the mold. b) Fluorescent microscope image of the coculture bundle at day 12. Schwann cells starts to migrate alongside the axons. c) Microscope pictures of a neurite growing on top of the OECT channel.

  electrodes were produced following the BEL fabrication protocol involving peel-off technique patterning for the PEDOT:PSS. The device contains 10 electrodes of 50 µm diameter spaced by 150 µm in a rectangular geometry shown in the Figure 61.

Figure 61 .

 61 Figure 61. NoC images obtained from a fluorescence microscope. Calceïne AM (viability marker) was added to the biological marker for the microscope to detect the 3D structures. a), b) and c) are pictures from a control biological model inside the PDMS mold. At diffent scale, the 3D structures of the nerve bundles show the differentiation of the Motor neuron into axons, and the Schwann cell migration. d) is a pictures of the control NoC deposited on the OMEAs

Figure 62 :

 62 Figure 62: Brainstorm software plotting the electrodes stimulation and recording simultaneously.

  experiment. The work presented in this manuscript aimed to develop a multiparametric platform to monitor in vitro experiments. Electrophysiological monitoring was implemented into standard biological experiment to complement the optical method. Chapter 1 reviewed the OMEAs state-of-the-art. It explains why conducting polymers are of interest to interface with biology. Their fabrication protocol, purposes, and applications are detailed. It then concludes on the importance of electrophysiology in biological study.

Contents 1 .

 1 Liposome size and zeta-potential characterization 2. Effect of PEG addition on membrane quality 3. Membrane disruption by EtOH characterized by EIS monitoring 4. Effect of ATP on BHK plasma membranes that lack overexpression of P2X2 channels 5. Modelling of EIS data to extract membrane properties École Nationale Supérieure des Mines de Saint-Étienne NNT : 2021LYSEM018 Aimie PAVIA Design et fabrication de dispositifs organiques électroniques pour le diagnostic in vitro: du Petri dish vers un système multiparamétrique Spécialité: Bioélectronique Mots clefs : Électronique organique, Polymère conducteur, OECT, in vitro, Cellule neurale, matrix Résumé : Les études pharmacologiques sont primordiales dans le développement des traitements de pointe. Cependant, les tests in vitro faisant partie intégrante des essais précliniques ne sont pas prédictifs du système in vivo qu'ils tentent d'imiter. Deux axes de recherche sont investis pour répondre à cette problématique: 1) Élaborer un modèle biologique in vitro plus complexe. 2) Améliorer les méthodes de contrôle in vitro. Nous proposons un système de suivi multiparamétrique intégrant des dispositifs bioélectroniques, permettant la surveillance électrophysiologique complémentaire au contrôle visuel des expérimentations in vitro. Un état de l'art des microélectrodes organiques (OMEA) introduit le domaine des matériaux organiques. Puis, afin d'intégrer un suivi électrophysiologique dans des expériences biologiques usuelles, les OMEA sont assemblées sous la forme d'un outil utilisé en biologie cellulaire (la plaque 12-puits). Afin de contourner la restriction du nombre de canaux adressables par l'électronique, le design du transistor électrochimique organique (OECT) est adapté en architecture matricielle. Des étapes de fabrication des OECTs sont optimisées. La capacité des dispositifs bioélectroniques à monitorer des expériences biologiques in vitro est évaluée pour a) contrôler la viabilité d'une bicouche lipidique b) mesurer la formation d'une barrière cellulaire épithéliale, et c) détecter l'activité de cellules électrogéniques. Ces dispositifs bioélectroniques peuvent être intégrés dans les protocoles standards de la pharmacologie, et permettent un monitoring multiparamétrique de l'expérience in vitro.

Table 1 : Summary of Common Conducting Polymers Summary of Common Conducting Polymers

 1

Table 1 :

 1 Optimization attempts done from the Bel Protocol

	Step	BEL Fabrication	Optimizations	Key points
		protocol
	Metal patterning	S1813 lift off	Double stack SL1/Neg1	• Remove residues and sonicating step
				• Increase undercut to facilitate lift
				off and remove "ear rabbits"

Table 3 :

 3 PEDOT:PSS patterning protocol using etching technique over OSCOR 5001 patterned Excellent features definitions have been obtained, up to 2µm diameter PEDOT:PSS disc.

	resist

Table 4 :

 4 DE1 resist protocol over PEDOT:PSS organic material

	Step

Scientific contributions .......................................................................................................................

cm -2 ) comparing to many other types of sensors(Figure9(b)) 120 . In addition, enzyme-based

templates. Lab Chip 14, 424-430 (2014).

Surface Conformable OMEAs

To minimize the inflammatory response that happens during deep-insertion recording, electrocorticography (ECoG), as a less invasive method, with conformable OMEAs increasingly applied to monitoring the surface brain activities without damaging any tissue and providing real-time information before brain surgeries 112 . Besides the advantages of the conducting polymer coating described above, they are lightweight, can be easily adapted on substrates of different shape and bendability. The features of these OMEAs rely on their thin (1-10 µm) and flexible organic substrate (ie. parylene). They potentially capable to conform to the curvilinear surface of the brain, and even fit and attach to the brain sulci. Khodagholy et al. demonstrated PEDOT:PSS microelectrode array on parylene substrate (total thickness 4 µm) was capable to record epileptiform sharp-waves, and the activities they picked up match the silicon probe depth-recorded spectra 113 . Also, the array of PEDOT:PSS electrodes detected the same events and mapped out them with time-frequency analysis with high spacial resolution 113 .

Reproducibility and simultaneous recording

To increase repeatability and facilitate simultaneous recording through the multiple experiments and chips, I have fabricated several MEAs chips in a same batch, and on the same carrier: a wafer containing 12 MEAs. I have reevaluated and modified the fabrication protocol (described in the chapter 3) to increase their performance and their electrical characteristic's repeatability after previous work initiated on fabrication processes 19 . A different microfabrication approach has been developed to pattern organic materials for MEAs. Etching technique replaces the peel off steps used to pattern PEDOT:PSS. The aim is to increase repeatability of the device's electrical properties and topology for an industrial OMEAs processing approach.

By arranging the OMEAs in a 12 well plate format, the biological experiment is also treated equally from chip to chip. Regarding the biological experimental setup for electrical monitoring, measures were previously performed outside of the incubator or the microscope, as the hardware is bulky and non-sterile. This would create a harsh environment for the cell culture inside the wells, as temperature is decreasing, and sterility highly compromised during the electrical monitoring. To address this issue, a specific packaging was designed: from connection solutions to extraction of the electrical signal. Firstly, we replaced and reorganized all the connective pads from the different OMEAs chips: by gathering them in a PCI slot, we can connect easily to the hardware, via a printed circuit board (PCB). Secondly, I have designed a packaging system for the smart 12-well plate to remain in the incubator or the microscope during the biological experiment. As a result, the electrical characterization and monitoring can be performed inside an incubator or the biological hood during the time of the biological experiment. The smart 12-well plate and its biological matter remain in its sterile environment as shown later in this chapter in Figure 24. Similarly, while the optical monitoring is performed under microscope, electrical recording can be performed as the packaging allows placement and extraction of the data from a microscope chamber simultaneously.

Electronic hardware and set up

Compatibility with an optical microscope was a key aspect during the design of the platform, as the plate should be able to fit onto the stage of a standard microscope as seen Figure 24 a) b) c). We developed a specific holder fitted to the plateau of the microscope depicted in Figure 22. This holder had three main objectives:

• Maintaining the 12-well plate assembly inside the microscope and placing the OMEAs at focus length of the bottom lens

• Integrating the functional PCB inside the holder and allowing the flat cables to connect at its extremity in a stable way

• Enabling easy exchanges between different 12 well plates once the measurement done.

A prototype made of aluminum was extruded and assembled with the functional PCB and the PCI connectors. Flat wires extract the data from the 12 well plate within the microscope to the third banana connectors PCB as shown in Figure 23.

Lift-off technique for Conductive Tracks (CT): SL1 + Neg 1 double stack

An alternative process described in Table 2 for gold lift-off has been investigated. I have developed the double-stack resist protocol. to replace the former S1813 resist, I have used two different chemical resists from Orthogonal Inc: the SL1+Neg1. A double stack is a process involving two types of resists, spin coated one on top of the other: beneath, a non-sensitive to UV light resist, that usually is easily dissolvable to ease the lift of the second resist. The second one on top is photo patternable that define the design features. This allows to create a large and high undercut to improve the lift of the metal deposition. In parallel, it eliminates the need for sonication to completely lift the resist residues from the substrate that could occur during the S1813 step and prevent from "ear rabbits" effect. "Ear rabbits" is the name given by the gold residues formed by deposition of the metal on the resist edges profile. They stuck to the gold lines after lift of the resist and might leads to lines and encapsulation defects.

The double-stack has three main advantages for users:

1. easy lift of the resists thanks to the first layer of SL1.

2. tuning properties according to the feature's sizes and metal thickness. The thickness of the resist layer can be adjusted with the spin coating parameters. By doing so, the shape of the undercut can be perfectly controlled over the different photolithographic step 3. Fluorinated chemicals : Organic friendly Scanning electronic microscope (SEM) was performed to evaluate the resist patterning step. SEM pictures are shown in Figure 26.

The undercut geometry depends mainly on the dipping time into the development bath.

SL1 is a non-UV sensitive resist, spin-coated first on the substrate, and hard-baked. Following this step, the Neg1, a UV-sensitive resist, is also spin-coated over SL1 and soft-baked. The stack is then exposed through the photolithographic technique: the patterns are transposed from the mask to the Neg1 layer. After a first development bath in AZ 300 MIF, the openings are done on the unexposed Neg1 areas that dissolve. Then, the SL1 is dipped into Developer 100 bath, which dissolves the resist through the opening made on Neg1, in an isotropic manner.

It is worth noticing that control over SL1 dissolution rate is complex. Creating a controlled When elaborating a protocol in microelectronics, each step is characterized and optimized according to the equipment available. Both characterization and process tools are of importance to achieve functional devices. Furthermore, as the various machines available are used numerous studies and have passed the state of "brand new and perfectly functional", independent variability was to be expected. A manufacturing tolerance must be considered in the protocol. The first challenge is to ensure good conductivity and insulation between the two layers of conductive tracks where it is required. The metal overlaying part shown in Figure 30 will be investigated and described in this section. Indeed, in the matrix circuit, some node of conductive material needs to be connected through the insulation layer. And other nodes need to be perfectly insulated to avoid crosstalk between the lines. Monitoring each step of the protocol is a crucial aspect of structure optimization. To conclude the test design, two fabrication protocols were selected: the BEL one using the peel-off technique and the Etching technique to pattern organic material layers. A base process is then used for both, and the process splits in two different ways to pattern PEDOT:PSS, the first being an etching technique using OSCOR resist, and the second one the BEL peel-off technique as depicted in Figure 40.

Self-assembly of mammalian cell membranes on bioelectronics devices with functional transmembrane proteins

This chapter is based on the publication:

Introduction

Throughout the three years of PhDs study, I have elaborated and partially developed, three different types of fabrication processes for bioelectronic devices. As described in the previous chapter, each one of them had to be evaluated through 3 steps: surface topography, electrical characterization in PBS, and in vitro experiment. Topography and electrical characterization are discussed in previous chapter and provide the electrical properties of the device before any application. In this chapter, I test the behavior of the devices with biological matter. The bioelectronic devices' purpose is to interface with biology. The language transduced is the ionic flux from biological matter, to an electron flow in the electronic circuit.

Ionic flow is the essence of any biological event, taking place through their membrane and the proteins embedded in its lipid bilayer. Our organic-based device aims to detect such fluctuations and transduces this signal by interfacing the ions flux and converting it to electronic current 1 (i.e., electrophysiology)

Material and methods

Epithelial barrier experiment: MDCK II

To confirm the new matrix multi-stack protocol, and the etching process for organic patterning, the experiment follows previous publication and methodologies developed and usually applied at the BEL and BEST laboratories

Laboratory process: Peel-off technique

Like the industrial protocol, up until the second deposition of Cr/Au layer patterning, a second and third layer of PaC was deposited over the device, separated by a thin layer of 2% soap solution. The two layers were then patterned using Az10Xt under photolithographic patterning. Then, the same PEDOT:PSS solution was spin-coated and soft baked for one minute at 110°C on a hot plate. Finally, the sacrificial third layer of PaC was peeled-off, and the device hard-baked in the oven at 110°C for 30 minutes.

Glass wells were stuck on the matrix with PDMS, and sterilization was done in ethanol bath under the biological hood. To maintain a sterile environment as much as possible, electrical cables were cleaned and inserted into the in the hood and not moved, as well as

Spheroid's elaboration

Well plate + agarose

To create spheroids, a solution of 1% (w/v) agarose (Sigma Aldrich) in deionized water was prepared and autoclaved. After heating the solution for one minute in a microwave, 75 uL were added to each well of a 96 well plate. It forms a meniscus on the bottom of the well.

After 10 minutes at room temperature, the cool downed agarose forms a non-adherent round bottom well.

Co-culture assembly

75 000 Cells of hSCs and 75 000 hMNs cells were mixed and added in each well filled with 200 uL hMNs media. Cells were incubated at 37°C and half of the media is changed every 48 hours.

Spheroids were forming around the 3 rd day of co-culture in the well plate.

Cell restrictive mold / environment

Using Solidworks CAD, a specific PDMS mold was created to contain cells and was inspired from the original publication. Initially, a solid plastic reproduction mold was extruded. Then, PDMS 1:10 was mixed, and centrifuged at 3000rpm for two minutes. Once the bubbles are cleared, the preparation was poured into the mold and cured on a hot plate at 60°C for four hours. The PDMS mold was then extracted from the reproductive mold.

Nerve on chip

Device fabrication

The device design and protocol chosen for this experiment is the matrix made of 60

Structure assembly

Cell restrictive environment to MEA assembly and sterilization

The PDMS molds were stuck on the matrix device using a thin layer of fresh PDMS.

They were aligned on the OECTs and central electrode as seen in Figure 59 a. Glass wells were attached around the mold and on top of the device. They were fixed using again fresh PDMS as depicted in Figure 59 b. The assembly was then washed three times for 10 minutes with 2% antibiotic/antimycotic wash buffer (10 000u/mL penicillin + 10 000 µg/mL strep + 25 µg/mL Fungizone Gibco).

Matrigel and spheroids seeding

Once the spheroids were formed, they were implanted in the PDMS mold, at the bulb section. The Matrigel stored at -20°C was defrosted slowly in an ice bucket. When reaching 0°C, the Matrigel starts to liquify, and can be inserted into the mold over the spheroids. The devices were then incubated for 5 minutes at 37°C to let the Matrigel polymerize. Thereafter, 200 µL of media was added to the well for the motor neuron to develop.

During the first week, cells are cultured in Pre-myelination co-culture medium, allowing the motor neuron to develop and the neurites to grow alongside the mold. The premyelination media comprises a mix of hNs Medium with 10% Hyclone FBS and 1%Antibiotic/Antimycotic wash buffer (12389802, Fisher Scientific). The medium is changed every 48 hours.

Then for three weeks, the medium was replaced by myelination co-culture medium. It allows the SCs to differentiate and aggregate on around the MN already developed as seen in Figure 59c. The myelination co-culture medium is a mix of 10% Hyclone FBS (12389802, Fisher Scientific), 10ng/mL of rat-b-NGF (#7815-NG-025, R&D systems), and 50ug/mL of L-ascorbic acid (A92902, Sigma). Half of the myelination medium changed every 48 hours.

Conclusion

The epithelial barrier experiment was repeated two times: firstly, in Cambridge BEST laboratory, and secondly at the BEL laboratory from Gardanne. The devices used where made from the same batch at the Centre de Microélectronique de Provence's clean room. The results obtained delivered two conclusions:

1. The matrix multi-layer fabrication protocol is effective, and performs well over a weeklong in vitro experiment

The etching technique to pattern organic shows a defect in the device lifetime for long term monitoring

The nerve-on-chip experiment confirms this hypothesis. The OECT matrix devices used for the experiment were produced using the etching fabrication protocol. They failed similarly to the one used in the MDCK II experiment made using the same process. Nevertheless, the biological model was a success. Thanks to the extra MEAs used to process the stimulation and the recording, we have shown that the nerve bundle was producing electrophysiological activity upon stimulation.

OECTs have previously shown promising results as a tool to study epithelial barrier formation and disruption. When cells are directly seeded over its surface, it has delivered two main advantages: its transparency for optical monitoring, and its simultaneous electrical recording of barrier formation and disruption. To map the whole forming barrier and its potential local disruption, the matrix disposition allows a higher density of OECTs recording sites. We have demonstrated the efficiency of the microfabricated structure overlaying multistack of PaC and metallic lines layers. Plus, we had shown that adding an organic layer patterning over the multi-stack was possible when the peel-off technique was used.

Unfortunately, the etching step to pattern PEDOT:PSS over the multi-stack requires further investigation to improve the lifetime of the device.

Considering the multiple fabrication issues during etching steps, such as the presence of PEDOT:PSS residues, unusual behavior from the machines was initially suspected. • % ylim(hAx_trans,[-3.5,0]);

• % ylim(hAx_gd,[0,6.5]);

• % ylim(hAx_gm,[0,3.5]);

Supporting Information

Self-assembly of mammalian cell membranes on bioelectronic devices with functional transmembrane proteins

Han-Yuan Liu

Scientific contributions

Part of the work described in this thesis resulted in publications listed here below. Abstract :

Optical and Electronic Ion Channel Monitoring from Native Human Membranes

Drug and toxicity assays are necessary to develop advanced treatments as a way of improving diagnostics and treatment. However, the actual in vitro models are currently not on par regarding the in vivo system they attempt to mimic. Two research directions can be invested in to overcome this issue: 1) elaboration of more complex in vitro biological model and 2) improvement of the in vitro experiment monitoring. We propose a multi-parametric system approach integrating bioelectronic devices complementing optical monitoring. A review on Organic Microelectrodes introduces the field of organic materials. To include electrophysiology monitoring steps into usual biological experiments, the planar bioelectronic chips are assembled in the form of a standard cell biology tool: the 12-well plate. To overcome the number limitation of channels available for both input and output monitoring, Organic electrochemical transistor (OECT) designs were arranged and optimized in a matrix structure. The bioelectronic device's ability to monitor biological experiments in vitro is tested to monitor a) the transmembrane proteins in a lipids bilayer b) an epithelial barrier formation, and c) detect electrogenic cell activity. In conclusion, we have included bioelectronic devices into generic tools from the biological experiment is possible, and allows multiparametric monitoring of the in vitro experiment.