Graphene devices for cell bioelectronics

. José Manuel de la Cruz Sánchez

Dra. Elena del Corro García Prof. Jose Antonio Garrido Ariza

ICN2 ICREA- ICN2

Advances in neuroscience are possible thanks to the progressive development of new tools and techniques that offer researchers the ability to visualize and record more and more aspects of the nervous system. Among all these tools, electrodes and microelectrode arrays allow us to directly measure and study the electrical activity produced by the brain and other organs of the nervous system, with great spatial and temporal resolution. In addition, electrodes allow us to establish bidirectional communication with neural tissue, applying electrical stimulation pulses that can be used to study and explore different aspects of the brain or even to restore neurological abilities lost due to illness or accidents. The need for stable and biocompatible materials, but also capable of recording electrical activity with low noise and injecting enough current to stimulate neural tissue, has led researchers to explore new materials to manufacture electrodes intended to interact with the nervous system. Within this framework, we have explored the capabilities of different carbon-based materials to interact bidirectionally with nervous tissue. In this thesis, we have developed low-noise, single-layer graphene multielectrode arrays and used them to measure electrical activity in primary cortical cultures. We have also developed transparent and flexible single-layer graphene devices with a single macroelectrode and used them to measure electroretinograms, comparing them with the current state of the art for animal use, using a commercially available, clinically approved measurement equipment. In addition, taking advantage of the transparency of monolayer graphene, we have developed transparent microelectrode arrays, which allow us to obtain spatial information on the corneal potential. In this thesis we also present the fabrication of new reduced graphene oxide electrodes, which have allowed us to develop microelectrode arrays with high charge injection capacities and low electrical noise values. We have demonstrated that these microelectrode arrays are capable of allowing the growth and development of healthy primary hippocampal cultures and of communicating bidirectionally with them, performing measurements and applying stimuli simultaneously. Finally, to exploit the versatility of our graphene-based microelectrode arrays, we have explored three different techniques to guide and control the growth of neurons cultured on our devices, with the aim of developing new tools designed to study diverse neuroscientific problems using bottom-up neuroscience. Overall, the results presented in this thesis demonstrate that graphene-based electrodes, with their stability, biocompatibility and extraordinary electrical capabilities, are extremely valuable tools for conducting in vitro and in vivo neuroscience studies.

In a purely practical sense, understanding how our brain and nervous system work is one of the cornerstones to improve our quality of life. Studies show that in 2010 an estimated 180 million people were affected by brain diseases in Europe, which amounts to one third of the total population(DiLuca & Olesen, 2014), positioning brain diseases as the major health problem in developed countries. Furthermore, and to raise awareness about this issue, several studies have estimated the total cost of brain disorders, in Europe in 2010 only, in more than 800 billion euros (Gustavsson et al., 2011; Olesen et al., 2012). In addition, the huge complexity of the human brain makes brain disorders more difficult to analyze, diagnose and treat than the rest of diseases, making neuroscience research fundamental to address this issue (Morris et al., 2016). This perspective has pushed for the creation of big collaborative and multidisciplinary research projects such as the Human Brain project in Europe (Markram, 2012), the BRAIN initiative (Jorgenson et al., 2015) and the Allen Brain Atlas (Jones et al., 2009) in the USA. With these facts in mind, it is obvious that addressing the issue of brain diseases is not only an unquestionable emergency but a fascinating challenge for our society. Within this context, the field of bioelectronic therapies is gaining huge interest lately. Bioelectronics is a field of medicine in which electrical and electronic engineering solutions are used to treat different diseases; that has led, for example, to the creation of devices such as the pacemaker. The bioelectronics marked size was valued at USD 4.5 Billion in 2018 and is expected to reach USD 10 Billion by 2026 (Bioelectronics Market Size, Share, Trends, Opportunities & Forecast, n.d.). Inside the bioelectronics field, neuromodulation is the branch that deals with the alteration of neuronal activity by using electrical stimulation. The most prominent example of neuromodulation therapy nowadays is the use of Deep Brain Stimulation (DBS) to threat Parkinson’s disease. DBS therapy works by delivering electrical stimulation to the subthalamic nucleus to disrupt the anomalous electrical activity patterns that cause the motor tremors produced by Parkinson´s disease (Stoker & Greenland, 2018). Besides the treatment of Parkinson´s, DBS has been postulated as therapy for other diseases such as drug refractory epilepsy, obsessive-compulsive disorder or chronic pain (Deep Brain Stimulation - Mayo Clinic, n.d.). Apart from applications in the central nervous system, bioelectronic therapies in which the peripheral nervous system is stimulated are being consider to threat epilepsy, depression, anxiety and even Alzheimer´s disease (Groves & Brown, 2005). With all this information in mind is clear that, due to the growing cost of brain diseases, the aging population in developed countries and the favorable market projections for bioelectronic therapies and neuromodulation, there is a huge business opportunity in this market

Neural Activity Recording; Neuroscience; New Tools; Techniques; Electrodes; Microelectrode Arrays; Electrical Activity; Brain; Nervous System; Spatial Resolution; Temporal Resolution; Bidirectional Communication; Neural Tissue; Electrical Stimulation Pulses; Restore Neurological Abilities; Stable Materials; Biocompatible Materials; Low Noise; Current Injection; Carbon-Based Materials; Low-Noise Single-Layer Graphene Multielectrode Arrays; Primary Cortical Cultures; Transparent and Flexible Single-Layer Graphene Devices; Macroelectrode; Electroretinograms; Animal Use; Clinically Approved Measurement Equipment; Transparent Microelectrode Arrays; Corneal Potential; Reduced Graphene Oxide Electrodes; High Charge Injection Capacities; Low Electrical Noise Values; Primary Hippocampal Cultures; Bidirectional Communication; Measurements; Stimuli; Versatility; Neuron Growth Guidance; Neuron Growth Control; Bottom-Up Neuroscience; In Vitro Neuroscience; In Vivo Neuroscience.