MISFET-based biosensing interface for neurons guided growth and neuronal electrical activities recording
Introduction
The study of the neuronal network dynamics in brain tissue is a difficult task owing to the large number of closely packed neurons with complex synaptic connectivity and the difficulty of monitoring activity in the neural network at the single-cell resolution with non-invasive techniques. In principle, low-density cultures of nerve cells drastically reduce the network complexity but uncertainty on the synaptic wiring remains. Furthermore, while single cell recording can be easily obtained by patch clamp recording [1], it is very challenging to achieve multicellular recordings with this approach. By contrast, the use of cell non-invasive extracellular multi-electrode array (MEA) enabled recordings and stimulations of large cell numbers [2], [3]. Similarly, planar MEA were used for neurons culture for days and months without inflicting mechanical damage to the neuronal membrane [4]. This involves electrical measurements (voltage or current) across the cell membrane [5]. Nevertheless, such extracellular electrical interfacing of neurons was implemented with planar metal electrodes on insulating substrates [6], [7], [8], associated with organic transistors [9] or integrated with semiconductor devices using Complementary Metal Oxide Semiconductor (CMOS) technology [10], [11]. In all cases, the activity of a nerve cell – i.e. the action potential – is stimulated and/or recorded by interfacing individual nerve cells with electrical microdevices that locally generate and/or sense electrical field variations towards medical applications [12]. So far, efforts have been focused onto interfacing nerve cells in order to measure and interpret the neuronal action potential and greater sensitivity would be required to measure smaller cell signals. One of the first way to “communicate” with nerve cells or to make them inter-communicate using microelectrodes was achieved by Jerome Pine [13] and Guenter Gross [14]. Then, Peter Fromherz proposed to use MOS technology in order to integrate microelectrode arrays [15], emphasizing the following principle: (i) neuronal activity is elicited by capacitive stimulation from a silicon chip and (ii) neuronal activity is recorded by a transistor located on the same chip. Nevertheless, the signal-to-noise ratios were relatively small often preventing the distinction between single cell recordings and multi-unit activity. Inspired by Fromherz's seminal works [16], [17], Charles Lieber's team demonstrated enhanced signal-to-noise ratios using arrays of nanowire field-effect transistors connected to individual axons or dendrites of mammalian neurons [18]. In this report, each nanoscale junction was used for spatially resolving stimulations, and/or inhibitions of propagating neuronal signals with great detection sensitivity.
Moreover, neural patterning has attracted much interest in neural electronics [19] and neural engineering [20]. Compared with randomly cultured neurons, patterned neurons possess a much simpler network structure, which is easier for network modelling and signal analysis. In addition, the amplitude of detected action potentials of neurons is higher when neurons are located directly on the electrodes [6]. With these advantages, different protocols have been developed to perform and optimize neuron positioning and neurite growth. Thus, methods such as micro-contact printing [19], photodegradation [21], microfluidic structuration [22], biomolecule patterning [23] and photoresist processing [24] were applied for constraining neurons and guiding neurites on planar substrates.
In the present study, we have produced neural networks with defined topology on a substrate with appropriate integrated electrical contacts for each (or groups of) neuron(s). This bottom-up approach faced three objectives: (i) non-invasive electrical interfacing of individual neuronal cell bodies, (ii) control of neurites outgrowth to form topologically defined and stable networks, and (iii) the integration of both defined networks and extracellular interfacing in a same, dedicated system. Our transistor-based biosensing interface solved these three challenges at once.
Section snippets
Design and fabrication of biosensors
Considering a SiO2/Si3N4 pH-sensitive chemical field effect transistor (pH-ChemFET) technology [25], N-channel metal-insulator-semiconductor field effect transistors (MISFET) were realized. The MISFET fabrication and encapsulation processes were previously described [26] but briefly reminded hereafter. Starting with a 6 inch, N-type (1 × 1013 cm−2), (1 0 0) silicon wafer, P-doped wells were realized by boron implantation (1 × 1015 cm−2) and, following, N-doped source and drain regions were formed by
Neuronal orientation
The first objective of our work was to generate on-chip guided growth of the neurons in order to increase their contact with our MISFET sensors. We first evaluated if SU-8 micro-channels above the transistors’ gates (Fig. 3) could induce the neuronal guided growth. To overcome the well-known SU-8 cytotoxicity [36], we proceeded to a four-step specific protocol necessary to turn the SU-8 biocompatible. The first step is chemical treatments with SU-8 developer in order to remove all resist
Conclusion
In this study, we developed a MISFET transistor array to record neuronal activities while using integrated SU-8 microfluidic structures in order to guide the neuronal growth. This lab-on-chip was tested for the organized growth of rat retina neurons and L. stagnalis snail pedal neurons. Then, the MISFET array was used for the recording of action potentials while using L. stagnalis pedal neurons. Neuronal activities were successfully triggered and inhibited by implementing a picrotoxin – GABA –
Acknowledgments
Amel Bendali received a PhD fellowship from the University “Pierre and Marie Curie” (Paris 06). All authors were supported by a CNRS grant (programme interdisciplinaire: interface physique chimie). Serge Picaud was supported by grants from the French “Fondation pour la Recherche Médicale” (FRM) and by the European Community's Seventh Framework Programme (FP7) under grant agreement n° 280433 (NEUROCARE project). The technological realizations and associated research works were partly supported
F. Larramendy was born in Brive-la-Gaillarde, France, in 1986. He received the M.S. degree in Microelectronic System Engineering from the Paul Sabatier University, Toulouse, France, in 2009. He joined the Laboratory for Analysis and Architecture of Systems (LAAS), National Center of Scientific Research (CNRS), Toulouse in 2009 as Ph.D. Student. He was in charge of the development of microsystems to measure neuronal activities. After obtaining his D.Sc. Degree in 2013, he joined the EUJO-LIMMS
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F. Larramendy was born in Brive-la-Gaillarde, France, in 1986. He received the M.S. degree in Microelectronic System Engineering from the Paul Sabatier University, Toulouse, France, in 2009. He joined the Laboratory for Analysis and Architecture of Systems (LAAS), National Center of Scientific Research (CNRS), Toulouse in 2009 as Ph.D. Student. He was in charge of the development of microsystems to measure neuronal activities. After obtaining his D.Sc. Degree in 2013, he joined the EUJO-LIMMS project in the Microsystem Materials Laboratory, Department of Microsystems Engineering (IMTEK), Freiburg, Germany, and the Laboratory for Integrated Micro Mechatronic Systems (LIMMS), Tokyo, Japan. Currently, he is in charge of the development of microprobes for brain insertion for biomedical applications.
A. Bendali received her BS degree in Physics in 2006 and her MS degree in Instrumentation for Biotechnology in 2008 from Institut National Polytechnique de Grenoble, France. She recently graduated her Ph.D. in Neuroscience at Université Paris VI under the supervision of Dr. Serge Picaud. Her research focuses on building networks of retinal neurons on multi-electrode arrays for the study of retinal signal processing and testing carbon-based materials such as diamond and graphene to design retinal prosthetic devices.
M.C. Blatché was born in Toulouse, France, in 1986. She received the Licence degree in Biotechnologies from Nice-Sophia Antipolis University, Nice, France in 2008. She joined the Laboratoire d’Analyse et d’Architecture des Systèmes (LAAS), Centre National de la Recherche Scientifique (CNRS), Toulouse in 2009, where she is in charge of the “Biology” technological platform and worked on the development of cell growth on microsystems.
F. Mathieu was born in Orléans, France, in 1972. He received the engineering degree in communication systems and electronics from the CNAM (Conservatoire National des Arts et Métiers), Toulouse, France, in 2003. He joined the Laboratoire d’Analyse et d’Architecture des Systèmes (LAAS), Centre National de la Recherche Scientifique (CNRS), Toulouse, in 2001, where he is currently in charge of the development and design of very low signal detection systems applied to the micro(nano)electromechanical systems area and its complete electronic treatment and control for automation.
S. Picaud was born in Paris (France) in 1961. He received a Ph.D. in Neurosciences from the University of Marseille in 1990. He was educated by leaders in the field of retinal physiology during doctoral and postdoctoral stays with Professor Wässle (Max-Planck Institute for Brain research, Francfurt, Germany) and Professor Werblin (University of California, Berkeley, USA). He is currently leading the team “retinal information processing” at the Vision Institute in Paris created by Professor Sahel. After studying retinal physiology and pharmacology with applications in neuroprotection, the team has moved towards strategies for restoring vision in blind patients having lost their photoreceptors. These strategies involve retinal prostheses or optogenetic therapy, which requires expression of photosensitive pump or ionic channels in selected retinal neurones.
P. Temple-Boyer was born in Montpellier (France) in 1966. He received his Engineer Master's Degree in electronic engineering from the Ecole Supérieure d’Electricité (Paris – France) in 1990 and his Master's Degree in microelectronics from the Université Paul Sabatier de Toulouse (France) in 1992. He joined the Laboratoire d’Architecture et d’Analyse des Systèmes of the French Centre National de la Recherche Scientifique (LAAS-CNRS) in 1992 and received the Ph.D. degree from the Institut National des Sciences Appliquées de Toulouse (France) in 1995. Since then, as a senior researcher, he has been working on the development of micro- and nanotechnologies.
L. Nicu was born in 1973 in Bucharest (Romania). After completing his Master of Electrical Engineering at the Paul Sabatier University of Toulouse (France) in 1997, he joined the Integrated Microsystems Group at the LAAS (Laboratory for Analysis and Architecture of Systems) of Toulouse where he obtained his Ph.D. in 2000 into the Micromechanical Structures field. From 2000 to 2003, he was R&D Engineer at Thales Avionics, Valence (France). His activities focused onto the development of micromechanical sensors for the civil and military navigation applications. Since 2003 he joined the NanoBioSystems Group at LAAS as a full time CNRS (National Center of Scientific Research) researcher where he currently works in two main research fields: the development of (1) new resonant bio(chemical)sensors using M(N)EMS technologies and of (2) cantilever-based microsystems for contact deposition of small amounts of biological samples for biochip applications.