Large array of GFETs for extracellular communication with neuronal cells

Graphene has already shown its high ability for biosensing. Solution-gated graphene field effect transistors, which showed very high sensitivity in electrolytes [1], have another biologically important application: recording neuronal activity. Such devices exhibit very high signal-to-noise ratio for extracellular measurements [2]. The aim of this work is to optimize and scale both fabrication procedure and measurement system. When working with biological samples, there is a need in a large number of devices. High density of the devices is also preferable. Therefore we fabricate the devices on 4’’ wafer, resulting in 50 chips, 11*11mm each. Each chip consequently embodies an array of 32 graphene FETs (see fig.1). The active area of the chip is around 2 mm2 while each GFET’s channel differs between 5 and 20 um with altered configurations. Such devices, when used with the already developed multichannel measurements system make possible simultaneous measurement and stimulation of all 32 transistors in a time-scale. This makes possible to measure not just discrete spikes, but even propagation of the action potential through the neuronal network

How to image cell adhesion on soft polymers?

Here, we present a method to investigate cell adhesion on soft, non-conducting polymers that are implant candidate materials. Neuronal cells were grown on two elastomers (polydimethylsiloxane (PDMS) and Ecoflex®) and prepared for electron microscopy. The samples were treated with osmium tetroxide (OsO4) and uranylacetate (UrAc). Best results can be achieved when the polymers were coated with a thin iridium layer before the cell culture. This was done to emphasize the usage of soft polymers as supports for implant electrodes. A good contrast and the adhesion of the cells on soft polymers could be visualized

Modulation of in vitro cortical networks by mechanical perturbation of individual neurons

Recent investigations of network responses to a stimulus point to the ability to modulate a network at the single neuron level. Different perturbation modalities, ranging from single-cell electrical stimulation to focal damage, modulate the local network’s firing dynamics and functional topology. With a growing body of evidence revealing that the target neurons respond to mechanical stimulation, we examined the neuronal network behavior upon mechanical stimulus. Specifically, we investigated to what extent a patch-clamp-induced mechanical perturbation influences the activity in the local network of dissociated rat cortical neurons. Simultaneous patch-clamp and calcium imaging experiments demonstrated that patch-pipette mediated mechanical stimulus induces calcium plateaus in targeted neurons, and on average in about 30% of neighbors in 0.185 mm2. Interestingly, the initiation and propagation of these responses are independent of the neuron’s spiking, as the calcium plateaus persisted after the pharmacological block of action potentials and synapses. However, combined electrophysiology experiments demonstrated a bursting activity during the mechanical perturbation, suggesting that the underlying mechanisms induce action potentials. To investigate the impact of patch-clamp-induced perturbation on the network’s functional topology, we assessed the effective network connectivity before and after the mechanical stimulus. Here we show that a single mechanical perturbation reduces the network’s ability to integrate information, with the recovery time of around 10 minutes. These findings indicate the potential use of mechanostimulation in network manipulation, while concurrently emphasizing the restrictions in the application of patch-clamp for network investigations

Mechanical stimulation of individual neurons selectively excites and modulates the local neuronal network

Mechanical stimulation is a promising means to modulate a neuronal network’s activity. In particular, wide-network mechanical stimulations demonstrated both neuron excitation and long-term modulation of neuronal excitability. Although simultaneous whole-network excitations reliably induce neuronal responses, it remains unknown if there is a cross-talk between mechanically affected neurons.To estimate whether the single-neuron mechanical stimulation induces network-level activity, we investigated in vitro cortical network responses during patch-clamp mechanical perturbation. Calcium imaging of the local neuronal network during mechanical perturbation of individual neurons demonstrated the responsiveness of about one-third of the close neighbors. Here we show that evoked calcium signals propagate from the soma to the neurites and the neighboring neurons up to a few hundred micrometers. The inter-neuron propagation is likely regenerative, while pharmacological investigations revealed that action potentials and chemical synapses do not participate in the process. On the other hand, electrophysiology measurements show bursting activity correlating with the mechanical stimulus, both in the target and responding neurons. In line with the recent findings of mechanostimulation’s neuromodulatory effect on the membrane, we further investigate whether the single-cell perturbation influences the local network. Our preliminary results indicate that single neuron mechanical stimulation transiently increases the excitability and gain exclusively in the neurons whose calcium signals responded to the mechanical stimulation of a nearby cell.Overall, our results demonstrate that an individual single-cell mechanical stimulation reliably evokes the responses within the local network and potentiates the pre-synapses within the responding assembly for further inputs. These indicate potential cross-talk in whole network mechanical stimulations and point to methodological restrictions of the golden standard in electrophysiology, the patch-clamp technique. We offer the time window in which these measurements are reliable

Effect of membrane deformation on electrical firing in rat cortical neurons during electrophysiological measurements

INTRODUCTION From patch-clamp to 3D nanoelectrodes, tight mechanical coupling with the neuronal membrane is essential to secure the high amplitude electrical recording. As a byproduct, both approaches induce membrane deformation. In the former, the membrane is acutely deformed by the glass pipette,while in the latter, the membrane spontaneously engulfs the 3D vertical nanostructure. OBJECTIVES In line with the discoveries pointing to the existence of mechanosensitive ion channels in neurons, we combined electrophysiology with functional imaging to test the effect of acute and chronic membrane deformation on rat cortical neurons’ electrical properties and firing dynamics.MATERIALS AND METHODS To estimate the effect of patch-clamp induced acute deformation, we combined semi-blind patch-clamp with calcium-imaging. Additionally,we utilized patch-clamp to investigate whether the long-term exposure to vertical topology on 3D nanoelectrodes influences the neurons’ electrical properties. All measurements were performed on rat cortical neurons starting from 2 weeks in culture.DISCUSSION AND RESULTS Calcium-imaging measurements during the formation of giga-seal have demonstrated that patch-clamp targeted neurons respond to the mechanical perturbation with plateau-shaped calcium signals (N = 29). Moreover,up to 100% of neurons in 0.185 mm 2 area responded in a similar trend.This finding suggests that acute deformation affects not only the targeted neuron, but also the immediate network. Furthermore, the comparison of neurons on flat surface and neurons on 3D nanoelectrodes showed no statistically significant difference in excitability and action potential firing.CONCLUSION Overall, these results recognize the effects of acute, patch-clamp mediated mechanical perturbation on the targeted neuron, as well as the immediate network. On the other hand, no changes were present with chronic membrane deformations during the spontaneous engulfment of vertical nanostructures. Keywords: electrophysiology, membrane, neuron

Wafer-scale fabrication of graphene-based field effect transistor arrays for extracellular measurements

The work is focused on the fabrication and analysis of graphene-based, solution-gated field effect transistor arrays (GFET arrays) in a large scale. The GFETs show extremely high electrolyte-gated transconductance promising exceptional biosensing capability. Signal-to-noise ratio (SNR) of the GFETs is analysed for different graphene areas. In the future we will apply these GFETs for extracellular recordings from neuronal and cardiac cells

Wafer-scale fabrication of graphene field effect transistors for neuronal interfacing

There are plenty of invasive methods for studying a neuronal network’s activities [1]. Of course, the invasiveness of the processes makes them undesired. In recent years, there has been vast research in the field of non-invasive neuronal interfacing and extracellular neuronal recordings [2]. Different methods (passive – MEAs and active – FETs) and different materials (carbon, silicon, PEDOT:PSS) have been used for the purpose.Graphene’s excellent electrical, mechanical and biological properties make it a perfect candidate for such a role. Firstly, liquid-gated graphene field effect transistors (GFETs, see fig. 1) show very high transconductance, and therefore sensitivity [3]. Secondly, graphene is a very stable and biocompatible material (fig.2). Thirdly, flexibility and bendability of graphene make it the most promising material for future bio-implantable devices [3].Therefore we established our 4-inch wafer fabrication process based on CVD-grown graphene (fig. 3a). Each fabricated wafer results in 52 biocompatible chips (fig. 3b). Each chip comprises 32 GFETs (fig. 3c). The size of graphene active area is varied in order to study the noise of the system. Each chip is measured on a multi-channel measurement system, which allows us to measure all the GFETs simultaneously. Thus, it is possible to measure not just single action potentials of the electrogenic cells, but even propagation of the potential through the network