Grant Agreement N°732032

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BrainCom Deliverable D1.1

Report on novel cortical µECoG and intracortical technologies

WP 1 Technology of neural interfaces

Task 1.3, 1.4 Surface conformal neural interfaces for preclinical studies; Penetrating soft neural interfaces for preclinical studies

Dissemination level PU Due delivery date 31/05/2018

Nature R Actual delivery date 24/07/2018

Lead beneficiary UCAM

Contributing beneficiaries UCAM, ICN2, CSIC

Document Version Date Author Comments

V1 18/5/2018 George Malliaras (UCAM)

V2 9/7/2018 Clement Hebert (ICN2)

V3 17/7/2018 Jose Garrido (ICN2)

V4 20/7/2018 Jose Garrido (ICN2) & George Malliaras (UCAM)

Ref. Ares(2018)3918747 - 24/07/2018

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Deliverable abstract

The scope of this deliverable is to summarize the activities in BrainCom related to the technology of epicortical probes and intracortical probes for mapping brain activity.

In the case of the epicortical probes, we have focused on i) the design, fabrication and characterization of arrays of graphene transistors (active probes) prepared on 10 micrometer-thick polyimide substrates and ii) design, fabrication and characterization of arrays of PEDOT:PSS microelectrodes (passive probes).

In the case of the intracortical probes, the focus has been on the development of different strategies for the insertion of the flexible microelectrode probes. To this end, a novel insertion procedure using bioresorbable polymers has been developed.

Deliverable Review

Reviewer #1: Jose A Garrido Reviewer #2: ..........................................

Answer Comments Type* Answer Comments Type*

1. Is the deliverable in accordance with

(i) the Description of Work?

Yes No

M m a

Yes No

M m a

(ii) the international State of the Art?

Yes No

M m a

Yes No

M m a

2. Is the quality of the deliverable in a status

(i) that allows it to be sent to European Commission?

Yes No

M m a

Yes No

M m a

(ii) that needs improvement of the writing by the originator of the deliverable?

Yes No

M m a

Yes No

M m a

(iii) that needs further work by the Partners responsible for the deliverable?

Yes No

M m a

Yes No

M m a

* Type of comments: M = Major comment; m = minor comment; a = advice

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Table of content

1. Introduction ............................................................................................................................. 4

2. State of the Art ......................................................................................................................... 4

3. Results and Analysis ................................................................................................................. 4

4. Conclusion ............................................................................................................................. 10

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1. Introduction

The objective is to design and fabricate state-of-the-art neural probes with PEDOT:PSS and graphene as the active layers for electrodes and transistors. Epicortical and intracortical designs are considered in this study.

Apart from the fabrication report, we present the assessment of the performance of the devices in vitro.

2. State of the Art

The state-of-the-art of PEDOT:PSS microelectrodes (passive sensor technology) is described in publications by the UCAM team1; it consists of a parylene-based microtechnology that yields flexible cortical probes that are highly conformal to the brain. In order to insert these probes intracortically in the brain, rigid, SU8 shuttles have been used until now. Here we have gone beyond the state of the art by increasing electrode density and decreasing electrode size (at this stage down to electrode dimensions of 7x7 µm2) (hence gaining in spatial resolution) and by using a novel insertion shuttle based on bioresorbable polymers.

Graphene and PEDOT:PSS microtransistors (active sensor technology) are also developed, with the ultimate goal of being used for the multiplexing strategy; in contrast to the passive sensor technology (electrodes), transistors are superior for multiplexing strategies. The operation principle of this technology is to modulate the current of the channel of a field effect transistor thanks to the variation of potential induced by neuron activity. The transistors present the advantage over the microelectrode to pre-amplify the signal very close to the neurons, thus reducing the contribution of the noise introduced by the connection tracks. UCAM and ICN2/ CIBER teams reported very efficient PEDOT:PSS2 and graphene3 microtransistors able to record LFPs.

3. Results and Analysis

3.1 Epicortical technology

a) Passive sensor technology: microelectrodes

UCAM has fabricated and characterised PEDOT:PSS-based microelectrode devices. Figure 1a shows optical pictures of the 8x8 array probe with 16x16 μm2 active sites and an electrochemical impedance spectrum. The impedance recorded at 1kHz with this type of electrode is around 40 kΩ, thus implying a low noise level for high signal to noise ratio recording. 8x8 electrode arrays with active sites of 7x7 μm2 were also designed. Figure 1b shows that by increasing the PEDOT:PSS

1 J. Rivnay, H. Wang, L. Fenno, K. Deisseroth, and G.G. Malliaras, “Next-generation probes, particles, and proteins for neural interfacing”,

Sci. Adv. 3, e1601649 (2017) ; T. Someya, Z. Bao, and G.G. Malliaras, “The rise of plastic bioelectronics”, Nature 540, 379 (2016) ; D. Khodagholy, J.N. Gelinas, T. Thesen, W. Doyle, O. Devinsky, G.G. Malliaras, and G. Buzsáki, “NeuroGrid: recording action potentials from the surface of the brain”, Nature Neurosci. 18, 310 (2015).

2 D. Khodagholy, T. Doublet, P. Quilichini, M. Gurfinkel, P. Leleux, A. Ghestem, E. Ismailova, T. Herve, S. Sanaur, C. Bernard, and G.G. Malliaras, “In vivo recordings of brain activity using organic transistors”, Nature Comm. 4, 1575 (2013).

3 C. Hebert, E. Masvidal-Codina, A. Suarez-Perez, A.B. Calia, G. Piret, R. Garcia-Cortadella, X. Illa, E. Del Corro Garcia, J.M. De la Cruz Sanchez, D.V. Casals, E. Prats-Alfonso, J. Bousquet, P. Godignon, B. Yvert, R. Villa, M.V. Sanchez-Vives, A. Guimerà-Brunet, J.A. Garrido. “Flexible Graphene Solution-Gated Field-Effect Transistors: Efficient Transducers for Micro-Electrocorticography”, Advanced Functional Materials 28, 1703976 (2018); B. M. Blaschke, N. Tort-Colet, A. Guimerà-Brunet, J. Weinert, L. Rousseau, A. Heimann, S. Drieschner, O. Kempski, R. Villa, M.-V. Sanchez-Vivez, J. A. Garrido “Mapping brain activity with flexible graphene micro transistors”, 2D Materials 4 (2), 025040 (2017)

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thickness it is possible to obtain similar impedance value as that of the 16x16 μm2 active site electrodes.

a b

Figure 1: PEDOT:PSS microelectrode arrays. a) Photos of flexible electrode arrays with 16x16 μm2 sites and corresponding electrochemical impedance spectra. b) Layout of arrays with 7x7 μm2 sites

and spectra for different thickness of PEDOT:PSS.

These “soft” neuroprosthetic devices, made of thin films of organic materials (polymers) with similar chemical nature and properties as soft tissues, follow a known strategy for overcoming the limitations of traditional neural electrode technologies4. Reducing size and thickness (and thereby bending stiffness) as well as manipulating innate material properties (e.g. Young’s modulus) of implants have been shown to significantly attenuate the foreign body reaction5. Therefore, selection of soft materials and fabrication of flexible and elastic devices offers promise for extending the lifetime of implantable devices to match patient lifespans6. The use of conducting polymer electrodes enables the conduction of ions, which paves the way for lower impedance, hence allows the fabrication of smaller electrodes, which is one of the goals of BrainCom2.

b) Active sensor technology: microtransistors

Following the roadmap of BrainCom, ICN2 fabricated and characterised 6x11 transistor arrays with 20x20μm2 and 50x50 μm2 active sites. The fabrication procedure (see Figure 2) is as follows.

A 10 µm thick biocompatible polyimide (PI) layer is spin-coated on a 4” Si/SiO2 wafer. The first layer of Ti/Au (10 nm/100 nm) metal contacts are deposited and then structured by means of optical lithography. Afterwards, CVD graphene is transferred to the wafer using a PMMA wet etching process. The graphene active area of the sensors is then defined by oxygen plasma in a reactive ion etching system. A second metallization layer of is then evaporated and lithographically defined followed by a lift-off step. To electrically insulate the device, a 2-µm-thick SU-8 epoxy photoresist layer is spin-coated and defined in such a way that only the graphene area is left uncovered.

Finally, the PI probes are cut by reactive ion etching using a protective Aluminum mask with the shape of the implants. The flexible PI implant with the graphene sensors are then peeled and released from the sacrificial Si/SiO2 wafer.

4 T. Someya, Z. Bao, and G.G. Malliaras, “The rise of plastic bioelectronics”, Nature 540, 379 (2016).

5 R. Chen, A. Canales, and P. Anikeeva, “Neural recording and modulation technologies”, Nature Reviews Materials 2, 16093 (2017).

6 J.W. Jeong, G. Shin, S.I. Park, K. Yu, L. Xu, and J.A. Rogers, “Soft materials in neuroengineering for hard problems in neuroscience”, Neuron 86, 175 (2015).

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Figure 2: Fabrication procedure of flexible graphene transistors.

Figure 3 shows the design and layout of a typical microtransistor arrays (6x11) as well as images with the completed technology.

Figure 3: Design and layout of the graphene microtransistor arrays for epicortical applications, and images of the 4” wafer technology.

Figure 4 shows the characterization of a probe of graphene transistors (20x20μm2 and 50x50 μm2

active sites) in terms of the drain-source current vs gate bias (left graph) and the transconductance vs gate bias (right graph). The DC characterization indicates a good homogeneity, which is attributed to the fabrication technology matureness.

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Figure 4: DC characterization of a graphene-based microtransistor array for epicortical applications; transistor active sites are 20x20μm2 and 50x50 μm2. Left: drain-source vs gate bias transistor curves (Vds=100mV). Right: transconductance (normalized by the drain-source voltage) of the

devices shown in the left graph.

The noise performance of the graphene transistors is also assessed. Figure 5 shows the noise characterization of the same device shown in Figure 4. Values as low as 5μV rms can be obtained for 50x50cm2 transistors, at an optimum gate bias polarization. These values are low enough to record LFP signals, which have amplitudes in the range of few hundreds of microvolts.

Figure 5: Noise performance of graphene-based microtransistor array for epicortical applications; transistor active sites are 20x20μm2 and 50x50 μm2. Left: RMS noise of the drain-source current vs gate bias (Vds=100mV). Right: RMS noise normalized to the gate, representing the minimum signal

that could be recorded by the transistors.

Aiming at increasing the number and density of sensing sites in an array, a new multiplexing strategy based amplitude modulation (AM) of the neural signals is being developed by the BrainCom consortium. The use of such technique requires to operate the g-SGFETs at high carrier frequencies. The suitability of the g-SGFETs to be operated in this mode has been assessed; to this end, the transconductance of the graphene transistors has been calculated by recording the current response to a gate signal (sinusoidal of 10 Hz) when different carrier frequencies are used (the carrier signal is applied at the drain-source). Figure 6 shows the transconductance of the transistors for different carrier frequencies (up to ~500kHz), confirming that graphene SGFETs can be used in this multiplexing strategy.

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Figure 6: Tranconductance of graphene microtransistors operated in an amplitude modulation operation mode, in which a gate signal (10Hz) is modulated by a carrier signal (drain to source

voltage) with different frequencies.

In order to assess the performance of the transistors array for the recording of neural signals, a stimulation generator mimicking the neural activity has been provided by the MCS partners. Figure 7 shows the response of the transistor array to the simulated signal of hippocampal population spikes, and retina spikes. The recording with the transistor exhibit very good signal to noise ratio of 14 in the case of the hippocampus activity simulation and 6 for the retinal activity simulation.

Figure 7: Response of graphene transistors to the signal generated by a stimulation generator mimicking the neural activity. Left: signals simulating typical retina spikes. Right: signals simulating

hippocampal population spikes.

A more detailed analysis of the noise performance of graphene transistors, including a comparison with other technologies, will be provided in the new deliverable that has been requested after the 1st review meeting. This deliverable is expected to be completed by the end of July 2018.

3.2 Intracortical technology

UCAM fabricated penetrating flexible probes with PEDOT:PSS electrode. These probes were rigidified using a combination of PVA and PLGA. To coat the probes with these two polymers, they were first placed in a PDMS mold (see Figure 8a). Figure 8b shows the evolution of the polymers

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with time. It can be clearly seen that the addition of the PLGA to the PVA is improving the life time of the stiff coating which is compulsory for the insertion of the probe inside the cortex. UCAM has developed a suitable set of processing parameters that are compatible with the insertion procedure of the penetrating neural probes. In vivo validation data are shown in Figure 9.

a b

Figure 8: Implanted probes. a) Rigidification process of the soft penetrating probes. b) Evolution of the polymer coating with time under ambient (humid) conditions.

Figure 9: In vivo characterization of intracortical probes. (a) Picture of a probe lowered in the craniotomy performed in an anesthetized mouse. (b) Nissl staining of a 60 µm brain section showing

the trajectory (black arrowheads) of an implanted probe through the cortex and reaching the CA1 region of the hippocampus (yellow arrowhead). Bar = 250 µm. (c) Recorded data showing LFPs

measurements of 5 recording sites of the probe showed in b. Note the increased quality of the LFP signals recorded with time after the probe insertion, revealing the dissolution of the PVA polymer.

The LFP traces are arranged according to the probe sites layout (tip = bottom).

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4. Conclusion

During the first 18 months of the project, the technology teams (ICN2 and UCAM) have been working on further improving the fabrication of flexible arrays of microelectrodes (based on PEDOT:PSS) and microtransistors (based on graphene field effect transistors). Arrays of PEDOT:PSS microelectrodes with electrode dimensions as low as 7x7 µm2 have been fabricated, which are going to be used for high density cortical mapping. Using active sensor technology, arrays of graphene microtransistors (6x11 transistors per cortical device) have been fabricated and fully characterized in vitro. This technology will be at the core of the frequency multiplexing strategy that BrainCom is developing, with the goal of enabling brain mapping with a large number of recording sites.

In addition to the epicortical devices, intracortical technology has also been developed; in this respect, a polymer-based coating that turns the soft probe stiff for insertion inside the cortex has been explored. First in vivo experiments have been performed to validate the

Currently, these technologies are going to be tested in vivo (by the LMU partner) with the goal of achieving a first assessment of the technology, in particular to evaluate the signal-to-noise ratio figure of merit. The first in vivo assessment of both technologies is planned during September 2018.

The technologies summarized in this deliverable will be evolving during the next 18 months. In particular, efforts will be dedicated to further improve the homogeneity of the arrays, as well as the noise performance. Long-term stability assessment in chronic environment will be conducted as part of BrainCom.