Biomedical Microdevices (2022) 24: 31

https://doi.org/10.1007/s10544-022-00632-0

A flexible implantable microelectrode array for recording

electrocorticography signals from rodents
Suman Chatterjee1 · Tushar Sakorikar1 · Arjun BS1 · Rathin K. Joshi1 · Abhay Sikaria2 · Mahesh Jayachandra3 ·
Vikas V2 · Hardik J. Pandya1

Accepted: 29 August 2022 / Published online: 17 September 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Electrocorticography signals, the intracranial recording of electrical signatures of the brain, are recorded by non-penetrating
planar electrode arrays placed on the cortical surface. Flexible electrode arrays minimize the tissue damage upon implanta-
tion. This work shows the design and development of a 32-channel flexible microelectrode array to record electrocorticog-
raphy signals from the rat's brain. The array was fabricated on a biocompatible flexible polyimide substrate. A titanium/gold
layer was patterned as electrodes, and a thin polyimide layer was used for insulation. The fabricated microelectrode array
was mounted on the exposed somatosensory cortex of the right hemisphere of a rat after craniotomy and incision of the
dura. The signals were recorded using OpenBCI Cyton Daisy Biosensing Boards. The array faithfully recorded the baseline
electrocorticography signals, the induced epileptic activities after applying a convulsant, and the recovered baseline signals
after applying an antiepileptic drug. The signals recorded by such fabricated microelectrode array from anesthetized rats
demonstrate its potential to monitor electrical signatures corresponding to epilepsy. Finally, the time–frequency analyses
highlight the difference in spatiotemporal features of baseline and evoked epileptic discharges.

Keywords Flexible microelectrode array · Implantable device · Electrocorticography signal · Epileptic activities ·
Biopotential acquisition

1 Introduction among intracranial electrophysiology techniques (Dong

Electroencephalography (EEG) is a widely utilized electro-
physiological monitoring technique to record the electrical
activities of the brain for both research and clinical appli-
cations. Recently, the popularity of electrocorticography
(ECoG), compared to EEG, has increased due to relatively
higher spatial resolution and improved signal-to-noise ratio
(SNR). As an invasive technique (Li et al. 2020), ECoG
induces minimal inflammation during chronic recording
Tushar Sakorikar and Arjun BS contributed equally to this work.
* Hardik J. Pandya
hjpandya@iisc.ac.in
1
Department of Electronic Systems Engineering, Indian
Institute of Science, Bangalore, India
2
Department of Neurosurgery, National Institute of Mental
Health and Neurosciences, Bangalore, India
3
Center for BioSystems Science and Engineering, Indian
Institute of Science, Bangalore, India
et al. 2017; Jeong et al. 2021). Thus implantable electrode
arrays are widely used to record intracranial electrical activi-
ties for applications in tumour delineation and as a technique
for epileptic foci identification. However, the commercially
available implantable electrode arrays used for pre-surgical
diagnosis have electrodes with a diameter of ~ 4 mm and an
interelectrode distance of ~ 10 mm (Kaiju et al. 2017). These
electrodes offer a poor spatial resolution, for example, dur-
ing pre-surgical identification of epileptogenic foci (Rubehn
et al. 2009). Therefore, there is a need for an electrode array
with a higher density of electrodes to provide better spatial
resolution in mapping brain surfaces.
Traditional high-density electrode arrays, designed for
recording from either the cortical surface or a depth of
the brain, when fabricated on a rigid substrate, such as
silicon (elastic modulus ~ 160 GPa), exhibit a mismatch
of mechanical properties with brain tissues (elastic modu-
lus ~ 0.1 – 10 kPa) (Miller and Chinzei 2002; Scholten
and Meng 2015; Xu et al. 2019) and may result in tissue
inflammation and irreversible brain damage (Branner et al.

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2004; Lacour et al. 2010; Vince et al. 2005). Hence, there
is an increasing interest in electrode arrays fabricated on
flexible substrates that can conformally attach to the corti-
cal surface, enabling good contact between electrode and
cortex because of compatible mechanical properties (Xu
et al. 2019). Flexible substrates, such as polyimide (PI)
(Jeong et al. 2021; Xie et al. 2017), polydimethylsiloxane
(PDMS) (Li et al. 2020; Tybrandt et al. 2018), parylene
C (Lee et al. 2019; Li et al. 2020), poly (L – lactic acid)
(PLLA), polycaprolactone (PCL) (Xu et al. 2019), poly
(lactic – co – glycolic acid) (PLGA) (Yu et al. 2016), etc.
and their composites are used to fabricate electrode arrays.
Parylene C is a transparent polymer that is used as a
substrate for flexible ECoG recording devices. A 32-channel
ECoG recording device was fabricated using parylene C
as substrate as well as an insulation layer and validated by
recording from the somatosensory cortex of an anesthetized
rat (Khodagholy et al. 2011). Konerding et al. reported a
16-channel electrode array (area ~ 4 m ­ m2) on a 20 µm thick
parylene C substrate. The fabricated device was validated
by subdural recording from the primary auditory cortex of
a guinea pig (Konerding et al. 2018). Ganji et al. reported a
microelectrode array (3.25 mm × 2.85 mm) with PEDOT:PSS-
based 56 electrodes on a flexible parylene C substrate (Ganji
et al. 2018). A selective neural recording or stimulation
using Pt microelectrodes on parylene C substrate has also
been reported (Torres-Martinez et al. 2019). A transparent,
chronically stable 16-channel device was reported by Park
et al. in 2014 owing to the optically transparent nature of
parylene C. The device was fabricated using a graphene-
based ECoG array on a transparent parylene C substrate to
facilitate neural imaging and optogenetic applications (Park
et al. 2014). In a recent study, Yang et al. reported a parylene
C-based transparent, flexible electrode array fabricated using
PEDOT:PSS – ITO – Ag – ITO electrodes to record ECoG
signals from the primary visual cortex of an anesthetized rat.
This 32-channel array was designed to record ECoG signals
from the primary visual cortex of the left and right hemispheres
simultaneously (Yang et al. 2021). In a study, Konno et al.
reported a 32-channel recording of ECoG signals from the
primary and secondary visual cortex of free-moving rats. The
platinum electrode array (recording area ~ 2.5 mm × 2.5 mm)
and a reference electrode were fabricated on a parylene-C
[poly(chloro-paraxylylene)] substrate, separated as strips, and
implanted for acquiring spontaneous neural activities (Konno
et al. 2021). In 2022, Fedor et al. reported a high-density
recording of ECoG signals from the cortical surface of awake
mice in chronic studies. This array of 31 gold – iridium oxide
recording electrodes was patterned on a thiol-ene/acrylate
substrate and was encapsulated by parylene C (Fedor et al.
2022).
PDMS is recently drawing attention as a substrate for fab-
ricating flexible devices because of its lesser rigidity than PI
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Biomedical Microdevices (2022) 24: 31
and parylene C. Additionally, PDMS is optically transparent
and elastic (Renz et al. 2020). Its limitation includes poor
adhesion with metals and compatibility with few common
MEMS-based fabrication processes because of its thermal
and chemical properties. However, the poor adhesion of
PDMS with metals can be overcome by an intermediate par-
ylene C layer (Lee et al. 2020). Schiavone et al. reported a
soft electrode array (described as e-dura) to deliver electrical
stimulation to dorsal roots of the spinal cord using Pt-PDMS
composite as electrodes and PDMS as substrate and insula-
tion (Schiavone et al. 2020). An optically transparent, soft,
stretchable 16-channel electrocorticography array was used
for recording from the dorsal cortex of an adult mouse (Renz
et al. 2020). Lee et al. reported recording of visually evoked
potential in mice by a 16-channel flexible electrode array
fabricated on a PDMS substrate (Lee et al. 2020).
PI is one of the most widely used flexible substrates for
fabricating microelectrode arrays with the advantage of its
thermal stability. One of the earliest reports on PI-based
64-channel electrode array (dimension ~ 5 mm × 5 mm)
was fabricated and implanted to record surface evoked
potential from rat's brain in 2008 (Yeager et al. 2008).
In 2009, Rubehn et al. reported a 252-electrode array
(dimension ~ 35 mm × 60 mm) for recording ECoG signals
from the visual cortex of a monkey (Rubehn et al. 2009). An
ECoG recording from the cerebellum of a rat was reported
by Kato et al. using a chromium/gold array on PI substrate
in 2012 (Kato et al. 2012). A 16-channel ­IrOx-modified
microelectrode array integrated with micro-LEDs was reported
to study neural signals with photostimulation (Ji et al. 2018).
A flexible electrode array with eight highly-dense recording
electrodes with 1 mm × 1 mm dimension was fabricated on
PI and implanted on the visual cortex and motor cortex of
a moving rat in a chronic experiment for recording ECoG
signals (Jeong et al. 2021). The device was fabricated to fit
in a dimension so that tissue re-growth could be prevented.
Recently, Vomero et al. reported a 32-electrode array on PI
substrate. The focus of this report was to study the relationship
of conformability with the thickness of an implant without
applying any external force after deployment on a brain surface
and modeling the minimum area with holes in a structure
compared to the recording area to avoid inflammation or tissue
re-growth (Vomero et al. 2020).
Researchers have developed flexible electrode arrays to
record the brain's electrical response for studying different
animal models of neurological disorders. Competence of the
fabricated arrays to record ECoG signals was demonstrated
using in vivo experiments on animal models, e.g., mice (Ji
et al. 2020, 2019), rats (Dong et al. 2017; Yeager et al. 2008;
Yu et al. 2016), monkeys (Kaiju et al. 2017; Rubehn et al.
2009), etc. Such flexible electrode arrays were implanted on
the cortical surface (e.g., the motor cortex (Jeong et al. 2021)
and the primary somatosensory cortex (Li et al. 2020)) for
Biomedical Microdevices (2022) 24: 31
recording ECoG signals. Kaiju et al. reported recording of
somatosensory evoked potential from a macaque model
using a parylene electrode array (Kaiju et al. 2017). In simi-
lar studies, recording of the somatosensory evoked potentials
were reported using rodent models (Idowu et al. 2018; Lu
et al. 2016). A few research groups have reported the record-
ing of epileptic activities using flexible electrode arrays
(Jeong et al. 2021; Xu et al. 2019). The epileptic activities
were induced by the application of convulsants, e.g., bicuc-
ulline (Yu et al. 2016), penicillin (Dong et al. 2017; Xu et al.
2019), pentylenetetrazole (Niknazar et al. 2013), etc. or by
the electrical stimulation (Bortel et al. 2019). However, the
effect of antiepileptic drugs (AED) during seizures on brain
signals has not been widely studied.
As the PI exhibits excellent thermal stability and robust
chemical resistance and offers good adhesion to the depos-
ited metal on the film, it was used as the substrate and the
insulation material. Besides exhibiting robust physical and
chemical properties, durable polyimide films of various
thicknesses can be achieved reliably by varying spin-coating
parameters. Moreover, the flexibility of PI allows for achiev-
ing a conformal coverage of the cortical surface.
This work reports the design and development of a bio-
compatible, flexible, and high-density micro-electrode array
(MEA) for a simultaneous 32-channel recording of ECoG
signals. Though electrode arrays with higher electrode den-
sity (number of electrodes/ ­mm2 surface area of the record-
ing site) were reported (at the cost of the number of record-
ing electrodes), the electrode density of this designed MEA
is comparable to the electrode arrays reported for ECoG
recording in recent years (Fedor et al. 2022; Jeong et al.
2021; Konno et al. 2021; Renz et al. 2020; Rubehn et al.
2009; Yang et al. 2021; Yeager et al. 2008). The fabricated
Fig. 1  Illustration of the experi-
mental setup for recording elec-
trocorticography signals using a
flexible electrode array in a rat
Page 3 of 12 31
flexible MEA makes conformal contact with the cortical
surface. Two OpenBCI Cyton Daisy Biosensing Boards
were used for signal acquisition. A schematic of this in vivo
study is shown in Fig. 1. In acute experiments, we have
demonstrated that the fabricated MEA can record the base-
line ECoG signals, the induced epileptic activities, and the
recovered baseline activities after administering AED from
the cortical surface of an anesthetized rat. The higher elec-
trode density of the fabricated array reported in this study
enabled acquiring the distribution of neural activities from
the somatosensory cortex under three neurophysiological
conditions with high spatial resolution, especially during the
elicit and recovery of epilepsy. To our knowledge, a study of
these three conditions in rats using custom-fabricated micro-
electrode arrays using an affordable and efficient alterna-
tive acquisition system (OpenBCI Cyton Daisy Biosensing
Board) is not reported in the current literature.
2 Materials and methods
2.1 Design and fabrication of the flexible electrode
array
The 32-channel flexible MEA was designed for studying
ECoG signals from the brain surface of a rat. The record-
ing area was 2.4 mm × 3.6 mm, consisting of thirty-two
electrodes (electrode diameter: 100 µm) for simultaneous
recording of ECoG signals. 50 µm wide metal lines with
a minimum separation of 25 µm are designed to connect
the recording electrodes to the contact pads. The contact
pads are designed with a pitch (100 µm) so that these can
be connected to the FFC/FPC connectors of the designed
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31 Page 4 of 12
electrode interface board. The schematic of the MEA is
shown in Fig. 2a.
Standard microfabrication techniques were used to fabri-
cate the MEA. Polyamic acid, a precursor of polyimide (PI),
was spin-coated (at 500 rpm, 1500 rpm, and 500 rpm for 30 s,
Fig. 2  Microelectrode array (MEA) for recording ECoG signals.
a Schematic of the MEA (inset) the recording electrode array; b – f
Process flow for fabricating the flexible MEA: b Silicon wafer, c
Spin-coating of polyamic acid followed by curing to form polyimide
film, d Metal (Titanium/Gold (Ti/Au) of thickness: 30 nm/200 nm)
deposition followed by lithography and etching, e Spin-coating and
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Biomedical Microdevices (2022) 24: 31
60 s and 30 s, respectively) on a silicon wafer (3-inch diam-
eter) twice, followed by curing at 80 °C for 1 h and at 250 °C
for 2 h on a hotplate to form a PI film of thickness ~ 15 µm.
The thickness was confirmed by using a Dektak surface pro-
filometer ­(DektakXT® Stylus Profilometer, Bruker). The
curing of polyamic acid to form an insulation layer, followed by pat-
terning for opening windows at the electrodes and the contact pads, f
RIE to etch polyimide film to define the boundary followed by peel-
ing off the fabricated microelectrode array from the silicon substrate;
g MEA fabricated on polyimide substrate; and h SEM image of the
recording electrode array
Biomedical Microdevices (2022) 24: 31
silicon wafer provided structural support during fabrication.
Three mask processes were used to fabricate the MEA. A Ti/
Au layer of 30 nm/200 nm was deposited using the e-beam
evaporation technique (E-beam evaporator, Tecport Optics,
USA). The metal-deposited wafer was coated with MICRO-
POSIT S1813 photoresist followed by soft-bake (110 °C for
1 min), UV exposure (Mask aligner: MJB 4, SUSS Micro-
Tec, Germany), and development (in MF26A developer) for
patterning. The recording electrodes, contact pads, and the
connecting metal lines were realized by wet etching the metal
layers. After photoresist stripping, another layer of polyamic
acid was spin-coated (at 500 rpm, 2500 rpm, and 500 rpm
for 30 s, 60 s, and 30 s, respectively) and cured at 80 °C for
1 h and at 250 °C for 2 h on a hotplate to form a PI film of
thickness ~ 5 µm as an insulating layer and was patterned by
lithography ­(AZ® 4562 photoresist coating, exposure, fol-
lowed by development) followed by plasma ashing in reactive
ion etching (RIE) chamber (Reactive Ion Etching System,
Oxford Instruments) to open windows at the electrode sites
and the contact pads for facilitating electrical contact. After
that, the processed wafer was coated with ­AZ® 4562 pho-
toresist, followed by exposure and development. After lithog-
raphy, the PI layer was etched in an oxygen environment in
the RIE chamber (Oxford Instruments) to define the MEA
boundaries. The final step was to release the MEA from the
silicon wafer. The PI was peeled off the silicon wafer to real-
ize the MEA. The process flow for fabricating the MEA is
shown in Fig. 2b–f. The photograph of the fabricated flexible
MEA is shown in Fig. 2g. The SEM image of the recording
electrode array of the MEA is shown in Fig. 2h. As polyim-
ide is an insulator, charging of the substrate is observed in
the SEM image. However, imaging of the electrodes and the
metal lines are not affected.
2.2 Design and fabrication of the Electrode
Interface Board
An electrode interface board (EIB) was designed and fabri-
cated to connect the fabricated MEA with the signal acqui-
sition system. The EIB was designed with a dimension of
34.25 mm × 18.75 mm × 0.8 mm (l × w × h) and a weight of
1.8 g. The fabricated MEA was attached to the EIB using a
34-pin FFC/FPC connector. It has four sets of Relimate Con-
nectors (RMC) (1.27 mm pitch) with eight data channels,
and two ground and reference electrode pins to interface
the OpenBCI Cyton Daisy Biosensing Board. The EIB was
placed in the designed and fabricated EIB holder.
2.3 Design and fabrication of the EIB holder
The rats can show involuntary movements even under
anesthesia, which can further increase during the induced
Page 5 of 12 31
seizure. Moreover, the electronic components are suscepti-
ble to movement during handling, which may affect the sig-
nal integrity. An EIB holder was designed and developed to
house the implantable flexible device and fabricated EIB on
the rat's head. The EIB holder was designed with a smooth
slanting surface above the rat's head such that the rat does not
have access to the flexible MEA and the EIB. The EIB holder
was designed and fabricated in parts to enable a quick and
hassle-free assembly preventing any damage to the implant-
able MEA. Another major design constraint was to reduce
the weight of the EIB holder. The EIB holder was fabricated
using 3D printing to reduce the weight and allow the design
flexibility required for housing the flexible device. During
the 3D printing process, the infill density was kept as low as
20% to reduce the weight, and the infill pattern was selected
to be a triangle to obtain maximum strength.
The biocompatible poly-lactic acid (PLA) was used as
the printing material. The overall weight of the EIB holder
was 8.6 g. The engineering drawing and the exploded view
of the EIB holder with an EIB and the fabricated MEA are
shown in Fig. 3a and b, respectively.
2.4 Characterization of the fabricated MEA
2.4.1 Measurement of the electrode impedance
After fabrication of the MEA, electrical impedance spectros-
copy (EIS) was performed on the electrodes using a com-
mercially available LCR meter (LCR-8105G, GWINSTEK)
over a frequency range of 20 Hz to 10 kHz. The EIS was
performed after immersing the recording area of the MEA
in a phosphate buffered solution (PBS) of pH 7.4, and con-
tact pads were connected to the LCR meter for impedance
measurement. A 100 mV sinusoid signal trigger was used to
measure the impedance, and 200 data points were collected
logarithmically over the frequency range. The impedances
of the consecutive equidistant electrode pairs were meas-
ured and plotted to determine the average impedance of the
recording electrodes.
2.4.2 Bending test
A bending test was performed to evaluate the reliability of
the fabricated MEA. The MEA was connected to the EIB
and was fixed onto the indentation stage of a micromanipu-
lator. The MEA was immersed in the PBS (pH 7.4), keeping
the contact pads outside the solution. Five pairs of elec-
trodes from the middle of the array were characterized at a
frequency of 1 kHz with a trigger of 100 mV. The device
was bent by 60° using the periodic vertical movements of
the micromanipulator. The impedance was measured up to
250 cycles, which can be an extreme case of bending in an
acute experiment.
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31 Page 6 of 12
Fig. 3  Implantation of the fabricated flexible MEA on the corti-
cal surface of an anesthetized rat. a Engineering drawing of the EIB
holder design; b Exploded view of the 3D printed EIB holder, with
the schematics of electrode interface board (EIB) and flexible micro-
electrode array; c A photograph of the 32-channel fabricated array
2.5 Acute in vivo experiments
In vivo recordings were performed with healthy
male Sprague–Dawley rats (age: three months, and
weight: ~ 350 g) (n = 3) following the Institution Animal Eth-
ics Committee guidelines (IAEC Project Number: CAF/Eth-
ics/667/2019). Prior to surgery, the rat was anesthetized by
administering urethane (dose: 1.5 g/kg body weight) intra-
peritoneally (Fiáth et al. 2021). Subsequently, the rat was
placed in a stereotaxic frame with ear bars and a rat adapter.
Following positioning, a curvilinear midline incision was
placed with the posterior end of the incision curving to
the right ear. A skin flap was raised along with the peri-
osteum. The Coronal and Sagittal sutures were identified,
and a 5 mm × 4 mm craniotomy was performed using an
electric micro-drill with a 1 mm drill bit. The bone flap was
elevated from the underlying dura. The Temporalis muscle
was reflected inferiorly, and craniotomy extended laterally
to expose the brain surface. The dura was incised posteriorly
and raised as a flap. It was opened based anteriorly. The
recordings were obtained after placing the electrode array
in contact with the cortical surface (Fig. 3c). Two stainless
steel screws were drilled in the skull above the left hemi-
sphere and were used as ground and reference electrodes.
The fabricated flexible MEA was connected to the acquisi-
tion system using an FPC connector, soldered on the EIB.
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Biomedical Microdevices (2022) 24: 31
placed on the somatosensory cortex of the right hemisphere after cra-
niotomy and dural incision; d Closure of the craniotomy hole using
dental cement after MEA placement; e Immobilized rat in stereotaxic
apparatus after implantation of MEA; and f Zoom-in view of the EIB
holder
The experimental protocol involved ECoG acquisition
under three different conditions, i.e., baseline after surgery
followed by MEA placement, epileptic discharges post con-
vulsant administration, and neuronal responses post-AED
administration. An initial two-minute baseline ECoG signals
were recorded from the anesthetized rat after MEA place-
ment. Thereafter, crystals of bicuculline, a convulsant, was
topically applied to the exposed cortical surface to induce
epileptiform discharges (Yu et al. 2016). The craniotomy
defect was sutured, and the EIB holder was fixed on the rat's
head using dental cement (Fig. 3d–f). The topical application
of bicuculline resulted in epileptic activities. The epileptic
activities were recorded for two minutes from all electrodes,
followed by intraperitoneal application of phenytoin sodium,
an AED, with a dose of 20 mg/kg body weight (Soysal et al.
2011). The recovered baseline activities after AED adminis-
tration were acquired for two minutes. Once the acute exper-
iment is over, the acquisition system can be detached easily
from the EIB by removing berg connectors. After the acute
experiment, the rat was euthanized by cervical dislocation
after applying an overdose of anesthetic agent.
2.6 ECoG signal acquisition
ECoG signals were acquired using two OpenBCI Cyton Daisy
Biosensing Boards. These biopotential acquisition boards have
Biomedical Microdevices (2022) 24: 31
16-channel Analog to Digital Converter (ADC; ADS 1299)
embedded to acquire generated biopotentials. These OpenBCI
Cyton boards were interfaced with the flexible MEA via a dedi-
cated PCB housing 1.27 mm pitch berg connectors (Cyton Get-
ting Started Guide 2021; Daisy Getting Started Guide 2021).
OpenBCI Cyton Daisy Biosensing Boards is a scientifically vali-
dated biopotential acquisition device (OpenBCI Documentation
2021). The OpenBCI Cyton Daisy Board sampled the biopoten-
tials at 125 Hz and established wireless communication with a
computer using the BLE (Bluetooth Low Energy) module.
2.7 ECoG signal processing
The offline ECoG analyses were done using custom scripts
in MATLAB R2020b (Institutional academic license) with
EEGLAB (version 2020.0) (Delorme and Makeig 2004).
Considering the frequency range of interest for ECoG,
acquired biopotentials were filtered using a bandpass filter
(1 Hz – 60 Hz) (Xie et al. 2017) followed by a notch filter
to avoid 50 Hz power line interference (Bianco et al. 2019).
Finally, the filtered data was smoothened with a 10-point
moving average to remove high-frequency non-neural bursts.
Subsequently, the power spectrum was computed to
understand different frequency components present in
acquired biopotentials for three different scenarios: (i)
Baseline after MEA placement, (ii) Bicuculline induced
epileptiform discharges, and (iii) Anti-Epileptic Drug
(AED) affected baseline. The power spectrum was approx-
imated by a Welch periodogram involving three main
steps: (i) dividing the twenty-second signal into optimal
segments with 50% overlapping, (ii) windowing each
Fig. 4  Characterization of the fabricated flexible MEA. a Electrical
impedance spectroscopy of the fabricated MEA by measuring imped-
ance of 17 equidistant electrode pairs; and b Impedance measurement
Page 7 of 12 31
segment using a Kaiser window, and (iii) averaging the
periodograms of the segments (Kaiser and Schafer 1980;
Welch 1967). Additionally, the time–frequency analysis
was performed to understand the spectral changes in ECoG
signals as a function of time. This spectrogram was com-
puted by concatenating each segment's short-term Fourier
transform (STFT) with the Kaiser window, resulting in a
matrix.
3 Results and discussion
3.1 Impedance measurement of the fabricated MEA
The impedance of the consecutive equidistant seventeen
electrode pairs was measured by EIS over a frequency
range of 20 Hz to 10 kHz with a trigger of 100 mV after
immersing the recording area in PBS (pH 7.4). The aver-
age impedance of the recording electrodes was determined
as ~ 26 kΩ at 1 kHz, which is in the range for recording
ECoG signals (Kuzum et al. 2014; Rubehn et al. 2009).
The electrodes had impedances of 26 kΩ ± 3 kΩ at 1 kHz.
The impedance spectroscopy results of seventeen pairs of
electrodes in the MEA are shown in Fig. 4a.
3.2 Impedance measurement during bending test
A bending test was performed after immersing the recording elec-
trodes of the device in the PBS (pH 7.4) and connecting the MEA
to the EIB for impedance measurement at 1 kHz. The impedance
of the MEA for 250 bending cycles to study the mechanical stability
of the passivation layer
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31 Page 8 of 12
of the electrode pairs was measured from five pairs of equidistant
central electrodes and plotted after fifty bending cycles by 60°
using the periodic vertical movement of the micromanipulator.
The impedance was measured up to 250 cycles with an interval
of 50 cycles and normalized as ΔR R
, where R and ΔR denote initial
impedance and the change in impedance. The normalized imped-
ance of the electrode pairs is plotted. Figure 4b shows the normal-
ized impedance wrt the number of bending cycles, indicating no
significant change in impedance because of bending.
3.3 ECoG signals acquisition
The microelectrode array was placed on the exposed soma-
tosensory cortex of the right hemisphere to record ECoG
signals, and the screw electrodes were used as ground and
reference electrodes. All the electrodes in the array recorded
baseline signals from the cortical surface, as shown in Fig. 5a1
(blue traces). The baseline ECoG signals exhibited an ampli-
tude in the range of ± 100 µV. After acquiring baseline ECoG
signals for two minutes, bicuculline (a convulsant) was topi-
cally applied to induce epileptiform discharges, followed by
sealing craniotomy defect. Recording of ECoG signals showed
epileptiform discharges after ten minutes of application of
the bicuculline. These discharges are shown for all electrodes
in Fig. 5b1 (red traces). Figure 5b1 shows the epileptiform
discharges with significantly higher amplitude in a range
of ± 1500 µV. The induced epileptic activities show repeti-
tive high-frequency discharges, shown in Fig. 5b1, observed
by most electrodes. Higher epileptic activities were observed
at the site where the bicuculline crystals were applied. Finally,
phenytoin sodium, an AED, was administered intraperito-
neally, and monitoring of signals from 32-channels exhib-
ited a recovered baseline signal after four minutes of AED
administration. The pre-processed signals recorded post-AED
administration are plotted for all channels in Fig. 5c1 (green
traces). It is important to observe that epileptiform discharges
were suppressed, and low amplitude (± 50 µV) baseline was
restored due to phenytoin sodium (AED) application.
Correlation among the electrodes showed the spatial
nature of the electrical discharges. Baseline traces showed
partial localization, mainly involving bottom central elec-
trodes, which depicts the neural source underlying just
beneath the site of electrodes (Fig. 5a2). Additionally, locali-
zation of seizure activities was observed in Fig. 5b2. Finally,
the correlation analysis of the retrieved baseline showed less
correlation in the central areas, whereas there was a higher
correlation on the peripheral electrodes (Fig. 5c2).
3.4 Time–frequency analysis
The time–frequency analysis of the obtained ECoG signals
was done to understand the spectral and temporal features
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Biomedical Microdevices (2022) 24: 31
Fig. 5  ECoG signals acquired using fabricated flexible MEA. a1 ◂
Baseline ECoG signals from all 32 channels for twenty seconds
(Amplitude Scale: 200 µV); a2 Correlation results for initial baseline
traces; b1 Epileptiform discharges from all 32 channels for twenty
seconds (Amplitude Scale: 3000 µV); b2 Correlation results with
electrodes with dominant epileptic signatures shows high correlation,
indicating seizure onset; c1 Baseline ECoG signals after AED admin-
istration from all 32 channels for twenty seconds (Amplitude Scale:
100 µV); and c2 Correlation results for retrieved baseline traces
under different neurological situations. Temporal trace,
power spectrum, and time–frequency analysis are shown
for the baseline (Fig. 6a1–a3; blue traces), induced epilepti-
form discharges (Fig. 6b1–b3; red trace), and restoration of
baseline following IP administration of AED (Fig. 6c1–c3;
green traces). A significant difference was observed between
baselines and epileptiform discharges, specifically in time-
domain amplitude and spectral power (Fig. 6a1, b1, c1, a2,
b2 and c2). AED administration recovered the baseline; how-
ever, the amplitude of the recovered baseline was smaller
than the initial baseline. Pre-AED baseline (Fig. 6a1) and
post-AED baseline (Fig. 6c1) show a flat line as compared
to Fig. 6b1, which shows epileptic activities in the form of
recurrent high-frequency bursts. Clear delineation among
before and after AED administration baselines and epilep-
tiform discharges was observed in time–frequency analysis
(Fig. 6a3, b3, and c3).
This prominent difference can be observed by the pres-
ence of high amplitude components (represented in yellow
colour in Fig. 6b3) at the time of epileptiform discharges.
Moreover, during induced epileptiform discharges, the lower
frequency components' dominance was observed, as indi-
cated in the power spectrum (Fig. 6b2) and time–frequency
analysis plot, specifically for the region greater than 30 dB.
As a bandpass filter with a range of 1 – 60 Hz was used in
pre-processing of the acquired raw biopotentials, the power
spectrum and time–frequency analysis results (Fig. 6a2, a3,
b2, b3, c2, and c3) showed a sudden drop after 60 Hz.
3.5 Spatial analysis
Identifying the similarity of the neural responses among the
electrodes is vital as it further conveys information about
the source and progression of epileptic activities. Therefore,
correlation analysis was performed to quantify the similar-
ity among all the electrodes. The correlation between each
electrode is obtained with the averaged response from all
electrodes.
Figure 7 summarizes epileptic activities and the correla-
tion of each electrode following MEA electrode placement.
Figure 7a indicates that the majority of the epileptic activi-
ties are enclosed in the central region (site of topical appli-
cation of the convulsant), shown in the black dashed oval in
Biomedical Microdevices (2022) 24: 31 Page 9 of 12 31

13
31 Page 10 of 12
Fig. 6  Time–frequency analysis of acquired ECoG signal from corti-
cal surface using MEA in three conditions. a Baseline: a1 Recorded
baseline signal for a duration of forty seconds, a2 Power spectrum
of baseline recording for a duration of twenty seconds, a3 Spectrogram
of the baseline duration of twenty seconds; b Epileptic activities: b1
Recorded epileptic activities after ten minutes of bicuculline admin-
istration for a duration of forty seconds, b2 Power spectrum of the
Fig. 7a. This bilateral spread also confirms that the effect
of convulsant (bicuculline) is more in the central regions
and considerably less in the other areas, especially at the
corners. This pattern distribution of neural activities resulted
Fig. 7  Spatial analysis and Correlation analysis of obtained epilep-
tic activities. a Spatial electrical traces with black dotted line show-
ing bilateral spread of epileptic activities due to topical application
13
Biomedical Microdevices (2022) 24: 31
epileptic activities for a duration of twenty seconds, b3 Spectrogram
of the epileptic activities for a duration of twenty seconds; and c
Effect of the AED: c1 Baseline restoration after four minutes of AED
administration for a duration of forty seconds, c2 Power spectrum of
the recovered baseline for a duration of twenty seconds, and, c3 Spec-
trogram of the recovered baseline for a duration of twenty seconds
in similar values of correlation (in percentage), where cen-
tral electrodes possess high correlation compared to periph-
eral electrodes (Fig. 7b). As the bicuculline was applied on
the cortical surface at the central region of the MEA, the
of bicuculline crystals; and b Correlation results with electrodes with
dominant epileptic signatures shows high correlation, indicating sei-
zure onset zone is localized in the area affected by bicuculline
Biomedical Microdevices (2022) 24: 31
analysis further suggests that the epileptic activity spread is
generalized and progressing equally in all directions.
Thus, spatial analysis with a correlation confirms that the
developed MEA can record the electrocorticography signals
and justify temporal actions and valuable measures to inter-
pret neural epileptic behaviour for spatial insight.
4 Conclusion
A flexible, high-density micro-electrode array was devel-
oped for 32-channel ECoG signal recording from the cortical
surface of a rat's brain. The MEA was fabricated with Ti/
Au electrodes on a flexible polyimide substrate to facilitate
conformal interface at contacts. A packaging strategy using
a custom fabricated EIB holder that encloses the electrode
interface board (EIB) and connectors to the signal acqui-
sition module (OpenBCI Cyton Daisy Biosensing Board)
was developed. In an acute experiment, the MEA, alongside
the electronic modules, was used to study the effect of a
convulsant and an antiepileptic drug on neural responses.
The in vivo experiments on anesthetized rats demonstrated
the competency of the fabricated array to record brain-
waves from all thirty-two channels using interfaced Open-
BCI Cyton Daisy Biosensing Boards. The time–frequency
analysis of the pre-processed ECoG signals showed distinct
variations among three neurophysiological conditions, i.e.,
baseline signals, chemically induced epileptiform activities,
and recovered baseline after AED application. Furthermore,
correlation-based spatial analysis interprets the variabilities
and progression of epileptic activities. We plan to perform
extensive chronic experiments in the future using this flex-
ible MEA. We envisage this model may help in evaluating
and understanding the efficacy of AEDs in rodent models.
Acknowledgements Tushar Sakorikar acknowledges the Science and
Engineering Research Board, Government of India for the National
Postdoctoral Fellowship (PDF/2019/002894). Arjun BS acknowledges
the fellowship support from the Prime Minister's Research Fellowship
(PM/MHRD-20-16562.03) and grant support from the BIRAC SITARE
– GYTI 2021 Award (BT/BIRAC/SITARE-GYTI-0283).
Declarations
Conflict interests The authors declared no conflict of interest concern-
ing with this article.
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