2022 44th Annual International Conference of the IEEE Engineering in Medicine & Biology Society (EMBC) | 978-1-7281-2782-8/22/$31.00 ©2022 IEEE | DOI: 10.1109/EMBC48229.2022.9870987

2022 44th Annual International Conference ofthe IEEE Engineering in Medicine & Biology Society (EMBC)
Scottish Event Campus, Glasgow, UK, July 11-15, 2022

Acute in vivo Recording with a Generic Parylene MicroelectrodeArray Implanted with Dip-coating Method into the Rat Brain*
Huijing Xu, IEEE, Member, Kee Scholten, IEEE, Member, Wenxuan Jiang, IEEE, Student Member,Jessica-Lizbeth Ortigoza-Diaz, IEEE, Student Member, Zhouxiao Lu, IEEE, Student Member, Xin Liu,IEEE, Student Member, Ellis Meng, IEEE, Fellow and Dong Song, IEEE, Senior Member
develop a platform for rapid manufacture of standard andcustomized polymer MEAs for chronic neural recording andstimulation (Fig. 1). For researchers who are interested inready-to-use polymer MEAs, we have developed a Parylene
C-based MEA with generic electrode layout. This “standard”
penetrating Parylene MEA is suitable for both cortical andsub-cortical recordings in small animal models such asrodents.

Abstract—Flexible polymer-based microelectrode arrays(MEAs) can reduce tissue inflammation and foreign body
response and greatly prolong the lifetime of neural implants.
However, standard and customized polymer devices are onlyaccessible to limited groups. To better promote the development
and application of polymer MEAs, we have launched thePolymer Implantable Electrode (PIE) Foundry and developed a64-channel Parylene C-based MEA with generic electrodes
layout that can be used to record from both cortical andsub-cortical regions in rodents. In addition, a practical
dip-coating protocol for the insertion of the flexible standard
Parylene MEA is developed.

In addition, implantation of flexible MEAs often requires
special procedures such as the usage of specifically fabricatedshuttle devices [7] or precisely aligned stiffener [8]. Thesemethods are usually not easy to adopt for practitioners in thebroader community. In this study, we experimented different
insertion techniques and developed an easy-to-operate
dip-coating protocol using polyethylene glycol (PEG) for the
implantation of the generic Parylene MEA.

I. INTRODUCTION
Penetrating microelectrode arrays (MEAs) are one of themost commonly used tools for electrophysiological studies ofbrain functions. Microelectrodes placed adjacent to neurons
can directly monitor the activities of individual neurons. In
particular, invasive penetrating MEAs provide the best spatial
and temporal resolution for neuroscience studies. Thisadvantage makes them one of the most powerful tools forreal-time investigations of neuronal properties of different cell
types and a necessary instrument to study the correlation
between neural activities and behaviors [1]. The advancementof manufacturing metal-based microwire arrays and thefabrication of high-density silicon-based multi-shank arrays
made it possible to monitor unitary activities of large
populations of neurons simultaneously [2][3]. However, theperformance of rigid MEAs typically degrades over time andlongevity of recording remains one of the major challenges
faced by rigid MEAs [4].
Flexible polymer-based probes can significantly reduce
inflammation and tissue response and obtain stable long-term
recording from the brain [5][6]. However, limited accessibilityof polymer MEAs has largely restricted the development andapplication of this new technology to a few research groups
that is able to build their own devices. Supported by the NIH
BRAIN Initiative, we established the PIE Foundry** as ashared-resource center to build and test polymer MEAs forneuroscience researchers. The aim of the PIE Foundry is tob.

c.

d.

e.

f.

Figure 1. Examples of polymer devices developed and fabricated byco-founders of the PIE Foundry. a: A flexible Parylene neural probewith 3D sheath structure for enhancing tissue integration [5]. b: A
multi-shank Parylene MEA for simultaneously targeting for multiple
sub-regions of the rat hippocampus[9]. c: A cuff electrode withmicrofluidics to interfacing with peripheral nerve [10]. d: A Parylene
probe with eight tetrodes for chronic recordings from mouse striatum. e:
A 32 channel, multi-shank MEA for multi-regions recordings from themice hippocampus and, f: the standard Parylene MEA for cortical andsub-cortical recordings from rodent’s brain.

*Research supported by NIH U24NS113647-01.
H. J. Xu is with the Center of Neural Engineering, University of Southern
California, Los Angeles, CA 90089 USA (phone: 213-740-8061; email:huijingx@usc.edu).
K. Scholten, J Ortigoza-Diaz, X. Liu and E. Meng are with the Biomedical
Microsystem Laboratory, University of Southern California, Los Angeles,CA90089 USA (email: kscholte@usc.edu, ellis.meng@usc.edu).
W. X. Jiang, Z. X. Lu and D. Song are with the Center of NeuralEngineering, University of Southern California, Los Angeles, CA 90089
USA (email: jian393@usc.edu, luzhouxi@usc.edu, dsong@usc.edu)** http://piefoundry.usc.edu/
978-1-7281-2782-8/22/$31.00 ©2022 IEEEa.

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Omnectics x 2

7.69 mm

Cortex, 2 mm

7.50 mm

Hippocampus,2 mm
Ribbon Cable

PCB

Contact Pins

5mm

2mm

Parylene
180 µm
80 µm

15 µm
Diameter

120 µmPlatinum
500 µmParylene

ExposedElectrodes

1800 µm

Insolated
TracesFigure 2. Layout, structure and packaging of the PIE Foundry standard Parylene MEA. Picture on the left of the top row shows the size of rat cortex andhippocampus. The middle figure illustrates the overall geometry and dimension of the Parylene MEA. Pictures on the right shows a fully packaged MEA
and the zoomed-in view of four Parylene shanks. The bottom left figure shows detailed dimensions of the width of individual shanks, spacing between
shanks, spacing between electrodes and diameter of recording electrodes. The bottom right picture illustrates the structure of the Parylene MEA incross-section.

layer was then encapsulated by another layer of 10 µm thick
Parylene C. Electrodes and contact pad were subsequently
exposed by deep reactive ion etching (DRIE) with an oxygenplasma, and a second DRIE step defined the array outline andshape. Finally, the devices were released from the siliconwafer in a water bath. Connectivity of electrodes and traces
were evaluated with visual inspection under microscope andelectrochemical properties of each individual electrode wereevaluated using electrical impedance spectroscopy.

II. MATERIAL AND METHOD
A. The Standard Parylene MEA
The 64-channel standard penetrating probe array consistsof four 5 mm long Parylene shanks separated by a 500 µmdistance (center to center). To reduce tissue damage and easethe insertion of the flexible MEA, shanks are tapered in widthfrom 180 µm to 80 µm. On each shank, 16 platinum (Pt)electrodes with 15 µm diameter are evenly distributed along
both edges of the Parylene shank. The center-to-center verticaldistances between electrodes are 120 µm. The recording
region spans 1800 µm vertical distance which is enough tocover multiple layers of the rat cortex and large sub-corticalstructures such as the hippocampus.
Each individual electrode is connected to a small Pt
contact pad with 3µm thin Pt traces. Metal traces aresandwiched between two layers of 10 µm thin ParyleneC films.
The 8×8 mm2 Parylene pad that contains all 64 contact pads is
directly bonded to an 18×17 mm2 customized PCB using anovel polymer-ultrasonic-weld method [11]. This method
significantly reduced the size of the PCB and eliminated theusage of extra connectors such as the zero-insertion-force (ZIF)
connectors. Two stainless steel wires are soldered onto thePCB as ground wires. To minimize the size of the PCB, two36-pin Omnetics connectors are soldered on both sides of thePCB, to connect the Parylene MEA with data acquisition
systems. All soldering pins and the entire PCB are coated withepoxy (Epotek 302M) for insulation and protection.
The standard MEA was fabricated with conventional
microlithography techniques. As illustrated in Figure 2, a baselayer of 10 µm Parylene C was deposited on a silicon wafer
followed by the deposition of a 200 nm thick Ptlayer. The
recording site, connecting traces and contact pads werecontained in the metal layer and defined by lift-off. The metal

B. Dip-coating Protocol
Polyethylene glycol (PEG) with 8kDa molecular weight
was melted and heated to just above 100°C for sterilization.
The melted PEG was then kept between 75 to 80°Cona
hotplate. The Parylene MEA, together with its packaging, waspre-sterilized by soaking in 70% ethanol for 5 minutes. Afterthe device was fully dried, it was attached onto a manipulator
which can smoothly rise or lower the assembly. First, theentire device was lowered into the PEG solution until thebottom edge of the PCB was in contact with the solution. Thenthe device was slowly retrieved from the PEG at a speed about
20 µm/sec. A thin PEG film is often formed between Parylene
shanks. If the shanks in the array are aligned, no further
operation is needed. Otherwise, the shanks are submerged intopre-sterilized distilled water for 2 minutes. The thin PEG film
dissolves quickly, and the shank tips are lowered into the PEG
solution to re-coat (Fig. 3).
C. Animal Preparation
Sprague-Dawley (SD) rats older than twelve weeks andweighing between 250 to 450g were used for the implantationof the standard Parylene MEA. All procedures were approvedby the University of Southern California Institutional Animal
Care and Use Committee. During the implantation surgery,rats were anesthetized intra-operatively with a mixture ofisoflurane and air. Toe-pinch withdrawal reflexes were tested

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verify the depth of the insertion and to evaluate damages
caused to surrounding tissues by the implanted MEA.

a. Materials
Stereotactic
FramePEG, 8000

III. RESULTS
A. Insertion of the Standard Parylene MEAThe insertion of the flexible standard MEA with
dip-coated PEG was first tested using a brain phantom madewith 0.6% agarose gel. Two dip-coated devices with thin PEG
films between the shanks were inserted into the agarose to adepth of 4 mm (Fig. 4). The PEG film dissolved immediately
upon contact with the agarose gel and did not affect theinsertion of the Parylene MEA. Feasibility of the dip-coating
protocol was further tested on rat brains. Six Parylene MEAs
were dip-coated following steps described above and inserted
into the brain of anesthetized rats. Two devices had thin PEG
films between shanks and the remaining four devices were
coated twice to remove the PEG film. All six MEAs
penetrated brain tissue smoothly and were successfully
inserted without buckling. Brain slices (10 µm) collected fromthe brain of an animal following implantation of the Parylene
MEA for one month verified that the Parylene MEA wassuccessfully inserted to the depth of 3.00 mm. In addition, thepreliminary histology result shows that no severe immune
response was observed at the implantation site and theParylene MEA caused very limited damage to surrounding
neurons.

Hot Plateb. Coat of the Entire Probe
PCBMEA

Bottom Edgeof the PCB
PEG Film

PEG

c. Remove PEG Film

Bottom Edgeof the Ribbon
Cable

Stuck
Shanks
Water

Straight
Shanksd. Re-coat the Shank PartPEG

Parylene
Shanks
Only

Straight
Coating withno PEG Filma.

Figure 3. Procedure to dip-coat the Parylene MEA with PEG. a: Material
and equipment used for dip-coating. b: Coating of the entire device withPEG. The right photo shows a coated MEA with thin PEG films between
shanks. c: Steps to remove PEG film by water immersion. d: Procedure tocoat only the shanks with PEG. The right photo shows a properly coated
MEA with no excess PEG.

PCBMEAfrequently to ensure proper depth of anesthesia. Each animal
was fixed on a stereotactic frame with ear bars, and a 2×4 mm2
piece of skull above the dorsal hippocampus on the righthemisphere was removed. Both the dura and pia layers werecarefully removed before the insertion of the MEA. Smalldrops of saline were applied to the exposed brain surface toprevent drying of the tissue. The PEG coated Parylene MEA
was slowly inserted to a depth of 3.8 mm measured from thesurface of the skull. Signals from all 64 channels weremonitored during the insertion and the final depth of the MEA
was adjusted according to the quality of neural signals. Afterthe microelectrode array reached desired depth, dental cement
was applied to seal the bone window and fix the location of theMEA by attaching it to five anchor screws pre-secured on theskull. Two ground wires were twisted onto the screw that wasin contact with cerebrospinal fluid and tips of ground wires
were inserted into a small hole drilled above the cerebellum.

0.6% agarose
b.

2 mm

0.6% agarose

Figure 4. Insertion of dip-coated Parylene MEAs into brain phantom. a:Bench top testing setup. 0.6% agarose gel is used to mimic the texture of
the brain tissue. The photo on the right shows an un-coated Parylene
MEA. b: Photos on the left show the front and side view of a PEG-coated
Parylene MEA with a small block of PEG on the shank. Photos on theright show front and side view of the coated MEA after insertion into theagarose gel ~4 mm deep.

After all neural signals were collected, the animal wasdeeply anesthetized and perfused with 4% paraformaldehyde(PFA). The brain was carefully dissected and preserved in
PFA for histological studies.

B. In vivo Recording with the Standard Parylene MEA
Neural activities were recorded during the implantation of
two Parylene MEAs. One prototype MEA with 48 functional
electrodes was implanted to a depth of 3.0 mm. Unitaryactivities and local field potentials from both the cortex andD. Histology
Horizontal brain slices (10 µm thick) from one animal
implanted with the Parylene MEA for one month wereprepared using cryostat and stained with crystal violet to216

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Shank 3a.

b.

c.
µm

500 µmµm

200 µV
400 µsec

100 µV
200 msec

Figure 5. Acute in vivo recording with dip coated, standard Parylene MEAs from ratcortex and hippocampus. a: Data collected from a PIE Foundrystandard MEA implanted to a depth of 3.00 mm. 800 msec of high-pass filtered (>300 Hz) neural activity recorded by 16 electrodes on a single Parylene
shank is shown. Single spikes from the cortex recorded with superficial electrodes and complex spikes from the hippocampus recorded with bottom
electrodes were recorded simultaneously. Not functioning channels are shown as gray. b: Data collected from standard MEA implanted to a depth of 4.10mm. 34 units from multiple hippocampal sub-regions were recorded. Multiple units recorded with the same electrodes are color-coded. c: Nissl stain of 10µm horizontal brain slices collected from an animal implanted with the Parylene MEA for one month. The MEA was dip-coated and inserted to a depth of
4.05 mm during the surgery. Red arrows indicate locations of two Parylene shanks. No severe tissue response is observed and damage to surround tissue islimited to the width of the Parylene shank.

the hippocampus were recorded along an individual shank.
Another fully functional Parylene MEA was implanted to adepth of 4.10 mm. All four shanks reached the hippocampal
region and recorded complex spikes from hippocampal
pyramidal cells. A total of 34 units were recorded with thestandard Parylene MEA (Fig. 5).

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IV. DISCUSSION AND FUTURE WORKSIn this work, we designed and manufactured apolymer-based MEA with a standard electrode layout. A
practical dip-coating protocol for the insertion of flexible
polymer MEAs was also developed. To further evaluate
long-term performances of the Parylene MEA, we will
implant the MEA chronically and collect neural signals
frequently over a long period of time. Immuno-histochemistrystudies will be conducted to evaluate the tissue responses tothe MEA in more details.
One of the major goals of the PIE Foundry is to produce
and distribute polymer devices with arbitrary geometries, sizes
and channel counts for the broader neuroscience community.
To achieve this goal, we will design, fabricate, and test
different MEAs for different animal models and different
experimental paradigms and include them into the PIE
Foundry’s library of polymer devices. In addition, we will
collaborate with researchers from the neuroscience and neuralengineering community to develop highly customized
polymer devices for each group’s specific research need.
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