J. Micro/Nanolith. MEMS MOEMS 9共3兲, 031007 共Jul–Sep 2010兲
Three-dimensional flexible microprobe
for recording the neural signal
Chang-Hsiao Chen
Shih-Chang Chuang
Yu-Tao Lee
National Tsing Hua University
Institute of NanoEngineering and MicroSystems
Hsinchu 30013, Taiwan
Yen-Chung Chang
National Tsing Hua University
Institute of NanoEngineering and MicroSystems
and
Institute of Molecular Medicine
and
Department of Life Sciences
Hsinchu 30013, Taiwan
Abstract. We have designed, fabricated, and tested a novel threedimensional 共3-D兲 flexible microprobe used for recording the neural signals of lateral giant 共LG兲 on the escape system of American crayfish. We
report an electrostatic actuation process to fold the planar probes to be
the arbitrary orientations of 3-D probes for neuroscience application. The
batch assembly method based on electrostatic force techniques gave
more simple fabrication compared to others. A flexible probe could reduce both the chronic inflammation response and material fracture when
animal breathes or moves. Furthermore, the cortex corresponds to hypothetical cortical modules with mostly vertically organized layers of neurons. Therefore, the 3-D flexible probe suits to understand how the cooperative activity for different layers of neurons. Advisedly, we present a
novel fabrication for the 3-D flexible probe by using Parylene technology.
The mechanical strength of the neural probe is strong enough to penetrate into a biogel. At the end, the flexible probe was used to record
neural signals of the LG cell from American crayfish. © 2010 Society of PhotoOptical Instrumentation Engineers. 关DOI: 10.1117/1.3455409兴
Shih-Rung Yeh
National Tsing Hua University
Institute of Molecular Medicine
and
Department of Life Sciences
Hsinchu 30013, Taiwan
Subject terms: flexible; microprobe; neuron; parylene; three-dimensional.
Paper 09159SSRR received Nov. 30, 2009; revised manuscript received May 1,
2010; accepted for publication May 4, 2010; published online Jul. 23, 2010.
Da-Jeng Yao
National Tsing Hua University
Institute of NanoEngineering and MicroSystems
Hsinchu 30013, Taiwan
E-mail: djyao@mx.nthu.edu.tw
1
Introduction
A fundamental goal of neuroscience is to develop quantitative descriptions of the functional operations and their coordination within a neuron that enable it to function as an
integrated unit to process information. The communication
of information among neurons, or between neuron and
muscle, requires that a signal travels over a considerable
distance. Through electrical stimulation applied to nerves
and muscles, early electrophysiologists demonstrated that a
command from the brain to a muscle to generate a movement was mediated by the flow of an electric current along
nerve fibers, so-called an action potential.1 Hodgkin and
Huxley2 demonstrated that axons at rest are electrically polarized, exhibiting a resting potential approximately
−60 mV across a membrane. This action potential propagates along the length of the axon through local depolarization of each neighboring patch of membrane, causing that
patch of membrane to also generate an action potential.
Action potentials from neural cells have been measured
over a wide range. To record the neuron signal, a single
electrode, comprising a sharpened tungsten wire probe and
a capillary glass probe, was used traditionally,3 but it is
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J. Micro/Nanolith. MEMS MOEMS
difficult to achieve uniform geometries of such finished array probes. It is impractical to assemble, manually, individual probes into an array with a small controlled spacing,
although a linear array or a two-dimensional matrix has
been reported.4 The neural probes of the 3-D type could be
used for neural recording in a brain system in vivo with its
penetration into neural cells, with a probe of variable
length. In contrast to conventional techniques, several researchers have been developing a neural probe with a microelectromechanical systems 共MEMS兲 technique. Wise
made a silicon-based neural probe based on wet silicon
etching.5 The individual probes are released using an ethylene diamine pyrocatechol 共EDP兲 etch. During this EDP
step, the boron-diffused areas act as an etch stop.6 Neural
probes combined with monolithically integrated CMOS circuitry have also been demonstrated.7 The four 64-sites
probes have been assembled into a three-dimensional 共3-D兲
structure.8 The shape of neural probe from the University of
Michigan formed by diffusing the boron dopant process
was uncontrollable.6 The Utah neural probe consists of
hundreds of silicon needles which were fabricated by using
a dicing saw process followed by chemical etchant.9 However, the individual probes can support only one electrode
site.
Although rigid types of microelectrode have been devel-
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Chen et al.: Three-dimensional flexible microprobe for recording the neural signal
2 Design of Neural Probe
Before fabricating electrode arrays with an appropriate geometry, the mechanical strength of electrodes was calculated using ANSYS. Each probe must satisfy the following
design criteria:
1. The probe must be strong to minimize damage on
penetrating a bio-organism.
2. The probe must be sufficiently long, ⬎1 mm, to allow for the desired depth of penetration into a brain
cortex.
3. The exposed area of the surface material of an electrode must be polarized such as Au, Pt, or Ir, because
the application would be used for extracellular recording.
4. The interconnect spacing from electrodes to bonding
pads were kept to ⬍10 m, to both limit the crosstalk capacitance 共CC兲 and facilitate fabrication using
liftoff technology.
5. The use of polymer or glass as the substrate effectively eliminated the substrate capacitance 共CS兲 and
the light sensitivity since the polymer, glass was
nonconductive.
Fig. 1 Schematic of the planar probe in the 共a兲 flat and 共b兲 folded
configurations.
oped for recoding, but at in vivo recording the mechanical
mismatch between the stiffness probe and soft biological
organization will aggravate inflammation in the implantation sites. Although silicon has a Young’s modulus of
⬃170 GPa, brain tissue has ⬃3 kPa. This large mechanical
mismatch can contribute to shear-induced inflammation at
the implant site. This inflammation encourages the formation of a glial sheath. In response to brain injury, glial cells
such as astrocytes and microglia proliferate to form a glial
sheath.10,11 The biological sheath would encapsulate the
probe, which would isolate the electrode from surrounding
neurons with time.12 The duration recording of chronically
implanted probe of the Michigan array about four months,13
of the Utah array was about seven months.14 A flexible
probe could reduce both the chronic tissue inflammation
response and material fracture when the animal breathes or
moves.15,16
Recently, flexible neural probes have been fabricated
with polymer materials such as polyimide, Parylene, and
SU-8.15,17,18 Advisedly, we present a fabrication method for
a penetrating type of 3-D flexible probe by using Parylene
technology. Parylene is an inert, vapor-deposited polymer.
The Parylene-C is selected as the main structure material of
the flexible probe, which gives its excellent flexibility, mechanical strength with Young’s modulus ⬃3 GPa, and its
United States Pharmacopoeia 共USP兲 Class VI grade biocompatibility; pinhole-free coating at room temperature has
low moisture permeability and has been used in the medical
device industry as a biocompatible coating for implants.
We report an electrostatic actuation process to fold the
planar probes to be the arbitrary orientations of 3-D probes,
shown in Fig. 1. Our method differs from that of Shaar et
al.19 in the segments actuated by Lorentz force, and of
Takeuchi et al.17 actuated by the magnetic field method for
the batch assembly. Moreover, we improve the limit that
toxicity of magnetic materials for long-term and in vivo
recording application, which would be very useful for further development. At the in vivo recording system, the mechanical mismatch between the stiffness probe and soft bioorganization will aggravate inflammation.11 A flexible
probe could reduce both the chronic inflammation response
and material fracture when the animal breathes or moves.
Furthermore, the cortex corresponds to hypothetical cortical modules with mostly vertically organized layers of neurons. Therefore, the 3-D flexible probe suits to understand
how the cooperative activity for different layers of neurons.
Advisedly, we present a novel fabrication for 3-D flexible
probe by using Parylene technology.
J. Micro/Nanolith. MEMS MOEMS
In this paper we have designed a novel 3-D flexible microprobe used for recording the neural signals of LG of the
American crayfish. We report an electrostatic actuation process to fold the planar probes to be the arbitrary orientations of 3-D probes for neuroscience application. The
batch-assembly method based on electrostatic force techniques gave more simple fabrication compared to others.
The advantages of using the fabrication process of the 3-D
flexible neural probe as follows: the shank is longer than
3 mm, the flexibility, 3-D type, and excellent biocompatibility. Besides, the multiple electrode sites upon individual
probe could be designed in the future. A neural probe of
this size might be used to measure a neural signal; a long
probe is susceptible to bending when it contacts 共extracellular recording兲 the neuron cell. The flexible probe insert
into the cortex is difficult. Takeuchi presented a fabrication
process of the Parylene probes that were filled with polyethylene glycol 共PEG兲. In order to increase the stiffness of
a flexible probe during implantation, an embedded microfludic channel can be filled with a solid material that dissolves after implantation.20 The Parylene probes were
coated with PEG, a biocompatible material that is solid
state at room temperature, liquid state over 60 ° C and dissolves in water or body fluid.
3 Fabrication of Neural Probe
MEMS technology was used to develop the fabrication of
the flexible probe, the fabrication starting from Parylene_C
deposition and ending with PEG coating. The fabrication
process of the flexible probe was outlined in Fig. 2. The
process consisted of three steps: patterning the metal layer,
the structure layer, and etching the structure to release the
probe. Details of the processes are as follows:
1. RCA standard clean was used to remove particles and
ion contamination before the ⬃5 m Parylene_C is
first deposited on silicon wafer by using specialty
coating system.
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Chen et al.: Three-dimensional flexible microprobe for recording the neural signal
Fig. 3 SEM image of a fabricated 3-D flexible microprobe: 共a兲 The
probe has one electrode on the tip. The geometry of developed
probe is 3 mm long, 100 m wide, and 6 m thick. The close view
of the probe for which the electrode was isolated with Parylene. The
width of tip is 5 m, and the opening size of gold electrode is
2500 m2. 共b兲 The folding probe after electrostatic actuation.
Fig. 2 Fabrication process of the 3-D flexible probe.
2. The process steps define patterning of electrodes by
using the liftoff method. Cr/ Au were evaporated by
E-beam. The metal patterns are defined in an ultrasonic bath in acetone. The trace was defined with a
5 m width and 100 nm thickness. The embedded
electrodes add the lateral contact area with Parylene.
The O2 plasma treatment with reactive ion etching
共RIE兲 was utilized to etch part of the Parylene before
depositing the gold electrode. A treated recipe of
power 共100 W兲 at 60 s could improve the mechanical
properties efficiently. Additionally, the rough surface
added the adhesion between electrode and Parylene.
3. A second Parylene_C with a 1-m thickness layer
was deposited on top of the entire wafer as a passivation dielectric layer.
4. The 2500 m2 via was patterned in the insulation
layer, which—over the ends of the Au electrodes—
contact pads were opened to the Au layer using O2
plasma by RIE through a photoresist mask. This step
at the same time defined the probe shape. The etch
step went through the entire thickness of the Parylene
device layer on the wafer, which was etched 6 m,
stopping on the silicon.
5. The flexible probe was peeled from the wafer by using tweezers and fixed on the glass substrate.
6. The planar probes were folded by electrostatic force.
7. The folded probes were fixed by a PEG drop with
liquid state on a 60 ° C hotplate.
8. Finally, the probe was dipped with liquid-state PEG
on a 60 ° C hotplate. After the cooling process, the
PEG transfers to solid state, which enhances the
probe’s stiffness.
outermost section. The thickness of probe is targeted to be
6 m, but can be varied by depositing Parylene with various thicknesses. The probe length is typically 3 mm. The
size of the recording electrode sites are 2500 m2. The
forded probe was fixed with a PEG drop by using a pipette.
The flexible probe was coated with PEG by a dipping process, as shown in Fig. 3共b兲. The thickness of PEG was
⬃5 m. The dip-coating process has a conformal surface
on the implant site.
4.2 Electrostatic Field Assembly
The electrostatic field was used to fold the planar probe for
the batch assembly, which avoids the error of manual assembly. After fabrication, the planar probe was placed in a
vertical electrostatic field, which would stand up by varied
electrostatic voltages. The voltage in the generator was
gradually increased from 0 V to 8 KV, which folded the
probe up from a horizontal position to about vertical. It was
folded to near 90 deg when an external electrostatic field of
8 KV was applied, as shown in Fig. 4.
4.3 Mechanical Strength
The flexible probe would be used for in vivo recording of
the neural signal of the cortex in the future. The pure flexible probe was too difficult to be implant into a living organism due to its soft property. The PEG material was as a
supporting structure of the Parylene probe. We had to
verify that the overall neural probe structure is mechanically strong enough to penetrate test.
4 Results and Discussion
4.1 Probe Structure
A scanning electron microscope 共SEM兲 image of the completed neural probe is shown in Fig. 3. In the design, the
flexible probe consists of one electrode site on the probe
关shown on Fig. 3共a兲兴. The width of probe is 100 m at the
J. Micro/Nanolith. MEMS MOEMS
Fig. 4 Electrostatic batches assembly of the probe.
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Chen et al.: Three-dimensional flexible microprobe for recording the neural signal
Fig. 5 Photograph of Parylene probe coated with PEG penetrated
into biogel.
As shown in Fig. 5, the 3-mm Parylene probe was
coated with PEG, then inserted into and pulled back from
the biogel, smoothly, to simulate the hardness of the cortex
and without critical buckling failure or break off. After inserting the probe into the biogel, the PEG was a biocompatible material. It would be dissolved in body fluid with
time and the probe would become flexibile again.
4.4 Interface Impedance
The impedance of electrode influences the ability to record
minute neural signals. In order to choose the cutoff frequency of the neural signal recording system, electrochemical impedance analysis was used to measure the impedance
and phase between the work electrode and reference electrode. The larger impedance of the electrode abated was,
the electrical transmission would increase the noise, which
influences the signal-to-noise ratio 共SNR兲 of the spike. The
impedance of the microelectrode was obtained by submerging only the recording in saline solution 共NaCl 0.9%,
27 ° C兲. The impedance measurement 共HIOKI LCR meter,
3522-50兲 was built up with respect to a large reference
electrode 共Ag–AgCl electrode兲. The testing signal for impedance measurement was sinusoidal 共ac voltage 10 mV,
frequency 100 Hz– 10 kHz兲.
For a typical electrode with a 2500 m2 opening area,
the average measured impedance is ⬃566.5 K⍀ at 1 kHz
共nerve working frequency兲 and the phase shift is
−84 deg/ 1 kHz, tending to −90 deg representing a capacitance dominating the interface, with the electrode interface
being highly polarized,21 as shown in Fig. 6. At the higher
frequency, the impedance of electrode decreased gradually
Fig. 6 Interface impedances of gold electrodes on flexible probe
submerged in a saline buffer solution 共NaCl 0.9%兲, the average impedance of electrode is 566.5 KΩ / 1 kHz.
J. Micro/Nanolith. MEMS MOEMS
Fig. 7 Schematic of the electrophysiology system for recording the
crayfish. Pairs of silver wires are placed on the mechanosensory
primary afferent neurons for stimulation. The Parylene-based neural
probe contacts on a LG neuron cell are for the measurement of the
action potential.
because a capacitor effect tended to a short circuit. Additionally, the long-term stability of the neural probe was
tested immersing in the saline buffer solution 共NaCl 0.9%兲
by impedance measurement. The Parylene film still adhered
to the probe well after one month. The adhesion remained
tight with no noticeable degradation, visually, and the
change of interface impedance was ⬍1% for the duration.
4.5 Neural Signal Recording
To confirm the functionality of electrode on the flexible
probe, we measured the neural signal of the escape circuit
of a crayfish with our developed neural probe. The advantage of using the escape circuit from a crayfish is the large
diameter of an axon, ⬎10 m, and the ⬃1 mm length
level. The neuron is large enough to contact or have implanted in it a neural probe, to minimize the error of the
distance between the electrode and the measured neuron
cell. The schematic view of the electrophysiology system
共shown in Fig. 7兲 could be used to record the neuron signal
from microprobe.
Juvenile crayfish are kept in a water tank at room temperature. The 2 – 5 cm junior crayfish was selected for our
experiments. The crayfish lost the ability to move on the ice
after 10 min. A crayfish was pinned in a dish filled with van
Harreveld’s solution. The abdominal nerve cord of the escape circuits was exposed dorsally by removing the exoskeleton and separating phasic flexor musculature, which’s
using isolated abdomen preparation. In the escape circuits
of the crayfish, the mechanosensory primary afferents received the environmental excitation. Parts of the neural signal transmit directly to the LG through electrical synapses.
Pairs of silver wires were placed on the mechanosensory
primary afferent neurons for stimulation. The functions of
the electrophysiology system were divided into two parts:
one is electrical stimulation and the other one is the recording part. The electrical stimulation was produced by a
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Chen et al.: Three-dimensional flexible microprobe for recording the neural signal
mechanical stiffness of developed the flexible probe, PEG
was used on the surface of probe. The neural recording data
were used to demonstrate that the probes are capable of
recording data from an electrode using crayfish.
In future work, we would develop further completed
data acquisition software and measurement circuitry, including stimulating, recording, and bypassing functions
with fully integrated digital components realized in an industrial CMOS process. To continue to improve the SNR of
the neural signal would be important for a multipleelectrode measurement system. Future durability tests must
include a toxicity test, long–term signal recording, and
stimulation in neural cells.
Fig. 8 Extracellular recording signals of an LG neuron were measured from an electrode of the flexible neural probe by electrical
stimulation. The response amplitude of each spike was ⬃150 V,
and the response time ⬃1 ms.
Acknowledgments
This research was partially supported by the National Science Council in Taiwan through an NSC Grant No. 962627-E-007-002.
References
digital-to-analog converter card and amplifier circuits. The
amplitude and frequency of the input voltage was controlled by software in a personal computer.
With an appropriate packaging—the substrate was packaged with the biocompatible epoxy after wire bonding process 共Epoxy Technology, EPO-TEK 301-2兲 and its USP
Class VI grade biocompatibility, an electrode of a flexible
neural probe was used to measure the neuron signal on the
LG after electrically stimulating. In the extracellular recording experiment, the flexible neural probe was contacted
on the membrane of the LG to record the neural responses.
The neural signals from the dorsal side of a LG nerve fiber
were recorded on shocking the tailfin afferents. Figure 8
shows the recorded extracellular signals from our developed flexible neural probe. The alternating square waveform of the shock voltage was about ⫾3.7 V in a period of
0.1 ms. The responsive amplitude of the action potential
was ⬃150 V for 1 ms 共red curve兲. Nevertheless, the
smaller shock electrical stimulation, which was about
⫾3.6 V for 0.1 ms 共blue curve兲, could not induce the action
potential from LG nerve fiber because of the resting membrane potential toward to the threshold for activation is yet
to surmount. Additionally, the distances between the LG
neuron and the electrode varied under measurement, which
would influence the amplitude of the response spike. However, most neurophysiologists are only concerned with the
appearance of the spike instead of its response magnitude.
The results demonstrate the capability of our developed
neural probe for recording neural signals independently
from a single electrode.
5 Conclusion
In this paper, we designed and fabricated a 3-D flexible
microprobe for recording a neural signal, which would be
used for the measurement system in biomedical applications. The fabrication process of the implant flexible probe
was accomplished by using MEMS technology. We report
an electrostatic actuation process to fold the planar probes
to be the arbitrary orientations of 3-D probes. We present a
fabrication method for a penetrating type of flexible probe
by using Parylene technology. Moreover, to improve the
J. Micro/Nanolith. MEMS MOEMS
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Chen et al.: Three-dimensional flexible microprobe for recording the neural signal
Shih-Rung Yeh is an associate professor at
Institute of Molecular Medicine, National
Tsing Hua University, Taiwan. He received
his PhD from the Department of Biology,
George State University at Atlanta in 1997.
Being an electrophysiologist, he is interested in the the neural plasiticity of the
nerve system.
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Chang-Hsiao Chen is a PhD candidate at
the Institute of NanoEngineering and MicroSystems, National Tsing Hua University, Taiwan, when he received his MS in SOIbased microprobe techniques in 2006. His
research includes microprobe fabrication,
carbon nanotube treatment, and signal process technology.
Shih-Chang Chuang studied micromechanical system 共MEMS兲 at National Tsing
Hua University, Taiwan, where he graduated
in 2009. The subject of his master thesis
was the design and fabrication of flexible
neural microprobe for three-dimensional assembly.
Da-Jeng Yao is an associate professor at
Institute of NanoEngineering and MicroSystems 共NEMS兲, also an adjunct professor at
Department of Power Mechanical Engineering and Department of Engineering System
and Science, National Tsing Hua University,
Taiwan. He received his PhD from the Department of Mechanical and Aerospace Engineering at University of California at Los
Angeles in 2001. His research scope is to
combine his strong backgrounds 共MEMS
and thermal fluidics兲 in microscience research, including BioMEMS,
electronic nose, MEMS packaging, thermofluidic MEMS, and thinfilm property measurement.
Yen-Chung Chang is a professor at the Institute of Molecular Medicine and an adjunct
professor at the Institute of NanoEngineering and MicroSystems of National Tsing
Hua University, Taiwan. He received his
PhD from the Department of Biochemistry
and Biophysics at Iowa State University in
1985. His current research interests lie in
understanding the molecular mechanisms
underlying the formation and function of
synapses in the mammalian central Nervous System and the proteomics of subcellular structures, such as
the postsynaptic density and axon of central neurons.
J. Micro/Nanolith. MEMS MOEMS
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