Micro Multi-Probes Electrode Array for the Recording Retinal Neuron Signal

Abstract

This research is to design micro multi-probe electrode array (MEA) for retinal neuron signal recording system, which would be used for the measurement system on bio-medical applications. Based on the optimal geometry of MEA designed by ANSYS, the novel fabrication process of MEA was accomplished by using MEMS technology based on silicon-on-insulator (SOI) wafer, inherently has good controllable probe thickness. The multi-probes with a 16-sites recording electrode array has been manufactured, which has been past for strength test by force gauge and electrical performance test by using impedance measurement system. Neuron extracellular recording by using fabricated MEA has been shown, which was comparable with the one measured by traditional glass pipettes. MEA for recording retinal neuron signal would be performed for further development.

Full text

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中國機械工程學會第二十四屆全國學術研討會論文集 中原大學 桃園、中壢
中華民國九十六年十一月二十三日、二十四日 論文編號：E10-0006


Micro Multi-probes Electrode Array for Recording the Retinal Neuron Signal
Chang-Hsiao Chen
1 Institute of NanoEngineering and MicroSystems, National Tsing Hua University
2 Department of Life Sciences, National Tsing Hua University
NSC Project No.: NSC-95-2218-E-007-011


1, Shao-Wei Lu
2, Chung-Chin Chiao
2 and Da-Jeng Yao
1
Abstract
This research is to design micro multi-probe
electrode array (MEA) for retinal neuron signal
recording system, which would be used for the
measurement system on bio-medical applications. Based
on the optimal geometry of MEA designed by ANSYS,
the novel fabrication process of MEA was accomplished
by using MEMS technology
silicon-on-insulator (SOI) wafer, inherently has good
controllable probe thickness. The multi-probes with a
16-sites recording electrode
manufactured, which has been past for strength test by
force gauge and electrical performance test by using
impedance measurement system. Neuron extracellular
recording by using fabricated MEA has been shown,
which was comparable with the one measured by
traditional glass pipettes. MEA for recording retinal
neuron signal would be performed for further
development.

Keywords：MEA, MEMS, Retina

based on
array has been
1. Introduction
Light is one of the innumerable information. Most
animals possess a visual system which not only can
sense the streams of light but also can reconstruct the
original scenes. Generally speaking, vision of
mammalian is initially formed in the retina. It has
become increasingly clear that the nervous system
achieves its complex functionalities through the
cooperative activities by many neurons.
Retina is a thin film of tissue lining the backside
of the eye, which can be used to transform an image
through processed electrical signal up to the brain [1].
The retina is a layered structure and contains various of
cell types. To gain an understanding of this neural code,
MEA are needed to record signals simultaneously.
Typically, two-dimensional pattern was fabricated on a
glass substrate to form MEA. A combination of electron
beam lithography, photolithography and dry-etch
pattern transfer to release MEA in the transparent
conductor indium tin oxide (ITO) [2, 3]. In a later
approach, a pyramid shaped three-dimensional MEA
was demonstrated. Each electrode features a pyramid
shape composed of one (1 1 1) plane and two vertical
planes [4].
Advisedly, MEA will be used in vivo neuronal
recording in the future (Fig. 1). We present a novel
method to fabricate MEA by using SOI wafers, which
inherently has excellent control over the final probe
thickness. Because of the thickness of the probe could
be defined by thickness of device layer on the SOI
wafer. A mostly dry etch based process where the
buried oxide layer of a SOI substrate acts as an etch stop.
The process can improve upon uniformity and
manufacturability.

2. Design
The size of the MEA would be used to measure
neural signal of the retina, a long probe is susceptible to
bend when it penetrates into inner limiting membrane.
The mechanical strength intensity of the probe is very
critical. Besides, the material of the probe is silicon that
needs to be considered for crystal fracture. The
maximum stress of the probe must be smaller than yield
strength of silicon at around 7GPa.
Several kinds of geometry were applied to be
simulated by ANSYS_9.0 before designing fabrication
process. The shank geometry of the quarter cycle (pillar)
was the proper one that maximum stress could be
6.92GPa when supplied 24mN force on the tip (Fig. 2).

3. Fabrication
MEMS technology was used to develop the
fabrication of MEA, starting from a 25µm SOI wafer n
with 2µm buried oxide (100mm φ, 25µm Si/ 2µm SiO2/
400µm Si). The fabrication process of micro
multi-probe electrode arrays is outlined on figure 3. (a)
RCA standard clean to remove particles and ion
contamination. (b) A wet oxide silicon oxide film was
grown up as an isolation layer. (c) The process steps
define patterning of electrodes by using lift-off method.
Cr/ Au was evaporated by E-beam. The metal patterns
are defined in an ultrasonic bath in acetone. (d) A
plasma enhanced chemical vapor deposition (PECVD)
silicon nitride layer was deposited on top of entire wafer
as an intermediate dielectric. (e) Via holes from Au
electrodes and contact pads were opened to the Au-layer
using reactive ion etching (RIE) silicon nitride through a
photoresist mask. (f) The wet silicon oxide film was
etched by BOE. (g) The steps in the process define the
probe shape. The outline of the probes was etched on
the front side of the wafer in an inductively coupled
plasma reactive ion etching system (ICP) using SF6 and
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中國機械工程學會第二十四屆全國學術研討會論文集 中原大學 桃園、中壢
中華民國九十六年十一月二十三日、二十四日 論文編號：E10-0006


C4F8 (SLR-700). The etch step went through the entire
thickness of the silicon device layer on the SOI, which
layer was etched 25µm and stopping on the buried oxide.
(h) A double-sided mask aligner (Mask Aligner Model
500IR) was used to pattern a thick photoresist on
backside of wafer, which was etched the full 400µm
down to the buried oxide by using the ICP. (i) The
multi-probe was released by using BOE to remove
buried oxide and release probes.

4. Results
4.1 Probe structure
In a design, there are one to four probes with four
electrode sites on each probe, shown on figure 4. The
width of probe is 100µm at the outermost section. The
thickness of probe is targeted to be 25µm, but can be
varied by the device layer on the SOI wafer
specification. The probe length is typically 3mm and the
separation between probes is 200µm. The recording
electrode sites are 12µm × 12µm each distributed with a
51µm pitch. The lateral tip width is designed as 5µm.
The MEA yield of designed fabrication was reached up
around 98%.
4.2 Mechanical strength
Because of the micro probe electrode would be
used to measure neural signal of retina. We have to
verify that the overall neural probe structure is
mechanically strong enough to penetrate tofu pudding
and egg without failure since the hardness of tofu
pudding and egg is similar to bio-organs, shown on
figure 5 (a) & (b). Furthermore, the maximum fracture
strength of the MEA was measured by the force gauge,
shown on figure 5 (c) & (d). The results showed the
fracture strength is 24mN, which is the comparable
value with the one by simulation.
4.3 Interface Impedance
The impedance of electrode influences the ability
to record minute neural signals. In order to select the
cut-off frequency of neural signal recording system, the
electrochemical impedance analysis was used to
measure the impedance and phase between MEA and
reference electrode. The impedance of the MEA was
obtained by submerging only the recording in AMES
bio-electrolyte. The testing signal for impedance
measurement was sinusoidal (AC voltage 100mv,
frequency 100Hz~ 100KHz) with respect to a large
reference electrode (Ag- AgCl) as shown on figure 6.
For a typical electrode with 144µm2 opening area,
the average measured impedance is around 2.46MΩ at
1kHz (nerve working frequency) and phase shift is
-85.9°/1KHz tends to -90° representing an interface
capacitance. The measurement result means MEA
closely highly polarization [5], as shown in figure 7.
This impedance was comparable to those measured by
using traditional glass needle electrode on extracellular
recording. At the higher frequency, the impedance of
electrode decreased gradually because of capacitor
effect.
4.4 Neuron extracellular recording
Neuron extracellular recording were made from
the soma of ganglion cell of retina from New Zealand
rabbits as shown on figure 8. The slices of the retina
was immersed in AMES at 35~ 37℃. By shining light
stimulated impulse to retina with 35mS spaced, the
neural recording data could be used to demonstrate that
the probes are capable of recording data from all
individual electrode of MEA, which results were similar
compared with recording data by adopting traditional
glass pipette as shown in figure 9.

5. Conclusions
This research was to design MEA for retinal
neuron signal recording system. Based on the optimal
geometry of MEA designed by ANSYS, the novel
fabrication process of MEA was developed and was
accomplished by using MEMS technology based on SOI
wafer, inherently has good controllable probe thickness.
The probes are 3mm long and 100µm wide. The
multi-probes with a 16-sites recording electrode array
has been manufactured, which has been past for strength
test by force gauge and electrical performance test by
using impedance measurement system. The average
measured impedance was around 2.46MΩ at 1 KHz.
The neural recording data could be used to demonstrate
that the probes are capable of recording data from MEA.
Future work would be to develop a completed
measurement circuitry with fully-integrated analog and
digital components realized in an industrial CMOS
process. To improve signal-to-noise ratio of the neural
signal is very important on multi-electrode measurement
system.

6. References
1. Rodieck, R.W., The First Steps in Seeing, Sinauer
Associates, Sunderland, 1998.
2. Mathieson, K., Cunningham, W., Marchal, J.,
Melone, J., Horn, M., O'Shea, V., Smith, K.M.,
Litke, A., Chichilnisky, E.J. and Rahman, M.,
Fabricating High-density Microarrays for Retinal
Recording, Microelectronic Engineering, Vol.67-68,
pp. 520-527, 2003.
3. Dabrowski, W., Grybos, P., Hottowy P., Skoczen, A.,
Swientek, K., Grillo, A.A., Kachiguine S., Litke,
A.M. and Sher, A., Development of Front-end
ASICs for Imaging Neuronal Activity in Live Tissue,
Nucl. Instrum. Methods Phys. Res., A Accel.
Spectrom. Detect. Assoc. Equip., Vol.541, pp.
405-411, 2005.
4. Koo, K., Chung, H., Yu, Y., Seo, J., Park, J., Lim,
J.M., Paik, S.J., Park, S., Choi, H.M. and Jeong, M.
J., Fabrication of Pyramid
dimensional 8 x 8 Electrodes for Artificial Retina”,
Shaped Three-
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中國機械工程學會第二十四屆全國學術研討會論文集 中原大學 桃園、中壢
中華民國九十六年十一月二十三日、二十四日 論文編號：E10-0006


Sensors and Actuators A: Physical, Vol.130-131, pp.
609-615, 2006.
5. Fricke, H., The Theory of Electrolytic Polarization,
Philosophical Magazine, Vol.7, pp. 310-318, 1932.

7. Figures and Tables

Fig.1 Schematic view of the MEA penetrate retina.


Fig.2 ANSYS simulate the static state force 24mN.


Fig.3 Fabrication process flow for MEA


Fig.4 SEM image of MEA: (a) the micro 1/2/4 probes,
(b) micro 4probes× 4electrode array, (c) the
electrode was isolated by Si3N4, (d) tip width is
5µm, and 12×12µm Au electrodes

(a)
(b)

(c) (d)
Fig.5 MEA strength test: (a) for penetrating tofu
pudding (b) for penetrating egg without failure (c)
by force gauge (d) Max. fracture force is 24mN.

(a)(b)
Fig.6 (a) Electrochemical impedance analysis system. (b)
The impedance of the Au electrode was measured
by submerging in AMES solution with respect to a
large reference electrode (Ag- AgCl).

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中國機械工程學會第二十四屆全國學術研討會論文集 中原大學 桃園、中壢
中華民國九十六年十一月二十三日、二十四日 論文編號：E10-0006



Fig.7 Interface impedances and phase of MEA in AMES
electrolyte compare with glass pipette: (a) the
MEA average impedance is 2.46 MΩ/1KHz. (b)
phase shift is -85.9°/ 1KHz.






Fig. 8 Overview of a complete recording system: (a) the
retina of the rabbit, (b) MEA was bonded layout
PCB, (c) the ganglion cell of retina of New
Zealand rabbits, (d) MEA penetrating retina and
Au electrode near the ganglion cell, (e) X1,000
amplifier with band-pass filter 0.1~ 3KHZ, and
data acquisition system by LabVIEW software.


Fig.9 Extracellular recording to ganglion cell. (Light
stimulation impulse to retina was spaced 35ms. The
response amplitude of each spike was about 300µV,
and the response time about 1-2mS.)



微型陣列式電極應用於微型陣列式電極應用於
視網膜神經訊號紀錄視網膜神經訊號紀錄
1
、呂紹瑋、焦傳金
1
國立清華大學奈米工程與微系統研究所研究生
2
國立清華大學生命科學系研究生
3
國立清華大學生命科學系教授
4
國立清華大學奈米工程與微系統研究所教授
國科會計畫編號：NSC-95-2218-E-007-011

陳昶孝
23
、饒達仁
4

摘要
本研究主要提出利用新型微探針電極陣列紀錄
分析視網膜神經訊號，同時有益於生醫訊號應用。在
製程前先行使用ANSYS模擬最佳化的幾何形狀，本
篇研究並提出新型製程技術製作為電極陣列，其架構
於矽絕緣體接合晶圓上，因此將能有效控制微探針厚
度。十六組紀錄電極陣列在電性特性以阻抗分析儀進
行量測，機械強度使用微型力量感測儀分析。此外，
在神經訊號紀錄與傳統玻璃電極做了紀錄比較。微電
極陣列在未來視網膜應用將能有效大幅提升。

關鍵字：微電極陣列，微機電，視網膜。

CSME-5894