Sensors and Actuators B 137 (2009) 774–780

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Agitation of magnetic beads by multi-layered flat coils

Mitsuhiro Shikida a,∗ , Motoki Koyama a , Nobuhiro Nagao a , Rentaro Imai a ,
Hiroyuki Honda b , Mina Okochi b , Hiroyoshi Tsuchiya b , Kazuo Sato a
a
Department of Micro-Nano Systems Engineering, Nagoya University, Japan
b
Department of Biotechnology, Nagoya University, Froh, Chikusa, Nagoya 464-8603, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The beads were magnetically agitated inside a droplet by changing the external magnetic-field distribu-
Received 19 February 2008 tion. We used both the multi-layered flat coils and the permanent magnet for keeping enough magnetic
Received in revised form 25 July 2008 force for driving the beads and for forming a non-uniform magnetic-field. The temperature in the well
Accepted 16 November 2008
solution was kept below 35 ◦ C at the appropriate agitation drive currents by adding a heat-sink foil
Available online 21 January 2009
between the flat coils. The agitation performance was evaluated by using an enzymatic reaction. The
reaction efficiency increased linearly with increasing reaction time and was more than four times higher
Keywords:
with agitation than without agitation.
Agitation
Magnetic bead © 2009 Elsevier B.V. All rights reserved.
Enzymatic reaction

1. Introduction
Various micro-total analysis systems (␮-TAS), in which mechan-
ical and electrical components are integrated on the same chip,
have been developed by using micro-electro-mechanical-systems
technologies. Such systems are expected to reduce the amount of
reagent solutions and analysis time required and to enable the
on-site monitoring of chemicals. Generally, most systems need
complicated mechanical fluidic devices, such as valves and pumps,
for handling solutions, because they use continuous flow as a
reaction medium. A system can therefore become large, even if
fluid channels and reactors are integrated onto microchips [1–5].
Fabrication processes making the devices and typical mechanical
fluidic components developed for ␮-TAS application are described
in Ref. [6]. To miniaturize the system, different mechanisms based
on a droplet solution as a medium have recently been pro-
posed [7–11]. These mechanisms are, in principle, rather simple
and can achieve fusion and separation by electrowetting con-
trol. However, the method based on the electrowetting droplet
handling is difficult to extract the targeted sample from the
droplet.
Given these difficulties, we therefore previously proposed a
novel type of magnetic bead-droplet handling mechanism for
a ␮-TAS application [12–14]. The proposed mechanism has the
advantages that it does not require complicated fluid-control
devices for handling solutions and it can collect and extract
∗ Corresponding author.
E-mail address: shikida@mech.nagoya-u.ac.jp (M. Shikida).
the targeted samples effectively from a droplet. Beads are used
for sample collection [15–18] in the field of ␮-TAS. The use of
beads has extended to various applications, including mixing
[19,20], micro-channel formation to increase reaction efficiency
[21], surface modification [22] and screening at micro-reactor
array chips [23]. Gijs’s group has recently developed a unique
magnetic bead-handling method based on a planar coil. They
have also shown that their system can perform two-dimensional
manipulation of the beads on chips [24,25]. We used magnetic
beads as carriers of a chemical material and handled them in a
droplet configuration on the chip. We previously focused on han-
dling magnetic beads [12] and a biochemical reaction unit [13],
which is a key component of our ␮-TAS. To construct a palm-
sized biochemical-analysis system, we also recently developed
transportation and agitation mechanisms that apply rotary motion
[14].
As described above, droplet handling based on a combination of
beads and magnetic force makes it possible to collect and extract
targeted samples effectively from a droplet on a palm-sized system
and to reduce the amount of reagent. However, the biochemical
reaction inside the droplet takes a certain amount of time because
there is no physical actuation force inside the droplet available for
agitation of the beads in the systems. Generally, agitation is one of
the main problems in ␮-TAS applications, because the Reynolds
number becomes small as system size decreases (meaning that
the viscosity of a liquid dominates flow conditions). We there-
fore developed an agitation device constructed from multi-layered
flat coils and a permanent magnet to increase reaction efficiency,
and we evaluated its performance by using an enzymatic reac-
tion.

0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2008.11.056
 M. Shikida et al. / Sensors and Actuators B 137 (2009) 774–780
Fig. 1. Schematic view of agitation device.
2. Operation of palm-sized system
2.1. Overview of system
We previously developed a rotary-drive-type biochemical-
analysis system using a bead-cluster handling mechanism [14]. It
consists of a multi-well chip, two small magnets, another magnet
coupled with multi-layered flat coils, a rotary table, and a stepping
motor and controller. The two magnets and the flat coils with the
magnet are fixed on the rotary table. The two small magnets are
used for the transporting the beads, and the magnet with the set of
the coils is used for the agitating the beads. The multi-well chip con-
sists of eight wells arranged in a concentric fashion, bent channels,
and a glass-bottom plate. A droplet, which contains magnetic beads
with samples adhered to their surfaces, is placed in the first well.
Various types of reagent and dilution droplets are pipetted into the
other wells. A series of reactions is performed by transferring the
magnetic beads from one well to the next. The system performs two
operations: transportation and agitation. Details of the system are
given in Ref. [14].
2.2. Agitation principle
A schematic view of the agitation device is shown in Fig. 1. It
consists of three-layered flat coils, a permanent magnet, a heat sink,
and two aluminum foils. We used a custom-ordered flat coil pro-
duced by Fujikura Ltd., Japan. A photograph and schematic view of
the flat coil is shown in Fig. 2. Square-shaped coils were formed on
both sides of the polyimide film to increase the number of turns
Fig. 2. Photograph and schematic view of flat coil.
775
(14 turns). At first, copper foils were attached on both sides of the
polyimide base film by pressure bonding. The thicknesses of the
polyimide film and copper foil were 25 and 30 ␮m, respectively.
The coil pattern on both sides of the polyimide film was formed
by photolithography. The hole for an electrical connection between
the both sides of the coil patterns was formed at the center of them
by laser. After that, metal deposition was performed to connect
the both coil patterns. Finally, the surface of the coil patterns were
covered by a polyimide film, with thickness of 30 ␮m, as an elec-
trical insulation layer. The copper wiring had a width of 100 ␮m
and a height of 30 ␮m, and the space between the windings was
50 ␮m. The heat sink was made of aluminum alloy, and its size
was 6.5 mm × 6.5 mm × 6.0 mm. A commercially available magnet
(a Nd–Fe–B permanent magnet, produced by Niroku Seisakusho
Co. Ltd.) with magnetic flux density of 0.07 T and diameter and
thickness of 10 and 0.5 mm, respectively, was used in the agitation
device.
The agitation of the magnetic beads inside the droplet is per-
formed by controlling the magnetic-field strength by means of the
pair of three-layered flat coils and the permanent magnet. The mag-
netic force acting on the beads is expressed by
 
F = I × ∇ H
 (1)
 I and H
 are magnetic force per unit volume (N/m3 ), mag-
where F,
netization of the magnetic material (Wb/m2 ), and magnetic-field
strength (A/m), respectively. By partially differentiating Eq. (1), the
magnetic force per unit volume, Fx (N/m3 ), in the horizontal x-
direction can be written as [2]
∂Hx ∂Hx ∂Hx
Fx = Ix + Iy + Iz (2)
∂x ∂y ∂z
As shown in Eq. (2), the magnetic force acting on the beads
depends on the bead’s magnetization, which increases with
increasing magnetic-field strength. We therefore used a permanent
magnet having a large enough magnetic-field strength to maintain
enough actuation force for handling the beads. The magnetic force
also depends on the gradient of the magnetic-field, meaning that
the beads move to a position under higher magnetic-field strength.
The flat coils were used to form a magnetic-field gradient inside the
droplet. Generally, the magnetic-field strength induced by a flat coil
is much smaller than that of a permanent magnet, and it increases
776 M. Shikida et al. / Sensors and Actuators B 137 (2009) 774–780

Fig. 3. Magnetic-field strength pattern around a droplet produced by summation of a flat coil and permanent magnet.

with increasing number of turns of the coil and applied current.
To increase the magnetic force, we therefore piled the flat coils on
top of each other to form a tri-laminar structure. Considering the
above factors, we used both the multi-layered flat coils and the per-
manent magnet for keeping enough magnetic force for driving the
beads and for forming a non-uniform magnetic-field.
The distribution pattern of the magnetic-field strength at the
droplet is determined by the summation of the magnetic-fields
induced by the multi-layered flat coils and by the permanent mag-
net (see Fig. 3). The magnetic-field strength becomes a maximum
at the center of the droplet when the direction of the magnetic-
fields induced by both the flat coils and the permanent magnet are
equal. In this case, the magnetic beads move to the center of the
droplet (see Figs. 3(a) and 4 left). On the other hand, the beads
move to the circumference on the inside of the droplet when the
direction of the magnetic-field induced by the flat coil is reversed
(see Figs. 3(a) and 4 right ). The position of the beads in the droplet
can therefore be controlled by changing the magnetic-field distri-
bution, which is controlled by the direction of the applied current
to the coil (see Fig. 2). The beads were repeatedly moved to increase
agitation performance. The direction of the current was changed by
simply applying an alternating current to the flat coils, and repe-
tition cycle was varied by changing the frequency of the applied
current.
3. Experiment
3.1. Heat management
The magnetic force for agitating the beads is proportional to
the current applied to the coil. To operate the agitation device cor-
rectly, a high current, in the order of several hundred milliamps,
is required. However, this current generates heat and increases
the temperature in the droplet. Therefore, we evaluated the tem-
perature change due to the applied current to the flat coil in this
section. Direct measurement of the droplet temperature was diffi-

Fig. 4. Magnetic-field distribution produced by three-layered flat coil and beads location in droplet.
 M. Shikida et al. / Sensors and Actuators B 137 (2009) 774–780 777

Fig. 5. Relationship between temperature inside droplet and applied current.

cult because of the small size of the droplet. Therefore, we filled the
well with silicone oil (KF-96L-1.5cS, viscosity: 1.3 cP, produced by
Shin-Etsu Chemical Co., Ltd) and measured the temperature inside
the oil (instead of inside the droplet) to estimate the temperature.
A thermocouple temperature probe (IT-2000, produced by AS ONE
Co.) with the diameter of 1.0 mm was used. A function generator
(WF1944A, produced by NF Co.) and an amplifier (HSA4101, pro-
duced by NF Co.) were used for applying the current to the coil.
A polyimide film used as the coil substrate has excellent electri-
cal and thermal insulation properties. We therefore inserted pieces
of aluminum foil between the flats coils to effectively release the
heat. The silicone oil temperature decreased by 7–8 ◦ C when the
same driving current was used with the aluminum foil inserted.
We confirmed that the temperature could be kept below 35 ◦ C at
the appropriate driving currents (less than 1.2 A), as shown in Fig. 5.
According to these results, we set the applied current as 1.1 A in the
following agitation experiments.

3.2. Analytical curve for enzyme reaction

To evaluate reaction efficiency, we conducted an enzyme reac-

tion with or without agitation. Alkaline phosphatase (AP) was used
as the enzyme, and p-nitrophenyl phosphate (pNPP) as the sub-
strate. The AP decomposed the pNPP, and the color of the pNPP
solution changed to yellow. The enzymatic reaction activity of
the AP was detected by measuring the absorbance at a wave-
length of 405 nm. We used commercially available magnetic beads
(Dynabeads® MyOneTM Streptavidin T1, Dynal, Invitrogen Co.; bead
diameter: 1.05 ␮m.), which are composed of super-paramagnetic
polymer, and their surface was modified by streptavidin. The
streptavidin-modified magnetic beads were then labeled with
biotinylated alkaline phosphatase (B-2005, Vector laboratories)
by using chemical bonding between the streptavidin and biotin
molecules. The immobilization procedure for the enzyme consists
of the following five steps.
(1) Sample 50 ␮l of the Dynabeads® streptavidinTM Myone T1.
(2) Wash the beads with PBS three times.
(3) Add biotinylated alkaline phosphatase (1-h incubation).
(4) Wash twice with PBS.
(5) Re-suspend in PBS.
We initially investigated the dependency of the enzymatic reac-
tion on the amount of pNPP (N2770, Sigma) substrate present
in order to determine the amount of the substrate required to
evaluate the effect of agitation. We used a two-well chip and
placed two droplets in each well, as shown in Fig. 6. The diam-
Fig. 6. Enzymatic reaction sequence.
eter and height of the well were 7.0 and 3.0 mm, respectively.
The width of the channel between the two wells was 1.5 mm.
The left droplet contained the AP-labeled magnetic beads which
weighted 30 ␮g, and the right one contained the substrate. The
volume of the each droplet was 30 ␮l. The beads were collected
and extracted from the original droplet. They were then transferred
from the left to right and fused into the substrate droplet by using
a commercially available Nd–Fe–B permanent magnet (Shin-Etsu
Chemical Co., Ltd) with a magnetic flux density of 1.3 T and size of
2.0 mm × 2.0 mm × 2.0 mm. The agitation step was not performed
in this experiment. After the AP-labeled magnetic beads and the
pNPP substrate were reacted for 10 min, 30 ␮l of 3 M NaOH was
added to the reacted droplet to stop the reaction. Finally, the beads
were collected by the magnet, and only the reacted solution was
extracted by pipetting. The extracted solution was then placed in
a quartz tube, and its absorbance was measured by spectral pho-
tometer (V-530, JASCO Co.). As shown in Fig. 7, the absorbance
value initially increased linearly with increasing substrate density,
778 M. Shikida et al. / Sensors and Actuators B 137 (2009) 774–780

Fig. 7. Analytical curve showing relationship between absorbance of enzymatic

reacted pNPP and concentration of pNPP.
Fig. 9. Relationship between time and reaction efficiency.

before becoming constant at density above 1 mg/ml. According to

this result, we chose a substrate density of 10 mg/ml to evaluate the
effects of agitation.

3.3. Agitation effect

We performed an enzymatic reaction by manipulating the AP-

labeled magnetic beads selectively. The experimental condition and
procedures were almost the same of those described in Section
3.2, except agitation performance was added to the parameters
evaluated in this experiments. Two droplets were initially placed
in each well. The beads were collected and extracted from the
original droplet. Next, they were transferred and fused into the
substrate droplet by using the magnet. After the beads were trans-
ferred to the right-side droplet, the two-well chip was placed on
the agitation device. The beads were then agitated by changing
the magnetic-field distribution, as shown in Fig. 8. A NaOH solu-
tion was added to stop the enzymatic reaction, and absorbance
was measured by spectral photometer. The absorbance increased
linearly with increasing reaction time either with or without agi-
tation. As shown in Fig. 9, however, the rate of the absorbance
increment at the agitated sample was more than four times that
of the un-agitated sample. The agitation frequency was 0.1 Hz, and
continuous agitation was performed during the reaction. We then Fig. 10. Reaction dependency on agitation time.

investigated the dependency of the enzymatic reaction on agitation

time. The agitation times were varied from 1 to 10 min for the 10-
min. reaction time. Two different agitation waveforms, as shown in
Fig. 10, were used. The absorbance increases linearly as the ratio
between the agitation and reaction times increases. But it does
not depend on the agitation waveform. This is because the mag-
netic bead used was poorly dispersed. The single magnetic bead
used in the experiments was composed of Fe3 O4 particles with
a diameter of 20–30 nm, which were bound together by polymer
material. Therefore, the bead had a remanent magnetization; as a
result, the magnetic beads agglutinated together in the droplet. This

Fig. 8. Close-up images of attraction and repulsion of beads.

 M. Shikida et al. / Sensors and Actuators B 137 (2009) 774–780
agglutination by remanent magnetization disturbs the dispersibil-
ity of the beads. On the other hand, a single Fe3 O4 particle with a
diameter of 20–30 nm does not have a hysterisis property (i.e., no
remanent magnetization), because it is formed of a single magnetic
domain. We therefore think that the dispersibility will be improved
by applying a single Fe3 O4 particle as a bead for biochemical reac-
tions.
4. Concluding remarks
We investigated the performance of agitation of the beads
produced by multi-layered flat coils and magnetic beads. The inves-
tigation results can be summarized as three key points. First, the
temperature in the well solution was kept below 35 ◦ C at the appro-
priate agitation drive currents by adding a heat-sink foil between
the flat coils. Second, the beads were magnetically agitated inside
the droplet by changing the external magnetic-field distribution.
Third, the enzymatic reaction efficiency increased linearly with
increasing reaction time and was more than four times higher with
agitation than without agitation.
Acknowledgments
We would like to thank Dr. Nanao Horiishi of Toda Kogyo Corp.
and Mr. Osamu Nakao of Fujikura Ltd. for their useful suggestions
and help in the experiments. This research was partially sup-
ported by the 21st COE program (Micro- and Nano-Mechatronics
for Information-based Society) from The Ministry of Education,
Culture, Sports, Science, and Technology, and Grant-in-Aid for Sci-
entific Research (B) No. 18310093 from the Japan Society for the
Promotion of Science.
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Biographies
Mitsuhiro Shikida received BS and MS degrees in electrical engineering from Seikei
University, Tokyo, in 1988 and 1990, respectively. He received a PhD from Nagoya
University in 1998. From 1990 to 1995, he worked at Hitachi, Ltd., Tokyo. In 1995,
he joined the Department of Micro-System Engineering at Nagoya University as a
research associate. He was an assistant professor from 1998 to 2004 and has been
an associate professor since 2004. He joined the Research Center for Advanced
Waste and Emission Management in 2001, the EcoTopia Science Institute in 2004,
and Department of Micro-Nano Systems Engineering at Nagoya University in 2007.
His research interests include integration of micro-sensors and actuators for intel-
ligent systems, micro-fabrication of 3D microstructures for medical applications,
and micro-total analysis systems for biotechnologies. Dr. Shikida is a member of
the Institute of Electrical Engineers of Japan and the Japan Society of Mechanical
Engineers.
Motoki Koyama received a BS degree in mechanical engineering from Nagoya Uni-
versity, Japan, in 2007. He is currently working on an MS degree in Micro-Nano
Systems Engineering at Nagoya University. His research interests include micro-total
analysis systems for biotechnologies.
Nobuhiro Nagao received a BS degree in mechanical engineering and an MS degree
in micro-system engineering from Nagoya University, Japan, in 2004, and 2007,
respectively. His research interests include micro-total analysis systems for biotech-
nologies.
Rentaro Imai received a BS degree in mechanical engineering from Nagoya Univer-
sity, Japan, in 2006. He is currently working on an MS degree in Micro-Nano Systems
Engineering at Nagoya University. His research interests include micro-total analysis
systems for biotechnologies.
Hiroyuki Honda received BS, MS, and PhD degrees in chemical engineering from
Nagoya University in 1983, 1985, and 1988, respectively. He worked as an assis-
tant professor at Nagoya University from 1988 to 1990 and at the Tokyo Institute of
Technology from 1990 to 1992. He was an associate professor from 1993 to 2004
and became a professor in April 2004. His research interests include bioengineering,
medical engineering, bioinformatics, and biomolecule sensing. Dr. Honda is a mem-
ber of the Society of Biotechnology, Japan, the Society of Chemical Engineers, Japan,
the Chemical Society of Japan, the Japanese Cancer Association, the Japan Bioindus-
try Association, the Japan Society of Bioscience, Biotechnology, and Agrochemistry,
and the Japanese Society for Bioinformatics.
Mina Okochi received BS, MS, and PhD degrees in biological engineering from Tokyo
University of Agriculture and Technology in 1993, 1995, and 1998, respectively. She
was a research fellow of the Japan Society for the Promotion of Science (JSPS research
fellow) from 1996 to 1999. She worked as an assistant professor at Tokyo University
of Agriculture and Technology from 1999 to 2004. She has become an associate
professor in 2004 at Nagoya University. Her research interests include bioscience,
bioengineering, bioelectronics, and biosensors. Dr. Okochi is a member of the Society
for Biotechnology, Japan, the Chemical Society of Japan, the Electrochemical Society
of Japan, the Society of Chemical Engineers, Japan, The Molecular Biology Society of
Japan, and the Japan Bioindustry Association.
Hiroyoshi Tsuchiya received a BS and an MS degree in biochemical engineering
from Nagoya University, Japan, in 2004, and 2006, respectively. His research interests
include micro-total analysis systems for biotechnologies.
780 M. Shikida et al. / Sensors and Actuators B 137 (2009) 774–780

Kazuo Sato received BS degree from Yokohama National University in 1970, and
PhD degree from The University of Tokyo in 1982. He worked with Hitachi Ltd.
during 1970–1994. Since 1994, he is a professor of Micromachining and MEMS Lab-
oratory, Department of Micro/Nano Systems Engineering, Nagoya University. He
started MEMS research in 1983, and published 95 journal papers, 29 review arti-
cles, and 11 book chapters on MEMS. His research areas are micro/nano-physics in
anisotropic etching and mechanical properties of single crystal silicon, as well as
applied microsystems such as sensors and actuators. He co-chaired IEEE MEMS-97.
He is the Editor in Asia of Journal of Micromechanics and Microengineering (IOP).
He is a Fellow of JSME, a Senior Member of IEEJ, a member of JSPE and IEEE.