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 the targeted samples effectively from a droplet. Beads are used
for sample collection [15–18] in the field of ␮-TAS. The use of
Various micro-total analysis systems (␮-TAS), in which mechan- beads has extended to various applications, including mixing
ical and electrical components are integrated on the same chip, [19,20], micro-channel formation to increase reaction efficiency
have been developed by using micro-electro-mechanical-systems [21], surface modification [22] and screening at micro-reactor
technologies. Such systems are expected to reduce the amount of array chips [23]. Gijs’s group has recently developed a unique
reagent solutions and analysis time required and to enable the magnetic bead-handling method based on a planar coil. They
on-site monitoring of chemicals. Generally, most systems need have also shown that their system can perform two-dimensional
complicated mechanical fluidic devices, such as valves and pumps, manipulation of the beads on chips [24,25]. We used magnetic
for handling solutions, because they use continuous flow as a beads as carriers of a chemical material and handled them in a
reaction medium. A system can therefore become large, even if droplet configuration on the chip. We previously focused on han-
fluid channels and reactors are integrated onto microchips [1–5]. dling magnetic beads [12] and a biochemical reaction unit [13],
Fabrication processes making the devices and typical mechanical which is a key component of our ␮-TAS. To construct a palm-
fluidic components developed for ␮-TAS application are described sized biochemical-analysis system, we also recently developed
in Ref. [6]. To miniaturize the system, different mechanisms based transportation and agitation mechanisms that apply rotary motion
on a droplet solution as a medium have recently been pro- [14].
posed [7–11]. These mechanisms are, in principle, rather simple As described above, droplet handling based on a combination of
and can achieve fusion and separation by electrowetting con- beads and magnetic force makes it possible to collect and extract
trol. However, the method based on the electrowetting droplet targeted samples effectively from a droplet on a palm-sized system
handling is difficult to extract the targeted sample from the and to reduce the amount of reagent. However, the biochemical
droplet. reaction inside the droplet takes a certain amount of time because
Given these difficulties, we therefore previously proposed a there is no physical actuation force inside the droplet available for
novel type of magnetic bead-droplet handling mechanism for agitation of the beads in the systems. Generally, agitation is one of
a ␮-TAS application [12–14]. The proposed mechanism has the the main problems in ␮-TAS applications, because the Reynolds
advantages that it does not require complicated fluid-control number becomes small as system size decreases (meaning that
devices for handling solutions and it can collect and extract 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,
∗ Corresponding author. and we evaluated its performance by using an enzymatic reac-
E-mail address: shikida@mech.nagoya-u.ac.jp (M. Shikida). 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) 774–780 775

(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-
Fig. 1. Schematic view of agitation device. 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
2. Operation of palm-sized system was 6.5 mm × 6.5 mm × 6.0 mm. A commercially available magnet
(a Nd–Fe–B permanent magnet, produced by Niroku Seisakusho
2.1. Overview of system 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
We previously developed a rotary-drive-type biochemical- device.
analysis system using a bead-cluster handling mechanism [14]. It The agitation of the magnetic beads inside the droplet is per-
consists of a multi-well chip, two small magnets, another magnet formed by controlling the magnetic-field strength by means of the
coupled with multi-layered flat coils, a rotary table, and a stepping pair of three-layered flat coils and the permanent magnet. The mag-
motor and controller. The two magnets and the flat coils with the netic force acting on the beads is expressed by
magnet are fixed on the rotary table. The two small magnets are  
F = I × ∇ H
 (1)
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-  I and H
 are magnetic force per unit volume (N/m3 ), mag-
where F,
sists of eight wells arranged in a concentric fashion, bent channels,
netization of the magnetic material (Wb/m2 ), and magnetic-field
and a glass-bottom plate. A droplet, which contains magnetic beads
strength (A/m), respectively. By partially differentiating Eq. (1), the
with samples adhered to their surfaces, is placed in the first well.
magnetic force per unit volume, Fx (N/m3 ), in the horizontal x-
Various types of reagent and dilution droplets are pipetted into the
direction can be written as [2]
other wells. A series of reactions is performed by transferring the
magnetic beads from one well to the next. The system performs two ∂Hx ∂Hx ∂Hx
Fx = Ix + Iy + Iz (2)
operations: transportation and agitation. Details of the system are ∂x ∂y ∂z
given in Ref. [14]. As shown in Eq. (2), the magnetic force acting on the beads
depends on the bead’s magnetization, which increases with
2.2. Agitation principle increasing magnetic-field strength. We therefore used a permanent
magnet having a large enough magnetic-field strength to maintain
A schematic view of the agitation device is shown in Fig. 1. It enough actuation force for handling the beads. The magnetic force
consists of three-layered flat coils, a permanent magnet, a heat sink, also depends on the gradient of the magnetic-field, meaning that
and two aluminum foils. We used a custom-ordered flat coil pro- the beads move to a position under higher magnetic-field strength.
duced by Fujikura Ltd., Japan. A photograph and schematic view of The flat coils were used to form a magnetic-field gradient inside the
the flat coil is shown in Fig. 2. Square-shaped coils were formed on droplet. Generally, the magnetic-field strength induced by a flat coil
both sides of the polyimide film to increase the number of turns is much smaller than that of a permanent magnet, and it increases

Fig. 2. Photograph and schematic view of flat coil.

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. bution, which is controlled by the direction of the applied current
To increase the magnetic force, we therefore piled the flat coils on to the coil (see Fig. 2). The beads were repeatedly moved to increase
top of each other to form a tri-laminar structure. Considering the agitation performance. The direction of the current was changed by
above factors, we used both the multi-layered flat coils and the per- simply applying an alternating current to the flat coils, and repe-
manent magnet for keeping enough magnetic force for driving the tition cycle was varied by changing the frequency of the applied
beads and for forming a non-uniform magnetic-field. current.
The distribution pattern of the magnetic-field strength at the
droplet is determined by the summation of the magnetic-fields 3. Experiment
induced by the multi-layered flat coils and by the permanent mag-
net (see Fig. 3). The magnetic-field strength becomes a maximum 3.1. Heat management
at the center of the droplet when the direction of the magnetic-
fields induced by both the flat coils and the permanent magnet are The magnetic force for agitating the beads is proportional to
equal. In this case, the magnetic beads move to the center of the the current applied to the coil. To operate the agitation device cor-
droplet (see Figs. 3(a) and 4 left). On the other hand, the beads rectly, a high current, in the order of several hundred milliamps,
move to the circumference on the inside of the droplet when the is required. However, this current generates heat and increases
direction of the magnetic-field induced by the flat coil is reversed the temperature in the droplet. Therefore, we evaluated the tem-
(see Figs. 3(a) and 4 right ). The position of the beads in the droplet perature change due to the applied current to the flat coil in this
can therefore be controlled by changing the magnetic-field distri- 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
Fig. 6. Enzymatic reaction sequence.
(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 eter and height of the well were 7.0 and 3.0 mm, respectively.
streptavidin-modified magnetic beads were then labeled with The width of the channel between the two wells was 1.5 mm.
biotinylated alkaline phosphatase (B-2005, Vector laboratories) The left droplet contained the AP-labeled magnetic beads which
by using chemical bonding between the streptavidin and biotin weighted 30 ␮g, and the right one contained the substrate. The
molecules. The immobilization procedure for the enzyme consists volume of the each droplet was 30 ␮l. The beads were collected
of the following five steps. and extracted from the original droplet. They were then transferred
from the left to right and fused into the substrate droplet by using
(1) Sample 50 ␮l of the Dynabeads® streptavidinTM Myone T1. a commercially available Nd–Fe–B permanent magnet (Shin-Etsu
(2) Wash the beads with PBS three times. Chemical Co., Ltd) with a magnetic flux density of 1.3 T and size of
(3) Add biotinylated alkaline phosphatase (1-h incubation). 2.0 mm × 2.0 mm × 2.0 mm. The agitation step was not performed
(4) Wash twice with PBS. in this experiment. After the AP-labeled magnetic beads and the
(5) Re-suspend in PBS. 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
We initially investigated the dependency of the enzymatic reac- were collected by the magnet, and only the reacted solution was
tion on the amount of pNPP (N2770, Sigma) substrate present extracted by pipetting. The extracted solution was then placed in
in order to determine the amount of the substrate required to a quartz tube, and its absorbance was measured by spectral pho-
evaluate the effect of agitation. We used a two-well chip and tometer (V-530, JASCO Co.). As shown in Fig. 7, the absorbance
placed two droplets in each well, as shown in Fig. 6. The diam- 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- netic bead used was poorly dispersed. The single magnetic bead
min. reaction time. Two different agitation waveforms, as shown in used in the experiments was composed of Fe3 O4 particles with
Fig. 10, were used. The absorbance increases linearly as the ratio a diameter of 20–30 nm, which were bound together by polymer
between the agitation and reaction times increases. But it does material. Therefore, the bead had a remanent magnetization; as a
not depend on the agitation waveform. This is because the mag- 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) 774–780 779

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Acknowledgments Biographies

We would like to thank Dr. Nanao Horiishi of Toda Kogyo Corp. Mitsuhiro Shikida received BS and MS degrees in electrical engineering from Seikei
and Mr. Osamu Nakao of Fujikura Ltd. for their useful suggestions University, Tokyo, in 1988 and 1990, respectively. He received a PhD from Nagoya
and help in the experiments. This research was partially sup- 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
ported by the 21st COE program (Micro- and Nano-Mechatronics research associate. He was an assistant professor from 1998 to 2004 and has been
for Information-based Society) from The Ministry of Education, an associate professor since 2004. He joined the Research Center for Advanced
Culture, Sports, Science, and Technology, and Grant-in-Aid for Sci- Waste and Emission Management in 2001, the EcoTopia Science Institute in 2004,
entific Research (B) No. 18310093 from the Japan Society for the and Department of Micro-Nano Systems Engineering at Nagoya University in 2007.
His research interests include integration of micro-sensors and actuators for intel-
Promotion of Science.
ligent systems, micro-fabrication of 3D microstructures for medical applications,
and micro-total analysis systems for biotechnologies. Dr. Shikida is a member of
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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 cles, and 11 book chapters on MEMS. His research areas are micro/nano-physics in
PhD degree from The University of Tokyo in 1982. He worked with Hitachi Ltd. anisotropic etching and mechanical properties of single crystal silicon, as well as
during 1970–1994. Since 1994, he is a professor of Micromachining and MEMS Lab- applied microsystems such as sensors and actuators. He co-chaired IEEE MEMS-97.
oratory, Department of Micro/Nano Systems Engineering, Nagoya University. He He is the Editor in Asia of Journal of Micromechanics and Microengineering (IOP).
started MEMS research in 1983, and published 95 journal papers, 29 review arti- He is a Fellow of JSME, a Senior Member of IEEJ, a member of JSPE and IEEE.