Design of Flow Channel with Surface Electrodes to Detect
Dielectrophoretic Movement of Floating Myoblast
Shigehiro HASHIMOTO, Daisuke HASEGAWA, Yusuke TAKAHASHI, Ryota MATSUZAWA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT
A micro flow channel with the surface electrodes has been
designed to detect the dielectrophoretic movement of abiological cell in vitro.
A pair of asymmetric surface
electrodes of titanium (thickness of 200 nm) were incorporated
in the flow channel by the photolithography technique: atriangular electrode with the tip angle of 0.35 rad, and arectangular electrode of the flat edge as reference. The
rectangular cyclic alternating electric current was introduced
between the surface electrodes. The suspension of C2C12(mouse myoblast cell line) was injected into the flow channel,
and the flow rate was controlled by the pressure head between
the inlet and the outlet. The selected system design (the shapeof the electrode, electric rectangular alternating current wave(±7.5 mA, 0.3 μs of period), and the flow velocity of 0.3 mm/s)
is able to detect dielectrophoretic movement of floating C2C12.
Keywords: Biomedical Engineering, Dielectrophoresis, Surface
Electrode and C2C12.

of dielectrophoresis is calculated by the equivalent dipole
moment method.
F = π εm r3 Re [K (ω)] ▽ E2

(1)

In Eq. 1, r is the radius of the particle, εm is the dielectric
constant of the surrounding medium, and E is the maximum
value of the electric field. Re [K (ω)] represents the real partof the function expressed by the following equation.
K (ω) = (εp* －εm*) / (εp* + 2 εm*)

(2)

In Eq. 2, εp* is the complex dielectric constant of the particle,
εm* is the complex dielectric constant of the solution.
With the aid of the micromachining technique [27], many kindsof micro systems were designed, in which dielectrophoresis wasapplied to sorting of biological cells floating in the medium inthe previous studies. In the present study, a micro flow
channel with the surface electrodes has been designed to detectthe dielectrophoretic movement of a biological cell in vitro.

1. INTRODUCTION
The movement of a biological cell suspended in the medium isgoverned by several factors: electric force, Van der Waals force,
affinity of surface, and pressure. Various methods have been
applied to control the movement of cells in vitro: the flow [1],slits [2, 3], the magnetic field [4], the gravitational field [5, 6],
and the electric field [7]. These methods might contribute toseveral applications of manipulation of cells: arrangement ofcells to make a tissue, sorting of cells, and measurement of thecharacter of cells.
Movement of a charged particle depends on the electric field.
The effect is applied to the electrophoresis device. When aparticle is subjected to a non-uniform electric field, a force acts
even on a non-charged particle, because the polarization
generates in the particle.
The phenomenon is called
dielectrophoresis [8–26], which depends on the several
parameters: the electrical property of the particle, shape and sizeof the particle [8], the electrical property of the medium, andfrequency of the electric field.
Electrophoresis is aphenomenon, in which a particle is moved by the Coulomb
force between the charge of the particle and the electric field.
Dielectrophoresis [9], on the other hand, is a phenomenon, in
which a particle moves due to the interaction between theelectric field and the charge induced in a neutral particle, when
the particle is placed in a non-uniform electric field. The force

2. METHODS
Surface ElectrodeThe titanium film was used for the surface electrode. Titanium
was coated on the glass plate (36 mm × 28 mm × 1 mm,
Matsunami Glass Ind., Ltd., Japan). The thickness of coating
is 200 nm. The tip angle of the triangle shape of the one of thesurface electrodes is 0.35 rad. The other reference electrodehas a flat edge. The distance between electrodes is 0.1 mm(Fig. 1). The shortest connecting line between electrodes isvertical to the flow direction.

Detail of A
Fig. 1: Dimension of surface electrode: unit mm.

Deposition of Titanium
Before the deposition of titanium, the surface of the glass plate
was hydrophilized by the oxygen (30 cm3/min, 0.1 Pa) plasma
ashing for five minutes at 200 W (20 Pa) by the reactive ion
etching system (FA-1, Samco International, Kyoto, Japan).
Titanium was deposited on the surface of the glass plate with
200 nm thickness in the electron beam vapor deposition
apparatus (JBS-Z0501EVC JEOL Ltd., Tokyo, Japan).
Photomask A for Triangle Tip of Electrode
Before the deposition of titanium, the surface of the plate washydrophilized by the oxygen (30 cm3/min, 0.1 Pa) plasma
ashing for five minutes at 200 W (20 Pa) by the reactive ion
etching system (FA-1). The positive photoresist material of
OFPR-800LB-20cP (Tokyo Ohka Kogyo Co., Ltd, Tokyo,
Japan) was coated on the titanium with the spin coater (at 3000
rpm for 30 s). The photoresist was baked at the hotplate at 338
K for one minute and at 368 K for three minutes, successively.

Coating

UV light
Developing

Etching

Fig. 3: Photolithography process for surface electrodes.

20 mm
Fig. 4: Photomask B (left), and surface electrodes (right).

The pattern of the electrode with the flow channel was drawn
on the mask with a laser drawing system (DDB-201K-KH,
Neoark Corporation, Hachioji, Japan). The photoresist wasdeveloped with tetra-methyl-ammonium hydroxide (NMD-3,
Tokyo Ohka Kogyo Co., Ltd., Kawasaki, Japan) for two
minutes, rinsed with the distilled water, and dried.
Fig. 5: Dimension of photomask C for flow channel: unit mm.
The titanium coated plate was etched with the plasma gas using
RIE-10NR (Samco International, Kyoto, Japan). For etching,
the gas of SF6 (50 cm3/min at 1013 hPa) with Ar (50 cm3/min at
1013 hPa) was applied at 75 W at 4 Pa for seven minutes.
Photomask B for ElectrodeThe titanium coated glass plate was hydrophilized by theoxygen (30 cm3/min, 0.1 Pa) plasma ashing for five minutes at
100 W by the reactive ion etching system (FA-1). The
negative photoresist material of low viscosity (SU-8 2: Micro
Chem Corp., MA, USA) was coated on the titanium with thespin coater (at 3000 rpm for 30 s) (Fig. 2). The photoresist
was baked at the hotplate at 338 K for one minute and at 368 K
for three minutes, successively.
The photomask A was mounted on the surface of SU-8, and thephotoresist was exposed to the UV light through the mask in themask aligner (M-1S, Mikasa Co. Ltd., Japan) for 15 s. The
photoresist was baked at the hotplate at 338 K for one minuteand at 368 K for three minutes, successively. The photoresist
was developed with SU-8 Developer (Micro Chem Corp., MA,
USA) for two minutes. The glass surface with the micro
pattern was rinsed with IPA (2-propanol, Wako Pure Chemical
Industries, Ltd.) for one minute, and with pure water for one
minute. After drying, the plate was etched with the plasma gas
using RIE-10NR. For etching, the gas of SF6 (50 cm3/min at
1013 hPa) with Ar (50 cm3/min at 1013 hPa) was applied at 75
W at 4 Pa for seven minutes. The areas at the bands of bothends were covered with the polyimide tapes (protect photoresist
material from ultraviolet light) to leave the part of titanium
coating for extended electrodes.
Coating

UV light
Developing

Etching

Fig. 2: Photolithography process for photomask B.

Surface Electrodes
The titanium coated glass plate was hydrophilized by theoxygen (30 cm3/min, 0.1 Pa) plasma ashing for five minutes at
100 W by the reactive ion etching system (FA-1). The positive
photoresist material of OFPR-800LB-20cP was coated on thetitanium with the spin coater (at 3000 rpm for 30 s) (Figs. 3&4).
The photoresist was baked at the hotplate at 338 K for one
minute and at 368 K for three minutes, successively. The
photomask B was mounted on the surface of SU-8, and thephotoresist was exposed to the UV light through the mask in themask aligner (M-1S) for 15 s. The photoresist was developedwith tetra-methyl-ammonium hydroxide (NMD-3) for two
minutes, rinsed with the distilled water. After drying, the plate
was etched with the plasma gas using RIE-10NR. For etching,
the gas of SF6 (50 cm3/min at 1013 hPa) with Ar (50 cm3/min at
1013 hPa) was applied at 75 W at 4 Pa for seven minutes.
Lower Plate of Flow Channel
To make the groove of the flow channel (width of 0.5 mm,
length of 18 mm, and depth of 0.04 mm), the photomask C (Fig.
5) was made by the same process as the photomask A. The
surface of the electrodes was hydrophilized by the oxygen (30cm3/min, 0.1 Pa) plasma ashing for five minutes at 200 W (20Pa) by the reactive ion etching system (FA-1). The negative
photoresist material of low viscosity (SU-8 100) was coated on
the surface electrodes with the spin coater (at 1300 rpm for 30 s,
and at 2700 rpm for 120 s, successively) (Fig. 6). The coating
condition was adjusted to make the layer of thickness(correspond to the height of the flow channel) between 20 μm
and 40 μm. The thickness of the layer (SU-8 100) on thesurface electrode was measured by the stylus (with 3×10－5 N)of the contact profilometer (Dektak XT-E, Bruker Corporation).
(Fig. 10) The photoresist was baked at the hotplate at 338 K forten minutes and at 368 K for seventy minutes, successively.
The photomask C was mounted on the surface of SU-8, and thephotoresist was exposed to the UV light through the mask in themask aligner (M-1S) for 20 s. The photoresist was baked at
the hotplate at 338 K for seven minutes and at 368 K for fifteen
minutes, successively. The photoresist was developed with
SU-8 Developer for eight minutes. The surface with the micro
pattern was rinsed with IPA, and with pure water. The
photoresist was baked at the hotplate at 368 K for twenty
minutes.
Upper Plate of Flow Channel
PDMS was poured into the mold of poly-methyl-methacrylate(Fig. 7) with the curing agent. The volume ratio of PDMS tocuring agent is ten to one. After degassing, PDMS was baked
at 353 K for two hours in the oven (DX401, Yamato ScientificCo., Ltd). The upper plate has holes with the diameter of 5mm for the inlet and the outlet of the suspension.
Flow Channel
The upper plate was exposed to the oxygen gas (0.1 Pa, 30
cm3/min) in the reactive ion etching system (FA-1) (50 W, 20 Pa,
for two hours). Aminopropyltriethoxysilane (APTES) was
coated on the upper plate. After ten minutes, the upper plate
was adhered on the lower plate under the pressure of 0.5 N/m2
by sandwiching between plates of poly-methyl-methacrylate.
The value of the pressure was selected between 0.25 N/m2 and
0.8 N/m2 to control the height of the flow channel between
plates. The plates were baked in the oven at 353 K for ten
minutes. A rectangular parallelepiped channel of 18 mm
length × 0.5 mm width × 0.035 mm height is formed between
upper and lower plates (Fig. 7). The flow channel is placed on
the stage of the inverted phase-contrast microscope (IX71,
Olympus Co., Ltd., Tokyo) (Fig. 7).
Electric Stimulation
The electric stimulation (E) of the alternating rectangular cyclic
wave (0.3 μs < period < 10 μs; ±5 V, ±10 V, or ±15 V) was
generated with an electric stimulator. The stimulator wasconnected to the titanium film electrode, and the electric
stimulation was applied to the medium of cells. An electric
resistance (R) of 2 kΩ is serially inserted between the electrodeand the stimulator (Fig. 8). The electric signal between theterminals of the resistance (V) is monitored by an oscilloscopeduring electric stimulation applied between the titanium surface
electrodes (Fig. 11b).
Upper plate

Developing

Coating

UV light
Lower plate

Voltage: V
Resistance:R
Stimulator: E
Flow channel
Microscope
Fig. 8: Flow channel with electric circuit.

Cell
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was used in the test. D-MEM(Dulbecco’s Modified Eagle Medium) containing 10% FBS(Fetal Bovine Serum) and 1% penicillin/ streptomycin was used
for the medium.
Before the flow test, the inner surface of the flow channel washydrophilized by the oxygen (30 cm3/min, 0.1 Pa) plasma
ashing for two minutes at 50 W by the reactive ion etchingsystem (FA-1). The bovine serum albumin solution was
prefilled in the flow channel, and incubated for thirty minutes at
310 K in the incubator.
Before the flow test, the cells were exfoliated from the plate oftheculturedishwithtrypsinincludingEDTA(ethylenediaminetetraacetic acid), and suspended in the medium.
The suspension of cells was poured at the inlet of the flow
channel. The flow occurs by the pressure difference between
the inlet and the outlet, which was kept by the gravitational
level of the medium (< 5 mm).
Each cell passing between the electrodes was observed by the
inverted phase-contrast microscope (IX71), and recorded by thecamera (DSC-RX100M4, Sony Corporation, Japan), which is
set at the eyepiece of the microscope. The movement of thecell was analyzed by “Kinovea (Ver. 8.23, Commons
Attribution)” at the video images: 30 frames per second. To
trace the movement of the cell, the coordinates are defined asthat in Fig. 9. The flow direction is defined as x. The
perpendicular direction from the reference electrode to the tip of
the triangular electrode is defined as y. The origin is adjusted
at the tip of the triangular electrode.
Both the position (x0,y0) and the velocity (v0) of each cell were measured at x =－0.4
mm (upstream from the tip of the electrode) as the initial value.
The shifted distance (Δy) was defined as the difference between
the maximum value of y coordinate and minimum value of ycoordinate according to the stream line of each cell.

ChannelFig. 6: Photolithography process for flow channel.

3. RESULTS
Fig. 10 shows the tracings of the stylus on the layer of SU-8 on
the surface electrode. The tracings shows that the thickness oflayer of SU-8 is 34 μm.

30 mm

30 mm

30 mm

Fig. 7: Mold for upper plate of flow channel (left), upper plateof flow channel on lower plate with electrodes (middle), flow
channel on stage of microscope (right).

Fig. 11 exemplifies tracings of the voltage of electric
stimulation of rectangular cyclic waves: the voltage between
terminals of the electric stimulator (E in Fig. 11a), and thevoltage between terminals of the resistance of 2 kΩ (V in Fig.
11b). The tracings show the following values: the period of 1
μs, and the amplitude of ±15 V. The electric current iscalculated from the electric voltage divided by the electric
resistance (2 kΩ). Fig. 11b shows the rectangular alternating
electric current (±7.5 mA) flows the electric circuit.

E:
20 V

In Fig. 12, each colored line shows the movement of each cellin the stationary fluid around the triangle electrode with electric
stimulation (rectangular wave, period of 1 μs, amplitude of ±15
V). Every cell near the lower plate moves to the triangle
electrode by dielectrophoresis.

Time:
0.4 μsFig. 13 shows cells in flow channel around the triangularelectrode. Every cell passing near the tip of the electrodechanges the flow direction near the electrode.
Fig. 14 shows the shifted distance (Δy) in relation to theamplitude of the rectangular alternating voltage (E). The markshows the mean value of Δy, and the bar shows the standard
deviation. The shifted distance increases with the increase of
the amplitude of the voltage. Fig. 15 shows the relationshipbetween the shifted distance (Δy) and the initial velocity (v0) of
the cell. The shift decreases with the initial velocity. Fig. 16
shows the relationship between the shifted distance (Δy) and theperiod (T [μs]) of cyclic rectangular alternating electric
stimulation. The shift decreases with the period of cyclic
stimulation.
Fig. 17a shows the relationship between the shifted distance(Δy) and the dimension of cell (Dy [μm]) at the period of 0.33
μs of the amplitude of ±15 V. At the non-circle shape of the
projected area of the cell, both the length of Dy (the verticaldirection to the flow direction: the electric field direction) andDx (flow direction) was measured at each cell (Fig. 17b). The
shifted distance (Δy) of each cell is compared with that (Δy0) of
the cell with the standard diameter of 18 μm (Dy0), which flows
from the similar position (x0, y0) at the similar velocity (v0) withthe corresponding cell (Fig. 17c). Fig. 17c shows therelationship between the shifted distance ratio (Δy /Δy0) and thedimension ratio of the cell (Dy / Dy0). The bigger cell tends tomake the larger shift.

Fig. 11a: Voltage (E) tracings of rectangular wave: period of 1
μs, amplitude of ±15 V.

V:
20 V
Time:
0.4 μsFig. 11b: Tracings of voltage (V) of rectangular electric
stimulation: period of 1 μs, amplitude of ±15 V: 7.5 mA (2 kΩ).

Electrode
0.4 mmyx

ΔyElectrode

Tracings of cell movement

Cell

v0

Fig. 9: Movement of cell flowing near tip of electrode.

Fig. 12: Tracings of movement of 18 cells in stationary fluid
around triangle electrode with rectangular alternating electric
stimulation: unit, μm; period of 1 μs, amplitude of ±15 V.
Cell

Normaldirection

34 μm

Electrode

10 μm

Tangential direction

0.1 mmFig. 10: Tracings of stylus on layer of SU-8 on surface
electrode.

0.2 mmFig. 13: Cells in flow channel around triangle electrode.

Shifted distance (Δy [mm])Amplitude of voltage (E [V])Fig. 14: Relationship between shifted distance (Δy [mm]) andamplitude of voltage (E [V]): mean (circle) ± standard deviation(bar), n = 4.

transparent. The shift might depend on the passing route of
the cell. The dielectrophoretic effect might be highest adjacentto the tip of the electrode. The shift might depend on the sizeof the particle [8]. The voltage between electrodes higher than
±15 V might enlarge the shift. The amplitude of the electric
stimulation has been limited within the threshold value toprevent electrolysis [12] in the present study. Figs. 13-17
show the movement of each cell in the x-y plane. The
movement of the direction (z) perpendicular to the flow is verysmall, because the height of the channel is 0.035 mm. The
movement of cells between surface electrodes depends on themorphology of surface electrodes (the angle of the tip) [13–16],
which relates to non-uniformity of electric field. The higher
slope of electric field with non-uniformity is necessary toenlarge the movement of cells around the electrode. In theprevious studies, dielectrophoresis has been tried to apply on
the biological cell manipulation technology [8–26]. The micro
grooves were used for trapping of flowing cells in the previous
study [5]. To apply the present system for cell sorting, boththe initial position and the velocity should be controlled before
the non-uniform electric field in the flow channel.

5. CONCLUSION
A micro flow channel with the surface electrodes has beenFig. 15: Relationship between shifted distance (Δy) and initial
velocity (v0). Shift decreases at higher velocity.

Shifted distance (Δy [mm])Shifted distance (Δy [mm])Period (T [μs])Fig. 16: Relationship between shifted distance (Δy [mm]) andperiod (T [μs]) of electric stimulation: 0.2 mm/s < v0 < 0.3 mm/s
(triangle), 0.3 mm/s < v0 < 0.4 mm/s (circle): mean (mark) ± SD(bar): n = 4.

4. DISCUSSION
The dielectrophoresis was applied to the cell sorting system as
minimally invasive method in the previous studies [10, 11].
The shift distance has been very small (< 0.1 mm) in the present
experiment. If the shift movement of the cell adjacent to thetip of the electrode is enlarged at the downstream by the inertial
movement, the method can be applied to the cell sorting. The
relationship between the stream line of the cell and the positionof the side wall of the flow channel should be considered for thecontinuous movement of the cell at the downstream. In
another way, the accumulated shift by the multiple electrodes
might enlarged the shift movement of the cell. In the present
study, the dielectrophoretic movement of the cell was tried to beobserved by the optical microscope.
The electrodes
arrangement with the shorter distance has not been designed,
because the electrodes in the present study are not opticallyDimension of cell (Dy [μm])Fig. 17a: Relationship between shifted distance (Δy [mm]) anddimension of cell (Dy [μm]): 0.33 μs, ±15 V.

Fig. 17b: Size of cell: Dy (vertical direction, electric field
direction) and Dx (flow direction).
50 μm
Shifted distance ratio (Δy /Δy0)

Dimension ratio of cell (Dy / Dy0)
Fig. 17c: Relationship between shifted distance ratio (Δy /Δy0)
and dimension ratio of cell (Dy / Dy0).

designed to detect the dielectrophoretic movement of abiological cell in vitro.
A pair of asymmetric surface
electrodes of titanium (thickness of 200 nm) were incorporated
in the flow channel by the photolithography technique. The
selected system design (the triangular shape of the electrode,electric rectangular alternating current wave (±7.5 mA, 0.3 μsof period), and the flow velocity of 0.3 mm/s) is able to detect
dielectrophoretic movement of floating C2C12.

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