Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

Analysis of Dielectrophoretic Movement of Floating Myoblast near Surface
Electrodes in Flow Channel
Shigehiro HASHIMOTO, Ryota MATSUZAWA, Yuji ENDO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTparticle, when the particle is placed in a non-uniform electricfield.

The dielectrophoretic movement of the floating biological cell
near the surface electrodes has been analyzed in the micro flow
channel in vitro. A pair of asymmetric surface electrodes oftitanium (thickness of 200 nm) were incorporated in the flow
channel by the photolithography technique: a triangularelectrode with the tip angle of 0.35 rad, and a rectangularelectrode of the flat edge as reference. The cyclic alternating
electric current of the square wave (between 0.25 μs and 0.3 μsof periods) 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
experimental results show that the absolute value of theamplitude of the acceleration by the electric field, which isperpendicular to the flow direction, increases with the diameterof the cell.

With the aid of the micromachining technique [27], many kindsof micro systems were designed.
In some systems,
dielectrophoresis was applied to sorting of biological cells
floating in the medium. In the present study, a micro flow
channel with the surface electrodes has been designed toanalyze the dielectrophoretic movement of a flowing biologicalcell in vitro.

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. The micro flow channel withsurface electrodes was manufactured by the photolithography
technique.

Keywords: Biomedical Engineering, Dielectrophoresis, Surface
Electrode and C2C12.

1. INTRODUCTION
The movement of a biological cell suspended in the medium isgoverned by several factors: movement of the medium, gravity,
electric force, Van der Waals force, and affinity of surface.
Various methods have been applied to control the movement ofcells in vitro: the flow [1], the shear field [2, 3], the filter, theslit [4–6], the magnetic field [7], the gravitational field [8–10],
and the electric field [11]. These methods might contribute toseveral applications of manipulation of cells [12, 13]: detectionof targeted cells [14–16], sorting of cells [17–21], arrangementof cells to make a tissue, and measurement of the character ofcells [22]. Movement of a charged particle depends on theelectric field. The effect is applied to the electrophoresis
device [23]. When a particle is subjected to a non-uniform
electric field, a force acts even on a non-charged particle,
because the polarization generates in the particle.
Thephenomenon is called dielectrophoresis [8], which depends on
the several parameters: the electrical property of the particle,
shape and size of the particle [24], the electrical property of themedium, and frequency [25] of the electric field.
Electrophoresis is a phenomenon, in which a particle is movedby the Coulomb force between the charge of the particle and theelectric field. Dielectrophoresis [26], on the other hand, is aphenomenon, in which a particle moves due to the interaction
between the electric field and the charge induced in a neutral

Electrode

Cell
Electric field
ElectrodeFig. 1: Dielectrophoretic movement of cell in non-uniform
electric field.

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

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 thickness of thelayer (SU-8 100) on the surface electrode was measured by thestylus (with 3×10－5 N) of the contact profilometer (Dektak
XT-E, Bruker Corporation).

I

E
Electrode

Flow Channel
The upper plate of polydimethylsiloxane has holes with thediameter of 5 mm for the inlet and the outlet of the suspension.
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.
A rectangular parallelepiped channel of 18 mm length × 0.5 mm
width × 0.035 mm height is formed between upper and lower
plates. The flow channel is placed on the stage of the inverted
phase-contrast microscope (IX71, Olympus Co., Ltd., Tokyo).

Electric field

V

ElectrodeFig. 2b: Electric circuit: alternating electric rectangular wave(E), resistance (R), voltage (V), and electric current (I).

Electric Stimulation
The electric stimulation (E) of the alternating rectangular cyclic
wave (0.25 μs < period (T) < 0.3 μs; －15 V < Ea < +15 V)
was generated with an electric stimulator (Fig. 2a). The
stimulator was connected to the titanium film electrode, and theelectric stimulation was applied to the medium of cells. An
electric resistance (R) of 2 kΩ is serially inserted between theelectrode and the stimulator (Fig. 2b) to measure the electric
current (I < ±7.5 mA). The electric voltage (V) between theterminals of the resistance (R) is monitored by an oscilloscopeduring electric stimulation applied between the titanium surface
electrodes (Fig. 2b).

vy

Electrode
y
vx
x

Cell

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.

O

ElectrodeFig. 3: Two-dimensional movement of cell flowing near tip ofelectrode: position (x, y), velocity (vx, vy) of cell.

Before the flow test, the inner surface of the flow channel washydrophilized by the oxygen plasma ashing. The bovine
serum albumin solution was prefilled in the flow channel, andincubated 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).

E

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. 3. The main flow direction of the medium isdefined as x. The perpendicular direction from the referenceelectrode to the tip of the triangular electrode is defined as y.
The origin is adjusted at the tip of the triangular electrode.

Ea
0

R

t
T

Fig. 2a: Voltage (E) tracings of rectangular wave: time (t),period (T), amplitude (Ea).

3. RESULTS
Fig. 4 exemplifies the microscopic image, which shows cells

14Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

flowing near the tip of the triangular electrode.

y [μm]

Fig. 5 shows the tracings of movement of twenty cells. Fig. 5ashows movement of cells without the electric field. Every cellmoves at the constant velocity according to the flow to the x
direction. The period of rectangular cyclic wave is 0.3 μs in
Fig. 5b, and 0.25 μs in Fig. 5c. Every cell shows the
movement perpendicular to the flow direction near the
triangular electrode. The movement at 0.25 μs (period) islarger than that at 0.3 μs in the alternating electric field.
Fig. 6 shows the tracings of velocity of twenty cells: vx (Fig. 6a),and vy (Fig. 6b). The period of rectangular cyclic wave is 0.3
μs. The velocity vx is almost constant (0.2 mm/s < vx < 0.7mm/s). The velocity vy is almost zero, except near the
triangular electrode.

x [μm]
Fig. 5b: Tracings of movement of 20 cells [μm]: period of 0.3
μs, amplitude of ±15 V.

Fig. 7 shows the tracings of velocity of twenty cells: vx (Fig. 7a),and vy (Fig. 7b). The period of rectangular cyclic wave is 0.25
μs. The velocity vx is almost constant (0.2 mm/s < vx < 0.6mm/s). The velocity vy is almost zero, except near the
triangular electrode. The maximum absolute value of vy islarger at 0.25 μs of the period than at 0.3 μs of the period,
although vx of the both periods are in the similar range.

y [μm]

Flow
Electrode
Cell
0.1 mmx [μm]
Fig. 5c: Tracings of movement of 20 cells [μm]: period of 0.25
μs, amplitude of ±15 V.

Fig. 4: Cells in flow channel around triangle electrode.
y [μm]

Fig. 6a: Velocity (vx) tracings of 20 cells: period of 0.3 μs,
amplitude of ±15 V.

x [μm]
Fig. 5a: Tracings of movement of 20 cells [μm]: without
electric field.

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

wave is 0.25 μs. The absolute value of the peak velocity near
the tip of the triangular electrode increases with the radius of
the cell.

vy [mm / s]Fig. 6b: Velocity (vy) tracings of 20 cells: period of 0.3 μs,
amplitude of ±15 V.

x [μm]
Fig. 8: Velocity (vy) tracings: : 9.1 μm, y0 = 22 μm, (circle), 6.6μm, y0 = 24 μm, (triangle), 5.9 μm, y0 = 22 μm, (square): periodof 0.25 μs, amplitude of ±15 V.

Fig. 7a: Velocity (vx) tracings of 20 cells: period of 0.25 μs,
amplitude of ±15 V.

Fig. 9: Acceleration (αy) tracings of 20 cells: 9.1 μm (circle),
6.6 μm (triangle), 5.9 μm (square): period of 0.25 μs, amplitudeof ±15 V.
Radius (r) [μm]Fig. 7b: Velocity (vy) tracings of 20 cells: period of 0.25 μs,
amplitude of ±15 V.

Fig. 8 exemplifies the tracings of velocity (vy) of three cells,
which have variations on the radius of the cells. The period ofrectangular cyclic wave is 0.25 μs. The absolute value of thepeak velocity near the tip of the triangular electrode is higher atthe bigger cell than at the smaller cell.
Fig. 9 shows the tracings of the acceleration (αy) of 20 cells.
The period of rectangular cyclic wave is 0.25 μs. The cells areaccelerated to the apart direction (the direction perpendicular to
the flow) from the tip of the triangular electrode before passing
through the tip of the electrode, and decelerated after passing
through the tip of the electrode.

Acceleration (αy) [mm / s2]
Fig. 10: Relationship between acceleration (αy) and radius (r):period of 0.25 μs, amplitude of ±15 V.

Fig. 10 shows the relationship between the acceleration (αy) andthe radius (r) of the cell. The period of rectangular cyclic

16Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

4. DISCUSSION

was introduced between the surface electrodes.
Theexperimental results show that the amplitude of the accelerationby the electric field, which is perpendicular to the flow direction,
increases with the diameter of the cell.

The dielectrophoresis was applied to the cell sorting system as
minimally invasive method in the previous studies [12, 28].
The shift distance [15, 26] has been very small (< 0.1 mm) in
the present experiment. If the shift movement of the cell
adjacent to the tip of the electrode is enlarged at thedownstream by the inertial movement, the method can beapplied to the cell sorting. The relationship between thestreamline of the cell and the position of the side wall of theflow channel should be considered for the continuous
movement of the cell at the downstream. To apply the present
system for cell sorting, both the initial position and the velocity[29] should be controlled before the non-uniform electric fieldin the flow channel. In another way, the accumulated shift by
the multiple electrodes might enlarged the shift movement of
the cell [30].

ACKNOWLEDGMENT
The authors thank to Mr. Daisuke Hasegawa for his assistanceof the experiment.

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5. CONCLUSION
The dielectrophoretic movement of the floating C2C12 (mouse
myoblast cell line) near the surface electrodes has been
analyzed in the micro flow channel in vitro. A pair ofasymmetric surface electrodes of titanium (thickness of 200 nm)
were incorporated in the flow channel by the photolithography
technique: a triangular electrode with the tip angle of 0.35 rad,and a rectangular electrode of the flat edge as reference. The
rectangular cyclic alternating electric current (0.25 μs of period)17

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