Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

Design of Flow Channel for Cell Sorter by Dielectrophoresis
with Photolithography TechniqueShigehiro HASHIMOTO, Ryota MATSUZAWA, Yuji ENDO, Takeru NOJI
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/
Kiyoshi YOSHINAKA
Health and Medical Research Institute, National Institute of Advanced Industrial Science & Technology
Tsukuba, 305-8564, Japan
ABSTRACT

shape and size of the particle [25], the electrical property of themedium, and the electric field (the amplitude and the frequency)[26].

The flow channel for cell sorter in vitro has been designed bydielectrophoresis with photolithography technique. A pair ofasymmetric surface electrodes of titanium (thickness of 200 nm)
were incorporated just before the branching flow channel: atriangular electrode with the tip angle of 0.35 rad, and arectangular electrode of the flat edge as reference. The cyclic
alternating electric current of the square wave (between 0.25 μs
and 0.3 μs of periods) was introduced between the surface
electrodes. The suspension of C2C12 (mouse myoblast cellline) was injected into the flow channel, and the flow rate wascontrolled by the pressure head between the inlet and the outlet.
Experimental results of the pilot test show that the absolutevalue of the amplitude of the acceleration by the electric field,
which is perpendicular to the flow direction, increases with theradius of the cell.
Keywords:
Biomedical
Engineering,
Cell
Dielectrophoresis, Surface Electrode and C2C12.

Electrophoresis is a phenomenon, in which a particle is movedby the Coulomb force between the charge of the particle and theelectric field. Dielectrophoresis [27], 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
particle, when the particle is placed in a non-uniform electricfield.
With the aid of the micromachining technique [28], many kindsof micro systems were designed.
In some systems,
dielectrophoresis was applied to sort biological cells floating in
the medium. In the present study, the flow channel for cell
sorter in vitro has been designed by dielectrophoresis withphotolithography technique.

Sorting,
2. METHODS
Lower Plate
The titanium film was used for the surface electrode. Titanium
was coated on the glass plate. The thickness of coating is 200
nm. The tip angle of the triangle shape of the one of thesurface electrodes is 0.35 rad (Fig. 1). The other referenceelectrode has a flat edge. The distance between electrodes is
0.1 mm. The shortest connecting line between electrodes isvertical to the flow direction. The micro flow channel withsurface electrodes was manufactured by the photolithography
technique.

1. INTRODUCTION
The movement of a biological cell suspended in the medium isgoverned by several factors: movement of the medium, gravity[1], electric force [2], Van der Waals force, and affinity ofsurface.
Various methods have been applied to control the movement ofcells in vitro: the flow [3], the shear field [4, 5], the filter, theslit [6–8], the magnetic field [9], the gravitational field [10, 11],laser [12], and the electric field [13]. These methods might
contribute to several applications of manipulation of cells [14,15]: detection of targeted cells [16–18], sorting of cells [19–22],
arrangement of cells to make a tissue, and measurement of thecharacter of cells [23].

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㸫5 N) of the contact profilometer (Dektak
XT-E, Bruker Corporation).

Movement of a charged particle depends on the electric field.
The effect is applied to the electrophoresis device [24]. Whena particle is subjected to a non-uniform electric field, a force
acts even on a non-charged particle, because the polarization
The phenomenon is called
generates in the particle.
dielectrophoresis [10]. The force of dielectrophoresis depends
on the several parameters: the electrical property of the particle,

Upper Plate
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 in the reactiveionetchingsystem(FA-1),
andcoatedwith

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Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

Aminopropyltriethoxysilane. 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-methylmethacrylate (Fig. 2). 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 placedon the stage of the inverted phase-contrast microscope.

Electrodevyyvx

Electric Stimulation
The electric stimulation of the alternating rectangular cyclic
wave (0.25 μs < period (T) < 0.3 μs; 㸫15 V < amplitude (Ea)
< +15 V) was generated with an electric stimulator. The
stimulator was connected to the titanium film electrode, and theelectric stimulation was applied to the medium flow. An
electric resistance (R) of 2 kΩ is serially inserted between theelectrode and the stimulator to measure the electric current (I <±7.5 mA).

Cell

O

x

Flow

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.

ElectrodeFig. 1: Two-dimensional movement of cell flowing near tip ofelectrode: position (x, y), and 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, andwas 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 were suspended in themedium. The suspension of cells was poured at the inlet of theflow channel. The flow was made by the pressure difference
between the inlet and the outlet, which was kept by thegravitational level of the medium (< 5 mm).
Each cell passing between the electrodes was observed by the
inverted phase-contrast microscope, and recorded by the video
camera, which is set at the eyepiece of the microscope. The
contour of each cell was traced, and the projected
two-dimensional area (S) at the image of each cell was
calculated using “Image J”. The equivalent radius (r) was
calculated by Eq. 1.
S =πr210 mm
Fig. 2: Upper and lower plates sandwiched between plates ofpoly-methyl- methacrylate.

(1)

Photomask A for Branched Flow Channel
The branched flow channel for dielectrophoretic sorting of cells
has been designed (Fig. 3). Before the deposition of titanium,
the surface of the glass plate was hydrophilized by the oxygen
plasma ashing by the reactive ion etching system. The
positive photoresist material of OFPR-800LB-20cP (Tokyo
Ohka Kogyo Co., Ltd, Tokyo, Japan) was coated on thetitanium with the spin coater. The photoresist was baked at the
hotplate.

The movement of the cell 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 aredefined as that in Fig. 1. The main flow direction of themedium is defined as x. The perpendicular direction from thereference electrode to the tip of the triangular electrode isdefined as y. The origin is adjusted at the tip of the triangularelectrode. The components (vx, and vy) of velocity were
calculated at the tracings of each cell. The components (αx,and αy) of acceleration of the velocity was calculated at thevelocity tracings of each cell.

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), rinsed with thedistilled water, and dried.

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Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

Electrode

Electrode
Flow

Cell
Cell
Flow

Classified cell

0.1 mmFig. 3: Dielectrophoretic classification of cells by branchedflow channel.

Fig. 4: Cells in flow channelElectrodearound triangle electrode.
Fig. 4: Cell is passing adjacent to tip of electrode.

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.

3. RESULTS
Fig. 4 exemplifies the microscopic image, which shows cells
flowing near the tip of the triangular electrode. The movie
shows the sifted movement of the flowing cell passing adjacentto the tip of the electrode by dielectrophoresis.
Fig. 5 shows the relationship among parameters displayed bythree-dimensional graph (RINEARN Graph 3D ver. 5.6):
maximum acceleration (αymax), radius (r), and initial position(y0). The period of rectangular cyclic wave is 0.3 μs in Fig. 5a,and 0.25 μs in Fig. 5b. The absolute value of the maximum
amplitude of the acceleration (αymax) of the velocity of each cell
during passing over the tip of the electrode is displayed in Fig. 5.
The absolute value of the maximum amplitude of theacceleration (αymax) is high, when the cell flows along thestreamline (y0 = 0) through the tip of the electrode. The
absolute value of the maximum amplitude of the acceleration(αymax) by the electric field, which is perpendicular to the flow
direction, increases with the radius of the cell (r).

Fig. 5a: Relationship among maximum acceleration (αymax),
radius (r) , and initial position (y0): period of 0.3 μs, amplitudeof ±15 V.

Fig. 6 shows the design of the flow channel. A cell flows fromleft to right. After passing between electrodes, the cells areclassified at the bifurcation into two branches. The number in
Fig. 6 shows dimension with the unit of millimeter. Fig. 7
shows the manufactured photomask for the flow channel withbifurcation. The branch pattern was successfully drawn on themask.

Fig. 5b: Relationship among maximum acceleration (αymax),
radius (r) , and initial position (y0): period of 0.25 μs, amplitudeof ±15 V.

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Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

The movement of cells between surface electrodes depends on
the topography of surface electrodes (the angle of the tip)
[38–40], which relates to non-uniformity of the electric field.
The higher slope of electric field with non-uniformity isnecessary to enlarge the movement of cells around the electrode.
In the previous study, variation of electrodes was used forsorting of cells [41].
In principle, dielectrophoretic effect increases with the diameterof the particle [26]. The present experiment shows that theacceleration of the cell along the electric field (perpendicular to
the flow direction) increases with the radius of the cell (Fig. 5).

Bifurcation
Flow
Fig. 6: Design of branched flow channel: unit mm.

The rectangular fluctuating voltage between electrodes [15, 35,42] higher than ±15 V enlarged the lateral shift of the flowing
cell. The amplitude of the electric stimulation has been
limited within the threshold value to prevent electrolysis [43] inthe present study.
Fig. 5 shows the two-dimensional movement of each cell in thex-y plane projected to the microscopic video image. The real
movement of each cell is three dimensional. The movement of
the perpendicular direction to x-y plane is very small, because
the height (0.035 mm) of the flow channel is small comparedwith the width (0.5 mm) of the flow channel in the present
experiment. The position of the streamline of the target cell
was tried to be controlled before passing through the electricfield area in the previous study [22].
In the previous studies, dielectrophoresis was tried to be applied
to the biological cell manipulation technology [14, 44]. It wasused to measure the mechanical properties of biological cells[45]. The micro grooves, on the other hand, were used fortrapping of flowing cells to distinguish mechanical properties offlowing cells in the previous study [11]. The cell sorting
technology would be applied to tissue engineering [46] (e.g.,
bio-actuator made from myoblast [47, 48]). The cell sorting
technology would also be applied to diagnostics in medicine [49,50]. The technology was also applied to yeast cells [51–53].
Another microflow system was applied for cell sorting [54], andcell salvage [55].

10 mm
Fig. 7: Photomask for branched flow channel in dish.

4. DISCUSSION
Dielectrophoresis was applied to the cell sorting system as
minimally invasive method in the previous studies [13, 29–33].
The shift distance [17, 27] was very small (< 0.1 mm) in theprevious experiment. If the lateral 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 lateral shift can be amplified
by accumulation with the multi-electrodes system on the flow
channel [24, 34]. Spiral Microchannel was used for sorting in
the previous study [35]. Ratchet Microchannels were also
applied to sorting device [36]. 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[37] should be controlled before the non-uniform electric fieldin the flow channel.

5. CONCLUSION
The flow channel for cell sorter in vitro has been designed bydielectrophoresis
withphotolithography
technique.
Experimental results of the pilot test show that the absolutevalue of the amplitude of the acceleration by the electric field,
which is perpendicular to the flow direction, increases with theradius of the cell. As application of the results of the pilot test,
the branched flow channel has been designed for cell sorting.

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

In the present study, the dielectrophoretic movement of the cellhas been tried to be observed by the optical microscope. The
couple of electrodes arrangement with the shorter distance eachother has not been designed, because the electrodes in thepresent study are not optically transparent [2]. The shiftdepends on the passing route position of the cell relative to theposition of the tip of the electrode. The dielectrophoretic
effect is highest adjacent to the tip of the electrode [38–40].

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