2018 IEEE-EMBS Conference on Biomedical Engineering and Sciences (IECBES)

Design Of DC-Dielectrophoresis Microfluidic

Channel For Particle and Biological Cell Separation
Using 3D Printed PVA Material
Poh Fong Lee
Boon Yew Teoh Yi Leng Thong
Dept. Mechanical Engineering
Dept. Biomedical and Mechatronic Dept. Biomedical and Mechatronic
Universiti Tunku Abdul Rahman
Engineering Engineering
Selangor, Malaysia
Universiti Tunku Abdul Rahman Universiti Tunku Abdul Rahman
leepf@utar.edu.my
Selangor, Malaysia Selangor, Malaysia
teohby@utar.edu.my
Yang Mooi Lim
Dept. Pre-clinical Sciences
Universiti Tunku Abdul Rahman
Selangor, Malaysia
limym@utar.edu.my

Abstract— In this study, the separation of cultured cancer
cells from polystyrene beads can be performed by
implementing direct current dielectrophoresis (DC-DEP) in
microfluidic LOC devices. The microchannels are designed for
a continuous separation of cells and polystyrene beads.
Simulation and empirical studies are carried out onto three
different microfluidic LOC designs to understand and
determine the separation efficiency of cancer cells (Raji cells)
from fluorescent polystyrene beads. For simulation, the flow
pattern inside the microchannels are studied and compared.
All the microfluidic separation designs allowed laminar flow
and there are no vortexes formed, which contribute to no
trapping of cells in the channel. The microfluidic LOC devices
are fabricated using 3D-printed PVA as scaffold to form the
microchannels. PVA was chosen to fabricate the scaffold due
to its ease of removal from the PDMS. The optimum separation
efficiency difference between outlet 1 and 3 found in the
empirical study is 20.85% in design C, with the application of
three pairs of electrodes that produce more dielectrophoretic
forces to the sample fluid consists of Raji cells and fluorescent
polystyrene beads. The separation of Raji cells and fluorescent
polystyrene beads using DC-DEP are successfully performed
by all three microfluidic LOC devices. Design C produce the
best results due to the higher dielectrophoretic forces in the
channel.
Keywords—Microchannel, DC-dielectric, particle separation.
devices, particles are manipulated to move towards or away
from the electrodes. This DEP forces are control by the
strength of the electric charges and distance between the
electrodes [6-7]. In this study, microfluidic with
microchannels are fabricated using 3D printed scaffold and
PDMS. This method allows wrapping of electrodes on the
3D printed scaffold which ease the fabrication process. Three
different design was studied to test the efficiency of
separation with different DEP strength.
II. DC-DEP THEORY
A. Separation of particles using DC-DEP
First The DEP force in a non-uniform electric field E,
that act on to the sphere-shaped particles with radius a, which
suspended in a medium with permittivity εm is given by
(1)
where ∇|E|^2 is the gradient of the square of the electric
field. The real part of the Clausius-Mossotti (CM) factor is
Re (fCM). The polarizability between particles and medium
is described by the CM factor and is given by

I. INTRODUCTION
In recent decades, different particle separation techniques
have been applied on micro- and Nano-particles in
bioscience, biomedical and other fields. Microfluidic is one
(2)
of the latest technique used to manipulate biological particles
[1]. Microfluidic utilise minimum amount of sample size due
to the size of the device, which provide a great advantage for
biological particle study. Combination technique of using where the complex permittivity is represented by
microfluidic channel and DC-DEP are often used in study of and . The complex permittivity is defined as
the chemical, biological and biomedical researches [2].
Particle control or separation assisted with DC-DEP
electrical fields have few advantages include: greater
controllability, higher efficiency in manipulating (3)
bioparticles, ease of operation, and minimum damage to the
targeted bioparticles [3]. Dielectrophoresis (DEP) was to
describe the transnational motion of particles due to the Based on the values of CM factor, fCM, the particles can
application of non-uniform electrical fields [4-5]. With the encounter either p-DEP or n-DEP effect.
particle flowing through the microchannel of the microfluidic

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 2018 IEEE-EMBS Conference on Biomedical Engineering and Sciences (IECBES)

Polystyrene particles usually express n-DEP, but when
the particles are submerged in a buffer solution which is not
very conductive, p-DEP can be expressed by small diameter
particles. Yet, living cells can only exhibit n-DEP when a
direct current field is applied, and the viability of cells also
affects the values of CM factor [8].
The electric conductivity of particles, σp, and medium,
σm, determined the CM factor, fCM and it is given by
dissolve the PVA scaffold. After 24 hours, PVA material
was dissolved and microchannel was form in the
microfluidic device. Electrodes were then insert into the
empty channel. Overall step to fabricate the microfluididc
device are shown in Fig 2.

(4)

Most studies implement DC-DEP with two origins of the

particle’s dipole, where two external electrodes are drenched
in the reservoirs. Then an electric field is applied to generate
the flow. With the used of external electrodes, a high voltage
is needed in DC-DEP so that sufficient DEP force is
generated. However, this DEP force will cause a severe
increase in temperature by Joule heating effect inside the
microchannels. This will result in a bubble formation that
will disturb the operation of the LOC.

III. METHODOLOGY
There are three different microfluidic designs developed
in this study. Each design has different configuration with
different number of electrodes as shown in Fig 1.
SolidWorks 2015 software was used to sketch and draw all
three microfluidic design. The designs are simulated with
ANSYS, Inc., Canonsburg, PA, USA software. Parameters
such as flow rate, flow pattern were simulated. With the
simulated results, all three microfluidic devices were
fabricated. Empirical results was collected using different
flow rate and electrophoretic charges. The efficiency of the
separation was compared in between the three designs. Raji
cells mixed with fluorescent polystyrene microspheres was
used to study the separation efficiency microfluidic device.
A. Design of microfluidic device
All three microfluidic designs (design A, B and C) are
shown in Fig 1A, 1B and 1C. For the design A, one pair of
extended microchannels located at the middle of the
microfluidic device for electrodes connection purpose.
Whereas for design B and C have two pairs and three pairs of
extended microchannels respectively.
Fig. 2. Overall steps to fabricate the microfluidic device. 3D printed
scaffold work as a scaffold and then dissolved/sacrifice to form the
microchannel.
C. Sample preparation
The mixture sample of Raji cells (~20µm) and used in
this project are prepared using tissue engineering technique.
Raji cells were revived from the -80°C refrigerator and kept
in the CO2 incubator for a week. RPMI medium was
changes every two days to allow the cells to rejuvenate.
Once the cells are healthy (as shown in Fig 3) enough to
perform experiments, a sample mixture is prepared by
adding 5 ml of Raji cells with 5 ml of diluted fluorescent
polystyrene microbeads (as shown in Fig 4). The fluorescent
polystyrene beads are diluted from a concentration of 3.6 x
106 beads/ml to a lower concentration of 3.6 x 104 beads/ml.

Fig 1. Design of the mcirofluidic device. A) Single pair of electrode. B) Two

pairs of elctrodes. C) Three pairs of elecctrodes. Fig. 3. Raji cells suspended in culture medium after revive from storage.

B. Fabrication of microfluidic device

The three designs of microfluidic device were printed
with 3D printer using polyvinyl alcohol (PVA). The 3D
printed plastic scaffolds are then suspended in liquid PDMS
and the polymer were left to cure overnight. After the PDMS
was cured, the device was soaked in water for 24 hours to

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 2018 IEEE-EMBS Conference on Biomedical Engineering and Sciences (IECBES)
Fig. 4. Mixture of Raji cells and polystyrene beads for study of separation
efficiency.
The separation efficiency of Raji cells are performed by
counting Raji cells collected at each outlets by using
haemocytometer. Mixture sample consists of 5ml of Raji
cells and 5ml of fluorescent polystyrene beads is allowed to
flow through the microfluidic device with the application of
different voltage. The collected samples at different voltage
applied are pipetted and mixed with Trypan blue dye on the
haemocytometer then viewed under the inverted microscope
for cell counting. The results were then compare with the
original mixture for separation efficiency.
Besides, the separation efficiency of the fluorescent
polystyrene beads are determined by filling the mixture
sample into a 96 well plate. The intensity of the beads in the
well plate are determined by using a spectrometer plate
reader. The separation efficiency of the beads are determined
as the number of beads passed through the device over the
reference number of beads.
D. Experimental setup
Copper electrodes are connected at the extended
microchannels in the middle of the microfluidic device to
induce dielectrophoretic force, where the electrode pairs
are supplied with positive and negative power source
respectively. Different DC voltage range from 0.5V,
1.0V, 1.5V, 2.0V and 2.5V are used on each microfluidic
design to obtained different separation efficiency of Raji
cells.
By setting a constant flow rate of 1 ml/min using a
syringe pump, the mixture sample containing Raji cells
and fluorescent polystyrene beads is injected into the
inlets of the microfluidic device through silicon tubing.
The mixture sample that passed through the three outlets
of the microfluidic device are collected in the beakers
that served as reservoirs. Overall view of the experiment
setup are shown in Fig 5.
Fig. 5. Overall setup of the experiment.
978-1-5386-2471-5 ©2018 IEEE
IV. RESULTS AND DISCUSSIONS
The simulation results of the three microfluidic designs
are performed and the results provide the optimum flow rate
for the laminar flow formation. Hence, experiment of the
microfluidic devices were perform using 1ml/min flow rate.
Besides, the empirical results performed using the fabricated
microfluidic devices are also discussed.
A. Simulation results
Simulations are performed on the three microfluidic
designs to assist the study of separation efficiency of Raji
cells. For the simulation of each design, a fixed laminar
inflow rate of 1.67x10-8 m3/s is allowed to flow through the
microchannels from the assigned inlets. Water is set as the
material used in the simulation to imitate the mixture sample
fluid flow. Through observation, the water travelled in a
laminar stream at a lower velocity at the inlets, then higher
velocity when travelling passed the middle part of the design.
This is due to there are two laminar flows from each inlet
later being centralized to one microchannel passing through
the extended microchannels which catered for electric
connection. Besides, particles in laminar flow are exposed to
shear gradient lift force where linear stream of water
particles can be seen from the inlets to the outlets. The lift
force are define as:
(5)
Where
= Lift force
= maximum fluid velocity
= fluid density
= diameter of particle
= dimension of channel
From the simulation results, it can be said that the
laminar flow in the microchannels of all three designs are
dominated by the viscous force with the Reynolds number
approximately 1 [9]. Particles in laminar flow experienced
gradient force due to the curvature of the velocity profile
where particles experience different velocity magnitudes at
either side that makes the particles rotates. However, the
shear gradient lift force does not depend on the particle
rotation but depend on Reynolds number. The laminar fluid
flow can be defined by Reynolds number. Laminar flow was
shown in Fig 6.
(6)
Where
= Reynolds number
= Length of channel
= viscosity of fluid
= fluid density
= average velocity of flow
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 2018 IEEE-EMBS Conference on Biomedical Engineering and Sciences (IECBES)

Fig. 7. Separation percentage of cells from each outlet for design A.

Fig. 6. Simulation results of the three designs, all forming laminar flow in
the microfluidic channel.

B. Separation efficiency

Since all three microfluidic devices consist three outlets, Fig. 8. Separation percentage of cells from each outlet for design B.
the comparison on the separation efficiency of Raji cells by
collecting results from each outlet. Cells collected are
compare to the original mixture for comparison. From the
result obtained from all outlet, at lower voltage applied
(0.5V, 1.0V and 1.5V) Fig 7,8,9, separation efficiency is low.
This is due to the voltage applied was too low, which leads
to weak dielectrophoretic forces to separate cells from the
beads even the number of electrodes increases from design A
to C. However, as the voltage applied increases from 1.50V
up to 2.50V, design C gives the highest separation
percentage of Raji cells (27.83% at 2.50V) while design B
and design C give significantly lower separation efficiency
of 22.19% and 9.07% respectively. Design C consists highest
number of electrodes that provide the highest
dielectrophoretic forces which able to separate Raji cells
Fig. 9.Separation percentage of cells from each outlet for design C.
from the polystyrene beads. Table 1 shows the percentage of
cells collected in each outlet from each design. Percentage of
cells may be lower due to the cells being trapped in the
microchannel. V. CONCLUSION
Design Outlet 1 Outlet 2 Outlet 3 The separation of Raji cells from the fluorescent
polystyrene beads are presented through simulations and
A 12.70% 4.82%
9.07% empirical studies using three DC-DEP microfluidic designs.
B 22.19% 14.94% 5.88% By looking at the results obtained from simulations, all the
microfluidic separation designs allowed laminar flow and
C 27.83% 17.02% 6.98% there are no vortexes formed.
To further verify the simulation, all three microfluidic
TABLE I. Percentage Of Cells Collected In Each Outlet From Each devices are fabricated and experiments are performed to
Design
study the separation of Raji cells. The scaffolds were
fabricated using PVA then are used to form microchannels
PDMS (Microfluidic device). All three microfluidic
devices give a successful separation of Raji cells from the
empirical studies. The highest separation efficiency of Raji
cells is showed from the empirical results of design C with
the highest number of electrodes (27.83%). With a higher

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 2018 IEEE-EMBS Conference on Biomedical Engineering and Sciences (IECBES)

number of electrodes, a higher dielectrophoretic forces will Manipulation. International Journal of Molecular Sciences, 15(10),
pp.18281-18309.
be produced, causing more Raji cells to be separated from
[4] Huang, Y., Wang, X., Becker, F. and Gascoyne, P., 1996. Membrane
the fluorescent polystyrene beads, thus results in higher changes associated with the temperature-sensitive P85gag-mos-
separation efficiency of Raji cells. dependent transformation of rat kidney cells as determined by
dielectrophoresis and electrorotation. Biochimica et Biophysica Acta
ACKNOWLEDGMENT (BBA) - Biomembranes, 1282(1), pp.76-84.
[5] Gascoyne, P. R. C., 2002, Cell Dielectric Properties as Diagnostic
This project was supported by UTARRF 2017. Vote Markers for Tumor Cell Isolation, in Tumor Markers — Physiology,
number IPSR/RMC/UTARRF/2017-C2/T07. Pathobiology, Technology, and Clinical Applications, E. P.
Diamandis, editor. Chapter 53.M. Young, The Technical Writer’s
Handbook. Mill Valley, CA: University Science, 1989.
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