Research Article

Insulator-based dielectrophoresis of
microorganisms: Theoretical and
experimental results
Dielectrophoresis (DEP) is the motion of particles due to polarization effects in nonu-
niform electric elds. DEP has great potential for handling cells and is a non-destructive
phenomenon. It has been utilized for different cell analysis, from viability assessments to
concentration enrichment and separation. Insulator-based DEP (iDEP) provides an
attractive alternative to conventional electrode-based systems; in iDEP, insulating
structures are used to generate nonuniform electric elds, resulting in simpler and more
robust devices. Despite the rapid development of iDEP microdevices for applications with
cells, the fundamentals behind the dielectrophoretic behavior of cells has not been fully
elucidated. Understanding the theory behind iDEP is necessary to continue the progress
in this eld. This work presents the manipulation and separation of bacterial and yeast
cells with iDEP. A computational model in COMSOL Multiphysics was employed to
predict the effect of direct current-iDEP on cells suspended in a microchannel containing
an array of insulating structures. The model allowed predicting particle behavior, path-
lines and the regions where dielectrophoretic immobilization should occur. Experimental
work was performed at the same operating conditions employed with the model and
results were compared, obtaining good agreement. This is the rst report on the
mathematical modeling of the dielectrophoretic response of yeast and bacterial cells in a
DC-iDEP microdevice.
Keywords:
Dielectrophoresis / Electrokinetic / Microorganisms / Microuidics / Modeling
DOI 10.1002/elps.201100168
1 Introduction
Signicant advances in microfabrication technology in the
past two decades have allowed the development of
miniaturized devices for biological applications. Due to
their small sizes and attractive advantages, lab-on-a-chip
systems are being employed in many diverse elds, for
instance, medical diagnoses, biological assays, food safety,
water monitoring, etc. In particular, there is an increasing
interest in the development of microdevices for the
separation and analysis of a wide array of bioparticles, from
macromolecules to cells. This research spurt is promoted by
advantages that come with working on the microscale:
shorter separation times, lower costs, higher performance in
terms of selectivity and sensitivity, and smaller required
quantities of samples and reagents.
Higher selectivity and sensitivity in shorter separation
times can be achieved with the implementation of dielec-
trophoresis (DEP), which has been proved to be an efcient
technique with a great potential for miniaturization. DEP is
the motion of particles due to polarization effects in the
presence of a nonuniform electric eld [1]. Traditionally,
DEP has been carried out employing arrays of microelec-
trodes and alternate current (AC) electric elds. Electrode-
based DEP allows obtaining high gradients of the squared
electric eld employing low applied electric potentials.
However, signicant electrolysis might be present, and
massive parallel systems aimed to produce high throughput
are prohibitive due to the high cost of electrode fabrication.
Cummings and Singh proposed a different approach for
the implementation of DEP [2]. In their work, they
employed an array of cylindrical posts embedded in a
microchannel made with an electrical insulating material.
Hector Moncada-Hernandez
1
Javier L. Baylon-Cardiel
1
Victor H. Pe rez-Gonza lez
1
Blanca H. Lapizco-Encinas
2
1
BioMEMS Research Group,
Tecnolo gico de Monterrey,
Campus Monterrey, Monterrey,
Mexico
2
Microscale Bioseparations
Laboratory, Centro de
investigacio n y de Estudios
avanzados del IPN Unidad
Monterrey, PIIT, Apodaca,
Mexico
Received March 15, 2011
Revised May 14, 2011
Accepted May 16, 2011
Colour Online: See the article online to view Figs. 15 in colour.
Abbreviations: AC, alternate current; CM, ClassiusMossotti;
DC, direct current; DEP, dielectrophoresis; EK, electrokinetics;
EP, electrophoresis; iDEP, insulator-based DEP
Correspondence: Dr. Blanca H. Lapizco-Encinas, Microscale
Bioseparations Laboratory, Centro de investigacio n y de Estu-
dios avanzados del IPN Unidad Monterrey, Via del Conocimiento
201, PIIT, Apodaca, NL, Mexico
E-mail: blapizco@cinvestav.mx
Fax: 152-811-156-1741
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Electrophoresis 2011, 32, 25022511 2502
This technique is known as insulator-based DEP (iDEP). In
iDEP, the electric potential is applied across an array of
insulating structures, employing only two external electro-
des, generating an electric eld that straddle the
array of structures. When the electric potential is
applied, the electric eld lines are distorted among the
insulating structures, creating the nonuniform electric eld
necessary for DEP to occur. Another advantage of this
technique is that the exerted DEP force on a particle
depends greatly on its dielectric properties, as well as on its
shape and size. Due to their complexity, biological particles
can be regarded as particles consisting of several layers of
different thickness, each one having different dielectric
properties [3]. Moreover, the shape of the particle can be
approximated employing different geometries, such as
spheres and ellipsoids corresponding to the shapes of
Saccharomyces cerevisiae and Escherichia coli cells, respec-
tively [4]. There have been studies by different research
groups reporting values of dielectric properties of the
mentioned microorganisms [5, 6].
Important mathematical modeling efforts have been
devoted to further develop insulator-based dielectrophoretic
microdevices. In 2003, Cummings and Singh demonstrated,
experimentally and theoretically, the ow regime of
Streaming DEP and Trapping DEP in insulator-based
microuidic devices employing direct current (DC) electric
elds. Streaming DEP occurs when electrokinetic (EK) ow
overcomes DEP, and DEP overcomes diffusion. Trapping
DEP is observed when DEP is superior than both EK ow
and diffusion, causing particle immobilization on the
insulating posts. Streaming DEP was demonstrated
employing uorescent particles inside diamond-, square-
and circular-shaped posts in different array alignments.
Trapping DEP was demonstrated employing circular posts
by evaluating theoretical isopotentials experienced by the
particles under a combined electrokinetic and dielec-
trophoretic potential [2]. Later, in 2008, Davalos et al.
demonstrated that iDEP microdevices produced in a poly-
mer-based platform may be employed to separate and
concentrate particles as with glass microdevices. They also
showed that by employing different surface coatings, the
performance of the polymer microdevice may be enhanced
by reducing the electric eld magnitude needed to achieve
particle trapping. In [7], simulation of trapping regions was
carried out by analysis of the ratio between the gradient of E
2
and the magnitude of the local electric eld. Kwon et al.
investigated the EK and DEP forces exerted on particles in a
microchannel employing numerical simulations and
proposing an improved geometry to increase particle
transport in EK streamlines. Their work describes the
theoretical background of EK and DEP discussing
the criteria for characterizing particle ow in terms of the
applied electric potential and its intensity when it is
heterogeneous [8]. Chen et al. studied the mechanism of
particle transport and separation for a sawtooth channel.
Since motion of a single particle in a DC-iDEP device was
not fully understood, their study was limited to a single
innitesimal particle. The dielectrophoretic immobilization
of the particles was analyzed in terms of a dimensionless
parameter, which accounted for the electrophoretic and
dielectrophoretic mobility ratio and the electric eld gener-
ated in the channel [9]. Baylon-Cardiel et al. studied the
manipulation of particles employing DC and AC signals
[10, 11]. A mathematical model was developed to predict the
regions of dielectrophoretic immobilization of polystyrene
microspheres employing DC-iDEP, in particular, the effect
of suspending medium properties and magnitude of the
applied DC potential was studied; obtaining great agree-
ment with experimental results [11]. The model was then
extended to AC signals with low frequencies (0.21 Hz),
where three different waveforms were employed: sinusoidal,
half sinusoidal and sawtooth. The results demonstrated that
highly controlled manipulation of bands or groups of
particles is possible employing low frequency AC-iDEP
systems.
Recently, several studies on the implementation
of iDEP for the manipulation and concentration of biologi-
cal particles have been made. Chou and Zenhausern
employed AC electric elds that successfully concentrated,
separated and lysed cells in a microdevice with a pair of
triangular hurdles that created a constriction in the
channel [12]. Suehiro et al. developed a lter, employing AC
elds and glass beads as insulating structures to create a
nonuniform electric eld, in order to remove S. cerevisiae
from water and separate viable from non-viable cells [13].
Cho et al. employed a membrane as an insulating
structure and E. coli cells were forced to ow through it; the
cells were then attracted to the regions of high eld intensity
due to the effect of DEP [14]. Lapizco-Encinas et al.
successfully separated and concentrated E. coli, B. subtilis,
B. cereus, B. megaterium and Tobacco Mosaic Virus in an
array of insulating posts employing DC electric elds
[1517]. In a more recent report with this system, the
simultaneous concentration and separation of E. coli and
S. cerevisiae cells was achieved in less than two minutes [18].
Viability assessments of live and dead microalgae cells and
selectivity analysis of iDEP devices have also been
performed [19, 20].
A novel DC-iDEP system was reported by Pysher and
Hayes for the simultaneous concentration and separation of
viable and non-viable B. subtilis, E. coli and S. epidermis cells.
The microchannel employed has an array of size-varying,
sawtooth-shaped insulating structures, located at each side
of the channel. The varying constriction generates an
increasing gradient, which favors the trapping of particles
along the channel, according to differences in the size of the
cells and their dielectric properties [21]. A microdevice with
an insulating hurdle that continuously separates white
blood cells from cancer cells was proposed by Lis group
[22, 23]; the microdevice performed the separation through
manipulation of the cells trajectories by means of streaming
DEP. Church et al. continuously separated S. cerevisiae form
E. coli cells manipulating trajectories inside a serpentine-
shaped microchannel, employing DC electric elds to
Electrophoresis 2011, 32, 25022511 Microuidics and Miniaturization 2503
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
promote electrokinetic ow and AC elds to develop an
AC/DC DEP force to focus each cell type in the turns of the
channel [24, 25].
In the present work, the manipulation and separation of
different biological particles is achieved, by employing a
microdevice for iDEP consisting of a microchannel with an
array of cylindrical insulating structures. A computational
model developed in COMSOL Multiphysics (COMSOL,
Burlington, MA, USA) was employed to predict the effect of
DEP on the different types of cells; the model takes into
account the dielectric properties of the cell membrane, wall
and cytoplasm. Experiments for the manipulation, concen-
tration and separation of E. coli and S. cerevisiae were
carried out employing DC electric potentials in the range
(6001200 V). Mathematical modeling of these experiments
was performed to predict the pathlines of the cells and the
magnitude of the regions of dielectrophoretic immobiliza-
tion of cells within the array of insulating structures. An
analysis of cell dielectrophoretic and electrokinetic mobi-
lities was performed in order to understand the mechan-
isms behind DC-iDEP immobilization of biological cells.
This is the rst report on the mathematical modeling of the
dielectrophoretic response of yeast and bacterial cells in a
DC-iDEP microdevice.
2 Theoretical background and
mathematical model
2.1 Theory of electrokinetics
The motion induced on a particle due to the electrical force
exerted on it can be expressed in terms of the time-
dependent EK velocity as:
~u
EK
m
EK
~
E 1
where m
EK
is the electrokinetic mobility and
~
E is the spatially
nonuniform electric eld. The mobility m
EK
takes into
account the contribution of electrophoretic (EP) motion of
the particle and the electroosmotic (EO) motion of the
suspending medium in terms of their respective mobilities
as:
m
EK
m
EP
1m
EO
2
2.2 Theory of dielectrophoresis
2.2.1 Spherical particles
The dielectrophoretic force on a spherical particle with a
radius R exerted by an electric eld with magnitude E is
dened as:
~
F
DEP
2pR
3
e
m
Ref
CM
HE
2
3
where e
m
is the permittivity of the suspending medium, and
f
CM
is the ClassiusMossotti (CM) factor, which account for
particle polarizability. The CM factor is dened in terms of
complex permittivities as
f
CM

e

p
e

m
e

p
12e

m
4
where subscripts p and m refer to the particle and
suspending medium, respectively. For an applied DC
potential, the CM factor expression can be simplied to [26]:
f
CM

s
p
s
m
s
p
12s
m
5
which is only dependent on the conductivities of the
particle and the suspending medium. It is important to
observe that Eq. (5) does not have an imaginary part,
therefore Re[f
CM
] 5f
CM
, for the DC case.
A biological particle, such as a cell, has a complicated
structure as it contains several different layers, each with
different dielectric properties. Thus, the evaluation of the
CM factor is not straightforward and an equivalent model,
which accounts for the dielectric and morphological prop-
erties of each particle layer, must be employed. A simple
model for a particle consists of a sphere (the cell cytoplasm,
with conductivity s
cyto
and radius R
cyto
), surrounded by two
concentric dielectric shells (the cell membrane, with
conductivity s
mem
and radius R
mem
and the cell wall, with
conductivity s
wall
and radius R
wall
). Cell dimensions and
dielectric properties are shown in detail in Table 1 and
Fig. 1. In the case of DC electric elds, the effective
conductivity of the particle that refers to the two-shell model,
where only cytoplasm and membrane are considered (the
innermost layers), can be expressed as in [27]:
s
p;2shell
s
mem
Rmem
Rcyto
_ _
3
12
scytosmem
scyto12smem
_ _
Rmem
Rcyto
_ _
3

scytosmem
scyto12smem
_ _
_

_
_

_ 6
Then, considering the cell wall properties and the
equivalent conductivity calculated with Eq. (6), the total
equivalent conductivity of the particle can be obtained by
evaluation of the expression [27]:
s
p;3shell
s
wall
R
wall
Rmem
_ _
3
12
s
p;2shell
s
wall
s
p;2shell
12s
wall
_ _
R
wall
Rmem
_ _
3

s
p;2shell
s
wall
s
p;2shell
12s
wall
_ _
_

_
_

_ 7
Moreover, the contribution of the DEP force to the
motion of the particle can be expressed in terms of the
dielectrophoretic velocity:
~u
DEP
m
DEP
HE
2
8
where m
DEP
is the dielectrophoretic mobility. For a spherical
particle of radius R, the mobility m
DEP,s
is dened as:
m
DEP;s

R
2
e
m
Ref
CM

6Z
9
where Z is the suspending medium viscosity and the
subscript s refers to spherical particles, where the f
CM
is
given by Eq. (4) and the spherical particle conductivity
considering the three-shell model is given by Eq. (7).
Electrophoresis 2011, 32, 25022511 2504 H. Moncada-Hernandez et al.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
2.2.2 Prolate ellipsoidal particles
For a prolate ellipsoidal particle with axes a
1
4a
2
5a
3
(see
Fig. 1B), where the major axis (a
1
) is aligned with the
electric eld, the dielectrophoretic force exerted can be
dened as [28, 29]:
~
F
DEP;pr
4pa
1
a
2
2
e
m
Ref
CM
HE
2
10
with a CM factor, for an applied DC electric potential,
expressed by
f
CM;i

s
p
s
m
3s
m
1s
p
s
m
L
i

11
where the subscript pr refers to prolate particles, L is the
depolarization factor, which leads to three dipole moments,
one along each axis of the prolate ellipsoidal particle; the
subscript i refers to the x-, y- and z-axis [28, 29]. For this
model it is considered that the prolate major axis (a
1
) is
aligned with the electric eld, therefore, there is no net
torque exerted on the particle and the dielectrophoretic force
can be estimated considering the dipole moment along the
main axis of the prolate [29], where the depolarization factor
employed is:
L
1

a
2
2
2a
2
1
e
3
ln
11e
1 e
_ _
2e
_ _
12
with the following expression for eccentricity:
e

1
a
2
a
1
_ _
2

13
Similar to the model employed for spherical cells, the
expressions for ellipsoidal prolate particles considering the
multiple layers model were developed for the conductivity of
prolate-shaped cells [27, 30]. According to the two-shell
model that comprises the cytoplasm and cell membrane
properties, and considering an applied DC electric potential
[26], the equivalent conductivity of these layers is dened by
the expression:
s
p;2shell
s
mem
s
cyto
1l
mem
s
cyto
1s
mem
=l
cyto;1
s
mem
1l
mem
s
cyto
1s
mem
=l
cyto;1
_ _
14
where the subscript 1 refers to the length along the main
axis of the prolate particle (a
1
), l
cyto,1
is the length of the
inner layer (the cytoplasm) along the main axis (a
1
), as
shown in Fig. 1B, and l
mem
is the thickness of the cell
membrane. As in the case of the sphere model, the
expression for the three-shell prolate model is now obtained
by considering the cell wall properties and the resultant
equivalent conductivity of the two-shell model [27]:
s
p;3shell
s
wall

s
p;2shell
1l
wall
s
p;2shell
1s
wall
=l
cyto;1
1l
mem

s
wall
1l
wall
s
p;2shell
1s
wall
=l
cyto;1
1l
mem

_ _
15
where l
wall
are the thickness of the cell wall layer (see
Table 1). The drag force for a prolate ellipsoidal particle with
its major axis aligned with the electric eld can be estimated
as [29, 31]:
~
F
Drag;pr
16Z~upa
2
1

1 e
2
p
e
11e
2
ln
11e
1e
_ _
2e
16
where ~u is the particle velocity. Finally, considering Eqs. (8),
(10) and (16), and assuming a DC electric potential, it is
possible to obtain the expression for the dielectrophoretic
mobility for a prolate particle with its major axis (a
1
) aligned
with the electric eld [2729]:
m
DEP;pr

a
2
2
e
m
12Ze
11e
2
ln
11e
1 e
_ _
2e
_ _

s
p;3shell
s
m
3s
m
1s
p;3shell
s
m
L
1

_ _
17
where the prolate particle conductivity considering the
three-shell model is given by Eq. (15). This equation is
equivalent to that derived by Kirby when the condition
a
1
52a
2
is satised [29].
2.3 Mathematical model
The geometry of the iDEP device, schematically presented in
Fig. 1D, was modeled with COMSOL Multiphysics software.
For this model, biological particles with different sizes and
shapes were considered. The microchannel considered has a
depth of 20 mm, which was not considered in this model since
the electric potential applied does not change signicantly
within the direction of the depth, this has been demonstrated
in the previous reports from our group [11].
Table 1. Values of dielectric properties of cells employed for
simulations
Dielectric parameters employed for the model
S. cerevisiae
cells [35]
E. coli Top10F
cells [36]
Cytoplasm conductivity
(s
cyto
)
0.5 S/m 7 10
3
S/m
Membrane conductivity
(s
mem
)
2.5 10
7
S/m 1 10
6
S/m
Cell wall conductivity
(s
wall
)
1.4 10
2
S/m 5 10
1
S/m
Cell size 5 mm diameter
a)
1.5 mm length,
0.75 mm width
Cell membrane
thickness (l
mem
)
4.86 nm
a)
8 nm
Cell wall thickness
(l
wall
)
133.6 nm
a)
50 nm
CM factor 0.2020 0.4021
Electrokinetic mobility
(m
EK
)
2.931 10
8
m
2
/Vs
b)
3.335 10
8
m
2
/Vs
b)
Dielectrophoretic
mobility (m
DEP
)
2.983 10
19
m
3
/V
2
s 3.433 10
20
m
3
/V
2
s
a) Values were adjusted from [35] according to the size of cells
employed in this work.
b) Values taken from [33].
Electrophoresis 2011, 32, 25022511 Microuidics and Miniaturization 2505
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A triangular mesh was built in order to solve the Laplace
equation using the Lagrange element implemented in the
software. Finite element analysis was applied in a triangular
mesh consisting of 538 500 elements and 1 093 200 nodal
points. The electric potential f was approximated by a
polynomial of low order at each mesh point. From the
numerical solution it is possible to obtain values for the
parameters describing the time-dependent motion of
the particles, such as the distribution of the electric eld
~
E
and the gradient of E
2
inside the array of insulating struc-
tures. Applying concepts of electrokinetics, dielectrophoretic
and electrokinetic velocities, pathlines can be estimated
within the microchannel.
In order to achieve immobilization of a particle due to
DEP, its contribution to the particle motion must overcome
other transport mechanisms, such as diffusion and EK
motion. In this study, it is assumed that the main contri-
butions to the ux of particles
~
j along the microchannel
come from EK and DEP, as [8, 32]:
~
j % ~u
EK
1~u
DEP
18
It can be assumed that particle immobilization is
achieved in the regions were the condition
~
j
~
E 0 is
satised. In terms of the EK and DEP mobilities, this
condition can be expressed as:
m
EK
~
E cm
DEP
HE
2

~
E 0 19
where c is a dimensionless correction factor which accounts
for effects of unconsidered phenomena and measurement
errors. This expression can be rearranged as a ratio in order
to nd a condition for particle dielectrophoretic trapping,
Eq. (20) shows this relation [11]:
cm
DEP
HE
2
m
EK
E
2

~
E41 20
By applying these concepts, the mathematical model
developed for this study is able to predict the regions where
dielectrophoretic immobilization of the cells will occur
for a given applied potential. Electrokinetic mobilities
of cells were obtained experimentally in a previous
report by our group [33] and dielectric properties of
the cells were obtained in the literature as reported in
Table 1.
3 Materials and methods
3.1 Biological cells
E. coli Top10F (PTA-5668, ATCC, VA, USA) were cultured
in LuriaBertani Broth (LB Broth, L3022, Sigma-Aldrich,
MO, USA) at 371C in a shaker incubator for 15 h to achieve
late log phase cells for a concentration of 6 10
8
cells/mL
veried by OD measurements at 600 nm. Cells were
centrifuged at 7000 rpm for 10 min and the pellet was re-
suspended in DI water. Cells were labeled with Syto
s
9
bacterial stain (Invitrogen, Carlsbad, CA, USA) that
uoresces green (ex/em 483/503 nm). A volume of 3 mL of
uorescent dye (5 mM solution in DMSO) was added for
every milliliter of cell culture and incubated at room
temperature for 30 min. Labeled cells were centrifuged
at 7000 rpm for 10 min, washed thrice with DI water to
remove any excess dye and nally re-suspended in the
buffer solution to reach the desired cell concentration
(6 10
8
cells/mL).
S. cerevisiae (24858, ATCC, VA, USA) were cultured in
YM Broth (271120, BD Difco, MD, USA) at 301C in a shaker
incubator for 18 h to achieve late log phase to a cell
concentration of 6 10
6
cells/mL, veried by OD measure-
ments at 600 nm. Yeast cells were labeled with Syto
s
9
following the same protocol used with E. coli cells. After
labeling, yeast cells were re-suspended to reach the desired
cell concentration (6 10
6
cells/mL).
Figure 1. Schematic representation
for cell shapes and experimental
setup. (A) sphere multishell model,
(B) ellipsoid prolate multishell
model, (C) experimental setup show-
ing a top view of the glass micro-
device and (D) top view of
microchannel employed.
Electrophoresis 2011, 32, 25022511 2506 H. Moncada-Hernandez et al.
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3.2 Buffer solution
A 0.5 mM KH
2
PO
4
(P2462, DEQ, NL, Mexico) solution in
DI water (00068, CTR, NL, Mexico) was prepared as
suspending media, resulting in solution with a pH of 7
and a conductivity of 5 10
4
S/m. The conductivity and pH
were measured with a multiparameter bench meter HI 255
(Hanna Instruments, Woonsocket, RI, USA) and a Orion 3
Star Benchtop pH meter (Thermo Scientic, Beverly, MA,
USA), respectively.
3.3 Experimental setup
A schematic representation of the experimental setup is
shown in Fig. 1. Experiments were conducted in a
microuidic chip consisting of two square Schott D263
glass wafers (50.8 50.8 mm sides, 1.1 mm thick, Howard
Glass, Worcester MA, USA) using standard photolithogra-
phy, wet etch and bonding techniques. Soft photolithogra-
phy was employed along with wet etch to create the
microchannels and the arrays of insulating structures on
the bottom glass wafer. Holes were drilled on the top wafer
and then bonded with the bottom wafer in order to get inlet
and outlet reservoirs for the microchannels.
The microdevice contains eight microchannels that are
20 mm deep, 10.12 mm long and 2 mm wide with an array of
cylindrical posts (Fig. 1C). The posts have a diameter
of 440 mm that are arranged 520 mm center to center
and transverse the entire depth of the microchannel
(Fig. 1D). There are 32 posts arranged in eight rows of four
posts each. The rst and last rows of posts have a dove-
tailed geometry in order to avoid particles from collapsing
against the posts and plugging the system, as shown in
Fig. 1D.
A high-voltage sequencer model HVS448 (LabSmith,
Livermore, CA, USA) was used to apply DC electric poten-
tials by employing platinum-wire electrodes. Visualization
of dielectrophoretic behavior of microorganisms was
performed employing an inverted epiuorescence video
microscope for microuidics model SVM340 (LabSmith). A
4 microscope objective was used for all experiments. The
microscope is equipped with a light module with a
conguration of blue light LEDs that excites at a wavelength
of 480 nm. The lter used in the microscope allows detect-
ing of light above a wavelength of 515 nm, which is appro-
priated for detecting the Syto
s
9 uorescent stain employed
in this work. The use of a personal computer for the
operation of the high-voltage sequencer and the micro-
uidics microscope is required. Prior to experimentation,
the microchannel was lled with buffer solution and a
sample of 80 mL of cells. Afterwards, electrodes were place in
the inlet and outlet reservoirs in order to generate a DC
electric eld across the microchannel. Dielectrophoretic
behavior of the cells was recorded in the form of videos and
pictures using the microuidics microscope and the
computer.
4 Results and discussion
As discussed in the theoretical background and mathema-
tical model section, three main mechanisms affect the
motion of particles in DC-iDEP systems: electrophoresis,
electroosmotic ow and dielectrophoresis. Previous work by
our group, employing microdevices made from the same
substrate (Schott D263), have reported the measurements of
electrokinetic mobilities of particles with size similar to the
cells employed in this project [33, 34]. The dielectric
properties of cells were introduced to the model to
determine the particle conductivity values employing the 3
shell model. Dielectrophoretic mobilities for the cells were
calculated according to Eq. (17).
Table 1 shows values of EK and DEP mobilities for both
types of cells obtained considering a suspending medium
with s
m
50.005 S/m and pH57. It should be noted that
dimensional units of m
EK
are different from those of m
DEP
.
The reason for this is that ~u
EK
depends on the electric eld
and ~u
DEP
on the gradient of E
2
, therefore, a comparison
between both magnitudes is not straightforward.
In Fig. 2, the predictions of the trapping regions for
yeast cells, suspended in a 0.5 mM K
2
HPO
4
solution,
obtained with the mathematical model show how the
magnitude of these regions and the velocity pathlines of the
cells change with increments of 200 V for the applied DC
potential, ow direction is from left to right. In this gure
the magnitude of the trapping condition, Eq. (19), is shown
by the color scaled y-axis. In theory, when this value is
greater than 1, the trapping condition is satised and cells
are immobilized. Figure 2A shows no trapping region with
f5200 V, because the trapping condition has not been met
and the velocity pathlines shown in red indicate that the
cells are owing. It can be observed how the pathlines focus
when passing through the rst row of posts due to the
interaction between dielectrophoretic and electrokinetic
forces. In Fig. 2B, small and weak trapping regions are
observed between the posts, modifying the original particles
pathow. Nonetheless values of trapping conditions are
nearly above one, therefore, particle trapping was not
signicant according to the experimental observations. In
Fig. 2c at f5600 V the trapping condition is satised at the
regions closer to the constriction between the posts (area
shown in blue). Just prior to the immobilization regions, it
is possible to observe how the pathlines stop or collide.
When a velocity pathline ends, it means that the trapping
condition is satised, i.e., its magnitude is greater than one
and the cells will be dielectrophoretically immobilized.
Figure 2d shows the regions where the trapping condition is
satised when the applied potential is increased to 800 V,
demonstrating that the trapping regions grow with the
applied potential, reaching a maximum at the post borders
at the constriction (tiny region shown in light green). Figure
2E andF shows the magnitude of the trapping region and
cell velocity pathlines with applied potentials of 1000 and
1200 V, respectively. It can be observed how the pathlines
stop just prior to the trapping regions. Additionally, the
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& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
immobilization regions are moving farther away from the
constriction (to the left); this is due to increasing negative
DEP force, where the maximum is located at the narrowest
area between the posts. Yeast cells under DC elds exhibit
negative dielectrophoretic behavior, and are therefore
repelled from the eld maxima, shifting the trapping region
to the left as the applied potential increases.
In order to further understand how the magnitude of
the applied DC-potential affects the dielectrophoretic trap-
ping of the cells, the gradient of E
2
at the constriction was
studied. In Fig. 2G the x-component of the E
2
gradient at
f51200 V is plotted in a magnication of a constriction
from the rst row. From this gure, it is possible to observe
how the magnitude of this component of the gradient
exhibits positive and negative values. The positive region is
located to the left of the constriction and the negative one is
positioned to the right of the constriction. It is important to
note that the gradient maxima for both regions are reached
next to the post edges as pointed out in Fig. 2G. Cells exhibit
the maxima repulsion from these small regions. Dielectro-
phoretic immobilization will only occur at the positive
gradient region (shown in red, left to the constriction), this is
because in this region, DEP mobility and EK mobility are
opposite as shown by the arrows in the gure. Cells that
are able to pass the constriction will not be immobilized at the
negative gradient region (shown in blue), since both DEP and
EK mobilities go from left to right, and cells will keep moving.
4.1 S. cerevisiae cells
The behavior of yeast cells was studied through modeling
and experimentation. Modeling results are presented in
terms of the magnitude of the trapping condition (Eq. 20)
with a correction factor of 100. Figure 3A and B shows the
experimental results and the estimated trapping zones for
yeast cells under a DC potential of f600 V. Yeast cells
exhibit negative DEP and are being repelled from the zones
of higher eld gradient, thus, being concentrated in bands
just before the constriction created by the insulating posts.
In Fig. 3C and D, it can be observed that by increasing the
applied potential to 800 V the magnitude of the trapping
condition increases, modifying the shape and size of regions
where S. cerevisiae cells are concentrated. When the potential
is increased to 1000 V, it can be observed that experimentally
well-dened bands are formed in the second row of posts
(Fig. 3E). Also in Fig. 3F at f51000 V, it is shown that the
size of the trapping regions increased and the magnitude of
the trapping condition reaches values up to 4 at the
constriction. Trapping zones depicted in Fig. 3B, D and F
reveal that a trapping condition between 1.5 and 4 is needed
to immobilize yeast cells with the present geometry and
applied potential. This value is referred to the zone at which
cells are completely trapped, the closest to the constriction
between the posts (Fig. 3A, C and E).
It is important to note that in the experimental results,
cellcell interactions are observed, particularly in the bands
of immobilized cells at the rst row, where long pearl-chain
formations are present. This is a phenomenon that the
mathematical model does not account for, and it is the
reason why the experimental bands of trapped particles at
the rst row look thicker than the bands predicted with the
model. Additionally, an opposite behavior is observed at the
second row, since the experimental bands of trapped parti-
cles look thinner than the bands predicted with the model.
The reason for these results is that the cells concentrate
Figure 2. Model simulation for velo-
city pathlines and trapping condition
values obtained at the constriction
area between the posts (plotted in
the y-axis) for yeast cells under a DC
potential: (A) f5200 V; (B) f5400 V,
(C) f5600 V, (D) f5800 V, (E)
f51000 V and (F) f51200 V. (G)
Magnication of the x-component
of the E
2
gradient with f51200 V,
showing the positive and negative
magnitudes of this component. The
gradient maxima are reached next to
the post edges as pointed in the
image. Includes a representation of
direction of the electrokinetic and
dielectrophoretic velocities.
Electrophoresis 2011, 32, 25022511 2508 H. Moncada-Hernandez et al.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
signicantly at the rst row, precluding the cells from
reaching the second row, i.e. a lower cell concentration is
present at the second row and since fewer cells are available,
the bands are thinner.
4.2 E. coli cells
The results obtained for E. coli cells are shown in Fig. 4,
where modeling results are presented in terms of the
trapping condition magnitude (Eq. 20) employing a
correction factor of 500. In Fig. 4A and B the dielectro-
phoretic response of the cells with f5800 V is observed,
these cells were modeled as prolate-shaped particles. As with
yeast cells, bacteria cells exhibited negative DEP and were
repelled from the zones of higher eld gradient; none-
theless, this potential is not enough to fully immobilize
bacterial cells. The shape of the trapping region plotted in
Fig. 4B is in agreement with the experimental results in
Fig. 4A. Figure 4C shows that by increasing the potential to
1000 V, the regions where E. coli cells are concentrated are
pushed back, shifted to the left and farther away from the
constriction, achieving a greater concentration of cells.
When the potential is increased to 1200 V, experimental
results in Fig. 4E show an even higher cell concentration,
and the band of trapped cells increased and shifted to the
left. Figure 4F shows that at f51200 V, the trapping region
changes its shape, increasing in the middle part, which
agrees with the experimental results in Fig. 4E. Trapping
zones showed in Fig. 4B, D and F show that the predicted
trapping regions agree with the region where bacteria cells
are immobilized. It is important to mention that the
trapping condition range for E. coli is lower (with a
maximum of 2.5) than that obtained for yeast cells (with a
maximum of 4). Therefore, a weaker repulsive force was
observed, requiring greater electric potentials to achieve
particle immobilization.
4.3 Separation of a mixture of E. coli and S. cerevisiae
cells
By analyzing Figs. 3 and 4, it is observed that S. cerevisiae
and E. coli cells exhibit different trapping behavior for a
given potential under the present geometry. Since their
trapping regions are different, there is potential for
achieving their separation from a mixture. Experimental
and modeling results for the separation of a mixture of
E. coli and S. cerevisiae cells are included in Fig. 5. For
f5800 V the experimental results in Fig. 5A show that
bacterial cells are trapped closer to the constriction than
yeast cells, which is expected due to a larger cell size for
Figure 3. Experimental and theoreti-
cal results for the trapping of
S. cerevisiae suspended in a 0.5 mM
of KH
2
PO
4
solution considering the
spherical model and under a DC
potential. (A) and (B) yeast cells with
f5600 V, experimental and simula-
tion results, respectively. (C) and (D)
yeast cells with f5800 V, experi-
mental and simulation results,
respectively. (E) and (F) yeast cells
with f51000 V, experimental and
simulation results, respectively.
Figure 4. Experimental and theoreti-
cal trapping of E. coli suspended in a
0.5 mM of KH
2
PO
4
solution consider-
ing the prolate model and under a
DC potential. (A) and (B) bacteria
cells with f5800 V, experimental
and simulation results, respectively.
(C) and (D) bacteria cells with
f51000 V, experimental and simu-
lation results, respectively. (E) and
(F) bacteria cells with f51200 V,
experimental and simulation results,
respectively.
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& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
yeast. It is important to note that at the rst row of
posts, where a much greater concentration of cells is
present, it is not possible to distinguish two bands of
trapped cells; the bands of trapped E. coli and S. cerevisiae are
overlapping, but each cell type has its own trapping region
as shown in Fig. 5B. By observing the trapped cells at the
second row in Fig. 5A, two different bands of trapped cells
are present; to help visualization E. coli cells are circled and,
since they are smaller than yeast cells, have a weaker
uorescence and look like a cloud. Yeast cells are trapped
farther away from the constriction, have a stronger
uorescence and look like more dened particles (i.e. not
like a cloud). Figure 5B shows the predicted trapping
regions for S. cerevisiae cells in the upper half and the
trapping region for E. coli cells in the bottom half at
f5800 V (same as Fig. 5A). As expected, since yeast cells
are larger, the magnitude of the trapping region is greater
and has a different shape since it extends wider in the
y-direction (i.e. perpendicular to the eld). As mentioned
previously, the differences in shape between E. coli and yeast
cells (prolate versus spherical) affect the polarization leading
to differences in the form of the trapping regions.
Differences between the shape of the trapping regions in
the rst and second rows of posts are due to geometry of the
posts; having a dove tail at the rst row makes the eld
gradient smoother and the regions do not extend as much in
the y-direction. Better separation is obtained at f51000 V
as shown in Fig. 5C, where at the second row of posts it is
possible to observe two separated bands of cells, this
separation is achieved since cell concentration is lower at
the second row. As mentioned, the signicant cell
concentration at the rst row precludes many cells from
moving downstream in the microchannel, leading to lower
cell concentration at the interior of the post array. Figure 5D
shows the trapping regions obtained at f51000 V for yeast
(top image) and bacterial (bottom image) cells. As expected,
differences in size and shape are reected in their trapping
regions, where the regions for yeast cells extend wider in the
y-direction, which creates the illusion of a valley at the center
of the trapping region at the second row for yeast cells. It
must be noted, however, that particle size and dielectric
properties are not the only determinant parameters for
achieving a clear separation. Particle morphology (shape)
and uid mechanics effects also affect the system. There-
fore, regardless of the fact that yeast cells are much larger
than E. coli cells; the distance between separation bands will
not be proportional to the difference in size of the particles.
It is important to observe the agreement between the
predicted trapping regions and the shape of the concen-
trated particles bands obtained experimentally. This effect
can be attributed to the fact that, between the posts, ow
velocity increases while the regions of greater gradient
magnitudes are located at the edges of the posts, as shown
in Fig. 2G.
These results demonstrate the great potential that
mathematical modeling has to offer for the improvement of
DC-iDEP systems for cell manipulation. By carefully simu-
lating the dielectrophoretic response and behavior of the
cells, microdevices capable of performing complex cell
separations and analysis can be designed.
5 Concluding remarks
In the present work, the dielectrophoretic behavior of E. coli
and S. cerevisiae cells in a DC insulator-based dielectrophore-
tic (iDEP) device was studied through mathematical model-
ing and experimentation. The system considered was a
microchannel with an array of cylindrical insulating struc-
tures. A mathematical model built with COMSOL Multi-
physics was employed to predict and estimate the squared
electric eld gradient, dielectrophoretic mobilities, velocity
Figure 5. Experimental and predicted
concentration and separation of a
mixture of S. cerevisiae and E. coli
cells suspended in a 0.5 mM of
KH
2
PO
4
solution and under a DC
potential. (A) Experimental separa-
tion of a mixture of cells with
f5800 V; (B) simulation results of
trapping regions of yeast cells (sphe-
rical model) in the upper half image
and bacteria cells (prolate model) in
the bottom half image. (C) Experi-
mental separation of a mixture of
cells with f51000 V and (D) simula-
tion results of trapping regions of
yeast cells (upper half) and bacteria
cells (bottom half).
Electrophoresis 2011, 32, 25022511 2510 H. Moncada-Hernandez et al.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
pathlines and the magnitude of the regions of dielectro-
phoretic trapping for the cells. Experimental work, at the
same operating conditions used for the mathematical
simulations, was performed in order to validate the modeling
results. It was found that it is possible to predict the
dielectrophoretic behavior of cells employing a model that
considers the shape of the cell and the dielectric properties of
the cell wall, membrane and cytoplasm. The magnitude of
electrokinetic and dielectrophoretic mobilities was analyzed
and compared, and a condition for dielectrophoretic immo-
bilization was established. Trapping of the cells occurred in
the regions where the condition was satised. The regions for
dielectrophoretic immobilization of the cells predicted with
the model were in agreement with the experimental results.
This is the rst report on the mathematical modeling of the
dielectrophoretic response of yeast and bacterial cells in a DC-
iDEP microdevice. These results contribute towards the
advancement of the dielectrophoretic behavior and character-
ization of cells under DC electric elds, and will provide with
guidelines that can be used in the design of DC-iDEP systems
for cell manipulation.
The authors acknowledge the nancial support provided by
the grant CONACYT-CB-2006-53603, Catedra de Investigacion
CAT142 of Tecnologico de Monterrey and CINVESTAV-
Monterrey.
The authors have declared no conict of interest.
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