1.
Comparative Analysis of Microfluidic Biomolecular Separation
Techniques With An Emphasis on Dielectrophoresis
Word Count: 1264
Prepared for
Leo Banuelos
Biomedical Engineering Master’s Student
California Polytechnic State University
San Luis Obispo, California
Prepared by
Keyon Keshtgar
Biomedical Engineering
March 4, 2016

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Abstract
Biomolecular separations are required in many contexts in biochemical and biomedical
applications for the identification, isolation, and analysis for a sample of interest. Microfluidics is
uniquely suited for handling biological samples, and emerging technologies have become an
increasingly accessible tool for researchers and clinicians. This report aims to explore the
innovative techniques for microfluidic cell separation and manipulation, specifically using
dielectrophoresis (DEP). A working principle first explains what DEP is and also highlights
some of the techniques to insulate and eliminate fouling. Two DEP techniques will then be
discussed; screen­printed microfluidic DEP chip and contactless DEP chip. This report will
explain each technique, highlight key features, and describe the manufacturing process. This
paper is intended to help a master student at California Polytechnic State University, San Luis
Obispo to complete their literature survey.

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Introduction
The manipulation of molecules is essential in biological research, clinical diagnostics,
and cellular therapies. Separating heterogeneous molecules into subpopulations allows for the
identification of samples of interest and subsequent physical and biochemical analysis for the
purpose of scientific research. Molecule separation has long been a central theme in
microfluidics research. ​In general, microfluidic devices are uniquely suited for manipulating
biomolecules: low Reynolds number, laminar flow, fast fluid manipulation. In addition,
incorporating channels on the order of 10–100 µm enable the use of small sample volumes and
fabrication from poly­dimethyl­siloxane (PDMS) makes the devices simple to make, cheap, and
easy for imaging. ​The motivation to reduce the size, cost and complexity of this technology
would allow for a more effective usability for scientists and clinicians. ​This article focuses
specifically on a few microfluidic biomolecular separation techniques involving
dielectrophoresis after a brief description of dielectrophoresis.
Dielectrophoresis
Dielectrophoresis (DEP) cell separation is a
sorting technique that relies on the principle that when
a polarizable molecule, such as large biomolecules
and cells, are placed in a non­uniform electric field,
the field can exhibit a net force on that particle due to
an induced or permanent dipole. A particle is not
required to be charged because all particles exhibit a
dielectrophoretic activity in the presence of an electrical field.

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The strength of the force, however, strongly depends on the medium and particle's electrical
property, shape/size, and the frequency of the electrical field. ​Multiple electrical fields can be
utilized in order to separate a mixture of particles. ​Figure 1​ accurately demonstrates this method.
DEP can be positive, like that shown in (a), where the particle moves in the direction of
increasing electrical field. Similarly, DEP can be negative, like that shown in (b), where the
particle moves away from the high field regions. Alternatively, multiple fields may be created to
separate different shaped or sized molecules in subsequent branching arrangements, like that
shown in (c).
There is an alternative manner of carrying out DEP which is known as insulator­based
DEP (iDEP). In this technique, a voltage is applied using two electrodes that straddle an
insulating structures array. They do not lose their functionality despite fouling effects, which
make them more suitable for biological applications. iDEP systems can also be made from a
wide variety of materials, including plastics. As a result, it becomes possible for inexpensive
systems and increased potential for high throughput applications.
Another potential issue is fouling. While working with bio­particles, fouling of the metal
electrodes may distort the operation of AC­DEP devices. One of the possible solutions, is to
employ carbon electrodes because carbon has a much wider electrochemical stability window
than metals commonly used in thin film electrode fabrications, such as gold and platinum. This
allows for higher voltages to be applied in a given medium without electrolyzing it. It is
important to understand these challenges when choosing the appropriate method for separation.

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Discussion
Screen­Printed Microfluidic Dielectrophoresis Chip
After conducting research on possible techniques for biomolecular separation, a few
spark particular interest. The first is an inexpensive screen­printed AC­DEP chip used for the
separation of yeast cells conducted by a group of researchers at South China University of
Technology. The main components for this technique, such as the electrodes and channels, were
constructed using a layer­by­layer screen printing process, which is especially suitable for
high­throughput mass production. In order to reduce fouling of electrodes, carbon paste was used
to print a semi­3d structure which serves for
the same purpose. By doing so, the chip cost
is dramatically decreased and particle
trapping efficiency is increased. ​Figure 2​,
shows the screen printing procedure of the
microfluidic AC­DEP chip. A clean glass is
served as the substrate and the bottom layer.
Next, a patterned carbon ink is used as the
electrode and conductive wires. A UV
curable, dielectric composition for
microfluidic channels is rested upon the
carbon ink. Finally, a PDMS layer with inlet
and outlet ports is placed on top of the
assembly. The electrode pattern may be oriented in three different ways; an offset castellated

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orientation, non­offset castellated orientation, and an interdigitated line orientation. These can be
seen in the figure above as (5), (6), and (7). When a suspension solution flows into the
microchannel, yeast cells feel the positive DEP forces and are thus trapped onto the carbon
electrodes. Meanwhile polystyrene microspheres experience weak negative DEP forces and are
repelled from the electrode. As a result, the separation between yeast cells and the PS
microspheres occurs.
In comparison to conventional microfabrication processes, this method is
especially suitable for high­throughput mass production due to it’s low cost, simple operating
procedure and facile fabrication conditions. After successfully separating yeast and PS cells, the
study conducted by Hongwu Zhu and his research team concluded that this technique showed a
high capture rate and separation efficiency. It is believed that this approach can offer a great
promise toward the development of miniaturized, portable flow­through DEP chips for
diagnostic and clinical applications.
Contactless Dielectrophoresis Chip
A second, highly favored technique for biomolecular separation involves an alternative
approach, which is known as contactless dielectrophoresis (cDEP). Traditionally, DEP devices
include metallic electrodes patterned in a sample channel, which can be expensive to
manufacture and requires the need for a highly sterilized environment. cDEP utilizes DEP while
avoiding direct contact between electrodes, which eliminates the possibility of contamination. A
unique characteristic is that the electrical field is generated using fluidic electrode channels that
contain a highly conductive fluid. Another main advantage of this technique over existing

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methods is that cells can be sorted and characterised based on their intrinsic biophysical
properties without the need for labeling based on biomarkers. This method is useful for
applications such as sorting live cells from dead, isolating tumor initiating cells, separating
cancer cells based on stage, and looking at the effects of drugs on different cells.
The fabrication of the cDEP chip begins by using ion
etching to etch the channel design into a silicone wafer.
PDMS is then mixed and poured onto the wafer, which is
collectively heated to 100℃ for 45 minutes. After cooling,
inlet and outlet holes are punctured into the PDMS. The
channel side of the PDMS is then pressed onto a clean glass
slide. A low conductivity buffer is mixed with the
experimental cells, which are dyed using a fluorescent
membrane permeable dye. This mixture is sucked into a thin
tubing that fits onto the tip of a syringe on one end and is attached to
the inlet hole of the microfluidic chip on the other. Next, pipettes
heads are attached onto the electrode channels and filled with PBS with a small amount of
rhodamine B. Finally, this assembly (​Figure 3a)​ is attached to the necessary machinery (​Figure
3b)​ for testing and analysis. The schematic of this low
frequency continuous sorting device can been seen in ​figure
4​ where the two pairs of fluidic electrode channels compose
the source and sink electrodes respectively and are separated
from the sample channel made of a PDMS barrier.

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Conclusion
In conclusion, the ability to separate biomolecules in microfluidics using DEP is an
important tool in order to fully understand their subsequent physical and biochemical
characteristics for scientific research. This paper aimed to highlight a few different ways for
effectively using DEP to biomolecular separation. There are both strengths and weaknesses
associated with the screen­printed and contactless DEP chip. It should be noted that this paper is
only a rough guide to these approaches of microfluidic bioseparation and the reader should
consult other sources to provide a more complete understanding before selecting the most
optimal method.

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