1. 1
Microfluidic Platform to Detect Waterborne Pathogens Using DEP and
Magnetophoresis
Avinash Kumar, Nikhil Makaram, Zina Kurian
June 9th, 2015
Abstract
This microfluidic device will assist in water pathogen detection. The POC device will be low
cost, small, portable, disposable, and require minimal preprocessing to maximize usage in
developing countries where water sources are more likely to have contamination. This device
should allow multiplexed separation and detection of multiple bacterial pathogens, such as E.
Coli or Salmonella. Fabrication will be through standard direct method using a PDMS substrate.
The design will utilize a serpentine mixing channel, DEP focusing and separating electrodes, and
magnetic complexes to sort cells into channels based on characteristics. Then, using fluorescence
microscopy, we will detect expression of different bacterial pathogens from GFP target tags.
Through these methods, waterborne pathogens will be detected on site. This prevents future use
of contaminated supply by fulfilling two goals of fabricating a novel microfluidic lab-on-a-chip
device and detecting a minimum of 70% of pathogens within one small sample.

2. 2
I. Problem Statement
Water is a necessity for all living things. The World Health Organization (WHO)
estimates that about 3.4 million people die from water related diseases. The worst part is the
majority of deaths occur in children. (Berman, 2009) This is seen as a high cause of death in
developing countries that don’t have the proper infrastructure to handle water and protect it from
raw sewage and feces getting into the water supply and contaminating it. This can allow for
bacterial growth in the water supply that is then ingested by people who can catch diseases such
as cholera, typhoid fever, hepatitis A, gastrointestinal illness, respiratory illness, skin infections,
diarrhea, and death. (CDC, 2013 and Berman, 2009) The different types of bacteria that can
cause this are E. Coli and Salmonella. These bacteria can not only be transmitted through
drinking water but also through eating food prepared by infected individuals, or touching a sick
person and then touching your mouth. (Berman, 2009) Building infrastructure such as pipelines
can help provide clean water for places that need it. Unfortunately, there is no real way of
knowing that even the water that is being transported through these pipelines is in fact bacteria-
free.
There are different methods that can be implemented to determine if bacteria is present in
a system. In labs, it is easy to undergo immunostaining techniques (or other visual techniques) to
see if there is E. Coli or any other type of bacteria in a solution. In determining if a pathogen is
there in a system, there must be a proven technique for detection and separation of said pathogen
(or multiple pathogens) in a system. Our focus will be in determining a method of fabrication of
a microfluidics device that allows for detection of at least 70% of waterborne pathogens as well
as implementation of a pathogen separation technique. Detection can potentially be performed
through immunostaining light scattering, fluorescence microscopy, or qPCR. The separation
technique will be based on fabricated microfluidics system with dielectrophoresis and magnetic
labeling. The fabrication method will be soft lithography with multiple microchannels to allow
for separation of the pathogen from water. The separation technique will combine fluorescent
activated cell sorting with two independent force fields. This device attempts to provide a point-
of-care solution to the problem of contaminated water toxins.
II. Goals
The goals of this project are twofold. First, we aim to fabricate a device that will be able
to separate and detect specific pathogens in a water sample. This method should be rapid and low
cost. We want a device to be for point-of-care use. Specifically, cell sorting needs to have high
purity, high specificity, high target recovery, high throughput, and with no crossover
contamination. We want to be able to separate multiple different pathogens in a water sample.
Second, the purpose of this device is to create a biologically relevant model. This device should
enable diagnostics for users in remote areas for real-time monitoring. The use of this device
should provide pathogen-specific testing and enable a whole community to use this reliable
method. If water samples can be tested for deadly diseases, communities will know which water
sources are usable. This device should follow the WHO guidelines established for benefits of
water testing devices. This includes advantages such as portability, minimal processing using
separate lab equipment, independence from power supplies, low cost, low maintenance, rapid
testing, sensitivity, purity, and safe operation by everyone in order to meet quality standards.

3. 3
III. Background
This high-throughput multiplexing device includes miniaturization of elements required
for mixing and synthesis of flow particles. This includes pumps, micro-valves, and mixers in
order to separate into microchannels. There are two conditions that will be applied on this device
in order to separate bacterial pathogen particles from other non-target water particles:
dielectrophoresis and magnetophoresis. Nontarget cells will be separated in spatially different
outlets from target cell types. This concept is known as integrated Dielectrophoretic-Magnetic
Activated Cell Sorter (iDMACS).
Dielectrophoresis
In the presence of electric fields, particles will have dielectrophoretic activity. Polarized
particles move toward a region of the field. This activity is influenced by the surrounding
medium. This force comes from the electrode surface. The magnitude of the force depends on
the cell’s electrical properties and size1. Low medium conductivity is optimal for these
conditions. Different configurations of electrodes affect the control of targets. The height of
electrodes in three dimensions, as well as two dimensional placement and angle, can control the
force. It has been previously demonstrated that dielectrophoresis activated cell sorters will target
cells that are immunochemically labeled with synthetic tags because of the properties of DEP2.
Typically, this concept is extended for use with double input and multiple outputs for continuous
flow of multitarget operation.
For this type of separation, each target cell type is labeled with a unique DEP tag via
receptor-ligand binding in the hybridization chamber. Each tag is specifically chosen for the
potential pathogen being assessed. Inside the device, the apparent forces include hydrodynamic
force from the sample flow and the dielectrophoretic force from the nonuniform electric field
acting perpendicular to the electrodes. Target cells are deflected when they pass through the
electric field and the force from DEP is stronger than the hydrodynamic force. By implementing
two sets of electrodes at decreasing angles, cells with tag A will be dielectrophoretically
separated and guided to channel A. Cells with tag B for a different pathogen will continue along
the channel until being deflected by the second set of electrodes and passing into channel B. The
liquid motion is controlled by gravity based pressure driven flow, instead of operating by tubing
or valves. Having two electrode sets allow two angles to be produced, directing target cells to the
correct outlet. Nontarget cells will
also continue to move along the
device because they are not tagged
and unaffected.
Figure 1: Two electrode sets at different �1 and �2 allow separation at two outlets2.
1
Foudeh, Amir M., et al.
2
Kim, Unyoung, et al.

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This strategy is used often in microfluidics. The DEP force generated by electrodes is
governed by many equations as explained by Krali3. The fluid drag force is described by Stokes
law, FHD = 6π��α. Cells experiencing these forces are affected by the sum total of these forces.
This enables accurate size-based separation since the radius is in both of these equations. Further
calculations can predict the velocity of particles travelling in the channels, and thus the
concentration profile to show how particles with tag A will have a larger complex. This allows
target A particles to deflect efficiently into the appropriate channel because of a variation in
force magnitude. The electrodes provide adequate electric fields, while the consistent
microchannel height ensures cell velocities (and FHD) can be controlled. Also, there is a buffer
stream and sample stream at different inlets, so laminar flow is necessary to control the
segregation of the target streams. In a single pass through of the device, the particles should form
complexes, segregate based on active forces, and collect at
magnetic sites. Since we are using bacterial cells, the DEP
tag will be similarly sized with the target cell.
Figure 2: Diagram of separating electrodes. (a) shows continuous cell
separation. (b) is fluorescence microscopy image of a separation
channel coming in while (c) is the separated target cells in the correct
channels. 4
Magnetophoresis and Nanoparticle Complexes
All materials have their own magnetic field in the
form of each material’s particle’s electron’s spin. When an
electrical field is created over an area (by an electrode),
the electrons (especially the electrons on the surface of a
material) have small forces enacted on them that can cause
translation of a material over an area5. DEP commands a
linear magnetic response to materials which means all
materials will translate away from a DEP electrode
equally until they are neither exhibiting a positive or
negative DEP6. If there was a need to enact a stronger
force on a particle, there would be a need to use a different
method as DEP enacts a similar force on all materials.
Magnetophoresis is another method similar to DEP that causes an magnetic gradient
across an area. While most materials do have a magnetic permeability, only some materials can
be actuated by the magnetic field when it is applied on an area with different materials and
particles. This process allows for actuation of particles that are strongly affected by a magnetic
field to be moved with a non-linear force response to magnetic permeability of a particle7. The
magnetic permeability (or the dipole moment) is expressed in the equation, m = (4/3)πα3xHϕ
where B = μoH8. When a magnetic field is applied on a group of particles, the magnetic
permeability of the medium, particle, and magnetic field combine to express the magnetophoretic
3
Kralj, J. G., et al.
4
Park, S., et al.
5
Kirby, Brian J., et al.
6
Pethig, Ronald, et al.
7
Kirby, Brian J., et al.
8
Ramadan, Qasem, et al.

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force on a particle; This force dictates how much pull a magnet might have on a particle that has
a magnetic permeability. This is expressed as Fmag=(μmag,mmeff×grad(f))H9. The magnetic force
depends on all these different forces but due to their non-linear relationship, a small change in
any one of the variables can have a huge shift in the magnetic force. However this force is
usually very small when being impacted on particles in a medium. Due to the fact that most
materials that exhibit a magnetic permeability have values that are very small, using particles
that have higher values is how this process is exemplified in microfluidic systems.
Figure 3: Magnetic bead attaches to RNA and allows
for a stronger magnetic pull to magnet.10
Using magnetic particles with a high magnetic
permeability, this allows for a strong magnetic
pull and therefore can cause an object to be
actuated and possibly stopped in a high flow
solution. Using this principle, magnetic beads
can be conjugated with antibodies for different
pathogens so when they come near each other
or into contact, they will bind with each other
and will become a complex conjugate. When the complex conjugate comes near a magnetic
field, the magnetic bead creates a strong dipole moment which causes the attraction between the
magnet and the magnetic bead. That draws the complex conjugate to the magnet holding it in
place. This method would allow for anything attached to the magnetic bead conjugate to be held
in place in a high flow system.
Fluorescence Imaging
A method for detection of cells in a system is by fluorescent imaging. This method
involves illuminating an area that contains cells that are either tagged or leak fluorophores
through a microscope. The light source from the microscope is a higher intensity which causes
the fluorescent species to emit a longer wavelength light of lower energy which is viewed in the
microscope. Depending on the wavelength of the background and the inspected fluorophores,
that would dictate what color is viewed in the microscope. When viewed, the image is taken
through 2 filters which prevent the combination of excited and fluorescing rays. The first filter
only lets through light that fits the wavelength of the tagged species that is being viewed. The
second filter then lets in the light that the entire sample produces and it separates that light from
the emitted light from the tagged species11. That is what is seen in the final image. This type of
imaging technique is used today due to a number of favorable properties including: high contrast
(little to no noise seen after signal processing), high sensitivity, cheap, and easy to use12.
9
Kirby, Brian J., et al
10
Zaytseva, Natalya V., et al
11
Rice, George., et al
12
Alander, Jarmo T., et al

6. 6
IV. Methods and Materials
Figure 4: Schematic of Proposed Microfluidic System Design. Dual-inlet channel takes in both the solution of target
pathogens & associated tag + micro-bead complexes, and the subsequently run surfactant solution. Particles are then
separated through DEP into corresponding outlet channels where they are imaged after magnetic containment.
The design of the microfluidic device is made up of multiple inlet and outlet ports and
sequential regions of first DEP separating action and subsequent magnetic separation action
(Figure 4). The first inlet port is designed to bring in a pre-prepared solution composed of the
target RNA of the pathogens of interest along with a mixture of reporter and capture probe-
complexes corresponding to each pathogen. These complexes contain magnetic beads attached to
the capture probes and different liposomes attached to each of the reporter probes for each
pathogen type. Within the liposomes of these reporter probes are fluorophore molecules; each
liposome type contains one of three differently colored fluorophores to correspond with each
pathogen type in this tri-pathogen detecting microfluidic system. The mechanism of action of
these reporter and capture probes can be seen in the example of Figure 3 mentioned earlier. As
the solution runs through the serpentine portion of the first inlet channel, the probe-complexes
hybridize with their appropriate target pathogens. Then these hybridized complexes pass through
the DEP region and are separated into each of their corresponding outlet channels based on the
respective electrical properties of each complex. Afterward, these complexes are attracted to
magnets at the end of each of their outlet channels and are collected there. At this point, a
surfactant solution is sent through the second inlet to wash over all the liposomes in each of the
complexes of the outlet channels, causing them to erupt to release the differently colored
fluorophores within so that each pathogen can be fluorescently imaged for detection and
quantification.
The creation of the microfluidics system will be an inexpensive, easy to build system.
The main portions of the system are the different buffer solutions, the surfactant, the liposome
tags, the magnetic beads, and fabricating the microfluidic system. Each provides a function in
separation and detection of the pathogens in the system. The fabrication method involved for the
device will be soft lithography. This is a popular method due to the inexpensive nature of it as
well as the ability to work in combination with hard material fabrication. The combination of
photolithography and dry etch lithography will be used to obtain a master pattern which will then
be to create a mold made up of PDMS (polydimethylsiloxane) and a silicon wafer. The structure
(once constructed) will be cured and the PDMS will be peeled off to produce a replica of the
system. Any extra holes needed to be made (for injecting buffer solutions) can be fabricated
too13. The device being made will have two inlets; one for a combination of magnetic beads,
water and the waterborne pathogens mixed, and the liposome tags while the other inlet will have
a surfactant that cause the liposome tags to burst releasing fluorophores. A special part of the
13
Zaytseva, Natalya V., et al

7. 7
device is the serpentine region where hybridization of the magnetic beads, liposome tags, and
pathogen occurs. The region is serpentine to allow for time for the binding to occur.
There will be 3 outlets with magnets placed in each outlet channel. The magnets will
have a strong enough pull that it can stop the magnetic bead complexes that will be travelling
through the channel. There will be 3 types of magnetic beads of different sizes (this way the DEP
separation will separate the complexes based on size into the right outlets). The magnetic beads
will be streptavidin coated superparamagnetic beads14 created as determined by a third party. The
magnetic beads will be bound with the DNA capture probes during this process. Liposome tags
will be made using a modified version of a reverse phase evaporation technique. They will also
be given a DNA capture probe that will allow them to bind to the target pathogen when going
through the hybridization process in the serpentine region. The surfactant solution will be a
running buffer OG solution. An imaging software (by Roper Scientific Inc) and Pro Express
software combined with Photometerics CCD camera is how detection of the different types of
pathogens attached to liposome tags15.
V. Potential Problems
There are limitations with this solution. While it is ideal to create a low cost device, there
may be elements that are impossible to miniaturize on the chip. There may be post or pre
processing for each sample required. We aim to minimize this condition of necessary lab
operations. There may be concerns of unnecessary capture with external magnetic pads. We
chose to use magnets to hold the target cells in place so that the complexes can be easily
collected or imaged. These external magnets do not need to be manually moved. However, this
can also be done by letting the target pathogens accumulate at the outlet. This may allow easier
withdrawal of cells using a flow-through solution after processing.
Additionally, this design should give the exact concentration of cells based on GFP
expression. Cell viability is usually measured via flow cytometry. An alternative would be to just
give a YES/NO signal if a pathogen is present in the sample. This would most likely be faster,
and fulfill the criteria for detection. However, our solution chose to implement various
concentrations as a user output because small concentrations of pathogens such as nitrate are
sometimes allowed and not toxic at that level. Length of the device is another critical factor. A
longer channel length ensures purity of separation. However, this increases size and cost of the
device. Other parameters include electrode spacing, dielectric constant, and viscosity of solution.
Electrode material is also up to debate. Our solution uses platinum plated electrode arrays3. It is
also practical to use a low operating voltage at the electrodes because the viability of the cells is
sensitive and it is limited by electrolysis. This is a minor pitfall because throughput would be
increased with higher voltage, but cells may die. Quantum Dots are another strategy for
detection, but fluorescence imaging has been shown to be more reliable for all cell types16. The
most obvious problem that we hope to avoid is the reduction of purity in separated outlets. This
likelihood is reduced by capturing pathogens at magnetic collection points, and overall by using
two force fields to double the separation idea. These potential problems are minimized with our
current strategy. This device has the best characteristics for optimal separation and detection for
the requirements desired. It will serve its purpose efficiently.
14
Zaytseva, Natalya V., et al
15
Zaytseva, Natalya V., et al
16
Deerinck, Thomas J.

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