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JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 18, NO. 2, APRIL 2009 363
Meniscus-Assisted High-Efficiency MagneticCollection and Separation for EWOD
Droplet MicrofluidicsGaurav J. Shah and Chang-Jin CJ Kim, Member, IEEE, Member, ASME
AbstractThis paper describes a technique to increase theefficiency of magnetic concentration on an electrowetting-on-dielectric (EWOD)-based droplet (digital) microfluidic platformoperated in air, i.e., on dry surface. Key differences in the forcescenario for droplet microfluidics vis--vis the conventional con-tinuous microfluidic systems are identified to explain the rationalebehind the proposed idea. In particular, the weakness of themagnetic force relative to the beadsubstrate adhesion and theliquidair interfacial tension is highlighted, and a new techniqueto achieve high-efficiency magnetic collection with the assistance
of the interfacial force is proposed. An improvement in collectionefficiency (e.g., from 73% to 99%) is observed with the newtechnique of meniscus-assisted magnetic bead collection. In ad-dition, isolation of the magnetic species from a mixed sample ofmagnetic and nonmagnetic beads is demonstrated. Comparisonwith other related reports is also presented. [2008-0232]
Index TermsCollection, droplet microfluidics, electrowetting-on-dielectric (EWOD), magnetic beads, meniscus, separation.
I. INTRODUCTION
T HERE HAS BEEN an increasing interest in microflu-idic systems over the last couple of decades [1], [2],particularly in lab-on-a-chip for numerous biochemical ap-plications such as DNA analysis [3], protein detection [4],and cell-based assays [5]. One important step in the samplepreparation of many of these applications is the separationor concentration of the specific cells or molecules of interestfrom complex mixtures [6], [7]. Various mechanisms, based onmagnetic, optical, mechanical, and electrical principles, havebeen reported [8]. Magnetic concentration and separation onlab-on-a-chip devices have been increasingly popular for theirunique advantages over other mechanisms [9]. Unlike electricmechanisms, for instance, magnetic interactions are generallyunaffected by surface charges, pH, or ionic concentration.Magnetic manipulation is possible using an external magnetthat is not in direct contact with the fluid, not requiring complexstructures or electrical circuitry.
Although the native magnetic properties have been exploredat times (e.g., magnetic separation for red blood cells [10]),
Manuscript received September 5, 2008; revised December 20, 2008. Firstpublished February 20, 2009; current version published April 1, 2009. Thiswork was supported in part by the National Aeronautics and Space Administra-tion through the Institute for Cell Mimetic for Space Exploration (CMISE) andin part by the National Institutes of Health through the Pacific Southwest RCEunder Grant AI065359. Subject Editor A. J. Ricco.
The authors are with the Mechanical and Aerospace Engineering Depart-ment, University of California, Los Angeles, CA 90095 USA.
Color versions of one or more of the figures in this paper are available onlineat http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JMEMS.2009.2013394
most biological species do not exhibit useful magnetic behavior.The much more common approach is to selectively label thespecies of interest to superparamagnetic beads [also referred toas magnetic beads (MBs)], which respond strongly to magneticfield gradients [11]. The beads surface can be suitably modifiedto achieve specific binding and subsequent isolation of thebound targets such as proteins [12], [13] and cells [14], [15].
Since conventional microfluidic devices are based on contin-
uous flows of liquids through microchannels, most reports of
magnetic concentration and separation for microfluidics have
been on such a platform. In a typical MB-based immunoas-
say or cell separation assay (e.g., [12] and [14]), antibody-
conjugated MBs (MB-Abs) passing through a channel are
trapped from flow using an external magnet. Next, the protein
or cell sample is flowed through the same channel, where the
specific target proteins or cells bind to the trapped MB-Abs.
The unbound proteins and cells are subsequently washed away
by flowing a wash buffer. A detection antibody, labeled perhaps
with a fluorescent tag, may then be flowed, followed by the
wash buffer to remove the unbound detection antibodies. The
MBs are subsequently released into flow by removing the ex-
ternal magnet to send them to the subsequent steps downstream.Due to its simple design, low power consumption, and repro-
grammable fluid paths, droplet-based or digital microfluidics
driven by electrowetting-on-dielectric (EWOD) [2], [16], [17]
is an attractive platform technology to develop microfluidic
devices and systems on for many applications. Unlike contin-
uous flows through channels, fluids are handled in the form
of droplets (hence, droplet or digital microfluidics) driven by
electrically controlling the wetting property of the surface
(hence, electrowetting) typically of a dielectric in recent years
(hence, EWOD, as opposed to the regular electrowetting that
had been known on metal surfaces for many years). Although
some biochemical applications of EWOD have been shown[18], [19], many are yet to be accomplished. Target separation is
one of the key steps in making EWOD more powerful as a lab-
on-a-chip platform. Mechanisms explored for target separation
and concentration inside the droplet include electrophoretic
[20], dielectrophoretic [21], [22], and magnetic [23], [24].
Magnetic concentration using just a permanent external mag-
net is an attractive option due to its simplicity. Analogous to
the steps in continuous microfluidics, concentration steps for
digital microfluidics would require cycles of adding sample or
wash-buffer droplets and removing the unbound proteins and
labeled Ab as outgoing droplets. The collection efficiency of
MBs in these steps eventually determines the sensitivity of
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detection and is therefore of critical importance. The next
section highlights the different force scenario in droplet mi-
crofluidics vis--vis continuous microfluidics, which exposes a
fundamental difference in the way the MBs are concentrated
and forms the rationale for the proposed technique.
On EWOD droplet microfluidic platform, collection of
MBs with good efficiency has so far been limited to oilenvironment [25][27]. When the device is immersed in oil
[28][30] rather than dry in air [16], [31], [32], a thin layer
of oil present between the hydrophobic device surface and
the aqueous droplet [33] greatly reduces the resistance against
droplet sliding, making most of the basic EWOD operations
much less challenging. The thin oil layer also separates the MBs
from the device surface, preventing adhesion of MBs on the
surfaces and easing their collection. Despite the conveniences,
there have been some concerns, as listed in the following,
regarding the use of oil, particularly in biological applications.
1) Even though oil prevents adsorption of proteins onto the
hydrophobic surface, there is the possibility of protein ad-sorption and entrapment (emulsification) at the wateroil
interface [34]. This may lead to the loss of proteins that
could adversely affect the assay and/or detection, besides
contamination of the surrounding medium.
2) If sample droplets need to be dried on chip, such as the
multiplexed MALDI-MS on EWOD platform [35], im-
mersing in silicone oil is incompatible. Not only is drying
difficult for droplets in oil, but a trace of silicone oil on
the dried surface can also interfere with detection signals.
3) If the application requires certain events to be performed
directly on the device surface, such as hybridization of
nucleotides on the surface or reading with electrochemi-
cal sensors patterned on the surface, the thin layer of oilcould hinder the performance.
4) The surrounding silicone oil may restrict the exchange
of gases between the droplets and the atmosphere, which
may be unfavorable for biological cells in the droplet. In
particular, it could lead to a buildup of carbon dioxide in
the droplets, which may suffocate the cells.
5) Ensuring that the oil does not leak makes the packaging
of the device much more challenging.
6) The devices developed for in-air operations, which are
far more demanding, work for both in air and oil but not
vice versa. General-purpose, or ideally universal, EWOD
devices should function without having to immerse in ortreat with oil.
In this paper, we consider the case where the surrounding
medium is air. Comparison with other reports [24], [36] that
have obtained good magnetic concentration on EWOD in air
without using the technique reported in this paper will be
provided later in Section IV.
II. THEORY
A. Forces on an MB in an Aqueous Droplet in Air on an
EWOD Surface
The interfacial force at the liquid menisci, while absent incontinuous flows, is a major force at play in droplet microflu-
Fig. 1. Unlike continuous flow, droplet microfluidics on a hydrophobic sur-face entails a different force scenario for MB. For an MB on the surface at thereceding meniscus, the vertical component of the interfacial force F pullingthebead away from thesurface is much strongerthan thevdW forceFA holding
it back. For an MB diameter of 15 m, the vertical component Fmag,z ofmagnetic force turns out to be smaller than either FA or F,z , even with thestrongest permanent magnet available.
idics. While the advancing meniscus of a droplet picks up
particles on the dry surface into the droplet [37], our interest
is with the receding meniscus, which keeps the particles inside
the moving droplet instead of leaving them behind on the dry
surface. As will be discussed hereinafter, the force with which
the receding meniscus pulls the MB away from the surface turns
out to be much stronger than other forces including magnetic.
Consider an MB located at the receding meniscus of the
droplet, in the presence of an external magnet. Fig. 1 showsthe forces of primary interest in this case: the surface adhesion
force viz. van der Waals (vdW) force FA, the magnetic forceFmag, and the liquid interfacial tension F . Other forces onthe MB like gravity, viscous drag, and buoyancy turn out to
be much smaller than these three.
The vdW force between sphere 1 and surface 2 in medium 3,
as shown in Fig. 1, is given by
FA =A132RMB
6H2, H RMB (1)
where A132 = Hamaker constant and H = separation. Typi-cally, H is assumed to be 0.51 nm, and A132 can be esti-mated based on individual material properties [37], [38]. Values
for the Hamaker constant in vacuum for polystyrene (A11 =7.8 1020 J) and protein (A11 = 7.3 10
20 J), used tocalculate the aforementioned three-component Hamaker con-
stant, are quite similar [38]. Based on these, the vdW force
for a polystyrene- or protein-coated bead over a typical
EWOD surface (Teflon or Cytop) in water is estimated to be
109 1010 N.The magnetic force acting on a magnetic dipole inside a
magnetic field is given by
Fmag = (m )B = 12
V B2 (2)
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Fig. 2. Simulation results to estimate magnetic force. (a) Magnetic flux density near a cylindrical NdFeB magnet, 12.7 mm thick and 12.7 mm in diameter(half cross section shown). (b) z-component of the magnetic force acting on a 1-m-radius MB placed in this magnetic field as a function of distance z from themagnets face and distance r from the magnets axis of rotation. It should be noted that the order of magnitude ofFmag,z is 10
1010
12 N.
Fig. 3. Maximum z-component of interfacial force at the receding meniscusF,z,max acting on a 1-m-radius MB over an EWOD surface (contact angle = 110) versus the contact angle of MB surface . F,z,max is maximizedover the angle , as shown in the inset. It should be noted that the order ofmagnitude ofF,z,max is 10
710
8 N.
where V = volume of the magnetic particle, = magneticsusceptibility, and B = magnetic flux density.
A Finite Element Method Magnetics (FEMM) software sim-
ulation was used to estimate the magnetic flux density and its
gradient that is 1 mm from the surface of an NdFeB perma-nent magnet [Fig. 2(a)]. Assuming the magnetic susceptibility
of a polystyrene microsphere with iron oxide core as 0.1
and (over)estimating the magnetic volume to be the entire MB
volume, the magnetic force on it turns out to be 1010 to1012 N [Fig. 2(b)].
Last, the force due to interfacial tension and its z-componentare given by
F = 2RMBlg sin sin( ) (3)
F,z = F cos (4)
where lg = liquid-vapor surface tension, = contact angleon the MB surface, = contact angle of the surface, and =
angle determined by the immersion of the MB into the liquid,as shown in Fig. 3 (inset).
As the receding meniscus sweeps across the MB, the vertical
component of the liquid interfacial force F,z pulling the MBaway from the surface into the droplet exceeds the surface-
adhering forces. Fig. 3 shows the maximum values (F,z,max)of F,z (maximized over ) generated using MATLAB forvarying values of assuming = 110 (typical value onEWOD surface). Typical values of this force range from
107 to 108 N, which are 23 orders of magnitude higherthan either the magnetic or the vdW force, implying that the
interfacial force will dominate.
B. Proposed Idea: Meniscus-Assisted MB Collection
and Separation
By keeping in mind the force scenario described earlier, the
new technique of meniscus-assisted MB collection is pro-
posed, which is extended from [23]. Fig. 4 shows the concept
of the proposed technique. When a magnet is introduced, the
MBs suspended in the droplet move toward it [Fig. 4(a-1)],
but many of those on the surface do not. Magnetic force is
ineffective in moving the MBs that are already in contact with
the device surface (by Fmag,z or gravity) against the surfaceadhesion force. As a result, no significant region of the droplet
becomes MB free. Therefore, when the droplet is cut as partof the wash step, many MBs will be lost in the outgoing droplet
[Fig. 4(a-2)], making the wash steps inefficient. Acknowledging
that meniscus crossing is unavoidable for droplet microfluidics,
we instead propose to utilize the powerful liquid interfacial
force to sweep the particles off the surface into the droplet, so
that the magnetic force will move the suspended particles to
one side. To achieve this, the droplet is moved toward the left
by EWOD [Fig. 4(b-1)]. As the meniscus moves, it sweeps the
MBs on the surface back into suspension, so that they can be
collected by the magnet [Fig. 4(b-1), inset]. After all the MBs
have been swept up by the receding meniscus [Fig. 4(b-2)],
the droplet is moved back to the right of the magnet so
that the MBs are collected on the left side of the droplet[Fig. 4(b-3)]. The droplet is subsequently split [Fig. 4(b-4)]
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Fig. 4. Schematic description of the meniscus-assisted MB collection tech-nique. (a) Existing practice, where a magnet attracts the MBs to one side in astationary droplet before splitting it. (a-1) When a magnet is introduced over thedroplet, some MBs move toward the magnet, but other MBs on the surface donot move effectively. (a-2) As a result, many MBs are left in the droplet that ismeant to be depleted of MBs. To overcome the problem, the magnetic attractionshould be performed quickly before the MBs settle on the surface [24].(b) Proposed procedure, where the droplet follows a prescribed path relative
to the magnet during the magnetic attraction, before splitting the droplet.(b-1) As the droplet is moved to the left, (inset) the receding meniscus sweepsthe MBs off the surface and into suspension, allowing the magnetic force to col-lect the now resuspended MBs. (b-2) Droplet is moved leftward, collecting allthe MBs to under the magnet. (b-3) When the droplet is moved back to the rightof the magnet, the MBs are concentrated near the receding meniscus. (b-4) Withthe MBs collected on the left side, the droplet is split. Most of the MBs are inthe (left) collected droplet, while the (right) depleted droplet is nearly MBfree. Compare with (a-2).
into the collected droplet (left) that contains most of the
collected MBs and the depleted droplet (right) from which
the MBs have been depleted. The collection efficiency, i.e., the
fraction of MBs present in the collected droplet after the
cut [Fig. 4(b-4)] was drastically enhanced using meniscusassistance.
Fig. 5. Schematic diagram to show how the proposed technique can be usedto increase the relative concentration of MBs in a droplet containing a mixtureof (dark circles) MBs and (light circles) non-MBs. (a) When a magnet isintroduced over the device, many MBs move toward the magnet. However,the non-MBs and some MBs, particularly those touching the surface, do not.(b) Droplet is then moved leftward. (Inset) As the beads are swept off thesurface and resuspended by the receding meniscus, the MBs are collected bythe magnetic force, but the non-MBs are not. (c) Droplet is moved further tothe left until all the beads on the surface are swept up by the receding meniscus.(d) Droplet is moved back to the right of the magnet. While the MBs arecollected on the left side, the non-MBs distribute uniformly across the droplet.(e) Droplet is split using EWOD, so that (d) most of the MBs are in the left(collected) droplet, while the non-MBs are distributed roughly in proportionto volumes. (f) By adding buffer and repeating steps (b)(e), the non-MBs canbe serially diluted to increase the purity of MBs.
The proposed technique can be further used to purge out
nonmagnetic species in a droplet containing a mixture of
magnetic and nonmagnetic particles. As shown in Fig. 5, a
droplet containing both MBs and non-MBs is taken through the
steps (ae) of meniscus-assisted MB collection. As the droplet
is moved leftward, [Fig. 5(b) and (c)], all the beads on the
surface are swept into suspension by the receding meniscus.
While the MBs get collected by the magnet [Fig. 5(c)], the
non-MBs distribute across the droplet. The droplet is moved
back to the right of the magnet [Fig. 5(d)] and split [Fig. 5(e)].Most of the MBs are retained in the collected droplet, while the
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non-MBs are distributed between the collected and depleted
droplets in proportion to their volumes. The depleted droplet
can be removed, and after adding a buffer droplet to the
collected droplet [Fig. 5(f)], the same steps can be repeated.
With each iteration, the non-MBs are removed in the form of
depleted droplets (depleted of MBs) while retaining most of
the MBs in the collected droplet (where MBs are collected),thus decreasing the concentration of non-MBs against MBs.
It is worth mentioning that the focus of our idea is on MBs
at the receding meniscus, which is typically not subject to
any voltage (since the electrode underneath is grounded).
Therefore, we do not expect electromechanical forces to be a
factor in particle detachment at the receding meniscus. Electro-
mechanical agitation at the advancing meniscus, where the
voltage is applied, is plausible. However, in our experiments,
most of the EWOD voltage drop occurs across the dielectric
layer (particularly since our dielectric and hydrophobic layers
are relatively thick compared, for example, to that in [37] and
[39]), and not across the droplet, even at 1 kHz frequency.
For different device geometry and material properties,
though, this phenomenon could indeed be more prominent, as
noted in [39].
III. MATERIALS AND METHODS
A. Device Fabrication and Droplet Actuation
Lithographic thin-film microfabrication processes were used
to fabricate the parallel-plate EWOD device (Fig. 1). Square
driving electrodes of 11.2 mm width were defined from an in-
dium tin oxide (ITO) (140 nm) layer over a 700 m-thick glasssubstrate (TechGophers Corporation). Cr/Au (15/150 nm)
was deposited and patterned to define the contact pads andelectrode labels for easier visualization. Next, a silicon ni-
tride layer (1000 nm) was deposited using plasma-enhanced
chemical vapor deposition (PECVD) and patterned to define
the dielectric layer. A Cytop (Asahi, Inc.) layer (1000 nm)
was spin coated on top and cured at 200 C to make the
surface hydrophobic. Some ITO-coated (140 nm) 1.1-mm-thick
glass substrates (Delta Technologies, Inc.) were used to prepare
the reference substrate. A thinner PECVD Si3N4 layer
(100 nm) was deposited and patterned on it to expose the ITO
for electrical ground connection, followed by Cytop (100 nm)
deposition. Double-sided tape (0.1 mm thick; 3M, Inc.) was
used as the spacer between the two substrates sandwiching thedroplet(s).
Droplet actuation was achieved by applying voltage typically
of70 V ac at 1 kHz to EWOD electrodes. Electronic control
for the actuation sequence was controlled using LabVIEW with
the help of a digital I/O device. Magnetic force was provided
using a cylindrical permanent magnet (NdFeB, 12.7 mm thick
and 12.7 mm in diameter) placed on top of the EWOD device
(Fig. 1).
B. Reagents and Samples Used
For MBs, Dynal-Invitrogens Dynabeads were used in
two sizes4.5 m (M450 epoxy) and 1.0 m (MyOneStreptavidin T1) in diameters. Dynabeads have a ferromagnetic
core surrounded by a polystyrene shell, often coated with
proteins like streptavidin or antibodies, and are, by far, the
most commonly used MBs [9], [40] in biological applications
like immunoassays (e.g., [41]) and cell separation (e.g., [42]).
For nonmagnetic fluorescent beads (FBs), Nile-red fluorescent
(535/575 nm) carboxylate-modified polystyrene microspheres
from Molecular Probes, Inc. (now Invitrogen, Inc.) were used intwo sizes2.0 m (Fluospheres F8825) and 5.3 m (InterfacialDynamics 2-FN-5000) in diameters. To eliminate any other
additives present in the beads stock solutions, all beads were
washed twice and resuspended in phosphate-buffered saline
(PBS) for EWOD experiments.
C. Visualization and Image Capture
The EWOD device, with droplet(s) sandwiched between sub-
strates, was mounted on an inverted fluorescence microscope
(Nikon TE-2000 U) for visualization. A video camera (KR-222,
Panasonic) was used to capture the droplet actuation movies,
while bright-field and fluorescence still images were takenusing a cooled charge-coupled device (CCD) camera (Coolsnap
EZ, Photometrics).
Particle counting was performed using the images taken on-
chip by the cooled CCD camera. Before taking these images,
the droplet was shuttled across a few electrodes without the
magnet in place to break up (at least some of) the clusters,
particularly for MBs. The concentration of particles was also
kept small enough (< 104/L) to keep the direct countingmanageable, instead of transferring it to a different counting
device such as a hemocytometer [24], which could add to
the error.
D. Image Analysis Using ImageJ
Image analysis was performed using ImageJ software to
determine the droplet volumes and particle counts. Images just
after each droplet cut were analyzed to find the area of each
droplet, and the volume was estimated based on the area and the
known spacer thickness. Images from the cooled CCD camera
were analyzed for three cases of bead samples. Cases 1 and 2
contained only MBs (4.5 and 1.0 m, respectively), whileCase 3 contained a mixture of MBs (4.5 m) and FBs (2.0 and5.3 m). The cases are described in detail in the next section.
To count the 4.5-m MBs in Case 1, bright-field images
were used. By manual inspection, the intensity threshold andsize in pixel squared for each MB singlet, doublet, triplet, and
quadruplet were estimated. Based on these values, counts were
done using the automated particle counting function (Analyze
Particles) in ImageJ. Manual inspection was performed to add
any remaining clusters and/or beads adjacent to the meniscus
and other dark features and also to eliminate any spurious MBs
picked up by the program. The 1-m MBs used in Case 2are harder to visualize because they are barely visible at the
typical magnification (14) used for droplet visualization. At
higher magnifications, the field of view is too small to see the
entire droplet or significant regions of it at a time. In this case,
therefore, images of droplets taken at a higher magnification
(10) were stitched together and used for counting. Incases where automated counting seemed unable to identify
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Fig. 6. (Case 1a) Without meniscus assistance, only moderate collection efficiency is achieved for 4.5-m MBs. Images (a)(d) depict the actuation sequence,while images (e) and (f) show the collected and depleted droplets, respectively. (a) Magnet (the right edge shown in black on the left side of the photograph)is introduced near the droplet containing 4.5-m MBs. (b) and (c) After 30 s, the droplet is cut by EWOD with the magnet in place. The left-side (collected)droplet stays in place, while the right-side (depleted) droplet leaves out of the view to the right. (d) After the cut, the left-side (collected) droplet does containmajority of the MBs. (e) Collected droplet is magnified to show the MBs more clearly. (f) Depleted droplet, which should ideally be MB free, contains many MBs.The cut droplets are shuttled back and forth without magnet to spread the MBs to help in the image analysis.
the MBs correctly, manual counting was performed. For
Case 3, the intensity threshold and size (in pixel squared) of
FBs were determined by manual inspection (as in Case 1).
Unlike MBs, FBs are not prone to forming clusters. Therefore,
only singlets and doublets were counted using the ImageJ
function. Manual inspection was then performed to add orsubtract any unaccounted or incorrectly picked FBs.
IV. EXPERIMENTAL RESULTS AND DISCUSSION
In order to demonstrate the proposed idea described in
Section II, two sets of experiments were performed. First, the
difference in collection efficiency between without and with
meniscus sweep was highlighted for MB concentration, testing
two different sizes of MBs (as shown in Fig. 4). Second, MB
separation was demonstrated using meniscus-assisted serial pu-
rification, whereby nonmagnetic FBs were depleted while MBs
were retained (as shown in Fig. 5). In all experiments, a strong
permanent magnet described earlier was used to maximize themagnetic force in the presence of surface and interfacial forces
that were stronger. Similar results can be expected with smaller
magnets, where the interfacial and surface forces will dominate
over the magnetic force even more.
A. MB Collection Without and With Meniscus Sweep
Case 1: MB Collection With 4.5-m MBs: The effect ofmeniscus assistance on collection efficiency was demonstrated
using 4.5-m MBs by performing MB collection without(Case 1a) and with (Case 1b) the proposed meniscus assistance.
Case 1aWithout Meniscus Assistance: Fig. 6 shows the
case of MB concentration without employing the meniscussweep. An NdFeB magnet is placed over the EWOD device
to the left of the droplet containing 4.5-m MBs [Fig. 6(a)].Many of the MBs move toward the magnet due to the magnetic
force. However, this force is ineffective in moving the MBs
that are touching the device surface, as per the discussion in
Section II. After about 30 s, therefore, when the droplet is
cut [Fig. 6(b) and (c)], not all the MBs are collected in theleft-side (collected) droplet [Fig. 6(d)]. Fig. 6(e) shows a
clearer image of the collected droplet with a magnified view.
More importantly, many MBs are (undesirably) present in the
right-side (depleted) droplet, as seen in Fig. 6(f), leading to a
moderate collection efficiency (73%).
Case 1bWith Meniscus Assistance: Next, MB collection
was performed using the proposed technique of meniscus-
assisted MB collection described before (see Fig. 4). A fresh
droplet from the same sample was used. As in Case 1a, the
NdFeB magnet is placed over the EWOD device to the left
of the MB-containing droplet [Fig. 7(a)]. The droplet is then
moved under the magnet and back using EWOD. In the process,
the receding meniscus sweeps the MBs off the surface back into
solution, allowing them to be attracted leftward by the magnet
[Fig. 7(b)] (this sweep sequence typically takes less than 10 s).
The droplet is subsequently split by EWOD [Fig. 7(c)], with
most of the MBs (99%) collected in the left-side (collected)
droplet [Fig. 7(d)]. Fig. 7(e) and (f) shows the collected droplet,
which has a greater fraction of MBs than in Case 1a, despite a
smaller volume fraction. The difference in the two cases is more
apparent when we compare Figs. 6(f) and 7(f). The right-side
(depleted) droplet is nearly MB free in Fig. 7(f), while the one
in Fig. 6(f) still contains many MBs. For the case of 4.5-mMBs, therefore, one can see that meniscus sweeping helps
achieve a very high collection efficiency (99%), which is asignificant improvement over the no-sweep Case 1a.
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Fig. 7. (Case 1b) Collection efficiency is drastically improved with meniscus assistance for 4.5-m MBs. Images (a)(d) depict the actuation sequence, whileimages (e) and (f) show the collected and depleted droplets, respectively. (a) Magnet is introduced near the droplet containing 4.5- m MBs. Some MBs move tothe left, but once they touch a surface, they can no longer be pulled by the magnet. (b) Droplet is moved under the magnet and back out, so that the meniscussweeps the MBs into the solution (see Fig. 4). After the meniscus sweep, the MBs are mostly collected to the left by the magnetic force. (c) Droplet is then cut byEWOD. The MBs can be seen collected at the left meniscus, just before the cut. (d) Left-side (collected) droplet contains most of the MBs. (e) Collected droplet,magnified to show the MBs more clearly. (f) Depleted droplet contains far fewer MBs than Case 1a, indicating a much improved collection efficiency. The cutdroplets are shuttled back and forth without magnet to spread the MBs to help in the image analysis.
Fig. 8. (Case 2a) Without meniscus assistance, only moderate collection efficiency is achieved for 1-m MBs. Images (a)(d) describe the actuation sequence,while images (e) and (f) show the collected and depleted droplets, respectively. (a) Magnet is introduced near the droplet containing 1- m MBs. (b)(c) After30 s, the droplet is cut by EWOD with the magnet in place. (d) After the cut, the left-side (collected) droplet does contain majority of the MBs. (e) Collecteddroplet, magnified to show the MBs more clearly. (f) Depleted droplet, which should ideally be MB free, contains many MBs. The MBs are clearly visible only inthe magnified photographs due to the smaller size.
Case 2: MB Collection With 1.0-m MBs: In order todemonstrate its applicability to the typical range of MB
sizes (15-m diameter), similar experiments as Case 1 wereperformed for smaller (1.0-m-diameter) MBs, i.e., MB col-lection without and with meniscus assistance.
Case 2aWithout Meniscus Assistance: Fig. 8 shows thecase for 1.0-m MBs collected without meniscus assistance.
Following the same steps as that in Case 1a, similar collection
efficiency is observed. The magnet is introduced to the left
[Fig. 8(a)]. Although some MBs move, the magnetic force is
ineffective in pulling the MBs that touch the surface. As in
Case 1a, when the droplet is cut [Fig. 8(b) and (c)], a majority of
MBs are collected in the left-side (collected) droplet. However,many MBs are left in the right-side (depleted) droplet. Fig. 8(e)
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Fig. 9. (Case 2b) High collection efficiency using meniscus assistance for 1-m MBs. Images (a)(d) describe the actuation sequence, while images(e) and (f) show the collected and depleted droplets, respectively. (a) (Left) Magnet is introduced. (b) Droplet is moved under the magnet and back out. Themeniscus-swept MBs are collected at the left meniscus. (c) Droplet is cut by EWOD. (d) Left-side (collected) droplet contains most of the MBs. (e) Collecteddroplet. The MBs can be seen in the magnified view. (f) Depleted droplet. Very few MBs can be seen, indicating high collection efficiency in this case similar toCase 1b. The MBs are clearly visible only in the magnified photographs here due to their smaller size.
TABLE ISUMMARY OF IMAGE ANALYSIS FOR CASES 1 AN D 2, COMPARING THE RELATIVELY POO R COLLECTION EFFICIENCY WITHOUT MENISCUS
ASSISTANCE (1A AN D 2A) AND THE HIG H COLLECTION EFFICIENCY WIT H MENISCUS ASSISTANCE (1B AN D 2B)
and (f) shows the collected and depleted droplets, respectively.
The smaller MBs, in this case, are harder to see, except in the
further magnified views in (e) and (f).
Case 2bWith Meniscus Assistance: MB collection was
then performed using meniscus assistance (see Fig. 4), as shown
in Fig. 9. A fresh droplet from the same sample as Case 2a is
pipetted onto the EWOD device. The magnet is placed over
the device to the left of the droplet [Fig. 9(a)]. Similar to
Case 1b, as the droplet is moved under the magnet and back out
using EWOD, the receding meniscus sweeps and resuspends
the MBs in the droplet, allowing them to be collected to the left
by the magnet [Fig. 9(b)]. The droplet is then split by EWOD
[Fig. 9(c)], with most of the MBs (> 98%) collected in theleft-side (collected) droplet [Fig. 9(d)]. Fig. 9(e) shows the
collected droplet, which has a greater fraction of MBs than
in Case 2a despite a smaller volume fraction. The right-side
(depleted) droplet is nearly MB free in Fig. 9(f), while the one
in Fig. 8(f) still contains many MBs.
Thus, for 1.0-m MBs as well, meniscus sweeping helps
achieve a very high and significantly improved collection ef-ficiency (> 98%) compared to the no-sweep Case 2a.
B. Summary of Results for Cases 1 and 2
The image analysis results for all the four cases described
earlier are summarized in Table I (see Section III for details
of the image analysis). For all cases, an EWOD device with1.2-mm 1.2-mm electrode arrays was used, with an initial
droplet volume of650 nL. Because significant evaporation
occurred over the course of setup, experiment, and imagecapture, all the data are presented in the form of numberof MBs and volume fraction, rather than concentration and
absolute volume. Images used for particle counting were takenafter moving the droplet over a few electrodes without the
magnet in place, so that the MBs were somewhat spread on
the surface. MB counting and area calculation were performedusing ImageJ software. For both Cases 1 and 2, the number of
MBs in the collected droplet was higher in the case of b (i.e.,with the meniscus sweep) despite a smaller volume fraction
of the collected droplet in this case. It should be pointed outthat well below 1% of the particles were left behind after the
receding meniscus across the surface. On the few occasions
that it was observed, surface heterogeneities (e.g., defects inhydrophobic layer, dirt, etc.) were usually responsible.
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SHAH AND KIM: MAGNETIC COLLECTION AND SEPARATION FOR EWOD DROPLET MICROFLUIDICS 371
Fig. 10. (Case 3) Image sequences showing three successive cycles of meniscus-assisted serial purification steps. MBs (4.5 m) mixed with nonmagnetic FBsof two sizes (2.0 and 5.3 m) suspended in PBS are used. Each cycle consists of a meniscus sweep, followed by droplet cut, and buffer addition, performed onthe same initial droplet at different locations. (a)(d) Sweep-and-cut 1: (a) Magnet is introduced at left of the initial droplet. (b) Droplet moved under magnet andback to sweep the MBs with the meniscus and collect them to the left with the magnet. (c) Droplet is cut, with most of the MBs on the left side of the droplet.(d) After the cut, the (left) collected droplet contains most of the MBs, while the FBs are distributed across both the collected and depleted (not seen) droplets.
(e) A buffer droplet is added to the collected droplet from (d), and the combined droplet is moved to a different location. (f)(i) Sweep-and-cut 2: Stepscorresponding to (b)(e). (j)(l) Sweep-and-cut 3: Steps corresponding to (b)(d).
The MBs swept up by the meniscus enter the liquid droplet
along with the fluidic movement within the droplet. Since the
liquid recirculates within the moving droplet in a 3-D flow
pattern [43], the fluid packets near the meniscus not only travel
toward the opposite device surface but also flow into the body
of the droplet, dragging the MBs with them. We observed that
practically all of the MBs that swept off the device surface and
accumulated near the receding meniscus are already inside the
liquid droplet rather than at the meniscus. Even if some MBs
were stuck at the meniscus, they would soon get dragged into
the liquid with the flow from the meniscus into the droplet,
before being collected magnetically.
It should be noted that the MBs used in these experiments
have hydrophobic surfaces, as per the product information
from the manufacturers [44], [45] (the exact contact angle with
water is not available). According to Fig. 3, the interfacial
force would be stronger for more hydrophilic particles (such
as glass microbeads used in [37], and some other commer-
cially available MBs), suggesting that the meniscus-assisted
technique will continue to be effective for their collection as
well. The interfacial force is expected to be slightly lower for
beads that are more hydrophobic (e.g., the superhydrophobic
beads used in [37]), although Fig. 3 suggests that it would be onthe same order of magnitude. The relatively greater tendency
of superhydrophobic beads to stay adsorbed on the droplet
meniscus [37] could also limit collection efficiency. However,
superhydrophobic beads are extremely rare in use and hardly
available commercially (in fact, [37] prepared them in-house
due to the difficulty in finding superhydrophobic spherical
particles). As such, the case of superhydrophobic beads has
not been considered in detail here.
C. Comparison to Other Reports not Using
Meniscus Assistance
It should be mentioned that MB concentration has been
reported without meniscus assistance [24], [36], where the
collection efficiency was significantly higher than that in
Cases 1a and 2a. However, there are some important differences
in the experimental procedures. Wang et al. [24] reported much
higher collection efficiency (> 91%) than Cases 1a and 2a,even without meniscus sweep. Acknowledging that particle
adhesion becomes prominent as the magnetic particle solution
stays longer on the surface, which would lower the collection
efficiency primarily because the MBs settle down on the sur-
face, they reduced the time between sample loading and cutting
execution to less than a minute. However, many practicalapplications may require much longer duration between these
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Fig. 11. (Case 3) Bright-field and fluorescence image pairs for the collected and depleted droplets, before and after each sweep-and-cut operation. (a) and (b)Initial droplet containing MBs (4.5 m) mixed with FBs of two sizes (2.0 and 5.3 m). After sweep-and-cut 1, the [(c) and (d)] collected droplet 1 contains mostof the MBs, while the [(e) and (f)] depleted droplet 1 contains mostly FBs. After adding a buffer droplet to the collected droplet 1, followed by sweep-and-cut 2,(g) most of the MBs are retained in the collected droplet 2, (h) but it now contains fewer FBs. (i) Depleted droplet 2 has very few MBs and (j) mostly FBs. Afterbuffer droplet addition to the collected droplet 2, followed by sweep-and-cut 3, (k) the MBs are still retained in the collected droplet 3, while (l) the FBs are furtherreduced. Depleted droplet 3 has (m) very few MBs and (n) mostly FBs. The fluorescence illumination was kept on during the bright-field images so that the MBsappear darker than the FBs. Irregularly shaped random dirt particles in the bright-field images are to be ignored.
steps. A decrease in collection efficiency was also reported for
smaller magnetic particles, since the adhesion force relative to
the magnetic force becomes more dominant for them. As a
more generalized scenario, therefore, we did not impose such
special conditions for the experiments in this paper. There wastypically a few (210) minutes gap between sample loading
and droplet actuation. We also show that the high collection
efficiency does not decrease when MB size is reduced from
4.5 to 1 m. Using the interfacial force to overcome surfaceadhesion, therefore, we mitigate the constraints of execution
time and particle size for high collection efficiency (99%, asdescribed in [24]).
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SHAH AND KIM: MAGNETIC COLLECTION AND SEPARATION FOR EWOD DROPLET MICROFLUIDICS 373
TABLE IISUMMARY OF SERIAL PURIFICATION BEFORE AND AFTER EACH SWEEP-AN D-CUT OVE R THREE CYCLES. WITH EACH SWEEP-AN D-CUT,
TH E NUMBER (#) OF FBS IN THE COLLECTED DROPLET FALLS ROUGHLY IN PROPORTION TO THE VOLUME FRACTION BY THE CUT
Another important difference of our test conditions from
[24] and [36] is that the beads were carefully washed prior
to experiments. Most MBs used for biological applications are
supplied in stock solutions containing additives for stability and
long-term storage, which are supposed to be washed off prior
to use, as per protocol. Additives and other constituents in the
medium, which are required at times for actuation of biological
samples, may cover the beads surface, affecting the surface ad-
hesion FA and leading to a different force scenario that does notrequire the meniscus assistance. To be conservative and moregeneral, this paper deals with only the purely physical method
of meniscus sweeping without entailing chemical addition and
any associated undesirable side effects. The force estimates and
results presented here are based on the washed beads suspended
in buffer, with presumably little or no surface coverage from the
constituents of the medium.
D. MB Separation Using Meniscus-Assisted Serial
Purification (Case 3)
Next, it is demonstrated that the MBs (or magnetically la-
beled targets) in a droplet containing a mixture of magneticand nonmagnetic species can be separated by meniscus-assisted
serial purification. This is demonstrated using 4.5-m MBsalong with nonmagnetic FBs of two sizes2.0 and 5.3 m,suspended in PBS. Pure PBS is used as the dilution buffer for
each serial purification step. An EWOD device with 1-mm
1-mm electrodes array was used for these experiments. The
sequence of EWOD actuations performed is shown in Fig. 10.
Bright-field and fluorescence images at each stage are shown
in Fig. 11.
Case 3Serial Purification: A PBS droplet containing
both MBs and FBs (initial volume 650 nL) is pipetted
onto the device [Fig. 10(a)], along with two droplets of PBS(1000 nL each) at separate locations (not shown). Bright-
field and fluorescence images are taken for the initial droplet
(Fig. 11(a) and (b), respectively). Next, the magnet is in-
troduced, and the droplet is taken through the steps of
Fig. 10(a)(d) (sweep-and-cut 1), as in Fig. 5. Bright-field
and fluorescence images of the left (collected) and right (de-
pleted) droplets are taken (Fig. 11(c)(f), respectively), after
which a buffer droplet is merged with the collected droplet
[as in Fig. 5(e)]. The combined droplet is then taken to a
different location [Fig. 10(e)] where steps of Fig. 10(e)(h) are
performed (sweep-and-cut 2), and images are taken for the
collected and depleted droplets (Fig. 11(g)(j), respectively).
A second buffer droplet is merged with this collected droplet,and the sequence of steps is repeated at a different location
Fig. 12. Decreasing trend of the relative concentration of FBs:MBs in thecollected droplet, over serial purification cycles. Over three cycles of sweep-and-cut, the number of FBs falls to under 10% of its original value, while mostof the MBs are retained (the plot is based on Table II).
[Fig. 10(i)(l)] (sweep-and-cut 3) [depleted droplets are out
of the view in Fig. 10(d), (h), and (l)]. Bright-field and fluores-
cence images are taken for the collected and depleted droplets
(Fig. 11(k)(n), respectively) at the end of these steps.
E. Summary of Results for Case 3
Table II presents the summary of results for Case 3. Sincethe droplet volume changes due to evaporation over the course
of setup, experiment, and image capture, the data are presented
in the form of number of beads and volume fractions, instead
of concentration and absolute volumes, as in Cases 1 and 2.
It can be seen that the number of FBs steadily falls with
each purification cycle, roughly in proportion to the droplet
volume fraction. From the initial droplet to the collected droplet
after sweep-and-cut 3, the number of remaining FBs drops to
below 10%.
While the FBs are relatively easy to count using the fluo-
rescence images, the only way to count the MBs is using the
bright-field images. This is made difficult due to the presenceof FBs, which show up in these bright-field images as well.
As such, MB counting was performed for one case (collected
droplet of After sweep-and-cut 3, since this has the fewest
FBs) and assumed to be the same (475) for all collected
droplets. This is a reasonable approximation based on the
results of Case 1b, where 99% of MBs are retained using the
technique. The trend for the decreasing FB:MB ratio is shown
in Fig. 12 based on these calculations.
V. CONCLUSION
This paper has highlighted the relative dominance of the
liquid interfacial force in droplet microfluidic systems, suchas an EWOD device, and the challenge that such dominance
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posed for magnetic concentration. Based on the force scenario,
we proposed to use the interfacial force at the droplet meniscus
to assist the magnetic collection. Significant improvement in
collection efficiency (from 73% to 99%) was observed
using the proposed technique. We also demonstrated the use
of the technique to purify the MBs by washing the non-MBs
off through serial purification while retaining the MBs in themain droplet. The MBs are often used as specific labels to con-
centrate or separate biological targets, e.g., cells, proteins, and
DNA/RNA, from a sample. Since collection efficiency of the
MBs is directly proportional to the effectiveness of this process,
the proposed technique is expected to help improve mag-
netic concentration and separation on EWOD, making EWOD-
based droplet microfluidics a more powerful lab-on-a-chip
platform.
ACKNOWLEDGMENT
The authors would like to thank Prof. D.-K. Jung of Dong-A
University, Busan, Korea, and Dr. J. Gong and Dr. P. Sen of theMicro- and Nano-Manufacturing Laboratory, UCLA, for their
helpful suggestions and discussion. The authors would also like
to thank J. Zendejas and the rest of the staff at the UCLA
Nanoelectronics Research Facility for their support during the
microfabrication.
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Gaurav J. Shah received the B.S. degree fromIndian Institute of Technology Bombay, Mumbai,India, in 2003, and the M.S. degree and the Ph.D. de-gree in mechanical engineering from the Universityof California, Los Angeles, in 2005 and 2008, re-spectively. His Ph.D. work focused on integratingparticle manipulation techniques for effective sep-aration and concentration of target species on an
electrowetting-on-dielectric-driven droplet microflu-idics platform for biochemical applications.
His research interests include MEMS and mi-crofluidics and their applications in biochemistry.
Chang-Jin CJ Kim (S89M91) received theB.S. degree from Seoul National University, Seoul,Korea, the M.S. degree from Iowa State University,Ames, and the Ph.D. degree in mechanical engineer-ing from the University of California, Berkeley, in1991.
In 1993, he joined the faculty of the Universityof California, Los Angeles (UCLA), where he is
currently directing the Micro and Nano Manufac-turing Laboratory and is a founding member of theCalifornia NanoSystems Institute. Since joining
UCLA, he has developed several MEMS courses and established a MEMSPh.D. major field in the Department of Mechanical and Aerospace Engineering.He is currently serving on the Editorial Advisory Board for the IEEJ Trans-actions on Electrical and Electronic Engineering and the Editorial Board forthe JOURNAL OF MICROELECTROMECHANICAL SYSTEMS. His research in-terests are MEMS and nanotechnology, including the design and fabrication ofmicro/nanostructures, actuators, and systems, with a focus on the use of surfacetension.
Prof. Kim has served on numerous technical committees and panels, in-cluding Transducers, the IEEE International Conference on MEMS, and theNational Academies Panel on Benchmarking the Research Competitiveness ofthe U.S. in Mechanical Engineering. He is currently serving on the Devicesand Systems Committee of the ASME Nanotechnology Institute as Chair. Hewas the recipient of the Graduate Research Excellence Award from Iowa State
University, the 1995 TRW Outstanding Young Teacher Award, the 1997 NSFCAREER Award, the 2002 ALA Achievement Award, and the 2008 SamueliOutstanding Teaching Award.
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