2009 JMEMS Shah Meniscus Magn Collection EWOD

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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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364 JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 18, NO. 2, APRIL 2009

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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SHAH AND KIM: MAGNETIC COLLECTION AND SEPARATION FOR EWOD DROPLET MICROFLUIDICS 365

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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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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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.

Documents

2009 JMEMS Shah Meniscus Magn Collection EWOD