2009 mems shah ewod conduit

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HIGH-PURITY SEPARATION OF RARE SPECIES IN DROPLET

MICROFLUIDICS USING DROPLET-CONDUIT STRUCTURES

Gaurav J. Shah and Chang-Jin CJ KimMechanical and Aerospace Engr. Dept., University of California, Los Angeles (UCLA), CA, U.S.A.

ABSTRACT

We present a technique to achieve high-purityisolation of the target species in electrowetting-on-dielectric (EWOD)-based droplet microfluidics byforming a long and slender liquid path between dropletsthat acts as a conduit for actively transported targetspecies, while minimizing non-specific transport due todiffusion and fluidic movement. By stabilizing the conduitchemically (e.g., using surfactants) and controlling thefluid by EWOD, we demonstrate, as an example, veryhigh magnetic bead (MB) collection efficiency (> 99%)while eliminating ~97% non-magnetic beads (nonMBs) injust one separation step.

INTRODUCTIONConcentration/separation is critical in manybiochemical assays, particularly where purity of isolatedtarget species (TS) (e.g. specific cells, proteins, DNA,either by themselves or bound to beads) is vital to theireffectiveness. Unlike continuous microfluidics where TSare immobilized while wash-buffer is flowed through thechannels to remove impurities, purification in dropletmicrofluidics (e.g. by EWOD) typically involves serial(i.e., repeated steps of) dilution of the non target species(nonTS) [1],[2]. In each wash step, a buffer droplet isadded to the sample. The TS is actively collected in oneregion of this combined (parent) droplet using one ormore differentiating properties, such as magnetic, electric,optical or dimensional (e.g., [3],[4]). The droplet is thensplit so that most of the TS are collected in one of thedaughter droplets.

The distribution of the nonTS between the twodaughter droplets, on the other hand, is governed by theiroriginal distribution in the parent droplet and non-specificphenomena such as diffusion and microfluidic movementthat occur during the purification step. If the nonTS wereuniformly distributed in the parent droplet, they would bedistributed between the daughter droplets roughly inproportion to their volumes. Thus, even though manynonTS from the parent droplet will be removed in theform of the depleted droplet (from which TS have been

depleted), this would still leave a significant proportion ofnonTS in the collected droplet (wherein the TS has beencollected). In order to improve the TS purity, therefore,TS is collected across the incoming buffer droplet [5],which is initially free from the nonTS (Fig. 1(1a-1b)). Thedroplet is then stretched (Fig. 1(1c)) and split (Fig. 1(1d))into the collected (left) and depleted (right) droplets.

Even though the original distribution of nonTS in theparent droplet is favorable for high purity (Fig. 1(1b)),their non-specific transport and redistribution of thenonTS during the subsequent collection and dropletsplitting (Fig. 1(1b-1c)) may lower purity of the TS in thecollected droplet (Fig. 1(1d)), entailing multiple wash

cycles (Fig. 1(2a-3a)). Two main mechanisms for the non-specific transport are: (a) diffusion and (b) microfluidicmovement.

Ficks law relates the diffusion flux (Jdif

) of a species

across a fluidic section to its concentration gradient:

difJ Dx=

where D is the diffusion coefficient determined by theparticle radius, temperature and viscosity of the medium.For a given particle size, concentration gradient andviscosity of the medium, the rate of diffusion-driventransport across a fluidic section varies directly with itscross-sectional area and inversely with its length.

The second mechanism for contamination is fluidics-driven transport, i.e. species transported due to the viscousforces during fluidic movement. Since the nonTS areusually not actively manipulated, they are therefore highlysusceptible to travel with the flow. Although appropriate

choice of droplet actuation sequence can reduce the flowinto the collected droplet, some flow is inevitable duringneck creation and pinch-off required for droplet splitting.As the droplet is stretched, the ensuing flow drags thenonTS along with it. This is particularly pronouncedalong the droplet meniscus, where flow velocity is higher[6].

NonTS contamination into the collected droplet dueto both the mechanisms described above could be reducedif a slender neck was created in the buffer droplet prior tosample introduction. While this neck could be a conduitfor active TS transport, its slender (long and narrow)structure would make it an excellent diffusion barrier.

Moreover, since little fluidic movement would be requiredfrom this pre-necked stage to split droplet formation, sucha slender conduit would also help minimize the fluidics-driven nonTS transport during the splitting.

Making a narrow, physical channel or other suchstructures is one way to achieve high purity [7], but hasdisadvantages like complexity in fabrication and lack ofre-configurability and control. Instead, we propose apurely fluidic conduit in this paper to overcome theseissues. The challenge, however, is that for pure DI wateror buffer media, such a slender liquid structure tends to behydrodynamically unstable. The addition of surfactants,for example, can make the thin liquid column more stableand may therefore stabilize the droplet-conduit

structure, forming the basis of the proposed idea.

PROPOSED IDEA: DROPLET-CONDUIT

The use of surfactants on EWOD has recentlyreceived much attention [8],[9]. However, addition ofsurfactants to the solution tends to impede dropletsplitting by stabilizing the neck. Although long necks areroutinely found during the cutting steps, their dimensionsand locations are not so controllable. Here, we reportachieving the controllability using a combination ofsurfactants and electrode modification. Specifically, asurfactant was added in a moderate concentration toincrease tendency for stable conduit formation, while a

stabilizing electrode (SE) was used to toggle betweenstability and breakage.

978-1-4244-2978-3/09/$25.00 2009 IEEE 471


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Fig. 1: Known technique of TS purification by serialdilution: (1a) Wash buffer is added to sample. (1b) TS

(dark) is transported and collected in one region of thedroplet. (1c) The droplet is then stretched and (1d) cut to

form collected (wherein TS are collected) and depleted(depleted of TS) droplets. Many nonTS (bright) areremoved with the depleted droplet, but some nonTS entercollected droplet due to diffusion, and/or fluidics-driven

transport. (2a, 3a) More wash-buffer is added and theabove steps are repeated to improve TS purity by serialdilution of the nonTS.

Fig. 2 illustrates the proposed idea. The typicalEWOD electrode layout is modified to incorporate aslender line electrode (i.e. SE) through the center of thesquare EWOD electrodes. The conduit-forming droplet(left) is stretched towards the mixed sample containing TS

and nonTS. Keeping the SE on, a slender droplet-conduitis created from the conduit-forming droplet (Fig. 2(a,b)),whose width is defined by the SE.

On merging with the sample, the TS are transportedacross the conduit using an active transport mechanism(Fig. 2(c)). However, very few nonTS can cross thediffusion barrier presented by the droplet-conduit. AfterTS transport, the SE is turned off, and the droplet isstretched further (Fig. 2(d)), breaking the conduit andcompleting the droplet split (Fig. 2(e)). It should be notedthat much lesser fluidic movement is involved in thiscutting operation as compared to Fig. 1, since the neckwas already formed. As such, the fludically driven nonTStransport into the collected region is much reduced. Thecollected droplet (left) contains the TS and very fewnonTS, most of which are left in the depleted droplet(right).

Fig. 2: Proposed technique for high purity rare TSseparation using droplet conduit. (a) TS are transported

to left edge of sample, while nonTS are randomlydistributed in the droplet. With SE on, the conduit-formingdroplet is stretched, (b) forming a slender conduit. Onmerging with sample, (c) the conduit allows active TStransport. But the slender conduit restricts diffusion-driven nonTS transport. (d,e) When droplet is stretched

with SE off, the droplet splits with minimal fluidicallydriven nonTS transport into the high purity collecteddroplet and the depleted droplet.

MATERIALS AND METHODS

The technique was demonstrated for high purity

collection of magnetic beads (MBs) from a mixed

population of MBs and non-magnetic beads (nonMBs) on

an EWOD device. MBs of 4.5 m diameter (Invitrogen)

were used as the TS, and fluorescent nonMBs of 5.2 m

diameter (Molecular Probes) were used as the nonTS.

Pluronic F68 surfactant of 0.15% w/v (Sigma-Aldrich) in

PBS was used for both the sample and the buffer droplets.

A standard two-plate EWOD device (e.g. [10]) was

used for the experiments (Fig. 3). For the bottom plate,

EWOD electrodes were patterned into the 140 nm ITO

layer on glass substrate. A 60 m wide line going throughthe middle of the 1 mm x 1 mm EWOD electrodes defines

the SE. A gap (1-1.5 mm) was left at the left end of the SE

to ensure the breakage of the conduit during droplet

splitting. Silicon nitride (1.1 m thick) was deposited by

plasma-enhanced chemical vapor deposition (PECVD) to

form the dielectric layer, and Cytop

(~1 m thick) was

spun-coated to form the top hydrophobic layer. For the

top plate, an unpatterned layer of ITO was coated with

100 nm silicon nitride and 50 nm Cytop

. Double-sided

tape (~100 m thick) was used as the spacer between the

plates.

Droplet actuation was achieved by sequential

application of voltage (70-80 Vac @1 kHz) to the EWODelectrodes. Electronic control for the actuation sequence

was controlled using LabVIEW (National Instruments)

via a digital I/O device (DAQPad 6507, National

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Instruments). For magnetic collection, an NdFeB

permanent magnet ( dia x thick) was placed on top

of the device to the left. Droplet actuation movies andlow-magnification (for a larger field-of-view) images

were captured by a video camera (Panasonic KR-222)

mounted onto the microscope (Nikon TE 2000U), while

better quality fluorescence images were taken using a

cooled-CCD camera (Photometrics Coolsnap EZ)

attachment.

Fig. 3: Schematic cross-section of EWOD device used.

RESULTS AND DISCUSSION

Experiments were performed to show high purity

separation of TS (i.e., MBs) from fluorescent nonTS (i.e.,

nonMB) using the slender droplet conduit. In order todemonstrate the utility of conduit for purification of rare

species, a low (around 1:20) TS:nonTS ratio was chosen.

Fig. 4 shows the image sequence for the experiment

performed. The sample droplet containing MBs and

nonMBs, along with 0.15 %w/v pluronic surfactant F68 is

placed on the right, while the conduit-forming (i.e.,

buffer) droplet, also containing the surfactant, isintroduced from the left (Fig. 4(a)). The magnet is

positioned at the left, so that the MBs collect at the left

meniscus of the sample droplet (see Fig. 4(a) inset). A

stable, slender conduit is formed by stretching the

conduit-forming droplet while keeping the 60 m wide SE

on (Fig. 4(b)). On merging with the sample (Fig. 4(c)), the

MBs from the sample are actively transported across the

conduit towards the magnet, to the leftmost edge of the

combined droplet, while most of the nonMBs remain

behind at the right (Fig. 4(d)). After all the MBs are

transported, the SE is turned off and the droplet is

stretched further (Fig. 4(e)) so as to split it into thecollected (left) and depleted (right) droplets (Fig. 4(f)).

Since the neck was already formed prior to the merging,

the splitting operation involves very little fluidic

movement.

To evaluate the purity of the separation, MBs (dark)

and nonMBs (fluorescent) are counted in the collected

(Fig. 5(a,b)) and depleted (Fig. 5(c,d)) droplets. Images

were taken using both cameras under fluorescent

excitation, but with some bright field illumination so as to

visualize non-fluorescent features as well. The bright

nonMBs can be easily distinguished from the background.

To distinguish the MBs from the other dark features in the

images, a magnet was introduced on one side of the

droplet, and the magnetically responsive collected at the

edge were counted. The original sample contained ~283

nonMBs and 16 MBs. Comparing Fig. 5 (a) and (b) it is

clear that much fewer nonMBs (bright) can be seen in thecollected droplet compared to the depleted droplet. The

fluorescent nonMB count in the collected droplet is less

than 10 while that in the depleted droplet is ~267. Thus,

~97% of nonMBs were kept out of the collected droplet.

Around 16 MBs were counted in the collected droplet

(Fig. 5(a)), while no (0) MBs were seen in the depleteddroplet (Fig. 5(b)), demonstrating that high purity

magnetic separation was achieved while maintaining high

collection efficiency (> 99%).

Fig. 4: Image sequence for high purity magnetic separation using droplet conduit structures: All droplets contain 0.15%pluronic F68 in PBS. (a) Magnet is positioned to the left of the sample and conduit-forming droplet, so that MBs (dark)are attracted to the left edge of sample (see inset). (b) Conduit is formed by stretching the droplet while the SE is on. (c)

Sample is merged with the conduit-forming droplet, (d) allowing MBs to pass through. After transport, very little fluidic

movement is involved as (e) the droplet further stretched with SE turned off, (f) cutting it into collected (MBs collected,very few nonMBs) and depleted droplets (depleted of MBs). Satellite droplets can be cleaned up by depleted droplet.

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Fig. 5: High-purity separation is obtained using droplet-conduit structures, as estimated by counting the MBs (dark) and

nonMBs (bright) in the collected and depleted droplets. Images were taken under fluorescent excitation but with somebright-field illumination so as to visualize non-fluorescent features as well. To count MBs (and not dirt etc.), a magnetwas introduced right next to a droplet edge, and the magnetically responsive MBs attracted to it were counted. Original

sample droplet had ~16 MBs and ~283 nonMBs. (a) All the MBs (~16) and very few nonMBs (

2009 mems shah ewod conduit

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