1120 JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 16, NO. 5, OCTOBER 2007

Sequential Field-Flow Cell Separation Method in aDielectrophoretic Chip With 3-D Electrodes

Liming Yu, Ciprian Iliescu, Guolin Xu, and Francis E. H. Tay

Abstract—This paper presents a sequential dielectrophoreticfield-flow separation method for particle populations using a chipwith a 3-D electrode structure. A unique characteristic of our chipis that the walls of the microfluidic channels also constitute thedevice’s electrodes. This property confers the opportunity to usethe electrodes’ shape to generate not only the electric field gra-dient required for dielectrophoretic force but also a fluid velocitygradient. This interesting combination gives rise to a new solutionfor the dielectrophoretic separation of two particle populations.The proposed sequential field-flow separation method consists offour steps. First, the microchannel is filled with the mixture ofthe two populations of particle. Second, the particle populationsare trapped in different locations of the microfluidic channels.The population, which exhibits positive dielectrophoresis (DEP),is trapped in the area where the distance between the electrodesis the minimum, while the other population that exhibits negativeDEP is trapped in locations of maximum distance between elec-trodes. In the next step, increasing the flow in the microchannelswill result in an increased hydrodynamic force that sweeps the cellpopulation trapped by positive DEP out of the chip. In the last step,the electric field is removed, and the second population is sweptout and collected at the outlet. For theoretical and experimentalexemplification of the separation method, a population of viableand nonviable yeast cells was considered. [2006-0157]

Index Terms—Cell separation, dielectrophoresis (DEP), micro-fluidic device, 3-D silicon electrodes.

I. INTRODUCTION

D IELECTROPHORESIS (DEP), which is the manipulationof neutral particles in a nonuniform electric field, is one

application in which microfabrication can play an importantrole. This technique gives the opportunity of fabrication, onthe same structure, of elements such as microchannels, valves,filters, reaction chambers, inlet/outlet holes, etc. As a result,lab-on-a-chip structures can be developed on a small area forparticle manipulation. The first report of a microfabricated DEPdevice was made by Masunda et al. [1].

Particle manipulation based on the DEP force requires asuitable gradient of the electric field to be generated. Thiscan be achieved using various solutions, such as micropat-

Manuscript received July 31, 2006; revised March 12, 2007. Subject EditorA. Ricco.

L. Yu and F. E. H. Tay are with the Institute of Bioengineering and Nanotech-nology, Singapore 138669, and also with the National University of Singapore,Singapore 119077 (e-mail: lmyu@ibn.a-star.edu.sg; ftay@ibn.a-star.edu.sg).

C. Iliescu is with the Institute of Bioengineering and Nanotechnology,Singapore 138669 (e-mail: ciliescu@ibn.a-star.edu.sg).

G. Xu is with the Institute of Bioengineering and Nanotechnology, Singapore138669, and also with Nanyang Technological University, Singapore 639798(e-mail: gxu@ibn.a-star.edu.sg).

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.2007.901136

terned electrodes that can be thin films [2], [3], 3-D extrudedelectrodes [4]–[6], or even a combination of thin films withextruded electrodes [7]. Another method used in producingelectric field gradients uses the changing of the phase of theapplied electric field [8], [9]—a method known as travelingwave DEP. Another method—isolating DEP or iDEP—uses thenonuniformities generated by a dielectric material placed in auniform electric field [10], [11].

The applications of DEP include manipulation of viruses[12], bacteria [13], or nanoparticles [14], and even trapping ofDNA by DEP was achieved [15]. However, most of the researchhas been focused on cell concentration [16] or separation [17].

In a DEP device, movement of the particles toward theregions with higher electric field strength is called positive DEP,whereas movement toward regions with lower electric fieldstrength is called negative DEP. The response of the particleto the electric field depends on the dielectric properties of theparticle relative to the medium.

Here, we report a new sequential separation field-flow tech-nique using a DEP chip with 3-D silicon electrodes. The tech-nique was made possible by the unique design of the device,where the electrodes are also the microfluidic channel walls,thus serving a double function. The first function is to generatepositive and negative DEP forces to trap two populations ofcells in different locations. The second function is to producea fluid velocity gradient, i.e., to have zones where the velocityis maximum and, also, to have fluidic dead zones. As a conse-quence, the resultant hydrodynamic force will first flush out thepopulation trapped by positive DEP. After the removal of theelectric field, the second population is collected at the outlet.

II. SEPARATION METHODS USING DEP

The DEP-based filtration technique offers general advantagessuch as the ability to process complex liquid suspensions withminimum risk of clogging when compared with the membranefilters. A detailed description of DEP separation methods inlab-on-a-chip devices is presented elsewhere [18]. In addition,Gascoyne and Vykoukal reviewed particle separation by DEPin [19]. These techniques can be summarized as follows: flowseparation, field-flow fractionation, stepped flow separation,travel wave DEP, and the ratcheting mechanism.

The flow separation method consists of flowing a particlesuspension solution over an electrode array. When there aremultiple populations that exhibit positive and negative DEP,one population will be trapped near the electrode, while theothers will be repelled into the center of the chamber tobe subsequently pushed by the flow toward the outlet. Flow

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YU et al.: FIELD-FLOW CELL SEPARATION METHOD IN A DIELECTROPHORETIC CHIP WITH 3-D ELECTRODES 1121

separators have been reported and demonstrated in [20] and[21]. A microfluidic device, with 3-D arrays of electrodesembedded in microchannels and with the use of a so-called “de-flector” structure (electrodes oriented at certain angle comparedwith the flow direction), is presented in [22].

Another method uses a fluid velocity gradient to separate par-ticles. Using an applied dielectrophoretic force field, differentparticles will be located at different regions within the fluidvelocity gradient and will travel with different velocities. Thisseparation method is known as field-flow fraction [23].

Another method that can be used for separation of bacteria,yeast, and plant cells uses castellated electrodes for cell trap-ping, two ports for outlet, and one port for inlet [24]. Particlesflow from the center of the array to one of the ports. Whenthe electric field is applied, positive and negative DEP forcesare experienced by each population of particles. Similar tofield-flow fraction, when the liquid is pumped, a hydrodynamicforce is applied to the cells, and if this force is larger than theDEP force, the cells will be swept out. The populations can beseparated and driven in opposite directions toward the two portsby selecting the correct frequency and electrical properties ofthe medium.

Traveling wave DEP can be also used as a separation tech-nique. A traveling electric field is generated by interdigitatedparallel electrodes usually connected in 3–4 periodic intervalswith different phases (0, 120, and 240 or 0, 90, 180,and 270). Related work was reported by Huang et al. [25](separation of yeast cells according with size), by Hughes et al.[26] (separation by changing the frequency of the electric field),and by Fuhr et al. [27] (“spiral electrode arrays”).

The ratcheting mechanism has been reported in two con-figurations. The first system uses a “Christmas tree” elec-trode and, as source for particle motion—thermal motion(Brownian motion). Early work in this area was performed byRousselet et al. [28]. The second method employs stackedratchets, which consists of two pairs of electrodes that arestacked one over the other. The populations are inserted be-tween these electrodes, and alternating the potential betweenthe upper and lower electrodes moves the particles. The methodwas presented also by Gorre-Talini et al. [29].

III. PRINCIPLE OF SEQUENTIAL FIELD-FLOW PARTICLE

SEPARATION METHOD IN A DEP CHIP

WITH 3-D ELECTRODES

The objective of the design is to achieve efficient separationof particle populations. The efficiency is achieved by creatinga structure where separation takes place over the entire crosssection of the fluidic flow. This ensures that particles flowing atany height within the channel are separated. The two distinctfeatures of the separation structure are periodic fluidic deadvolumes that are created by an undulating microchannel wallshape and an extruded electrode design that exerts a DEPforce parallel to the channel floor and ceiling, and normal tothe fluidic dead zones. A DEP force that is parallel to thechannel floor and ceiling ensures that there are no unnecessaryfriction forces on the particles to impede their motion andreduce the sorting efficiency. The force exerted on particles with

Fig. 1. Separation method: (a) insertion of the particles in the DEP chip,(b) cells separation using positive and negative DEP, (c) removing the firstpopulation by increasing the velocity of the fluid, and (d) the second populationis released after removing the electric field.

different dielectric properties can be a positive or negative DEPforce. Correct selection of the operating frequency allows thisdifference to be used in separating the particle populations.

The separation sequence consists of four steps presented inFig. 1. Initially, the channel is filled with the particle mixture[Fig. 1(a)]. At the optimal frequency, one population will expe-rience a negative DEP force that drives particles into the deadfluidic zones where they remain trapped [Fig. 1(b)—zone B].Simultaneously, the other population will experience a positiveDEP force that focuses them in the region of the channel crosssection where maximum fluid velocity occurs during the flow[Fig. 1(b)—zone A]. After the particles have segregated into thetwo regions within the channel, fresh buffer solution is pumpedthrough the channel. The population that was focused at thecenter of the channel where the velocity is greatest is swept outby the drag force exerted by the fluidic flow [Fig. 1(c)]. Thepopulation trapped in the fluidic dead zones remains trappedunder flow or no-flow conditions. The DEP force capturing thepopulation in the dead zones is then reversed, and the capturedpopulation can be swept out [Fig. 1(d)].

In the following section, we will discuss the formation of thefluidic dead zones by numerical (ANSYS) simulation of differ-ent wall structures. We will then discuss the extruded electrodedesign using electric field analysis (Maxwell simulation) andanalyze the DEP force and the gradient of the electric fieldthat are generated across the channel for different electrodedesigns. Finally, we analyze the cumulative effect of DEP andhydrodynamic forces for different electrode profiles.

IV. THEORETICAL ANALYSIS AND SIMULATIONS

A. Hydrodynamic Force

The flow of the fluid in the microchannel can play an impor-tant role in the separation performance of the dielectrophoreticdevice. In our special case, the shape of the channel wallsgenerates a specific velocity gradient in the fluid flowing inthe microchannel that is essential for separation. Fig. 2 showsan ANSYS simulation of the flow in microchannels definedby electrodes that extruded the walls with different geome-tries (semicircular, triangular, and rectangular), with a minimal

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Fig. 2. Velocity gradient of the fluid flow in a microfluidic channel with semicircular, triangular, and square electrodes.

channel width of 100 µm and a maximal opening of 300 µm.The simulations were performed for a flow rate of 0.05 mL/min.These geometries lead to a huge gradient in the flow velocitybetween the maximum and minimum wall separation regions,neglecting the boundary layer values (which are zero by defi-nition in the nonslip continuum model case). The low velocityregions will, from here on, be referred to as dead zones (let usapproximate with those areas where the fluid velocity is lessthan 10% of the average velocity at the center of the channel).For this reason, the hydrodynamic force

F = 6πηrν (1)

exerted on the particles situated in the dead zones is morethan one order of magnitude lower than in the channel at itsnarrowest region. In (1), η is the viscosity of the fluid, r is the

radius of the particle, and ν is the velocity of the fluid. Thehydrodynamic force is directly proportional to the velocity, andradius of the particle, hence decreasing the velocity by one or-der of magnitude, correspondingly decreases the hydrodynamicforce with the same amount.

The calculated hydrodynamic force for a yeast cell, whichis approximated by a sphere with a diameter of 7 µm, for amedium viscosity of 10−3 N · s/m2 (water) and a velocity of100 µm/s, is 6.6 × 10−12 N.

B. DEP Force

The DEP force acting on a spherical particle with radius r isgiven by [30]

F = 2πr3εmRe [K(ω)]∇E2 (2)

YU et al.: FIELD-FLOW CELL SEPARATION METHOD IN A DIELECTROPHORETIC CHIP WITH 3-D ELECTRODES 1123

Fig. 3. Frequency variation of the Re[K] for viable and nonviable yeast cellsin a suspending medium with a conductivity of 1 mS/m.

where εm is the absolute permittivity of the suspendingmedium, and ∇E is the electric field gradient intensity.Re[K(ω)] is the real part of the Clausius-Mossotti factor, whichis defined as

K(ω) =(ε∗p − ε∗m

)/(ε∗p + 2ε∗m

)

ε∗ = ε − j(σ/ω) (3)

where ε∗p and ε∗m are the complex permittivity of the particle andmedium, respectively. The complex permittivity for a dielectricmaterial can be described by its permittivity ε and conductivityσ, where ω is the angular frequency of the applied electricalfield E.

In the expression of the dielectrophoretic force (2), the termRe[K(ω)] (Re stands for “the real part of”) plays an importantrole. In (3), the difference of the real part of (ε∗p − ε∗m) canbe positive or negative (Fig. 3), giving either a positive ornegative DEP force. As a result, the movement of the parti-cles toward the areas of high (positive DEP) or low electricfield strength (negative DEP) is determined by the dielectricproperties of the particles and medium. Particle populations thatexhibit DEP forces of opposite polarity can thus be separated inthis way.

The complex permittivity of the particle is strongly de-pendent on the frequency of the generated electric field. Weconsider, for our experiment, viable and nonviable yeast cells.According to the model elaborated by Huang et al. [31] andapplied also by Talary et al. [32] and Hughes [18], we considerthe values of 60, 6, and 50, respectively, for the relative per-mittivities of the yeast cell wall, cytoplasmic membrane, andcell interior, respectively. For viable yeast cells, the conductiv-ities of the cell wall, membrane, and interior were 14 mS/m,0.25 µS/m, and 0.2 S/m, with thicknesses of the cell wall andmembrane of 0.22 µm and 8 nm, respectively. In contrast, thecorresponding conductivity values were 1.5 mS/m, 160 µS/m,

and 7 mS/m, respectively, for nonviable yeast cells (with thesame dimension of the cell wall and membrane). Employinga cell wall thickness of 0.22 µm, a cell membrane thicknessof 8 nm, and values of 7 and 6 µm for the radii of viableand nonviable yeast cells, respectively, the dependence ofRe[K(ω)] on frequency was derived for a suspending mediumwith a conductivity of 1 mS/m, as shown in Fig. 3. It can benoticed that, for low frequencies up to 50 KHz, viable yeastcells present a negative value of Re[K(ω)] which will result ina negative dielectrophoretic force, while nonviable yeast cellspresent a constant positive value. This range of frequencieswill allow separation of these populations. Another window offrequency can be between 10 and 200 MHz where the situationis opposite: the viable yeast cells will be trapped by positiveDEP, while nonviable yeast cells will experience negative DEP.For different conductivities of the medium, different frequencybands of cell separation can be achieved.

The electric field gradient and the DEP force calculated forthe same yeast cell, with a diameter of 6 µm positioned near theedge and in the center of the channel for all the three structurestudied, were presented in Table I. The calculations were madefor a permittivity of the medium of εm = 8.1 × 10−10 F/m anda Re[K(ω)] of 0.15 (maximum value for nonviable yeast cells)for a yeast cell suspended in aqueous solution with a conduc-tivity of 1 mS/m. These data show that the hydrodynamic forceand DEP force are in the same range.

A comparison between the electric field generated in a DEPstructure with planar electrodes and a device with extrudedelectrodes is presented in Fig. 4. The extruded electrode designwill generate an almost uniform electric field in the verticaldirection with a very small variation at the interface betweenthe corner of the electrodes and the glass [Fig. 4(b)]. Theuniformity of the electric field will determine a null DEP forcein the direction perpendicular to the electrode plane (the electricfield gradient is zero). A gradient of the electric field alongthe vertical direction—characteristic of the planar electrodestructure [Fig. 4(a)]—will generate a DEP force—FZ−DEP—inthis direction. For positive DEP, this force will be in the samedirection with the gravity force and will increase the sedimen-tation and the trapping of the particle to the floor (where thehydrodynamic force is null).

C. Considerations About Electrothermal Forces

In addition to the DEP force and hydrodynamic force,as aforementioned, there are also other electrohydrodynamicforces that act on the particles in a DEP device. An analysis ofthese forces is presented by Ramos et al. [33]. Most importantforces are electrothermal forces generated by high electricalfields that are used to manipulate small particles. The highelectric field can generate a large power density in the fluidsurrounding the electrode. Moreover, the nonuniform electricfield can generate a nonuniform power density which canfurther generate thermal gradient which is equivalent with localchanges in the density, viscosity, permittivity, and conductivitywithin the medium. These nonuniformities give rise to forces onthe fluid. Characteristic for our structure with 3-D electrodes, ascompared with the classical structure with coplanar electrodes,

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TABLE IELECTRIC FIELD GRADIENT AND DIELECTROPHORETIC FORCE

Fig. 4. Simulation of the electric field in vertical plane (perpendicular to electrode) for (a) a planar DEP structure and (b) extruded DEP structure.

is a low Joule effect. At the same applied voltage, the gradientof temperature is around ten times lower (we present a detailanalysis in [34]).

While in a DEP device with coplanar electrodes the DEPforce decreases with the distance from the electrodes, this forceis constant in vertical plane in our structure. As a result, fora particle that flows at a certain distance from the floor of themicrofluidic channel, an increase DEP force is required [34].Since the relationship between the DEP force and the appliedvoltage is

FDEP ≈ V 2 (4)

it results that an increased voltage is required for the particletrapping. Once the relationship between the gradient of temper-ature and the applied voltage (presented in [33]) is

∆T ≈ σV 2

k(5)

where k is the thermal conductivity, and σ is the electricalconductivity of the medium, it became very clear that therequirement for an increased force will generate an increasedgradient of temperature.

We can conclude that, for the DEP structure with 3-D elec-trodes, the gradient of temperature and the temperature areconsiderately reduced, and for this reason, the forces asso-ciated with the electrothermal effect could be ignored. Nev-ertheless, low variation of temperature in the structure with3-D electrodes makes it suitable for biological applications (thetemperature can affect the viability of biological samples).

D. 3-D Electrode Design Consideration

The theoretical selection of electrode design requires ananalysis of positive and negative DEP forces exhibited onthe particles (in our case, yeast cells) and also the generatedhydrodynamic force. Two zones are analyzed for each electrodetype: one where the positive DEP is experienced (in the areawhere the space of the channel is minimal) and the secondwhere the opening of microfluidic channel is maximal (thezone where negative DEP is generated).

For positive DEP, the variation of DEP force (for all ofthe mentioned types of electrodes) and hydrodynamic forceis presented in Fig. 5. For simplification, only one graph waspresented for the hydrodynamic force. We can observe thatthe triangular shape of the electrode gives a stronger DEPforce, with a maximal value near the tip of the electrode.The cells population, which experienced positive DEP, will betrapped between the tips of the electrodes; therefore, the areawhere the particles will be in contact with the electrodes (andat the same time with the microchannel walls) will be reduced.The triangularly shaped electrodes provide a wider zone wherethe modulus of DEP force is larger than the modulus of hy-drodynamic force, as compared to the semicircular or square-shaped ones. However, a comparison between the absolutevalues of DEP and hydrodynamic forces is not relevant dueto the fact that their direction is different. It is therefore muchmore relevant to analyze the resultant force (which is composedof DEP and hydrodynamic forces). For this purpose, we studiedthree cases: when the direction of the resulting force R is insidethe electrode area [Fig. 6(a)] or is parallel with the triangleedge [Fig. 6(b)] and when the direction of the resulting forceR is outside the electrode area [Fig. 6(c)]. In the former twocases, the particles cannot be released. The particles can be only

YU et al.: FIELD-FLOW CELL SEPARATION METHOD IN A DIELECTROPHORETIC CHIP WITH 3-D ELECTRODES 1125

Fig. 5. Variation of hydrodynamic force and positive dielectrophoretic forcefor different electrode profiles between electrodes tips for 100-µm channelwidth.

Fig. 6. Typical cases for triangularly shaped electrodes.

moved or rolled along the edge of the electrode in the regionswhere the hydrodynamic force is increased (high flow velocity)and, at the same time, where DEP force is weak. As a result, theparticles can be released. Improving the release of the particlescan be achieved in three ways: by decreasing the angle of thetriangle apex (in this way, the probability that the direction ofthe resulting force to be outside the electrode area increases),by decreasing the DEP force, or by increasing the velocity ofthe fluid (in effect, increasing the drag—hydrodynamic force).

In Fig. 6(b), the following condition for the maximal valueof the triangle apex (α) can be extracted:

tg(α/2) =FHD

FDEP. (6)

By using the values from the graph presented in Fig. 5, at adistance of 5 µm from the apex of the electrode, the maximalvalue of α is around 50.

However, decreasing the angle of the profile in the deadregions will be more akin to having a square electrode. Thesecond solution, decreasing the DEP force, can be achieved bya careful selection of some parameters such as conductivityof the solution (σ) or working frequency (that is equivalentto imposing a smaller value of Re[K(ω)]). Increasing thevelocity can be performed within some experimental limits dueto the increase of the velocity and force simultaneously in thearea where negative DEP is experienced. For the semicircular

Fig. 7. Directions of the resulted force for semicircular and square electrodesfor particle that exhibits positive DEP.

Fig. 8. Variation of hydrodynamic force and positive dielectrophoretic force(different electrode profiles) for population that experiences negative DEP upto 100-µm distance for the channel wall.

Fig. 9. Direction of the resulting force for semicircular for particle thatexhibits negative DEP.

electrode, the positive DEP force presents a lower value, butin this case, the resulting force will keep the particles in theregion situated near the electrode edge (where the velocity andalso the drag force are reduced)—as shown in Fig. 7(a). Forthe square electrode, the resultant force will keep the particlein contact with the electrode edge—as shown in Fig. 7(b).In addition, for these cases, a small positive DEP force isrecommended. For the population that experiences negativeDEP, the variation of forces is presented in Fig. 8. The values ofthe dielectrophoretic force are presented in the absolute value.It can be observed that, for the dead zone, the DEP force andalso the hydrodynamic force are very weak. Moreover, for theexample presented in Fig. 9 for the semicircular electrode, itcan be observed that the resultant force will keep the particlesin the “dead zone” region.

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Fig. 10. Schematic drawing of the DEP chip.

The cells population, which experienced positive DEP, willbe trapped between the tips of the electrodes; therefore, thearea where the particles will be in contact with the electrodes(and at the same time with the microchannel walls) will bereduced. From the velocity point of view, the maximal velocityis achieved in the channel with rectangular electrodes, and inthis case, the vertical plane that contains the maximal velocityvalue is not identical to the plane where most of the populationis trapped by positive DEP. For the rectangular design, thisplane is on the electrode corners.

V. 3-D DIELECTROPHORETIC CHIP DESCRIPTION

The separation method proposed in this paper is based on adielectrophoretic chip with 3-D electrodes. We described thefabrication process of the device and its application to yeastcell trapping in [6], and electrical and thermal characterizationis presented in [34], while a similar device, but with bulk andthin electrodes, was presented in [7]. An improved version ofthis chip is presented in Fig. 10. As can be observed, the thickelectrodes made from bulk silicon are sandwiched betweentwo glass dies. The electrodes’ surface forms the walls of themicrofluidic channels, and the glass die forms the ceiling andthe floor of the microchannels. Via holes were created in thebottom glass die, and a metallization connects the electrodesto the contact pads. The inlet and outlet connections to themicrofluidic channels are on the lateral sides of the chip, andthe sample is injected through classical syringe needles with adiameter of 0.41 mm.

The main steps of the fabrication process are presented inFig. 11. A 4′′ conductive silicon wafer, with a resistivity of0.005–0.01 Ω · cm and 300-µm thick, was anodically bondedto a glass wafer (Corning 7740) at 305 C with an appliedvoltage of 1000 V for 15 min [Fig. 11(a)]. In the next step[Fig. 11(b)], the patterning of electrodes was carried out. Forthe masking layer, a 2-µm-thick photoresist mask AZ7220(from Clariant) was applied on top of a 2-µm-thick PECVD-SiO2 layer (deposited on STS ProCVD system). The patterningof SiO2 layer was performed in a deep reactive ion etch-ing (RIE) inductive coupling plasma (ICP) system (AdixenAMS100_DE) using CHF3/He, while the final patterning ofsilicon electrodes was performed in a deep RIE ICP (AdixenAMS100_Si) using a classical Bosch process (in SF6/C4F8).

A small notching effect was noticed in the large expose area(inlet/outlet regions), but this phenomenon cannot affect thefunctionality of the device. A second wafer-to-wafer anodicbonding process was performed at 380 C using an appliedvoltage of 1200 V and an applied pressure of 500 N [Fig. 11(c)].Prior to the previous process step, in the top glass wafer, twochannels (400-µm wide and 200-µm deep) were etched usingan amorphous silicon mask in an HF 49%/HCl 37% solution(10/1) [35], [36]. The bottom glass wafer was thinned up to100 µm by wet etching process in the same solution[Fig. 11(d)]. The uniformity of the process was in an acceptablerange (5%). The roughness (Ra) of the generated surface afterthe wet etching process was 10 nm. Via holes were created bywet etching in the same solution through a Cr/Au (50 nm/1 µm)mask—as shown in Fig. 11(e) [37]. After removing the mask,another wet etching process of 1.5 min (equivalent to an etcheddepth of 10 µm) was performed mainly to remove the sharpnessof the edges and also to remove the nonuniform effects of thewet etching process. In this way, the risk of metallization stepcoverage issues over a sharp edge is eliminated. The metalliza-tion was performed using Cr/Au deposition. The patterning ofthe metal layer [Fig. 11(f)] was performed using an optimizedspray-coating process described in [38], with a mixture ofpositive photoresist AZ4620, methyl-ethyl ketone, and metoxy-propyl acetate.

An image with the fabricated DEP chip for sequential sep-aration of cells is presented in Fig. 12. The dimensions of thechip are 6 × 15 × 0.9 mm.

VI. TESTING

For testing of the system performance, two populations ofviable yeast cells and dead yeast cells were used. 100 mg ofyeast, 100 mg of sugar, and 2 mL of DI water were incu-bated in an Eppendorf tube at 37 C for 2 h. The cells werethen concentrated by centrifugation at 1000 r/min for 1 min.The supernatant solution was removed, and the cell pelletwas washed by adding 2 mL of DI water into the tube. Thecentrifugation and washing process was repeated three times.The cell culture was divided into two, and one populationwas boiled for several minutes in 5-mL phosphate bufferedsaline (PBS) (dead cells). Then, the cells were recollected bycentrifugation. Both populations were mixed and resuspendedin the separation buffer, which was a mixture of PBS and DIwater. The conductivity of the separation buffer was adjusted toabout 20 µS/cm−1 using NaOH. The final concentration of thecells was 107 cells/mL.

A function generator and a linear amplifier were usedfor drive signal generation of the dielectrophoretic chip. Thesuspension with cell populations was injected into the chip[Fig. 13(a)], and another buffer solution was prepared forremoval of the population that will be trapped.

The drive signal was increased from 0 to 25 V peak topeak gradually. The signal frequency was in the range of20–100 kHz. The trapping of the two populations by positiveand negative DEP was achieved for 20 V (peak to peak), asshown in Fig. 13(b). A stable equilibrium cell concentrationpattern was achieved in around 30 s. After separation of the

YU et al.: FIELD-FLOW CELL SEPARATION METHOD IN A DIELECTROPHORETIC CHIP WITH 3-D ELECTRODES 1127

Fig. 11. Main steps of the fabrication process: (a) anodic bonding, (b) deep RIE process, (c) SU8 bonding, (d) glass thinning, (e) via holes etching, and(f) metallization.

Fig. 12. DEP chip for sequential separation of cell populations.

Fig. 13. Yeast cell separation using sequential field-flow separation method.

population, a fresh buffer solution was flown through to col-lect the population that expresses positive DEP. The flow rateof 0.05 mL/min was assured by syringe pump (Cole Palmer

Fig. 14. Optical image with the ratio between dead (red color) and living(green color) yeast cells (a) before insertion of the solution in the DEP deviceand (b) after the separation process.

749000). The image in Fig. 13(c) shows the microchannel afterremoving the population trapped by positive DEP. After therelease of the electric field, the second population was alsoremoved, injecting a fresh buffer at a speed of 0.5 mL/min.

In order to test the efficiency of the DEP device, yeastviability was determined using the live/dead yeast cell viabilitykit from Molecular Probes (Invitrogen) following the methodas described in the instruction manual. Briefly, yeast cells(∼106 cells/mL) were stained with 20 µm of FUN 1 cell stainin sterile solution containing 4% D-(+)-glucose and 10 mMof Na-HEPES (pH 7.2). The cell suspension was then mixedthoroughly and incubated in the dark for approximately 1 h toallow sufficient amount of stain to diffuse into the cytoplasmand nucleus of the cells. After this, 10 µL of the stainedyeast suspension was loaded into the cell counting chamberof the hemacytometer. The cells were then observed with anOlympus IX71 microscope using the blue fluorescent cubewith an excitation wavelength of about 480 nm and emissionwavelength of > 520 nm for the FUN 1 cell stain.

The ratio of live to dead yeast cells was then determinedbefore passing the cell suspension into the DEP chip. Theinitial ratio between the percentage of live and dead yeast was42%/58% [an image is presented in Fig. 14(a)]. With the devicepowered at an ac voltage of 20 V, at a frequency of 20 kHz, thecell suspension was then pumped into it. After the separation

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process, the proportions of live to dead yeast cells were notedusing the fluorescence imaging technique together with thehemacytometer. As observed in Fig. 14(a), the ratio betweenliving and dead cells changes drastically in such a way thatthe percentage of the dead cells increases (up to 80%), whilethe percentage of living cells decreases (around 20%). Thisproved clearly that the separation of the two populations canbe achieved. Surely, the geometry of the electrodes and thegeometry of the microfluidic channel can be further optimized.

VII. CONCLUSION

A new sequential field-flow separation method in a DEP chipwith 3-D electrodes has been presented. A characteristic of theDEP device presented in this paper is that the geometry of thedefined electrodes is also the geometry of the microfluidic chan-nel’s lateral walls. This method successfully uses the variationof the geometry of the microfluidic channel that, at the sametime, generates the gradient of the gradient of the electric fieldto remove the population trapped by positive DEP, while thesecond population remained trapped in the velocity “dead zone”of the microfluidic channel. The removal of the electric fieldenables to the second population to be collected at the outlet.

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YU et al.: FIELD-FLOW CELL SEPARATION METHOD IN A DIELECTROPHORETIC CHIP WITH 3-D ELECTRODES 1129

Liming Yu was born in Zhejiang Province, China,in 1976. He received the B.S. degree and M.S. degreefrom Tsinghua University, Beijing, China, in 1999and 2002, respectively, and the Ph.D. degree fromthe National University of Singapore, Singapore,in 2007.

He is currently with the Institute of Bioengineer-ing and Nanotechnology, Singapore, and also withthe National University of Singapore. His researchinterests include MEMS technologies, bio-MEMS,and dielectrophoresis.

Ciprian Iliescu was born in Bucharest, Romania, in1965. He received the B.S. and Ph.D. degrees fromthe Polytechnic University of Bucharest, Bucharest,in 1989 and 1999, respectively.

He has more than 17 years of working experi-ence in microfabrication. While pursuing his Ph.D.degree, he was with Baneasa S.A. (IC company),Bucharest, where he was involved in the design andfabrication of pressure sensors. From 1997 to 2000,he collaborated with the Institute for Microtechnolo-gies, Bucharest, on projects related to magnetic sen-

sors. From 2001 to 2003, he was a Postdoctoral Fellow with the MicromachinesCenter, Nanyang Technological University, Singapore, where he was involvedin projects related to microphone, wafer level packaging of MEMS devices,and RF microrelay. Currently, he is a Senior Research Scientist with theInstitute of Bioengineering and Nanotechnology, Singapore. He is the authoror coauthor of more than 140 papers published in journals and conferenceproceedings. His current research projects are related to dielectrophoresis,electrical characterization of cells by impedance spectroscopy, transdermaldrug delivery using microneedles array, and microfabricated dialysis system.

Guolin Xu was born in Guangxi Province,China. He received the B.S. degree in mecha-tronics from Tsinghua University, Beijing, China,in 1992 and the M.S. degree in MEMS fromthe National University of Singapore, Singapore,in 1999. He is currently working toward thePh.D. degree at Nanyang Technological University,Singapore.

From 1992 to 1998, he was with Tsinghua Univer-sity, working on automation control. From 2000 to2002, he was with PBA Systems Pte. Ltd., Singapore.

He joined the Institute of Bioengineering and Nanotechnology, Singapore, in2002 as a Research Officer. His research interest includes microfluidic base rarecell isolation, biosample preparation, and lab-on-a-chip system using MEMStechnology.

Francis E. H. Tay received the Ph.D. degreefrom the Massachusetts Institute of Technology,Cambridge, in 1995.

He is currently an Associate Professor with theDepartment of Mechanical Engineering, Faculty ofEngineering, National University of Singapore. He isthe Deputy Director (Industry) of the Centre of Intel-ligent Products and Manufacturing Systems, wherehe takes charge of research projects involving the in-dustry and the Centre. He was the Founding Directorof the Microsystems Technology Initiative and had

established the microsystems technology specialization. He is also the MedicalDevice Group Leader with the Institute of Bioengineering and Nanotechnology,Singapore. He was the Technical Advisor in the Micro and Nano SystemsLaboratory, Institute of Materials Research Engineering, Singapore. He is alsothe Principal Investigator for several Agency for Science, Technology, andResearch projects. The most recent one is the “MEMSWear: IncorporatingMEMS Technology Into the Smart Shirt for Geriatric Healthcare,” which waswidely published by the local and overseas media and well received by thepublic. His research areas are MEMS, biotechnology, nanotechnology, andwearable devices.