Microelectronic Engineering 97 (2012) 369–374

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Microelectronic Engineering

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The quadrupole microelectrode design on a multilayer biochipfor dielectrophoretic trapping of single cells

S. Noorjannah Ibrahim a,⇑, Lynn Murray a, Volker Nock a, John J. Evans b, Maan M. Alkaisi a

a The MacDiarmid Institute of Advanced Materials & Nanotechnology, Department of Electrical and Computer Engineering, University Of Canterbury, Christchurch, New Zealandb The MacDiarmid Institute of Advanced Materials & Nanotechnology, Department of Obstetrics and Gynaecology and Centre for Neuroendocrinology, University ofOtago, Christchurch, New Zealand

a r t i c l e i n f o

Article history:Available online 28 May 2012

Keywords:Dielectrophoretic trappingQuadrupole microelectrodeMultilayer biochipSingle cells trappingIshikawa cancer cell

0167-9317/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.mee.2012.04.018

⇑ Corresponding author.E-mail addresses: siti.ibrahim@pg.canterbury.ac.n

(S.N. Ibrahim).

a b s t r a c t

This paper introduces a new quadrupole microelectrode design for trapping mass loading of single cellsusing dielectrophoretic (DEP) force. The DEP force profiles generated by the pattern which represent trap-ping regions on the biochip platform, were studied using finite element software Comsol Multiphysicsv3.5a. Arrays of the quadrupole microelectrode were patterned on a multilayer structure called sand-wiched insulator with back contact (SIBC) biochip platform, fabricated using photolithography technique.This platform consists of a 10 nm Nickel–Chromium (NiCr) and 100 nm Gold (Au) top electrode, 5 lmthick SU-8 2005 insulation layer and a bottom layer of 10 nm NiCr and 100 nm Au called the back contact.Access to the back contact is achieved via a microcavity located at the center of the quadrupole micro-electrode arrangement. Together, the DEP forces generated from the quadrupole microelectrode andthe microcavities are used to anchor single cells and maintain cell positions. The quadrupole designwas tested with polystyrene latex microbeads, and Ishikawa cancer cells suspended in media and flowedthrough microfluidic channels made from PDMS. Single cell trapping becomes evident when both quad-rupole microelectrode and back contact are biased using AC potentials of different phases. Experimentresults show that the quadrupole microelectrode trapped 22 percent of Ishikawa cancer cells and 17 per-cent of the polystyrene microbeads.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

A lab-on-chip (LOC) device with trapping capability can benefitlife science studies and is useful for real-time observation of singlecell responses to chemical stimulus. For example, trapping singlecells at specific locations allows systematic cell addressing andanalysis. It provides more control over labeling of individual cellsfor comparison purposes. Furthermore, successful trapping of sin-gle cells is also useful for studying cell morphological structureusing Bioimprint {Alkaisi, 2007 #1090} technique. In this study, acell manipulation technique known as the Dielectrophoretic(DEP) force is used for trapping single cells. DEP is very versatileand can trap single cells through various microelectrode designs[2,3] or by using traps and/or microwells [4–6]. Theoretically,DEP is the force exerted on cells caused by cell polarization thattranslates into cell movements. The average DEP force for sphericalparticle or cell is described as [7]:

< FDEP >¼ 2pr3emRejfcmðxÞjrEj2ðr;xÞj ð1Þ

ll rights reserved.

z, noorjannah@iium.edu.my

where r is the cell radius, x is the angular frequency and em is thesurrounding medium permittivity. From (1), cell translationalmovements are proportional to the »E2 (the gradient of non uni-form electric fields) while, the polarization direction is due to thedielectric properties of cell membrane and suspending medium.In order to simplify polarization calculations on cells, an equivalentmodel shown in Fig. 1(a) is used. In Fig. 1(a), cell polarization can bedescribed by the Clausius–Mossotti (CM) factor fcm (x) as:

½fcmðxÞ� ¼e�p � e�me�p þ 2e�m

ð2Þ

where the e�p ¼ ep � j rp

x and e�m ¼ em � j rmx represent the complex

permittivity of both particle and suspension media. Usually, theCM factor will have values between +1.0 and �0.5. Cell manipula-tion is categorized as nDEP (negative DEP) when the CM value isless than zero and cells are directed to the low electric field regionson the LOC [1]. Meanwhile, the pDEP (positive DEP) occurs whenthe CM value is more than zero and cells are directed to the highelectric field regions on the LOC. These distinct operation regionscan be used to trap cells in a controllable manner.

Microelectrode design plays an important role in cell move-ments and trapping on LOC or biochip. Various microelectrodedesigns can be used on biochip to accommodate the degree of

Fig. 1. (a) The equivalent model of the multi-shell model in (b). The equivalent model uses the homogeneous dielectric effective permittivity e�p .

370 S.N. Ibrahim et al. / Microelectronic Engineering 97 (2012) 369–374

electric field non-uniformity, size of particles and type of physicalmanipulations required. The four (quadrupole) and the eight (octu-pole) electrode arrangements for example, are useful in single celltrapping as they create higher concentrations of »E2 at the centerof electrode arrangement than the dipole electrode [8,9]. These»E2

concentrations manifest into strong DEP holding force. However,excessive exposure to electric fields can deteriorate cell viabilityon the biochip. Therefore, a microcavity or cell trap can reducethe DEP holding force dependency on»E2 but still maintain cellpositioning [4].

Here, we developed a new quadrupole microelectrode design totrap single cells during mass loading of cells inside a microfluidicchannel. The microfluidic channel regulates fluids flow i.e. cellmedia on the biochip. The new design consists of arrays of quadru-pole microelectrode and microcavity fabricated on a multilayerstructure called sandwiched insulator with back contact (SIBC)platform. In the microfluidic channel, DEP force generated fromthe quadrupole microelectrode is used to attract cells towardsthe microcavity. Meanwhile, DEP force generated by the back con-tact is used to maintain cell positioning even when the quadrupolemicroelectrode is not biased. Cell movements to the microcavityare controlled by the AC potentials connected to the quadrupolemicroelectrode and the back contact. The multilayer structure de-sign i.e. the SIBC platform, was fabricated using standard photoli-thography technique while the microfluidic channel wasfabricated using the softlithography technique. This paper de-scribes details of the quadrupole microelectrode design and fabri-cation including the simulations of DEP profile using ComsolMultiphysics v3.5a software.

2. Design and methodology

A quadrupole electrode trap is established by positioning fourelectrodes towards a common point [10,11] but adjacent elec-trodes (of the four electrodes) has to be connected to AC potentialsthat are 180� out of phase from each other [5] or to AC potentials ofdifferent phases. When array of quadrupole microelectrode is re-quired on biochip to trap mass single cells, the needs to isolate be-tween adjacent electrodes of the quadrupole arrangementincreases the complexity of fabrication process. Therefore, in thenew quadrupole design of Fig. 2(a), the adjacent electrodes areconnected together instead of having separate electrical connec-tions as described in [5]. This design allows AC potentials of differ-

ent phases to be connected between opposite electrode pairs and amicrocavity (cell trap) to be located at the center of the electrodearrangement.

Inside a microfluidic channel, there are other forces such as theBrownian motions, the electrohydrodynamics force and etc., whichcan affect the DEP holding force on cells. To overcome this prob-lem, the microcavity has to generate DEP force from within themicrocavity itself. Therefore, the microcavity needs to be an elec-trode and structured on a different layer from the quadrupole pairsshown by Fig. 2(b). Here, an insulator layer made of SU-8 2005 isused to separate the back contact and the quadrupole microelec-trode. With the new electrode arrangement and the multilayerstructure, various biasing combinations for trapping single cellscan be implemented on the biochip. In other words, the 180� phasedifference to create high DEP holding force can also be establishedbetween opposite microelectrode pairs or between the quadrupolemicroelectrode pair and the bottom of microcavity (back contact).This also means, the DEP force can still be exerted on trapped cellsthrough the back contact and the quadrupole microelectrode canbe left unbiased (floating).

One advantage of this alternate biasing between layers is to re-duce damages caused by electric field in order to retain cell posi-tioning. To realize a successful trapping event and single celltrapping protocol, it is important to identify the DEP trapping zoneon the platform. Numerical studies on the new quadrupole designwere conducted using Comsol Multiphysics v3.5a software todetermine the trapping zones and the supplied AC signals optimi-zation. With the new quadrupole design, it is expected that singlecells can be trapped by the quadrupole arrangement described in[5].

2.1. Simulations

In this study, electric fields generated by the new quadrupolemicroelectrode were simulated and DEP trapping regions wereidentified. Results were obtained by solving the Maxwell’s equa-tions in electrostatic approximations. In this approximation, thedielectric properties are ideal i.e. materials are considered to haveonly permittivity and zero conductivity. Then, the Laplace equa-tion, which relates the two-dimensional domain’s electric field dis-tribution, is simplified to E ¼ �ru, where u is the AC potentialconnected to the pattern.

Fig. 2. The new quadrupole biochip platform (a) on x-y plane and (b) on cross section of the SIBC platform.

Table 1Simulation parameters for the quadrupole microelectrode from [12].

Parameter Polystyrene microbeads

Relative permittivity 2.55Conductivity 0.5 lS/m (measured)Medium permittivity 78.5Medium conductivity 1.7 mS/m (DI water)CM factor estimated �0.4760269 (nDEP)Radius 5 lmFrequency 1 MHzPotential / 10 Vpp

S.N. Ibrahim et al. / Microelectronic Engineering 97 (2012) 369–374 371

Subsequently, studies on the supplied AC potentials configura-tion between the quadrupole electrodes and the back contact werealso conducted. AC potentials for biasing the back contact and thequadrupole microelectrode were set to have 0�, 90�, 180� and 270�degree phase different and unbiased condition (floating electrode)in order to identify the optimum working combinations. In theanalysis, it is expected that at least two well-defined maximumfields are generated to attract single cells at any given time. Param-eters used in the simulation are listed in Table 1.

2.2. Fabrications

The fabrication process for the new quadrupole microelectrodeis shown in Fig. 3(a). The back contact or bottom layer of the bio-chip is made of 20 nm NiCr and 100 nm Au layers deposited on topof a glass slide. Here, the NiCr layer was used as an adhesion pro-moter for Au to remain on the glass. Then, a 5 lm thick SU-8 2005layer was spin-coated on the substrate and arrays of 20 lm indiameter microcavity were structured. The SU-8 sandwiched layerfunctions as an insulator between the two metal layers. Mean-while, access to the back contact for biasing is allowed on the bio-chip platform through the microcavity. Subsequently, anothermetal layer that consists of 20 nm NiCr and 100 nm Au weredeposited on top of the SU-8 layer. Using a positive resist AZ1518, the final layer was patterned with four microelectrode arraysand each array consists of thirty quadrupole microelectrodes fortrapping single cells.

Then, the master microfluidic channel pattern or mould wasfabricated separately using a SU8-2100 layer on a silicon wafer.In this design straight channels are used to regulate fluids on thebiochip. Cells were introduced onto the biochip through a200 � 100 lm (width � height) microchannel made from PDMSmaterial, from a mixture of 10:1, silicone elastomer and its curingagent (Slygard 184 Silicone elastomer base & curing agent). Afterthe mixture was degassed inside a vacuum chamber, it was spreadonto the mould. Finally, the PDMS block was left on a hot plate at80 �C for 2 h to be fully cured. The microchannels block was thenmanually aligned with the biochip platform in preparation forthe DEP experiments shown in Fig. 3(c).

Fig. 3(b) shows the fabricated quadrupole microelectrode on theSIBC biochip. Each microelectrode arm is approximately40 � 80 lm (width and height) with the hyperbolic –shaped tippointing towards the central region to exert high DEP trappingforce on single cells. The microcavity is located at approximately10 lm from the quadrupole microelectrode tips.

2.3. Experiments

The quadrupole microelectrode trapping ability on the SIBCplatform was demonstrated using 10 lm polystyrene microbeads(Polyscience Inc.). Subsequently, DEP trapping on living cells wasconducted on Ishikawa cancer cells, suspended in media of variousrange of conductivity. The Ishikawa cancer cells, size between 20and 30 lm, were obtained from the Department of Obstetricsand Gynaecology, Christchurch School of Medicine, Otago Univer-sity. Measurements for the suspension conductivity were obtainedusing conductivity meter (Waterproof Cyberscan PC300) while thecell concentrations were calculated using hemocytometer. The sin-gle cell trapping was demonstrated using Ishikawa cancer cellssuspended in Eagle minimum essential media (Sigma Aldrich) di-luted 1:5 from their original concentration of 3.4 � 106 cell/mL.The suspension conductivity (reagle) for Ishikawa cells was8.04 mS/m. Meanwhile, the 1.45 � 106 cell/mL concentrationmicrobeads were suspended in sodium chloride (NaCl) media withconductivity (rNaCl) of 8.2 mS/m.

The DEP experiments setup consists of a function generator topower up the biochip, a microscope with mounted CCD camera

Fig. 3. (a) Fabrication process for the new quadrupole biochip design. (b) The fabricated quadrupole microelectrode structure. (c) The SIBC biochip and microfluidic channel.

372 S.N. Ibrahim et al. / Microelectronic Engineering 97 (2012) 369–374

to capture particle movements in microchannels and a syringepump to flow fluids into the microchannels. In order to preventany leakage from the microchannel, particles were flowed at0.5 lL/min and stopped once the microchannel was filled withfluid. Then cells were allowed to settle inside the microchannelfor about two minutes before switching ‘ON’ the function genera-tor. Using AC potentials of 10 V peak-to-peak (Vpp) with frequencyrange between 100 Hz and 5 MHz, DEP trapping and particlemovements into microcavities were observed and recorded. In or-der to highlight the back contact enhancement in trapping effi-ciency, the experiment was repeated on another platform ofquadrupole microelectrode fabricated without the presence ofthe back contact layer called the planar two-layer biochip.

Trapping efficiency of the SIBC biochip was defined as the per-centage of cells trapped by the DEP force of the total traps availableon the platform. Trapped single cells were counted after 10 minfrom when the AC potential was switched on. Then, the microchan-

Fig. 4. (a) Electric field intensities generated by the quadrupole microelectrode. High eleinside the microcavity. (b) The DEP force profiles. (c) Simulation results of the DEP traquadrupole microelectrode pairs and the microcavity.

nels were flushed with deionised (DI) water to clean and removeany residues. The experiments were repeated three times and re-sults represent the average trapping events. Results presented inthis paper were obtained from biasing combination that gave theoptimum DEP trapping force previously mentioned in the Simula-tion Results.

3. Results and discussion

3.1. Simulation results

The electric field intensity and the DEP trapping profiles areshown in Fig. 4(a) and (b).These results indicate that the regionof the strongest DEP force is proportional to the region of high elec-tric field i.e. between the microelectrode tips and the microcavityedge. There are four regions of high electric field generated by

ctric field regions occur at microelectrode tips while low electric field region occurspping force generated from combinations of AC signal superpositions between the

Fig. 5. The microbeads moved towards a microcavity in microchannels on the SIBC biochip at 5 MHz.

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the design which attract single cells towards the tips of microelec-trode. At the same time, the existence of low electric field regioninside the microcavity is useful to maintain cell positions insidethe microcavity.

In terms of AC potentials combination, results in Fig. 4(c) showthat the highest trapping force occurs when potentials of themicroelectrode pairs and the microcavity, have 180� phase differ-ence. In contrast, the weakest trapping force occurs when thereis no phase difference between the two AC potentials. Connectingthe quadrupole microelectrode with two potentials of 90� phasedifference produces the same trapping effects when connectingwith potentials that have 270� phase difference. In order to obtainoptimum results, the quadrupole microelectrode and the micro-cavity were then biased with AC potentials that have 180� phasedifference during the DEP trapping experiments. From the simula-tion results, it can be inferred that the new quadrupole microelec-trode design attracts single cells towards the centralmicroelectrode region provided the AC potentials used have differ-ent phases. Furthermore, the additional DEP holding force insidethe microcavity suggests that cells can remain inside themicrocavity.

3.2. Experiment results

Trapping single cells ability of the new quadrupole design wasdemonstrated using polystyrene microbeads and Ishikawa cancercells. Fig. 5 presents video1 segments of polystyrene microbeadsmovements towards the microcavity. Here, microbeads wereattracted to the microelectrode tips and then moved one by one intothe microcavity. Note that the quadrupole microelectrode pair isconnected to AC potential that has 180� phase difference from thepotential of the microcavity (back contact). The AC potentials createelectric fields minima inside the microcavity which attracts polysty-rene microbeads to the central electrode arrangement or nDEP.

The DEP trapping results on the SIBC biochip and the planartwo-layer biochip using the quadrupole microelectrode designare presented in Fig. 6. The results reveal that the SIBC biochipcan trap a higher percentage of single cells than the planar

1 Video of polystyrene microbeads movements into the microcavity is attachedSupplementary information.

two-layer biochip at 1 MHz using 10 Vp-p AC potentials. Note,the quadrupole microelectrode and the back contact were con-nected with AC potentials that have 180� phase differencethroughout the experiments. From the graph, the SIBC biochiptraps 22 percent of Ishikawa cells and 17 percent of polystyrenemicrobeads. Meanwhile the planar two-layer biochip managed totrap 5.56 percent of both type of particles. Apparently, DEP forceinside the microcavity which is generated by the back contact,has significant influence on trapping particle or single cells.

From Fig. 6, the SIBC biochip traps more living particles (cells)than non-living particles. One possible reason for the result is thatlives Ishikawa cancer cells have the tendency to adhere on surfacesthan the polystyrene microbeads. Therefore the multi-shell modelin Fig. 1(b) is expected to give more accurate numerical value ofDEP force provided dielectric properties of the cell membrane areknown. The results suggested that separation between lives cells

Fig. 6. Trapping results using the new quadrupole microelectrode on the SIBCbiochip and the planar two-layer biochip at 1 MHz.

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and dead cells can be conducted on the biochip. However, furtherinvestigations are needed to understand the different responses ofliving cells’ and non-living cells’ polarization on the biochip.

4. Conclusion

A new quadrupole microelectrode dedicated for trapping of sin-gle cells was designed and constructed on a multilayer LOC struc-ture called the SIBC biochip platform. In this study, successful DEPtrapping was demonstrated using polystyrene microbeads andIshikawa cancer cells. Results from simulation studies suggestedthat single cells’ movements towards cell traps are due to strongDEP holding forces generated at the four microelectrode tips ofthe new quadrupole design, whereas additional DEP force fromthe trap itself is used to maintain trapped cells’ positions. OptimumDEP trapping force occurred when the quadrupole microelectrodeand the microcavity are biased with AC potentials that have 180�phase difference.

Results from experiments also showed that the new quadrupoledesign on SIBC biochip is capable of trapping single cells. Evidenceobtained from the experiments validates the simulation resultswith 22 percent of Ishikawa cancer cells and 17 percent of thepolystyrene microbeads were successfully trapped. With furtherdevelopment to enhance trapping yield, the new quadrupolemicroelectrode has potential in facilitating study of single cellintracellular activities on a single biochip platform.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.mee.2012.04.018.

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