The Octupole Microelectrode for dielectrophoretic trapping of single cellsDesign and Simulation

S.Noorjannah Ibrahim1,2 and Maan M. Alkaisi11 The MacDiarmid Institute of Advanced Materials & Nanotechnology, Department of Electrical and Computer Engineering,

University Of Canterbury, Christchurch 8041, New Zealand 2Department of Electrical and Computer Engineering, Faculty of Engineering, International Islamic University Malaysia,

Kuala Lumpur, Malaysia email: noorjannah@iium.edu.my,siti.ibrahim@pg.canterbury.ac.nz

Abstract- Different microelectrode designs for a dielectrophoresis (DEP)-based lab-on-chip, have significant effect on the DEP force produced. Study on the microelectrode factor is essential, as the geometry and the numbers of microelectrode can influence particles and/or cells polarization. This paper presents one of the three new microelectrode designs, called the octopule microelectrode, in a study on the microelectrode factor of the DEP trapping force. The octupole pattern was constructed on a metal-insulator-metal layer structured on a Silicon Nitride(Si3N4)coated Silicon(Si) substrate. The first layer or back contact is made from a 20nm Nickel-Chromium(NiCr) and a 100nm gold (Au). Then, an insulator made of SU-8-2005 was spin-coated on the metal layer to create arrays of microcavities or cell traps. The third layer, where the octupole geometry was patterned, consists of 20nm NiCr and 100nm Au layers. The microcavities which were defined on the SU-8 layer, allows access to the back contact. Gradient of electric fields which represent the actual DEP trapping regions were profiled using COMSOL Multiphysics 3.5a software. Then, the microelectrode trapping ability was evaluated using polystyrene microbeads suspended in deionised (DI) water as the cell model. Results obtained from the experiment were in agreement with results from simulation studies where polystyrene microbeads concentrated at the trapping region and filled the microcavity.

I. INTRODUCTION The development of lab-on-chip (LOC) devices has evolved

tremendously in recent years. A LOC device with trapping capability can benefit life science studies and is useful for real-time observation of single cell responses to chemical stimulus. It provides more control over labelling of individual cells for comparison purposes. One vital question however, is how to precisely localize single cells on the LOC devices. Dielectrophoresis (DEP) is one of the cell manipulation techniques used on the lab-on-chip devices. This method utilizes the polarization effect between the dielectric properties of cell and suspension medium, and the supplied AC signals to initiate cell movements. On a DEP-based device, the DEP forces are generated by microelectrodes fabricated on the LOC platform. The fabricated microelectrodes can be customized to accommodate the

electric fields non-uniformity, the particle sizes and the type of physical manipulations required. Some examples of the common microelectrode designs for DEP device are planar [1], quadruple [2], interdigitated (IDE) [3] and planar ring arrays [4-6].

Fig. 1: a)The schematic of the octupole microelectrode pattern for trapping single cells. b) The cross section of the

microelectrode structure.

This paper presents a new microelectrode design for trapping single cells, called the octupole microelectrode. The motivation behind this work is to design a LOC device that is capable of trapping single cells, with minimum chemical interventions on the cells physical conditions. As illustrated in Fig. 1(a), the octupole design comprises of a pair of three-arm electrodes, two floating electrodes and a microcavity. The octupole microelectrode is structured on a multilayer LOC platform. As shown in Fig.1(b), the microcavity is made of an insulating material, SU-8-2005. The microcavity allows access to the back contact electrode at the bottom layer and acts as the cell trap. Meanwhile, the three-arm electrode and two floating electrode are patterned on the uppermost metal layer.

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Numerical simulations were conducted using COMSOL Multiphysics 3.5a software, to identify the DEP trapping regions on the device. In the simulations, electric field distributions that represent the actual DEP forces were solved in two-dimensional (2D) geometric domains with time-dependent electrostatics model mode. Results show that the V-shaped of octupole microelectrode tips produce high gradient of electric fields and generate strong DEP trapping forces on the LOC. Meanwhile the back contact creates a low DEP trapping force to anchor cell inside the trap. Hence, the trapping regions of the octupole design are from cumulative DEP forces generated from the electrode tips and the back contact. After fabrication, the microelectrode functionality was assessed using polystyrene microbeads suspended in DI water as the cell model. Experimental results obtained were in agreement with simulation results conducted earlier.

II. THE MICROELECTRODE DESIGN The DEP force is described as polarization effects on cell

due to non-uniform electric fields that manifests into translational movements. The octupole microelectrodes are designed to produce strong DEP force governed by [7] :

= []|| (1)

where Hm, R, |E|2 and Re[K(Z)] denote the medium permittivity, the cell radius, gradient of electric fields and the real part of Claussius-Mosotti (CM) factor. The CM factor is frequency dependent and is defined by:

(Z) = (2)

where H*p/m is the particle complex permittivity of cell and medium and defined as:

/ = / (3)

where V is the conductivity,H is the permittivity of cell or medium respectively, Z is the angular frequency and j =

1 . Cell movements due to DEP force can be categorised into positive DEP (pDEP) i.e., movements towards high electric fields intensity or negative DEP (nDEP) i.e., movements towards low electric fields intensity. As described in (2), due to CM factor dependency on the frequency of AC signals, particle trapping is in nDEP polarization when K(Z) 0.

From (1), the gradient of electric fields or |E|2 is proportional to the DEP force strength. Hence, in order to generate strong DEP forces, the designed microelectrode has to create high gradient of electric fields E2, on the LOC platform. This has points out the reason for designing eight electrode tips surrounding a microcavity, as the E2

concentrations from the octupole microelectrode manifest into strong DEP holding force. However, excessive exposure to electric fields can deteriorate cell viability on the LOC which leads to cell membrane rupture or cell death. Therefore, two floating electrodes are incorporated in the arrangement to reduce the electric field concentrations. The two floating electrodes generate induced electric fields [8] which are useful to reduce total amount of DEP forces exerted on cells and can minimize damages imposed on the cells.

III. METHODS AND MATERIALS A. Simulation The DEP forces generated by the octupole geometry were

profiled using finite element analysis software, COMSOL Multiphysics 3.5a. Results were obtained by solving the Maxwells equations in electrostatic approximations i.e., the dielectric properties were in ideal condition where materials were considered to have only permittivity and zero conductivity. The Laplace equation was simplified to = ,whereM is the supplied AC signals and therefore, the electric fields can be derived from:

D = . (4) where D, H and E denote as the electric displacement fields,

the permittivity of suspension medium and the electric fields respectively. Due to the multilayer structure, studies on the Mconfiguration between the octupole pattern and the microcavity with respect to its phase, were also conducted to estimate the performance of the microelectrode. Table 1depicts parameters used in the numerical simulations.

TABLE1 PARAMETERS USED IN THE OCTUPOLE MICROELECTRODE SIMULATIONS [9].

Parameter Polystyrene Microbeads

Relative PermittivityRelative ConductivityMedium PermittivityMedium ConductivityK calculated Radius Frequency Potential (M)Phase difference

2.550.5 S/m 78.51.7mS/m (DI water)-0.4760269 (nDEP)5m1MHz10Vpp0q,90q,270q,180q

B. Fabrication Arrays of the octupole microelectrode were fabricated using

photolithography technique on a metal-insulator-metal layer platform of Fig.1(b). The back contact or the bottom electrode is made of a 20nm NiCr and a 100nm Au deposited on top of a Si3N4 coated silicon substrate. Then, SU-8-2005 (a negative photoresist) was spin-coated on top of the back contact creating an approximately 5m thick layer. Finally, the third layer consists of a 20nm NiCr and a 100nm Au, was deposited

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on top of the SU-8 layer. As illustrated in Fig.1(b), the sandwiched SU-8 layer separates the back contact from the microelectrode arrays on the third layer but allows access to the back contact only through arrays of microcavity. Using a positive resist AZ1518, the third layer was patterned with arrays of the design. Any unwanted area was removed by wet etching after the AZ1518 resist exposure and development. Then, the photoresist was stripped using photoresist remover leaving behind arrays of the microelectrode pattern. Fig.2 illustrates the octupole microelectrode fabricated on 15mmx15mm Si3N4 coated Si substrate.

Fig.2:The fabricated 15mmx15mm LOC with arrays of octupole microelectrode.

C. Experiments The octupole microelectrode trapping ability were tested

using polystyrene microbeads (Polyscience Inc.) of 9-10 Pmsize, suspended in deionised (DI) water as the cell model. Before testing, the LOC of Fig.2 was placed on a glass holder for electrical connections. Then, a 10mmx10mmx1mm (Lenght x Width x Height) square spacer made of PDMS material was placed on top of the biochip to contain the suspension medium before dispersing the polystyrene microbeads using a micropitte. Then, a glass cover slip is placed on the spacer to level the medium.

The biochip was tested using 10 Vpp AC signals of various frequency (50 kHz to 5 MHz) supplied by a function generator(HP3312A). After the function generator was turned ON, movements of the suspended microbeads were observed using a microscope (Nikon eclipse 80i). Then, the movements were recorded using a camera (Nikon digital Sight DS-U1). Each video was recorded for 3 seconds every 3 minutes controlled by the ACT-2U software. Finally, the video segments werecompiled using commercial video editing software before thoroughly studied.

IV. RESULTS AND DISCUSSION D. Simulation Results Fig.3 illustrates the electric fields and the DEP force profiles

generated by the octupole microelectrode. The results were

obtained by setting the M between the microcavity and the microelectrode pattern with 180q phase different. The electric field intensities in Fig.3(a), indicate that the microcavity generate the low electric field region while the high electric field region occurs at the tips of microelectrodes. The DEP trapping region occurs at the central region of the octupole microelectrode i.e., surrounding the microcavity. Note that,DEP forces are also generated in between the floating electrodes pair and the three-arm electrodes that indicate the presence of induced electric fields generated by the floating electrodes.

Table 2 presents the DEP force values calculated between microelectrode tips and the microcavity edge i.e., the trapping region. The results show that the differences of M between microelectrode pairs and microcavity can affect the trapping region. The maximum calculated DEP forces occurred when the back contact is 180q out of phase from the microelectrode pattern on the third layer. Meanwhile, the minimum DEP force was resulted from connecting M of the same phase for both microcavity and the microelectrode pair. Interestingly, if the microcavity is not connected with M, the DEP force is stronger than connecting the microcavity with the same M of the same phase with the microelectrode pair.

TABLE 2 THE ESTIMATED DEP FORCE EXERTED ON MICROBEAD USING M OF DIFFERENT PHASE

CONNECTED TO THE BACK CONTACT.

TABLE 3 TRAPPING RESULTS USING THE OCTUPOLE MICROELECTRODE .

Frequency of M

50kHz

100kHz

250kHz

500kHz

1MHz

2MHz

5 MHz

Trapping in the microcavity

No No No No No Yes No

E. Experimental ResultsThe functionality test of the octupole microelectrode was

conducted using polystyrene microbeads. In the experiments, the microelectrode was supplied with M that has 180q phase difference from the M of the back contact. Table 3 depicts the results for trapping microbeads using the octupole design. Successful trapping of polystyrene microbead inside the microcavity was observed at 2MHz indicating nDEP force. Fig. 4 illustrates microbeads movement toward octupole trapping region at 2 MHz. During the first 60 seconds, microbeads moved toward the central region of the octupole microelectrode. The movements are due to the DEP forces generated at the tips of microelectrodes. At t= 300s of Fig.5(b) a microbead was successfully trapped inside the microcavity.

Back contact (M)

DEP Force (mean)(N)

No connection 2.75x10-14

No phase shift 2.94x10-1890 5.51x10-14

180 1.15x10-13

270 5.51x10-14

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Fig. 3:a) Regions of high and low electric field intensity generated by the octupole microelectrode. b) The DEP trapping region.

The microbeads however, tend to concentrate at the trapping region if the DEP forces were exerted for a longer period. Results from the experiment have demonstrated the octupole microelectrodes trapping ability and the presence of DEP trapping regions that surrounding the microcavity. Overall,microbeads movements to the trapping region are slow, due tothe low conductivity (V) of the DI water with measured V =1962 S/m. From the results, it can be deduced that with theexistence of high DEP force at the trapping region, single cellscan be held properly and stay inside the microcavity.

V. CONCLUSION Precise localization of cell on lab-on-chip devices can be

realized using the DEP force. In this paper, the octupole microelectrode on the multilayer structured LOC has demonstrated its ability to trap single cells using polystyrene microbeads suspended in DI water. Connecting the SIBC biochip with AC signals that has 180 phase difference between microelectrode pair and microcavity, has resulted with successful cell trapping at 2 Mhz.

Fig. 4: Movement of microbeads toward the trapping region of the octupole microelectrode at 2MHz.

REFERENCE[1] M. R. Tomkins, A. W. Jeffery, and A. Docoslis, "Observations and

analysis of electrokinetically driven particle trapping in planar microelectrode arrays," The Canadian Journal of Chemical Engineering, vol. 86, p. 609, 2008.

[2] L. F. Hartley, K. V. I. S. Kaler, and R. Paul, "Quadrupole levitation of microscopic dielectric particles," Journal of Electrostatics, vol. 46, pp. 233-246, 1999.

[3] M. Hywel, et al., "The dielectrophoretic and travelling wave forces generated by interdigitated electrode arrays: analytical solution using Fourier series," Journal of Physics D: Applied Physics, p. 2708, 2001.

[4] E. G. Cen, et al., "A combined dielectrophoresis, traveling wave dielectrophoresis and electrorotation microchip for the manipulation and characterization of human malignant cells," Journal of Microbiological Methods, vol. 58, pp. 387-401, 2004.

[5] L. C. Hsiung, et al., "A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells," Biosensors and Bioelectronics, vol. 24, pp. 869-875, 2008.

[6] L. Qian, et al., "Integrated planar concentric ring dielectrophoretic (DEP) levitator," Journal of Electrostatics, vol. 55, pp. 65-79, 2002.

[7] H. A. Pohl, Dielectrophoresis: Cambridge University Press, 1978. [8] S. Golan, et al., "Floating electrode dielectrophoresis,"

Electrophoresis, vol. 27, pp. 4919-4926, 2006. [9] M. P. Hughes, Nanoelectromechanics in engineering and biology:

Boca Raton, Fl: CRC Press, 2003.

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