2014 Hyuk Rok Gwon1 Suk Tai Chang2 ∗ Chang Kyoung Choi3 ∗ Jung-Yeul Jung4 ∗ Jong-Min Kim1 Seong Hyuk Lee1 ∗ 1 School

of Mechanical Engineering, Chung-Ang University, Heuksuk-dong, Dongjak-gu, Seoul, Korea 2 School of Chemical Engineering and Materials Science, Chung-Ang University, Heuksuk-dong, Dongjak-gu, Seoul, Korea 3 Department of Mechanical Engineering-Engineering Mechanics, Michigan Technological University, MI, USA 4 Technology Center for Offshore Plant Industries, Korea Research Institute of Ships and Ocean Engineering, KIOST, Daejeon, Korea

Received November 14, 2013 Revised April 1, 2014 Accepted April 3, 2014

Electrophoresis 2014, 35, 2014–2021

Research Article

Development of a new contactless dielectrophoresis system for active particle manipulation using movable liquid electrodes This study presents a new DEP manipulation technique using a movable liquid electrode, which allows manipulation of particles by actively controlling the locations of electrodes and applying on–off electric input signals. This DEP system consists of mercury as a movable liquid electrode, indium tin oxide (ITO)-coated glass, SU-8-based microchannels for electrode passages, and a PDMS medium chamber. A simple squeezing method was introduced to build a thin PDMS layer at the bottom of the medium chamber to create a contactless DEP system. To determine the operating conditions, the DEP force and the friction force were analytically compared for a single cell. In addition, an appropriate frequency range for effective DEP manipulation was chosen based on an estimation of the Clausius–Mossotti factor and the effective complex permittivity of the yeast cell using the concentric shell model. With this system, we demonstrated the active manipulation of yeast cells, and measured the collection efficiency and the dielectrophoretic velocity of cells for different AC electric field strengths and applied frequencies. The experimental results showed that the maximum collection efficiency reached was approximately 90%, and the dielectrophoretic velocity increased with increasing frequency and attained the maximum value of 10.85 ± 0.95 ␮m/s at 100 kHz, above which it decreased. Keywords: Active particle manipulation / Contactless dielectrophoresis / Movable liquid electrode DOI 10.1002/elps.201300566

Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction To date, DEP has been one of the important manipulation techniques in microfluidic systems. It has been widely used to manipulate particles and cells with different dielectric properties. DEP force also depends on a particle shape, size, and frequency of electric fields. In addition, DEP can manipulate and sort biological cells without a biochemical labeling or other bio-engineered tags, and any contact materials to solid

Correspondence: Professor Seong Hyuk Lee, School of Mechanical Engineering, Chung-Ang University, Heuksuk-dong, Dongjakgu, Seoul, Korea E-mail: [email protected]

Abbreviations: cDEP, contactless dielectrophoresis; CM, Clausius—Mossotti; iDEP, insulator-based dielectrophoresis; ITO, indium tin oxide; ODEP, optically induced dielectrophoresis; Tw-DEP, travelling wave dielectrophoresis

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surface of particles and cells [1, 2]. However, there are intrinsic limitations in conventional DEP techniques involving on-chip metal electrodes. Metal electrodes can be corroded by direct contact with fluid media, and any degradation due to metal corrosion, bubble formation, and fouling, substantially affects sensing accuracy and repeatability [3]. Secondly, it is difficult to manipulate particles and cells actively by using conventional DEP techniques because the particles and cells are collected or repelled around the patterning of metal electrodes only. To overcome these limitations of conventional DEP technology, many researchers have developed improved technologies. In particular, a contactless DEP (cDEP) technique was developed to resolve corrosion and fouling problems in metal electrodes [4]. An insulator-based electrodeless

These authors have contributed equally to this study.

Colour Online: See the article online to view Fig. 2 in colour.


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Figure 1. The operating concept of the liquid-drop electrode DEP technique with (A) AC field off; (B) AC field on.

dielectrophoresis (iDEP) was also developed to prevent direct contact between electrodes and solutions [5]. Lapizco-Encinas et al. [6] also demonstrated an iDEP for the manipulation of bacteria and inert particles, while Sano et al. [7] fabricated microfluidic devices based on iDEP and cDEP. However, it is difficult to use iDEP and cDEP to manipulate particles and cells actively owing to the use of prescribed patterns to collect particles. Alternatively, other DEP techniques such as stepping electric field DEP [8], travelling wave DEP (Tw-DEP) [9], and optically induced DEP (ODEP) [10, 11] have been proposed. ODEP facilitates active manipulation of particles and cells, but heat or bubble generation problems may occur owing to the focused light source. Although there have been many efforts to manipulate particles or biological cells in labon-a-chip (LOC) systems, and active manipulation of particles still remains a challenging task. In active cell manipulation, cells can be manipulated on any desired local positions without prepatterned metal electrodes at a desired time. This study suggests a new DEP system for effective particle manipulation by actively controlling the location of an electrically conductive liquid metal, which is used as the electrode instead of patterned surface electrodes. A newly designed microfluidic DEP device was fabricated to demonstrate this new technique for manipulating particles and cells. To determine the optimal DEP operating conditions, the theoretical complex permittivity of various yeast cells was calculated; it was required to calculate the real and imaginary terms of the corresponding Clausius–Mossotti (CM) factor. A new squeezing method was also introduced to make a thin PDMS layer between the electrode path and the medium. In experiments, DEP velocities and collection efficiencies were measured at different AC electric field strengths and frequencies.

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2 Materials and methods 2.1 Design and fabrication of a DEP system The prominent advantage of the new DEP system is active manipulation with the movable liquid drop. The operating concept of the new DEP system for active manipulation (positive DEP) is illustrated in Fig. 1. The liquid drop moves to desired position by operating syringe pump as shown in Fig. 1A. By applying an AC voltage to top and bottom ITO-coated glass, nonuniform electric fields can be generated inside the sample chamber. The liquid drop acts as the electrode, then, suspended particles are collected to the liquid electrode, which is located at a desired position as shown in Fig. 1B. The new DEP system consists of indium tin oxide (ITO)coated glasses at its top and bottom, SU-8 microchannels to guide movable electrodes, and a PDMS sample chamber (Fig. 2A). ITO-coated glasses are chosen because of optically transparent and electrically conductive characteristics. In order to make a thin insulating layer between the sample chamber and the microchannel, glass, photoresist, and PDMS can be possibly used. Among them, PDMS was chosen for fabricating a thin layer between the medium chamber and the microchannel due to its inherent characteristics. It is because PDMS is an elastomer widely used in microfluidic devices due to its low cost and ease of fabrication, while electrical resistivity of PDMS is relatively similar to that of glass and photoresist [12, 13]. A sample chamber was built by replicating a negative photoresist (SU-8, MicroChem, Newton, MA, USA) patterning mold. The microchannels for the electrodes were fabricated with a negative photoresist (SU-8 2050, MicroChem). The sample chamber and the channels were fabricated by using conventional photolithography



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Figure 2. (A) Schematic of a liquid electrode DEP chip, (B) proposed hot pressing method for fabricating a PDMS structure with a thin layer, (C) fabricated DEP chip, and (D) cross-sectional image of a sample medium chamber with a thin PDMS layer.

processes [14–16]. Note that a hot press process (25 kPa & 100°C) was employed to replicate the PDMS thin layer shown in Fig. 2D and the chemical gluing method [17] was used to bond the SU-8 channels and the PDMS sample chamber. This new bonding strategy is realized by anchoring amine-terminated silane on one PDMS substrate and epoxyterminated silane on the other PDMS substrate via silane coupling reaction followed by amine-epoxy bond formation. Then, conductive adhesive copper films (1181, 3M, St. Paul, MN, USA) were taped to the top and bottom ITO-coated glass surface for applying AC voltages. To achieve an airtightened connection between the plastic tubing and the PDMS channel, PDMS connection patches were prepared using a customized punching tool with a gage-16 grinded blunt needle (5 mm inner diameter). Flexible microbore plastic tubes (AAQ0413, Saint-Gobain, France) were inserted into each port. The T-shaped channel was fabricated to generate movable liquid electrode drops of mercury (261017, Sigma Aldrich, St. Louis, MO, USA) surrounded by air were positioned by using two syringe pumps (TJ-3A and LSP04–1A, Longer Pump, Boonton, NJ, USA). The height of the T-shaped channel was 100 ␮m. Mercury was chosen because of its easy handling, small contact angle change due to electro-wetting,  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and nonevaporation at this experimental condition. Alternatively, other conductive liquids (such as Eutectic GalliumIndium) can be used, considering that mercury is a hazardous material. The generation of the mercury drop of 8 nL in the T-shaped channel and its movement are shown in Fig. 3.

2.2 Theoretical analysis 2.2.1 Comparison between DEP force and friction force on a single spherical particle When a liquid electrode is moved in the channel, particles may migrate to the liquid electrode. However, the particles may also exhibit frictional resistance to such migration; therefore, a competitive relationship clearly exists between these two forces. Therefore, the particle can be only moved when the DEP force is larger than the friction force. In order to find the appropriate DEP force (operating conditions) for the present system, it is necessary to compare the two forces on the particles. Supporting Information Fig. 1A illustrates the friction force and the DEP force in the x-direction for a spherical particle in a medium. Other forces, such as the www.electrophoresis-journal.com

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Figure 3. (A) Generation of liquid-drop in a microchannel with T-shaped intersection and (B) movement of a liquid drop in a microchannel with controlled syringe pump.

electrical double layer interaction force, van der Waals force, and Brownian random force are possibly present but the magnitudes of these forces are very small compared to the DEP and friction forces [18]. In general, the dielectrophoretic force acting on a spherical particle in a nonuniform electric field is described by [1]: FDEP = 2␲a 3 ε0 εm Re{K (␻)}∇|E RMS |2 ,


where ε 0 and ε m are the vacuum permittivity (8.85 × 10−12 F/m) and unitless relative permittivity, respectively. a is the radius of the particle, ERMS is the root mean square electric field, and ␻ is the angular field frequency. Re{K(␻)} indicates the real part of CM factor where K(␻) is the particle polarizability that can be written as: K (␻) =

∗ ε ∗p − εm ∗ ε ∗p + 2εm

ε ∗p



∗ εm

where and are the complex permittivities of the particle and the medium, respectively. The complex permittivity ε ∗ can be expressed in terms of the relative permittivity, electrical conductivity, and radial frequency of the electric fields, i.e. ε ∗ = ε − ␻␴ i. Electro-rotation means the rotation of polarized particles suspended in a liquid owing to an induced torque in a rotating electric field. The torque is given by [1]: ⌫ = −4␲εm a 3 Im{K (␻)}|E RMS |2 ,


where Im{K(␻)} is the imaginary part of the CM factor. Meanwhile, the friction force on a spherical particle Ff can be expressed by: F f = ␮ f mp g ,


where ␮f , mp , and g are the friction coefficient, the particle mass, and the gravitational acceleration, respectively. For the PDMS surface in the wet state, the friction coefficient is well known as 2.5 [19]. As seen in Eq. (4), the friction force should be determined when the particle is stationary. At this condition, the friction force reaches its maximum value. Meanwhile, the DEP force in the x-direction (FDEP ,x ) can be easily estimated as follows: FDEP,x = 2␲a 3 ε0 εm Re{K (␻)}∇|E |2 sin ␪  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim



Supporting Information Fig. 1B shows the comparison of the DEP force and the friction force for a 5-␮m radius spherical particle. The estimated friction force is approximately 5.5 × 10−13 N, which is smaller than the DEP force (7.9 × 10−13 N) at x = 0. This supports the idea that a particle can be successively moved by overcoming the friction force. Also, the DEP force in the x-direction increases as the position of the liquid electrode moves in the positive x direction; however, after a distance of about 30 ␮m from the initial position, the DEP force in the x-direction starts decreasing because   the square of the electric field decreases more rapidly |E |2 . Consequently, within a distance of about 200 ␮m from the initial position, the DEP force in the x-direction is sufficiently large to overcome the friction force of the spherical particle.

2.2.2 Characteristics of the DEP force direction In general, for dielectric particles with a single layer, such as polystyrene or silica, the permittivity and conductivity are independent of frequency, and the CM factor of the medium can be easily calculated with respect to the frequency. The concentric shell model [16] is used to calculate the CM factor of biological cells due to their complicated internal structure [20]. For example, with the two layers cell (subscript m means medium), the dipole moment and effective polarizability are expressed by [21]:  ∗  ∗ ε − εm (6) ␣ = 3εm K (␻)12 = 3εm 12 ∗ ∗ ε12 + εm 

∗ ε12

  ∗ ε − ε1∗ 3 ␥12 + 2 ∗2 ε + 2ε1∗  ,  ∗2 = ε1∗  ε2 − ε1∗ 3 ␥12 − ε2∗ + 2ε1∗


where ␥12 = a1 /a2 is the radius ratio of two layers. This single shell model can be extended to the concentric shell model with multi-layers. The yeast cells examined are proposed to have four shells, as seen in Supporting Information Fig. 2, since the yeast cell consists of cytoplasm, organelles, organelle membranes, and plasma membranes. In this case, the four distinct complex permittivity values are replaced by one equiv∗ . The thickness of the yeast cell alent complex permittivity, εeq r is 2.95 ␮m. The dielectric properties and thicknesses of the layers are listed in Supporting Information Table S1 [22, 23]. A 25°C 20 mM KCl isotonic solution was used for the DEP experiments, and its unitless relative permittivity and electrical conductivity of the solution were 78 and 0.0028 S/cm, respectively [24]. Supporting Information Fig. 3A shows the calculated equivalent complex permittivity (permittivity and the conductivity) of the yeast cell by using the concentric shell model [16]. The real and imaginary parts of the CM factor for a yeast cell in the 20 mM KCl solution are calculated by using Eq. (2) based on the calculated permittivity and conductivity, as seen in Supporting Information Fig. 3B. The real part of the CM factor is associated with the magnitude and direction of the www.electrophoresis-journal.com


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DEP force. The positive or negative DEP phenomenon is determined based on the polarizability of the particle relative to its surrounding medium. The positive DEP phenomenon occurs when the CM factor is positive. It acts in the direction of increasing electric field gradient. On the other hand, negative DEP acts to repel the cell from regions with high electric field strength. For the 20 mM KCl isotonic solution, the positive DEP force increases until 1 MHz and then decreases rapidly beyond that frequency. It is anticipated that the friction force in the KCl solution is still negligible because its viscosity of 0.87 mPa s is similar to that of water, 0.89 mPa s [25]. The highest value of the real part of the CM factor is estimated as 0.91 around 100 kHz. Physically, the imaginary part of the CM factor is directly associated with the rotational motion of a particle, in particular, its angular velocity and direction. Therefore, the rotational motion could be removed by controlling the frequency to let the particles move near the surface efficiently. From Supporting Information Fig. 3B, it is seen that at approximately 100 kHz, the imaginary part of the CM factor is nearly zero, indicating that at this frequency, the rotational energy reaches a minimum resulting in the effective translational movement of a particle. From our numerical analysis, 100 kHz was found to be an optimal frequency in this system. This corresponds to a maximum in the positive DEP force and a minimum in the rotational motion of a cell.

3 Results and discussion 3.1 Electric potential drop For demonstration of active manipulation of particles in the present DEP system, AC voltages were applied by a function generator (33210A, Agilent, Santa Clara, CA, USA) and an AC power amplifier (A800, FLC Electronics, Partille, Sweden). The movable liquid-drop electrode and the yeast cells suspended in the medium were visualized using an upright bright field optical microscope (Moticam 2300, Motic, British Columbia, Canada). The dielectric layers of glass, PDMS, and DI water have intrinsic high impedance characteristics and frequency-dependent complex conductivity [24]. For this reason, the dielectric voltage drops of the applied voltage in the thin PDMS layer should be considered normal for operation of a cDEP system. In addition, similarly to the other contactless DEP systems [4, 26–29], the frequency dependency is manifested in this proposed system as well. Supporting Information Fig. 4 shows that the measured applied voltage with respect to frequency can be changed due to the drop of voltage through a PDMS thin layer surface. The applied voltage is a peak-to-peak voltage and the dropped voltages are RMS voltages. The dropped voltages across the thin PDMS layer in sample channel were measured with the general Wheatstone bridge circuit. The maximum measured voltage is measured approximately 750 V for the 800 V apparent voltages and remains constant up to 100 kHz,

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but it monotonically decreases after 100 kHz, owing to the bandwidth characteristics of the used power amplifier. The dropped voltage becomes low for the maximum voltage applied up to a frequency of about 30 kHz because of the high impedance characteristics of PDMS [30]. It has been reported that the minimum electric field is approximately 0.2 kV/cm (with about 100 ␮m electrode distance) for DEP manipulation [31]. According to other literature, the electric field of 2 kV/cm is the limitation electric field for avoiding substantial yeast cell killing [32], and the electric field of 0.72 kV/cm has no critical Joule heating problem in the yeast cells [33]. In Supporting Information Fig. 4, it is shown that the maximum dielectric dropped voltage for the 30-␮m thin PDMS layer is about 7.2 V at 100 kHz. Accordingly, electric field range 0.2 kV/cm (dropped voltage of 2 V) through 0.72 kV/cm (dropped voltage of 7.2 V) was selected in the present DEP system based on our parametric study and the literature on manipulation of yeast cells. In addition, a suitable operating frequency was determined in the range 1 to 100 kHz approximately.

3.2 Effect of applied voltage and frequency on cell manipulation efficiency In the present experiment, yeast cells were used to demonstrate active manipulation. To prepare a suspension of live yeast cells, 20 mg of yeast powder (Instant Dry Yeast, Vega, Istanbul, Turkey) was mixed with 1 mL of potassium chloride (KCl) isotonic solution (20 mM). The number of cells was 67 × 107 per mL measured by a standard Neubauer counting chamber (0640030, Marienfield, Lauda-Konigshofen, Germany). Suspended yeast cells were kept frozen prior to use and thawed at 37°C for the experiment. Figure 4 shows dielectrophoretic manipulation of yeast cells obtained at different applied voltages and frequencies. Without the existence of electric fields, yeast cells are initially well dispersed near the bottom surface of the PDMS medium chamber, whereas when electric fields are applied, they are successfully collected and aligned on the PDMS layer surface at which the liquid electrode is located in the channel. As noted previously, the collection efficiency of the particles is a very important factor, which may be analyzed to evaluate the system performance. This study defines the collection efficiency of yeast cells as follows:   nc × 100 (8) ␩c = 1 − nT where nc and nT represent the number of collected cells and the cell number initially suspended in the medium chamber, respectively. As the applied voltage increases from 100 to 800 V, better collection of yeast cells is observed. The 100 kHz condition yields better cell collection compared to other frequencies at 800 V. This indicates a clear effect of applied voltage and frequency on the particle collection. Figure 5A shows the estimated collection efficiencies


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Figure 4. Dielectrophoretic manipulation of yeast cells obtained at different applied voltages and frequencies.

of yeast cells with respect to applied voltages over 30 s for applied voltages in the range 100 to 800 V at the fixed frequency of 100 kHz. The collection efficiency increases with the applied voltage because the DEP force is proportional to the square of the dissipated voltage as indicated in Eq. (1). The maximum collection efficiency reached was 90%. This result is comparable with results for existing cDEP techniques reported in the previous literature [34]. This demonstrates extremely high collection efficiency and improved cell mobility using a movable liquid electrode cDEP design. The frequency effect on the CM factor should be also considered. As shown in Eqs. (1) and (2), DEP force depends on the electric field strength and the magnitude of the real part of the CM factor, which is related to the exerted frequency-dependent dielectric properties of particle and medium, as seen in Supporting Information Fig. 3.

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As demonstrated in Fig. 5B, the dielectrophoretic velocity of cells was measured for operation between 1 kHz and 1 MHz at the fixed AC apparent voltage of 800 V. To estimate the dielectrophoretic velocity of a cell, two digital images captured at different times were used. The focal plane was fixed on the PDMS thin layer because the yeast cells were distributed near the bottom surface owing to the gravitational effect. As shown, the dielectrophoretic velocity increases with frequency. The maximum value is 10.85 ± 0.95 ␮m/s at 100 kHz compared with 0.58 ± 0.11 ␮m/s measured at 1 kHz. Measured dielectrophoretic velocities of this study are comparable with the previous measurement for conventional DEP [35]. This result is attributed to low dissipated voltage and the low value of the real part of the CM factor at 1 kHz. This result supports the idea that collection efficiency is closely associated with the dielectrophoretic velocity and is also dependent on the frequency.



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applied voltage in a thin PDMS layer. The maximum dielectric dissipated voltage with the 30-␮m thin PDMS layer was about 7.2 V at 100 kHz. High values of dissipated voltage on the surface were required to manipulate yeast cells with DEP. The dielectrophoretic velocities and collection efficiencies of yeast cells were estimated with respect to a voltages and frequencies. The maximum collection efficiency reached was approximately 90%, indicating that the present system was well suited for particle collection. From the experimental results, it was found that the most effective frequency for positive DEP of yeast was approximately 100 kHz, and the maximum dielectrophoretic velocity was about 10.85 ± 0.95 ␮m/s at an applied voltage of 800 V. From the results, it was concluded that the new contactless DEP system would be useful for dynamic particle collection by using a movable liquid electrode. This work was partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0016837) (Lee), (2013R1A1A2012177) (Chang), and (2011-0008941) (Kim). In addition, this work was supported by the National Research Foundation Grant (NRF) (NRF-20100023917) funded by Korean Government (MEST) (Jung). The authors have declared no conflict of interest.

5 References [1] Pohl, H. A., Dielectrophoresis, Cambridge University Press, Cambridge 1978. [2] Kadaksham, J., Singh, K., Aubry, N., Electrophoresis 2005, 26, 3738–3744. Figure 5. Effect of applied E-field strength and frequency at liquid electrode DEP on collection efficiency for yeast cells, (A) collection efficiencies of yeast cells w.r.t input voltages and (B) dielectrophoretic velocity of yeast cells with respect to frequency for 800 V input voltage.

4 Concluding remarks This study proposed a new movable liquid-drop electrode DEP technique and experimentally demonstrated the active manipulation of yeast cells. The equivalent complex permittivity and the CM factor of yeast cells were numerically analyzed by using the concentric shell model with respect to frequency to facilitate the selection of optimal operating conditions. The highest value of the real part of the CM factor was estimated as 0.91 at around 100 kHz. The present DEP system is advantageous owing to its ability to control the local position at which the particles could be collected by properly manipulating a liquid drop. The experimental data clearly indicated an effect of the applied frequency on the drop of  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Development of a new contactless dielectrophoresis system for active particle manipulation using movable liquid electrodes.

This study presents a new DEP manipulation technique using a movable liquid electrode, which allows manipulation of particles by actively controlling ...
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