Anal. Chem. 2007, 79, 6975-6987
Dynamic Cell Fractionation and Transportation Using Moving Dielectrophoresis Chin Hock Kua,† Yee Cheong Lam,*,†,‡ Isabel Rodriguez,§ Chun Yang,‡ and Kamal Youcef-Toumi†,⊥
Singapore-MIT Alliance, Nanyang Technological University, Singapore 639798, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore 639798, Institute of Materials Research and Engineering, Singapore 117602, and Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
This study presents a new cell manipulation method using a moving dielectrophoretic force to transport or fractionate cells along a microfluidic channel. The proposed moving dielectrophoresis (mDEP) is generated by sequentially energizing a single electrode or an array of electrodes to form an electric field that moves cells continuously along the microchannel. Cell fractionation is controlled by the applied electrical frequency, and cell transportation is controlled by the interelectrode activation time. The applicability of this method was demonstrated to simultaneously fractionate and transport Saccharomyces cerevisiae yeast cells, both viable and nonviable, by operating at conditions under which the cells were subjected to positive and negative dielectrophoresis, respectively. Compared to the conventional dielectrophoresis (cDEP and traveling wave dielectrophoresis (twDEP), moving dielectrophoresis allows cells to be separated on the basis of the real part of the Clausius-Mossotti factor, as in cDEP, but yet allows the direct transportation of separated cells without using fluid flow, as in twDEP. This dielectrophoresis technique provides a new way to manipulate cells and can be readily implemented on programmable multielectrode devices. Cell separation technology using microfluidic devices has emerged as an efficient technology to purify target cells from a variety of environmental and biological samples from which the target cells can be isolated and subsequently collected for downstream testing. Numerous techniques have been developed to achieve this purpose, including electrophoretic, dielectrophoretic, acoustic,1 hydrodynamic,2 and bifurcation3 methods. As * To whom correspondence should be addressed. Tel: 65 6790 5866. E-mail:
[email protected]. † Singapore-MIT Alliance, Nanyang Technological University. ‡ School of Mechanical and Aerospace Engineering, Nanyang Technological University. § Institute of Materials Research and Engineering. ⊥ Massachusetts Institute of Technology. (1) Petersson, F.; Nilsson, A.; Holm, C.; Jo¨nsson, H.; Laurell, T. Lab Chip 2005, 5, 20-22. (2) Lutz, B. R.; Chen, J.; Schwartz, D. T. Anal. Chem. 2006, 78, 54295435. (3) Huang, L. R.; Cox, E. C.; Austin, R. H.; Sturm, J. C. Science 2004, 304, 987-990. 10.1021/ac070810u CCC: $37.00 Published on Web 08/17/2007
© 2007 American Chemical Society
one of the well studied and mature technologies, dielectrophoresis utilizes the response of polarized cells to the frequency of applied nonuniform electric field to manipulate cells. Recent efforts on dielectrophoretic techniques focus on improving the throughput and accuracy for continuous cell separation or to achieve single cell manipulation from a massive population. The former approach attempts to optimize the electrode design or to couple the dielectrophoretic force with other forces to achieve higher throughput and accuracy. Examples include the 3D-asymmetric microelectrodes,4 trapezoidal electrode,5 multistep separation,6 ring-dot traps,7 insulating blocks,8 DEP gate/deflector,9,10 and the electrosmear method.11 These techniques require the application of a continuous fluid flow to provide an additional dimension of force. There are also numerous attempts to couple the dielectrophoretic technique with other techniques to improve cell separation selectivity. These techniques include cell-targeted antibody,12 DEP markers,13 ultrasonic standing wave,14 AC electroosmosis,15 electrohydrodynamic flow,16 and field-flow fractionation17 methods. The latter approach involves employing programmable devices to position cells in a microfluidic chamber. Such devices function by generating pockets of electrical potential to allow single cells to be trapped and positioned from one electrode to another. Unlike (4) Park, J.; Kim, B.; Choi, S. K.; Hong, S.; Lee, S. H.; Lee, K.-I. Lab Chip 2005, 5, 1264-1270. (5) Choi, S.; Park, J.-K. Lab Chip 2005, 5, 1161-1167. (6) Aldaeus, F.; Lin, Y.; Amberg, G.; Roeraade, J. J. Chromatogr., A 2006, 1131, 261-266. (7) Taff, B. M.; Voldman, J. Anal. Chem. 2005, 77, 7976-7983. (8) Kang, K. H.; Kang, Y.; Xuan, X.; Li, D. Electrophoresis 2005, 26. (9) Holmes, D.; Sandison, M. E.; Green, N. G.; Morgan, H. IEE Proc.: Nanobiotechnol. 2005, 152, 129-135. (10) Du ¨ rr, M.; Kentsch, J.; Mu ¨ ller, T.; Schnelle, T.; Stelzle, M. Electrophoresis 2003, 24, 722-731. (11) Das, M. D.; Becker, F.; Vernon, S.; Noshari, J.; Joyce, C.; Gascoyne, P. R. C. Anal. Chem. 2005, 77, 2708-2719. (12) Yang, L.; Banada, P. P.; Chatni, M. R.; Lim, K. S.; Bhunia, A. K.; Ladisch, M.; Bashir, R. Lab Chip 2006, 6, 896-905. (13) Hu, X.; Bessette, P. H.; Qian, J.; Meinhart, C. D.; Daugherty, P. S.; Soh, H. T. Proc. Natl. Acad. Sci. 2005, 102, 15757-15761. (14) Wiklund, M.; Gunther, C.; Lemor, R.; Jager, M.; Fuhr, G.; Hertz, H. M. Lab Chip 2006, in press. (15) Zhou, H.; White, L. R.; Tilton, R. D. J. Colloid Interface Sci. 2005, 285, 179191. (16) Tuval, I.; Mezic´, I.; Bottausci, F.; Zhang, Y. T.; MacDonald N. C.; Piro, O. Phys. Rev. Lett. 2005, 95, 236002. (17) Gascoyne, P.; Satayavivad, J.; Ruchirawat, M. Acta Trop. 2004, 89, 357369.
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other methods that require fluid flow, programmable devices can manipulate cells in a stationary liquid. Two examples are a square electrode array on a CMOS chip18 and optoelectronic tweezers.19 Programmable devices have also been used to perform cell transportation and cell separation using an array of elongated electrodes.20-22 For example, in the dielectrophoretic cage-speed separation method,20,21 polystyrene particles of distinct sizes are separated in electrical potential cages by employing the principle that large particles travel faster than small particles. Programmable devices have also been used in electrophoresis to perform DNA migration,22 in which negatively charged DNA is sequentially attracted to programmable anodes. Optoelectronic tweezers19 have been used to transport polystyrene particles and to collect live human B cells using a conveyer belt concept. This device employs an optical projector system to construct programmable electrodes on a photovoltaic surface. This paper presents a method that is able to transport cells under positive or negative dielectrophoresis and also to simultaneously fractionate and transport cells having different dielectrophoretic affinities. Cell transportation is achieved by sequentially energizing one electrode or one array of electrodes at a time, in contrast to energizing multiple electrodes to form potential cages used by the existing methods.18,19 The cell fractionation technique presented in this work allows for fractionation and transportation of cells in the same direction, instead of separating a mixture of cells at both ends of a microchannel, as reported in other methods.21 Viable and nonviable yeast cells were used in this investigation as model particles to demonstrate the applicability of this technique in manipulating cells. Yeast cells have been widely used in numerous dielectrophoresis experiments, and they have well-characterized properties.23,24 Furthermore, this technique allows cells to be aligned along specific electrodes, which may be located at any part of the microchannel after separation. In addition, since only individual electrodes are energized for a short duration at a time, electrolysis and related problems such as ohmic heating that are associated with dielectrophoresis are avoided. MATERIALS AND METHODS Yeast Cells Preparation. Yeast cells, Saccharomyces cerevisiae (strain ATTC 18824), were grown at 30 °C in a culture medium of pH 6.5. The nonviable yeast cells were obtained by boiling viable yeast cells at a temperature of 75 °C for 15 min. Three types of samples were prepared; namely, a sample of viable yeast, a sample of nonviable yeast, and a sample of a mixture of viable and nonviable yeast. The cultured cells were suspended in 250 mM mannitol solution (M-9546 D-mannitol, Sigma) by centrifugation and resuspension of the yeasts at 5000 rpm in three consecutive steps. The conductivity of the mannitol solution was (18) Manaresi, N.; Romani, A.; Medoro, G.; Altomare, L.; Leonardi, A.; Tartagni, M.; Guerrieri, R. IEEE J. Solid-state Circuits 2003, 38, 2297-2305. (19) Chiou, P. Y.; Ohta, A. T.; Wu, M. C. Nature 2005, 436, 370-372. (20) Vulto, P.; Medoro, G.; Altomare, L.; Urban, G. A.; Tartagni, M.; Guerrieri, R.; Manaresi, N. J. Micromech. Microeng. 2006, 16, 1847-1853. (21) Medoro, G.; Vulto, P.; Altomare, L.; Abonnenc, M.; Romani, A.; Tartagni, M.; Guerrieri, R.; Manaresi, N. Proc. IEEE Sens. 2004. (22) Shaikh, F. A.; Ugaz, V. M. Proc. Natl. Acad. Sci. 2006, 103, 4825-4830. (23) Huang, Y.; Ho¨lzel, R.; Pethig, R.; Wang, X.-B. Phys. Med. Biol. 1992, 37, 1499-1517. (24) Huang, Y.; Wang, X.-B.; Tame, J. A.; Pethig, R. J. Phys. D: Appl. Phys. 1993, 26, 1528-1535.
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previously adjusted to 305 µS cm-1 (at 20.6 °C) by adding sodium chloride (GCE Laboratory Chemical). The conductivity was measured using an ION check65 conductivity meter (Radiometer Analytical). The pH of the mannitol solution was measured to be 5.0. The yeast samples were stained with 0.4% trypan blue (Invitrogen) using a 5:1 ratio. Trypan blue was used to differentiate between viable and nonviable yeast cells because the dye stains only nonviable yeast. Microfluidic Chip Fabrication. To determine the dimensions of the device to be fabricated, simulations were conducted. In general, our simulation results confirmed that a small electrode width and a low microchannel height generate a larger dielectrophoretic force, and a small interelectrode gap allows cells to travel less distance to reach the next electrodes. Due to fabrication limitation, we employed a 10-µm electrode width and 10-µm interelectrode gap for our device, which can be comfortably achieved using common photolithography techniques. Since the size of yeast cells were ∼4-10 µm, a microchannel height of ∼25 µm was selected such that the height was large enough for cells to pass through while small enough to generate a high dielectrophoretic force. The dielectrophoretic chip was fabricated in three layers, including an ITO plate, a spacer, and a microelectrode plate. The ITO plate was a 500-µm-thick glass precoated with a film of indium tin oxide (ITO) on one of the surfaces (the channel surface). Two L1-mm holes were drilled on the ITO glass to act as fluid inlet and outlet. The spacer was a layer of 25-µm-thick acrylic polymer (ARclear 8154, Adhesives Research) that has adhesive on both surfaces. A slot of ∼16 mm long × 2 mm wide was cut on the spacer using a laser cutting machine (M-300, Universal Laser Systems) to form the fluidic channel. The microelectrode plate was a 500-µm-thick glass wafer (Pyrex 7740). Electrodes were fabricated onto this glass wafer using a lift-off photolithographic process, as illustrated in Figure 1A. Briefly, the glass wafer was first cleaned in piranha solution for 10 min. It was then rinsed with deionized Millipore water, blown dry, and dehydrated in an oven for 10 min at 110 °C. Subsequently, a thin film of chromium was sputtered onto the glass wafer to act as an antireflection layer during the photolithography process. A layer of photoresist (AZ 7220, Clariant) was then spin-coated onto the glass wafer with a spread speed of 500 rpm for 5 s and a spin speed of 5000 rpm for 40 s. After soft baking, the photoresist was exposed and developed using AZ developer solution. After chromium and gold were sputtered onto the prepared glass surface, the lift-off process was performed by soaking the glass wafer in acetone and ultrasonic bathing it for ∼30 min until the electrodes were fully developed. After the lift-off process, the glass wafer was cleaned with acetone, IPA, and DI water and blown dry. The chromium and gold electrodes were measured to have an average thickness of 330 Å using a surface profiler (P-10, KLA Tencor). The chromium layer that was not covered with the gold layer was removed using chromium etchant solution. An image of the fabricated array of electrodes is shown in Figure 1B. The microelectrode plate carried a total of 60 independently excitable, 10-µm-wide electrodes spaced at 10 µm. The effective length of the electrodes is defined by the microchannel width, that is, ∼2 mm. The microfluidic chip was assembled by placing an adhesive spacer between the microelectrodes plate and the ITO plate. The
Figure 1. (A) Photolithography lift-off process to fabricate microelectrodes on the microelectrode plate: (1) sputtering of chromium as antireflection layer, (2) spin-coating of photoresist (AZ 7220, Clariant) at 5000 rpm for 40 s, (3) UV exposure, (4) development pattern, (5) sputtering of chromium, (6) sputtering of gold, (7) lift-off process, and (8) removal of chromium to obtain microelectrodes. (B) Fabricated microelectrodes, showing 30 independently excitable electrodes on the left and right edges. This microelectrode coated glass piece acted as the microelectrode plate for the microfluidic chip.
slot on the spacer formed the fluidic channel, which allowed the passage of the cell suspension. The channel height was measured to be approximately 24 µm, using the autofocus capability of a microscope (Eclipse TE2000-S, Nikon) with a motorized stage. The ITO layer on the ITO plate was electrically connected to the ground line on the microelectrode plate using conductive epoxy (CW2400 Circuitworks, Chemtronics). Experimental Setup. The microfluidic chip was attached onto a printed circuit board (PCB). The PCB had 62 independent via on the top surface and 31 via on the bottom surface. The microfluidic chip was electrically connected to the PCB through wire bonding (4534AD, Kulicke & Soffa). Each bonding pad on the microfluidic chip was connected to the corresponding via on the PCB. Each end of the PCB was connected to a 31-way edge connector (IBM PC AT, RS component). The final device consisted of 2 sets of 30 independently excitable inputs and a common ground. The assembly is shown in Figure 2A. An enlarged view of the microchannel and the electrodes is shown in Figure 2B. One of the edge connectors was connected to a series of 30 relays (HE3621A0510, Breed Electronics) through two 16-way IDC ribbon cables. The relays were soldered in parallel onto a PCB. The power lines of the relays were electrically connected to a function generator (33250A, Agilent). The control lines of the
relays were electrically connected to a digital I/O card (PCI-6509, National Instruments) installed on the PCI slot of a personal computer. The switching of the relays was controlled by a program written in LabView (National Instruments). The program was written to generate a sequential TTL signal to trigger the switching of the relays, which results in the generation of a moving electric field on the microelectrodes. The whole switching process was automatically repeated at the end of the cycle. The full experimental setup is as illustrated in Figure 2C. Cell Motion Tracking. The motion of the cells was recorded through a CCTV camera (Sony ExwaveHAD SSC-DC58AP) using a commercial video tape recorder. The analog video was converted to digital video files using the Movie Studio EzCoder 3.0 program. The final digital video files had 25 frames/s. The cells’ trajectories were traced using the Manual Tracking plug-in of the image processing software ImageJ. Experiment Preparation. A small amount (0.05∼0.1 mL) of yeast suspension was drawn into the syringe from the test tube. The suspension was later carefully dropped into the inlet of the microfluidic chip. A suction cap (Silicone Flat Pad stock no. 2273856, RS component) assembled on another syringe was used to draw the suspension into the channel by sucking from the outlet. Care was taken to ensure no gas bubble was formed inside the electrode area in the channel. Excess solution outside the inlet Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
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Figure 2. Experimental setup to generate moving dielectrophoresis: (A) Image of the microfluidic chip, assembled to a PCB. The microfluidic chip was formed by layering an ITO-coated plate on the plate carrying the patterned microelectrodes with a 25-µm-thick adhesive spacer. (B) Enlarged view of microelectrodes and microchannel showing independently excitable electrodes. (C) Schematic of the experimental setup. From a function generator, the electrical signal is routed to the respective microelectrodes through relays controlled by a computer. Video is recorded using a video tape recorder.
and outlet were carefully cleaned using clinical paper to avoid a pressure difference in the microchannel. The experiment was performed on a static fluid volume encapsulated in the bounded microchannel. The channel was cleaned by deionized water before testing on a different sample. MOVING DIELECTROPHORESIS THEORY Consider a microfluidic channel of which the top plate consists of a finite number of individually excitable electrodes, and the bottom plate consists of an infinite electrode acting as a common electrical ground for the top electrodes (see Figure 3). In this design, the length (along the z-axis) of each top electrode is significantly longer than their width (along the x-axis) and microchannel height such that the system can be treated as twodimensional-dependent on the x-y coordinates only. The electrodes are excited with a sinusoidal voltage with amplitude V0 and angular frequency ω. A moving electric field is generated when a single or an array of independently excitable top electrodes are energized sequen6978
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tially. Such system contains two independently controllable time parameters, which are the electric field angular frequency, ω, and the interelectrode activation time, T. The simulation images of the resulting moving electric field is shown in Figure 3. At arbitrary time zero, the second top electrode from the left is energized. The activation of the second electrode generates a nonuniform electric field. After time T s, the second electrode is de-energized, and the third electrode is energized. Subsequently, at time 2T s, the third electrode is de-energized, and the fourth electrode is energized. By repeating this energizing scheme, a nonuniform electric field is generated that moves from left to the right, as illustrated in Figure 3. Cells suspended in a liquid medium in this microchannel would experience a dielectrophoretic force due to the existence of the nonuniform electric field, E. Assuming that a finite-sized polarized cell can be represented as an equivalent dipole,25 the time-averaged (25) Jones, T. B. Electromechanics of Particles; Cambridge University Press: New York, 1995.
˜f cm )
˜ c - ˜ m ˜ c + 2˜m
(2)
where ˜c is the effective complex permittivity of the yeast cells, and ˜m is the complex permittivity of the suspending medium. The effective complex permittivity, ˜c, can be calculated using the smeared-out sphere approach23 by progressively simplifying the two-shell sphere to a simple homogeneous sphere, which is given by26
[
˜ c ) ˜ 1
) )
( (
) )
˜ 23 - ˜ 1 ˜ 23 + 2˜1 ˜ 23 - ˜ 1 γ123 ˜ 23 + 2˜1
[
and
˜ 3 - ˜ 2 ˜ 3 + 2˜2 ˜ 3 - ˜ 2 γ233 ˜ 3 + 2˜2
γ233 + 2
˜ 23 ) ˜ 2
Figure 3. Moving electric field is generated by sequentially energizing single (or array of) electrodes to form an electric field that moves from one end to other. There are two independently controllable time parameters in this proposed moving electric field method; that is, the electric field angular frequency, ω, and interelectrode activation time, T. The angular frequency is used to control the polarization of cells and, thus, the direction of DEP force. The interelectrode activation time is used to control the cells’ transportation speed. Note that there is no phase difference in the electric field, as in the traveling wave electric field.
dielectrophoretic force acting on a spherical cell with radius a and suspended in an aqueous medium with permittivity m is given by26
FDEP ) πma3Re[f˜cm]∇|E|2
(1)
where Re[f˜cm] is the real part of the Clausius-Mossotti factor and has a value between -0.5 and 1. For biological cells such as yeast, the Clausius-Mossotti factor, ˜fcm, can be approximated as the socalled two shells model given by (26) Morgan, H.; Green, N. G. AC Electrokinetics: colloids and nanoparticles; Research Studies Press: Philadelphia, 2003.
]
( (
γ123 + 2
]
(3)
(4)
where ˜1, ˜2, and ˜3 are the complex permittivity of the cells’ walls, cytoplasm, and nuclei, respectively. The complex permittivity is defined as ˜ ) - iσ/ω, where and σ are the permittivity and conductivity, respectively. The value of Re[f˜cm] can be controlled by changing the electric field angular frequency, ω, which results in either positive or negative dielectrophoresis on a cell. An example of an Re[f˜cm] plot for viable yeast cells and nonviable yeast cells is shown in Figure 4. When a cell is experiencing a positive dielectrophoresis, it is attracted to the high electric field gradient region. For the system shown in Figure 3, the cell would be attracted to the top edge of an energized electrode. Likewise, a cell would move in the opposite direction when it is experiencing a negative dielectrophoresis, that is, away from the high electric field gradient region. The moving electric field can be used to differentially transport cells across the microchannel. As illustrated in Figure 5, a cell experiencing positive dielectrophoresis moves toward the high electric field gradient region, that is, the cell is attracted to the energized electrodes. The cell is sequentially transported to the next electrodes due to the attractive force from the subsequently energized electrodes. Referring to Figure 5, at time t ) 0, when the second electrode from the left is energized, a cell experiencing positive dielectrophoresis is attracted to the energized electrode edge and is immobilized there. At time t ) T, when the third electrode is energized, the cell continues to move to the third electrode under the attractive force. Similarly, the cell moves to the fourth electrode at time t ) 2T. Thus, by sequentially energizing a single or an array of electrodes, the cell follows the electric field. Similarly, a cell experiencing negative dielectrophoresis can be transported using the same principle. However, in this case, the cell moves away from the high electric field gradient region, that is, the electrode edge. As illustrated in Figure 6, when the Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
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Figure 4. Predicted frequency response of viable and nonviable yeast cells for a medium conductivity of 305 µS cm-1. At an applied electrical frequency of 2 MHz, viable yeast cells experience positive dielectrophoresis with a Clausius-Mossotti (CM) factor of 0.42, whereas nonviable yeast cells experience negative dielectrophoresis, with a CM factor of -0.34.
Figure 6. Illustration of cell transportation using negative dielectrophoresis. At time t ) 0, the second electrode from the left is energized. A cell experiencing negative dielectrophoresis is repelled from the high electric field gradient region; that is, away from the energized electrode. The dotted circle represents the initial position of the cell. At time t ) T, the third electrode is energized. The cell is repelled farther from the third electrode. By sequentially applying an electric field that moves from left to right with a tempo T, the cell is sequentially repelled from the energized electrode edge on each successive step. The cell moves by leading the electric field.
Figure 5. Illustration of cell transportation using positive dielectrophoresis. At time t ) 0, the second electrode from the left is energized. A cell experiencing positive dielectrophoresis is attracted to the high electric field gradient region, that is, energized electrode edge, and is immobilized there. The dotted circle represents the initial position of the cell. At time t ) T, the third electrode is energized. The cell continues to move to the third electrode under the positive dielectrophoretic force. By sequentially applying an electric field with an angular frequency, ω, that moves from left to right with a tempo, T, the cell is sequentially attracted to the energized electrode edge on each successive step. The cell moves by trailing the electric field.
second electrode from the left is energized at time t ) 0, a cell experiencing negative dielectrophoresis is repelled from the energized electrode edge. When the cell reaches the third electrode at time t ) T, the third electrode is energized and the cell is repelled farther to the fourth electrode. Similarly, the cell moves to the fifth electrode at time t ) 2T. Thus, by sequentially energizing a single or an array of electrodes, a cell experiencing 6980 Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
a negative dielectrophoresis travels in front of the electric field. These motions are possible as long as the interelectrode activation time, T, is long enough for the cell to travel to the next electrode. This mechanism can be further exploited to achieve cell fractionation. As illustrated in Figure 7, the cell experiencing a negative dielectrophoresis always moves in front of the cell experiencing a positive dielectrophoresis. By sequentially energizing more than one adjacent electrode, the cell experiencing positive dielectrophoresis would be attracted to the electrode edge and remains there, while the cell experiencing negative dielectrophoresis is transported away. Their separation distance can be further amplified by sequentially energizing several adjacent electrodes. This occurs because the highest electric field gradient region appears at the first or last electrode edges, and the cell experiencing a positive dielectrophoresis is always attracted to those regions. Note that cell transportation can be achieved by switching on sequentially a minimum of one electrode, whereas cell fractionation would be better achieved by an array of electrodes. In this moving dielectrophoresis scheme, cells are separated on the basis of the electrical frequency response through the control of the time parameter ω. Like the conventional dielectrophoresis (cDEP), the separation is achieved on the basis of the real part of the Clausius-Mossotti factor. Cell transportation is achieved by sequentially energizing adjacent electrodes, and the
Figure 7. Cell fractionation and transportation under a moving electric field. Cell under a positive dielectrophoresis (hatched sphere) is attracted to the electrode edge, whereas cell under a negative dielectrophoresis (filled sphere) is repelled from the electrode edge. Cells are fractionated by consecutively switching on three electrodes. Cells are transported by sequentially energizing three electrodes along the microchannel. Note that all the energized electrodes have the same angular frequency, ω; this is different from the traveling wave electric field, which has a phase difference between different electrodes.
transportation speed is controlled through the time parameter T. There is no electrical phase difference. At any point in time (in the seconds range), the electric field strength is present around the single or array of energized electrodes only. The transportation efficiency of moving dielectrophoresis is determined by the transportation time required to move polarized cells from an energized electrode to the adjacent electrode. This transportation time can be estimated by solving the equation of motion caused by the net force acting on the cells. There are four dominant forces involved; namely, the dielectrophoretic force, FDEP; the fluid drag, Fdrag; the buoyancy force, Fbuoy, and the gravitational force, Fgrav. From Newton’s law of motion, the trajectory of cells under an energized electrode is given by
FDEP + Fdrag + Fbuoy + Fgrav ) ma
(5)
where m is the cell’s mass and a is the vector component of the cell’s instantaneous acceleration under these forces. For the configuration shown in Figure 3, the cells’ motion may be
considered as two-dimensional, where eq 5 can be expanded into x- and y-components, respectively, as
πma3 Re[f˜cm] πma3 Re[f˜cm]
∂ d2x dx |E|2 - 6πηaKx )m 2 ∂x dt dt
(6)
dy ∂ d2y |E|2 - 6πηaKy + (Fm - Fc)Vcg ) m 2 ∂y dt dt (7)
where ∂/∂x and ∂/∂y are gradient operators in the x- and y-directions, respectively; η is the viscosity of the fluid medium; Kx and Ky are the wall correction factors in the x- and y-directions, respectively;27,28 dx/dt and dy/dt are the instantaneous cell velocities in the x- and y-directions, respectively; Fm and Fc are the density of the fluid and the density of the cell, respectively; Vc is the volume of the cell; g is the acceleration due to gravity; and d2x/dt2 and d2y/dt2 are the instantaneous cell accelerations (27) Ganatos, P.; Weinbaum, S.; Pfeffer, R. J. Fluid Mech. 1980, 99, 739753. (28) Ganatos, P.; Pfeffer, R.; Weinbaum, S. J. Fluid Mech. 1980, 99, 755783.
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Figure 8. Transportation of viable yeast cells experiencing positive dielectrophoresis. Medium conductivity, electrical frequency, electrical potential, and interelectrode activation time were 305 µS cm-1, 2 MHz, 9.3 Vpp, and 2 s, respectively. The dark-field horizontal stripes were electrodes. The word “ON” represents the electrode that was energized at the corresponding time instance. Viable yeast cells can be seen to align with the energized electrode during motion. The corresponding concept is as illustrated in Figure 5. Video is available as Supporting Information on the journal website.
in the x- and y-directions, respectively. By simultaneously solving eqs 6 and 7, the trajectory of a cell under a single energized electrode can be predicted. The minimum time required for a cell to travel from its initial location to the adjacent electrode then determines the lower bound of the interelectrode activation time, Tmin. This time then governs the total time required to transport cells across the microchannel. However, because the electrical properties of individual cells could differ from each other, it is expected that there would be some scatter of Tmin. In addition, it is rather tedious to simulate and solve the equation of motion. Thus, instead of determining Tmin analytically, it was decided that Tmin would be obtained experimentally. It is worth noting here the differences between the proposed moving electrophoresis from the well-known traveling wave dielectrophoresis (twDEP). In traveling-wave dielectrophoresis, all of the electrodes are energized, and there are typically only three or four independently excitable arrays of electrodes. Each array of electrodes is related by a phase difference of 120° for a three-electrode array or 90° for the four-electrode array. Thus, a high electric field strength always exists on the whole microchannel throughout the operation, which may lead to an adverse reaction, such as electrolysis and electrode erosion. Moreover, in the traveling wave dielectrophoresis, the real part of the Clausius-Mossotti factor determines the levitation of the cells from the electrode plane, and the imaginary part of the ClausiusMossotti factor controls the translational movement of the cells along the electrode plane. Since the real and the imaginary parts of the Clausius-Mossotti factor cannot be controlled independently of each other, cells’ separation and transportation using twDEP are coupled. Cells can be separated and transported only when the real and imaginary parts of the Clausius-Mossotti factor are in the right combination, for example, the real part has to be 6982 Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
negative before it can be moved along the microchannel with the force induced by the imaginary part. RESULTS AND DISCUSSION Cell Transportation under Positive Dielectrophoresis. The sequence of motions of viable yeast cells when the electrode array was energized is shown in Figure 8. At an applied electrical frequency of 2 MHz, the viable yeast cells were observed to be attracted to the electrode edges. When a single electrode was sequentially energized, the viable yeast cells were observed to follow the energized electrode, moving from one electrode to another, as shown by the time series images in Figure 8. The interelectrode activation time was 2 s. The electrical voltage measured at the ribbon connector was 9.3 Vpp. The single-pass efficiency was 49.4%, calculated from a sampling size of 85 cells. The single-pass efficiency was defined as the percentage of cells transported after one cycle of moving electric field and was calculated by manually counting the number of cells before and after the application of the moving electric field from the video images. Assuming that the Clausius-Mossotti factor for yeast cells can be modeled by a two-shell sphere,23 the viable yeast cells suspended in a medium with conductivity of 305 µS cm-1 experience a positive dielectrophoresis at an applied electrical frequency of 2 MHz. The real part of the Clausius-Mossotti factor was estimated to be 0.42 at 2 MHz, calculated from the literature values.23,29 In this model, the viable yeast cells were assumed to have a wall thickness of 0.22 µm, a membrane thickness of 8 nm, and a cell diameter of 8 µm. The relative permittivity of the cell wall, cytoplasm, and nucleus were 60, 6, and 50, respectively. The (29) Talary, M. S.; Burt, J. P. H.; Tame, J. A.; Pethig, R. J. Phys. D: Appl. Phys. 1996, 29, 2198-2203.
conductivity of the cell wall, cytoplasm, and nucleus were 14 mS m-1, 0.25 µS m-1, and 0.2 S m-1, respectively. The ClausiusMossotti factor was then calculated from eqs 2, 3, and 4. For applied electrical frequencies from 1 kHz to 100 MHz, the estimated values of the Clausius-Mossotti factor are plotted in Figure 4. Indeed, the exact magnitude of the Clausius-Mossotti factor was not important for our experiment, other than that the viable yeast cells were experiencing substantial positive dielectrophoresis at the applied frequency of 2 MHz, that is, the Clausius-Mossotti factor with a large positive value. This was supported by our experimental observations that they were attracted to the electrode edge. This mode of transportation was based on the strategy as outlined in Figure 5. The viable yeast cells experiencing positive dielectrophoresis were transported by sequentially attracting the cells to the adjacent energized electrode edge. The cells moved by trailing the moving electric field. The major disadvantage of the transportation under positive dielectrophoresis, as compared to the transportation under negative dielectrophoresis, is that the cells have to be close enough to the energized electrode to be attracted to the electrode edge. As simulated in Figure 3, the dielectrophoretic force decays exponentially away from the energized electrode. Since the transportation under positive dielectrophoresis is based on the concept of pulling the cells far from the energized electrode to the edge of the energized electrode, the cells would be some distance away initially. The farther the cells are from the energized electrode, the smaller the dielectrophoretic force that the cells will experience. Thus, the transportation of cells under a positive dielectrophoresis scheme is sensitive to the initial position of the cells. This behavior is one of the major reasons for a relatively low single-pass efficiency (recorded above), as compared to the results based on the negative dielectrophoresis, which is discussed in the following section. Our experiments using viable yeast cells at different operating conditions showed varying single-pass efficiency, since the magnitude of Clausius-Mossotti factor differs in these cases, and thus, the magnitude of the dielectrophoretic forces also differs. Since viable yeast cells were attracted to the electrodes during operation, they had a higher tendency to adhere to the electrodes’ surfaces, in particular to the edges of the electrodes. This is due to the edge effect where the electric field strength is the highest at the electrode edge. Cell Transportation under Negative Dielectrophoresis. Due to the heat treatment, the protein content of the nonviable yeast cells was denatured. The nonviable yeast cells had a smaller cell diameter, a larger cytoplasmic membrane conductivity and a smaller nucleus conductivity, as compared to the viable yeast cells.23 Under the conditions of a buffer with conductivity of 305 µS cm-1 and an applied electrical frequency of 2 MHz, nonviable yeast cells are expected to experience a negative dielectrophoresis. Using the two-shell sphere model,23,29 we can estimate the Clausius-Mossotti factor to be -0.34. The nonviable yeast cells were assumed to have a wall thickness of 0.25 µm, a membrane thickness of 8 nm, and a cell diameter of 7 µm. The relative permittivity of the cell wall, cytoplasm, and nucleus were 60, 6, and 50, respectively. The conductivity of the cell wall, cytoplasm, and nucleus were 1.5 mS m-1, 160 µS m-1, and 7 mS m-1,
respectively. Using these values, the Clausius-Mossotti factor for nonviable yeast cells was then calculated from eqs 2, 3, and 4, and the corresponding frequency response from 1 kHz to 100 MHz is plotted in Figure 4. Figure 4 shows that at the applied frequency of 2 MHz, the nonviable yeast cells were experiencing substantial negative dielectrophoresis, that is, the Clausius-Mossotti factor has a large negative value. Our experimental observations support the prediction that the nonviable yeast cells were experiencing negative dielectrophoresis, in that the nonviable yeast cells were observed to be repelled from the electrode edge. Thus, we expect that the nonviable yeast cells would not exhibit any tendency to stick to the electrodes. Under these operating conditions, when a single electrode was sequentially energized, the nonviable yeast cells were repelled from the electrode edges and were continuously transported across the microchannel. As indicated by the “ON” label in Figure 9, the nonviable yeast cells moved in front of the energized electrode on each successive step, indicating that the nonviable yeast cells were repelled from the energized electrode. Unlike the cells transportation under positive dielectrophoresis, the cells transportation under negative dielectrophoresis had higher transportation efficiency and exhibited no tendency to adhere to the electrodes as expected. The single-pass efficiency calculated from a sampling size of 100 cells was 89.0%. The reason is that the cells experiencing negative dielectrophoresis were repelled from the high electric field gradient region. The cells that were lagging in motion during the previous excitation period would experience a large dielectrophoretic force when the next adjacent electrode was energized. This phenomenon allowed the nonviable yeast cells to be always transported. In general, cells under negative dielectrophoresis are transported by leading the moving electric field. The corresponding x-directional trajectories of viable and nonviable yeast cells at different initial positions from the edge of the upstream electrode are plotted over time in Figure 10. Three random candidates were picked from the viable and nonviable yeast cell populations. The curves indicate that the motion of viable yeast cells was sudden, meaning the cells rapidly accelerated to the next electrode and suddenly stopped when reaching the electrode. In contrast, the motion of the nonviable yeast cells was gradual. Cell Fractionation. Using the activation strategy outlined above and illustrated in Figure 7, the viable yeast cells were separated from the nonviable yeast cells. The operating conditions were the same as before; that is, medium conductivity of 305 µS cm-1 and applied electrical frequency of 2 MHz, but to achieve ample separation, an array of three-neighboring electrodes was energized at a time. Under these operating conditions, the viable yeast cells experienced a positive dielectrophoresis, and the nonviable yeast cells experienced a negative dielectrophoresis. When a single electrode was energized, the viable yeast cells were attracted to the energized electrode edges, and the nonviable yeast cells were repelled from the energized electrode. When the adjacent electrode was energized after 2 s, while keeping the first electrode energized, the viable yeast cells remained stationary at the initial electrode edge, and the nonviable yeast cells were repelled farther from the two energized electrodes. After another 2 s, the third adjacent electrode was energized together with the Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
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Figure 9. Transportation of nonviable yeast cells experiencing negative dielectrophoresis. Medium conductivity, electrical frequency, electrical potential, and interelectrode activation time were 305 µS cm-1, 2 MHz, 9.3 Vpp, and 2 s, respectively. The dark-field horizontal stripes were electrodes. The word “ON” represents the electrode that was energized at the corresponding time instance. Nonviable yeast cells can be seen to move in front of the energized electrode. The corresponding concept is as illustrated in Figure 6. Video is available as Supporting Information on the journal website.
Figure 10. Trajectories of viable and nonviable yeast cells at different initial positions from upstream electrode as traced from movie images with a time interval of 0.04 s. Marker “b” represents the viable yeast cells, whereas the marker “O” represents the nonviable yeast cells. Viable yeast cells experiencing positive dielectrophoresis moved in step behavior, that is, synchronized with tempo T. In contrast, nonviable yeast cells experiencing negative dielectrophoresis displayed a rather linear motion.
two energized electrodes, and the viable yeast cells remained at the same location and the nonviable yeast cells were repelled farther. Thus, after 6 s, the viable and nonviable yeast cells in the vicinity were separated a three-electrode distance, or ∼60 µm, away from each other. Thus, the viable and nonviable yeast cells had been fractionated by sequentially energizing three neighboring electrodes. 6984 Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
After 6 s, the first electrode was de-energized, and the fourth electrode was energized. The viable yeast cells were attracted to the second electrode, and the nonviable yeast cells were repelled from the fourth electrode. Thus, both the viable and nonviable yeast cells had been transported one electrode distance away, or 20 µm. By sequentially energizing an array of three-neighboring electrodes, the fractionated viable and nonviable yeast cells were transported along the microchannel. The viable yeast cells moved by following the last edge of the array of energized electrodes, whereas the nonviable yeast cells moved in front of the array of electrodes. It is worth noticing that the fractionation and transportation actions occurred simultaneously while the electrodes were energized. The result of the cell fractionation process with three electrodes energized is shown in Figure 11. Figure 12 shows the fractionation of viable and nonviable yeast cells with three electrodes energized at 50× optical magnification. The images show two bands of cells moving in the channel, where the cells on the top band displayed a yellowish color, and the cells on the bottom band exhibited a blue color. Since nonviable yeast cells were stained with trypan blue, these images confirm that the nonviable yeast cells move in front of the electric field. The separation distance can be increased by energizing more neighboring electrodes. It is worth noting that during the transportation process, there were viable yeast cells that moved in the direction opposite to the rest of the viable yeast cells on the previous electrode. The reason for such motion is that viable yeast cells under positive dielectrophoresis are attracted toward the energized electrodes. The fractionation efficiency may be
Figure 11. Fractionation of a mixture of viable and nonviable yeast cells under the same operating conditions as those in Figure 8 and Figure 9. The nonviable yeast cells experiencing negative dielectrophoresis moved in front of the energized electrodes, whereas the viable yeast cells experiencing positive dielectrophoresis moved behind the energized electrodes. When an array of three electrodes was activated, nonviable yeast cells were separated a three-electrode distance away from the viable yeast cells. The images were recorded at 20× optical magnification. The dark-field horizontal stripes were electrodes. The corresponding concept is as illustrated in Figure 7. Video is available as Supporting Information on the journal website. Time indicated was when there were always three electrodes energized.
further improved by repeating the moving dielectrophoresis process along the microchannel. The maximum velocity of the moving electric field in achieving higher fractionation efficiency is determined by the largest of the minimum interelectrode activation times, Tmin, of viable and nonviable cells, as depicted by eqs 6 and 7. Let Tmin(+DEP) and Tmin(-DEP) represent the individual minimum interelectrode activation time needed for cells experiencing positive dielectrophoresis and negative dielectrophoresis, respectively, to move from an energized electrode to the adjacent electrode. The minimum time required to achieve cell transportation during the fractionation process is then governed by the largest of these individual activation times or mathematically, max[Tmin(+DEP), Tmin(-DEP)]. In our experiment, the viable yeast cells required ∼1 s to travel from one electrode to the adjacent electrode, that is, Tmin(+DEP) ≈ 1 s, which can be determined from Figure 10. For nonviable yeast cells, as the cells were repelled from the electrodes, and the starting position of the cells was not necessary at the electrode edge. As such, Tmin(-DEP) could not be directly determined from Figure 10. We carried out separate experiments and determined that the minimum interelectrode activation time could be as low as 0.5 s, that is, Tmin(-DEP) ≈ 0.5 s. Thus, the fractionation had to be performed using an interelectrode activa-
tion time of at least 1 s. In practice, a multiplier was applied to this minimum time as a factor of safety. In our experiment, this multiplier was 2; that is, 2 s was employed as the interelectrode activation time. In moving dielectrophoresis (mDEP), fractionation of cell populations and transportation occur in the same direction. This is a significant advantage for integration purposes into a lab-ona-chip device, where subsequent analytical steps can be performed on the cells downstream; for example, cell detection, cell lysis, polymerase chain reaction (PCR), etc. The localized excitation scheme also avoids the possible electrolysis problem that might occur in conventional dielectrophoresis and traveling wave dielectrophoresis, in which all the electrodes are energized throughout the separation process, and hence, a large electrical current is present. In our scheme, any electrodes are energized less than a few seconds at any instance, thus avoiding electrolysis due to a long period of excitation. Effect of Joule Heating and Electrically Induced Fluid Flow. For microfluidic devices, the fluid medium temperature, θ, due to Joule heating from the electric field, E, can be found by solving the corresponding diffusion equation,30,31
km ∇2θ + σm|E|2 ) 0 Analytical Chemistry, Vol. 79, No. 18, September 15, 2007
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Figure 12. Fractionation of a mixture of viable and nonviable yeast cells at the same operating conditions as above, at 50× optical magnification. The viable yeast cells were yellow, whereas the nonviable yeast cells were dark blue. The dark-field horizontal stripes were electrodes. Viable yeast cells were trailing the electric fields, whereas nonviable yeast cells were leading the electric fields. These two bands of cells can be clearly seen at +8.48 s. Pictures at time +4.76, +5.68, and +6.00 s show the intermediate process, where several viable yeast cells (white arrow) were moving up when the electric fields were traveling down. Video is available as Supporting Information on the journal website. Time indicated was when there were always three electrodes energized. 6986
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where km is the thermal conductivity of the fluid medium and σm is the conductivity of the fluid medium. For our experimental conditions, km scaled as 1, σm scaled as 10-2, and E scaled as 106. Thus, the temperature change ∆θ was estimated to scale as 0.01 K, due to a device size that scaled as 10-6. In fact, a finite element (FEM) simulation on our device configuration using parameters km of 0.6 J m-1 s-1 K-1 and σm of 305 µS cm-1 gave a temperature change in the domain to be no more than 0.03 K. Therefore, the Joule heating from the electric field had a minimal effect on the experiments. Joule heating may be significant for a fluid medium with a high conductivity (which was not the case in our experiments), since the temperature changes proportionally to the conductivity.31 In addition, it is expected that the electric field that polarizes the cells would also polarize the fluid medium, which would result in an induced fluid flow. For instance, it was observed that at a voltage of 10 Vpp, frequency of 100 kHz, and medium conductivity of 21.5 µS cm-1, viable yeast cells exhibited local rotation and spinning at the electrode edges, which is believed to be caused in part due to the induced fluid flow. There are at least two phenomena related to such an effect: namely, AC electroosmosis and electrothermal flow.32-36 Both phenomena can have a detrimental effect on dielectrophoresis separation. Several experiments were conducted using L0.60-µm latex spheres (SKU S37495, Invitrogen) as tracer particles to investigate the presence of an induced fluid flow at different dielectrophoretic conditions. Fluid flow effects are expected to be dominant as compared to the dielectrophoretic forces for such a small particle size. The experimental observations indicated that the AC electroosmosis was significant at an electrical frequency below 50 kHz. The fluid flow induced by AC electroosmosis spanned as far as 80 µm from its origin and exhibited strong xand z-directional movements. Electrothermal effects were observed at a frequency larger than 100 kHz and induced a fluid flow spanning a 20-µm zone. The electrothermal flow circulated mainly in the x-y plane. However, at the frequency employed in this investigation and for cells with size in the micrometer range, the dielectrophoretic forces were dominant, with negligible AC electroosmosis and electrothermal effects. Thus, it is concluded that cell fractionation and transportation observed in this investigation (30) Castellanos, A.; Ramos, A.; Gonza´lez, A.; Green, N. G.; Morgan, H. J. Phys. D: Appl. Phys. 2003, 36, 2584-2597. (31) Jones, T. B. IEE Proc.: Nanobiotechnol. 2003, 150, 39-46. (32) Ramos, A.; Morgan, H.; Green, N. G.; Castellanos, A. J. Phys. D: Appl. Phys. 1998, 31, 2338-2353. (33) Green, N. G.; Ramos, A.; Morgan, H. J. Phys. D: Appl. Phys. 2000, 33, 632-641. (34) Green, N. G.; Ramos, A.; Gonzalez, A.; Morgan, H.; Castellanos, A. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2000, 61, 40114018. (35) Green, N. G.; Ramos, A.; Gonzalez, A.; Castellanos, A.; Morgan, H. J. Phys. D: Appl. Phys. 2000, 33, L13-L17. (36) Green, N. G.; Ramos, A.; Gonzalez, A.; Castellanos, A.; Morgan, H. J. Electrostat. 2001, 53, 71-87.
are primarily due to dielectrophoresis and not to the induced fluid flow. This conclusion is further supported by the experimental results that the cells moved linearly along the microchannel. An induced fluid flow would generate vortices around the electrode edge, resulting in a curved path for cells. CONCLUSION A new cell fractionation and transportation technique based on moving dielectrophoresis (mDEP) using a programmable microfluidic device has been developed. A mixture of viable and nonviable yeast cells was separated to demonstrate the feasibility of the approach. The mDEP parameters were selected whereby viable cells experienced positive DEP and nonviable cells experienced negative DEP. Cells experiencing negative dielectrophoresis traveled in front of the moving electric field, whereas cells experiencing positive dielectrophoresis moved behind the moving electric field. As a result, a mixture of viable and nonviable cells was fractionated on the basis of their dielectrophoretic affinity. Improved separation resolution could be achieved by carrying out the process repeatedly. One distinctive advantage of mDEP is that cells are transported unidirectionally, irrespective of whether they experience positive or negative dielectrophoresis. This characteristic makes mDEP more amenable to integration into a labon-a-chip device than other dielectrophoretic methods. The mDEP fractionation scheme is suitable for cell separation processes that require accuracy, but it is limited to low-volume processing, which is characteristic of microfluidic systems. As such, it is more applicable for identification of specific cells in a liquid sample than for its quantification. This method is anticipated to complement the existing single-cell cage method. For instance, a mixture of cells can be first separated and then transported and aligned to different locations using this scheme, and finally, individual rare cells can be positioned for further analyses using the potential cage. ACKNOWLEDGMENT This work was supported by the Singapore-MIT Alliance research fund. The fabrication of the microfluidic chip was sponsored by the Institute of Materials Research and Engineering, Singapore. The yeast cells were kindly donated by Prof. Prakash P. Kumar from the Department of Biological Sciences, National University of Singapore and Dr. Chen Hui from the Department of Pediatrics, National University of Singapore. The authors also thank Prof. Ronald Pethig for valuable discussion on yeast cell modeling. SUPPORTING INFORMATION AVAILABLE Four movies as mentioned in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 23, 2007. Accepted July 7, 2007. AC070810U
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