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Bidirectional field-flow particle separation method in a dielectrophoretic chip with 3D electrodes
Available online at www.sciencedirect.com
Sensors and Actuators B 129 (2008) 491–496
Bidirectional field-flow particle separation method in a dielectrophoretic chip with 3D electrodes夽 Ciprian Iliescu a,∗ , Liming Yu b , Francis E.H. Tay a,b , Bangtao Chen a a
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669, Singapore b National University of Singapore, Singapore Available online 22 November 2007
Abstract This paper proposes a bidirectional field-flow separation method in a dielectrophoretic chip with 3D electrodes. The DEP chip presents a sandwich structure glass/silicon/glass. The top glass layer assures two inlets and two outlets, disposed in cross, for the inlet/outlet of particle suspension and buffer solution, respectively. The silicon layer defines the walls of the microfluidic channels and at the same time the electrodes (rows of pillars with square cross-section) of the DEP device. The bottom glass presents via holes (one for each pillar) and a metallization layer which assures both the connections between pillars (a row of pillar being connected at one electrode) and also the connection of the electrodes with the PCB. The 3D electrodes structure that is used in this device is not only used for generating an uniform DEP force across the microfluidic channel but also for achieving a gradient of the velocity (and in this way a variable hydrodynamic force) in the microfluidic device. DEP and hydrodynamic forces are used in the separation technique of two particle populations. The method consists of four steps. First, the solution with the mixture of two particle population is inserted in the microfluidic chamber between the silicon pillars. Second, by applying an electric field the two populations are separated in different locations according to their electrical properties. In the third step one population is first collected at one outlet by flowing a fresh buffer solution. Finally, the second population is collected at the second outlet by flowing fresh buffer in the perpendicular direction. The device has been tested successfully with live/dead yeast cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Bio-MEMS; Dielectrophoresis; Cell separation; Microfluidic device
1. Introduction Separation of different cell populations is gaining more and more attention for lab-on-a-chip devices due to its potential important applications in biology and medicine. A successful separation of fetal cells from the mother blood can replace the amniocentesis technique used in Down syndrome detection. Also, in terms of cell culture and tissue regeneration, the separation of viable and non-viable cells is one important aspect. Dielectrophoresis (DEP) – the movement of neutral but polarisable particles in a non-uniform electric field – is one of the potential methods that can be used in cells separation [1,2]. An electric field gradient can be generated using three main methods. The first method utilizes microfabrication techniques to create electrodes of different shapes that can induce a change in the electric field magnitude across the particle. These electrodes 夽 This paper is part of the Eurosensors 2006 Special Issue Volume 127 Issue 1, 20 October 2007. ∗ Corresponding author. Tel.: +65 68247137; fax: +65 64789082. E-mail address: [email protected] (C. Iliescu).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.11.023
can be thin films [3,4], bulk electrodes that simultaneously define the microfluidic channel [5,6] or even a combination between a thin electrode and a bulk electrode [7]. The second method – travelling wave DEP – involves changing the phase of the applied electric field [8,9]. The last method is called “isolating DEP” or iDEP, as the electric field gradient is generated by a non-homogenous dielectric medium between the electrodes [10,11]. In previous work [12,13], we have proposed two separation methods in DEP devices with 3D electrodes. The important advantages of this structure are the low Joule effect and increased DEP force [14] within the whole volume of the microchannels which makes it very suitable for biological applications. This paper proposes a bidirectional separation method in a DEP chip with 3D electrode array. The DEP chip presents a sandwich structure glass/silicon/glass (Fig. 1). The top glass layer assures two inlets and two outlets, providing bidirectional input/output of fluid. The silicon layer defines the walls of the microfluidic channels and the electrode array (rows of prismatic pillars). The bottom glass presents via holes (one for each pillar) and a metallization layer which assures electrical connections between pillars (a row of silicon pillars being connected at one
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sists of a solution flowing with a particle suspension over an electrode array. When there are two populations that exhibit positive and negative DEP, one population will be trapped near the electrode while the other one will be repelled into the center of the chamber to be subsequentially pushed by the flow towards the outlet. Flow separators using thin electrodes had been reported in [15,16]. We have also reported a sequential field-flow separation technique in [12], separation under continuous flow method in [13] using DEP structure with 3D electrodes (described in [5]) and a field-flow method in an iDEP filtering chip in [17]. Another method uses a fluid velocity gradient (field-flow fraction) to separate particles [18]. Separation method using traveling wave DEP are reported in [19,20]. Early work using ratcheting mechanism (“Christmas tree” electrode) was performed by Rousselet et al. [21]. 3. Bidirectional separation in a DEP chip with 3D electrode array
Fig. 1. Schematic view of the DEP device.
electrode). The 3D electrodes structure that is used in this device is not only used for generating of an uniform dielectric force across the microfluidic channel but also for achieving a gradient of the fluid velocity (and subsequently a variable hydrodynamic force) in the microfluidic channel. 2. Separation methods Hughes in [1] and Gascoyne and Vykoukal in [2] gave a brief description of separation methods using dielectrophoresis. These can be summarized as: flow separation, field-flow fractionation, stepped flow separation, travel wave dielectrophoresis and the ratcheting mechanism. The flow separation method con-
The separation method using DEP chip with 3D electrode array can be described in four steps (Fig. 2). First, the solution with the mixture of two particle populations is injected into the microfluidic chamber. Secondly, by applying an electric field the two populations are separated into different locations according to their electrical properties. In the third step, a fresh buffer solution flows through in one direction, and one of the population is collected at one of the outlets. Finally, the second population is collected at the second outlet by flowing the fresh buffer in the perpendicular direction. As it was previously mentioned, in the second step the populations must be separated in different locations of the chip according to their electrical properties. This was achieved using dielectrophoresis. The DEP force acting on a spherical particle with radius r is given by [22]: F = 2πr 3 εm Re[K(ω)]∇E2
Fig. 2. Main steps of the separation technique.
(1)
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493
Besides the DEP force, the particles can also experience hydrodynamic forces due to the movement of fluid. The hydrodynamic force can be given by F = 6πηrv
Fig. 3. The electric field distribution in the DEP structure.
where εm is the absolute permittivity of the suspending medium, E is the electric field strength and Re[K(ω)] is the real part of the polarization factor, defined as [22]: K(ω) =
ε∗p − ε∗m ε∗p + 2ε∗m
(2)
where ε∗p and ε∗m are the complex permittivity of the particle and medium, respectively. Re[K(ω)] can play an important role: if it is positive, the particles will move to the regions with higher electric field (positive DEP). Alternatively, if it presents negative values, the particles will move to regions with lower electric filed (negative DEP). In our particular case, the particles that exhibit positive DEP will be trapped between the tips of the pillars of opposite electrodes (Zone A), while, for negative DEP, the particle will be trapped between the pillar tips of the same electrode (Zone B). This is confirm further by the simulation (using Maxwell software) presented in Fig. 3.
(3)
where η is the viscosity of the fluid, r is the radius of the particle and v is the velocity of the fluid. The hydrodynamic force is directly proportional to the velocity and radius of the particle. Decreasing the velocity by one order decreases the hydrodynamic force by one order. Therefore, the flow profile of the fluid inside the microchannel plays an important role in the movement of particles. Fig. 4 shows an ANSYS simulation of the flow through the electrode-pillars. As a result, these geometries lead to a huge gradient in the flow velocity. The zone with higher fluid velocity experiences larger hydrodynamic forces (Zone A) increases and the particles that are trapped using positive DEP force can be removed. While, in the low velocity regions (Zone B) the hydrodynamic force is too weak to remove the particles that experience negative DEP. 4. Fabrication of the DEP device Some of the major keys of the fabrication processes of the device are shown in Fig. 5. The process is similar with the fabrication of the DEP device presented in [5,7]. Here, we just give a brief description of the fabrication process. Four holes were first etched through in a glass wafer (Corning 7740) with a thickness of 500 m for the inlet–outlet access of fluid using a low-stress amorphous silicon/silicon carbide/photoresist mask (Fig. 5a) technique describe previous in [23]. After that a 4 in. silicon wafer with a thickness of 100 m was wafer-to-wafer anodically bonded to a 4 in. Pyrex glass wafer (Corning 7740) with a thickness of 500 m at 305 ◦ C with an applied voltage of 800 V for 20 min (Fig. 5b). Next, the microchannel walls and electrode array were defined in the silicon wafer using deep
Fig. 4. Simulation of the flow in the microfluidic device.
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Fig. 5. Fabrication processes of DEP chip: (a) wet etching through holes in a Pyrex glass wafer, (b) bonding of a thin heavy doped silicon wafer on glass, (c) DRIE of silicon wafer, (d) second anodic bonding process, (e) thinning of the bottom glass wafer (wet etching), (f) via holes (wet etching process) and (g) metallization.
Fig. 6. Picture (top view and bottom view) with the fabricated DEP chips.
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RIE technology through a silicon oxide mask (Fig. 5c). Next, the top glass wafer with two inlet/outlets was bonded to the patterned silicon wafer using second anodic bonding at 400 ◦ C with an applied voltage of 1200 V and an applied force of 1500 N (Fig. 5d). Subsequently, the bottom glass wafer was thinned from 500 m to around 100 m using an optimized wet etching process in a HF 49%/HCl 37% solution (10/1) (Fig. 5e) [24]. The uniformity of the process was in an acceptable range (5%) and the resulted surface roughness was also acceptable (10 nm). Next, the via holes were wet etched through the thinned bottom glass wafer in the same solution using a low-stress amorphous silicon mask [25], to provide a path to connect the outside metal electrodes and silicon electrodes (Fig. 5f). Finally, a Cr/Au metal layer was deposited on the bottom glass surface, to provide the electrical connection to silicon electrodes (Fig. 5g). The patterning of the metal electrodes in the bottom glass wafer with 100-m deep via holes was transferred from photoresist to metal using an optimized spray-coating process described in [26], with a mixture of positive photoresist AZ4620, MEK and PMGA. The fabricated DEP device is shown in Fig. 6. 5. Experiment The feasibility of the separation mechanism in the DEP chip with 3D electrode array was proved using populations of viable and non-viable yeast cells were used. 100 mg of yeast, 100 mg of sugar and 2 ml DI water were incubated in an Eppendorf tube at 37 ◦ C for 2 h. Next, the cell culture was divided into two with one population being boiled for several minutes in 5 ml boiling DI water (dead cells). Both viable and non-viable populations were mixed and resuspended in the separation buffer, which was a mixture of phosphate buffered saline (PBS) and DI water. The conductivity of the separation buffer was around 20 S cm−1 . The final concentration of the cells was 107 cells/ml. A function generator and a linear amplifier were used for the drive signal generation of the dielectrophoretic chip. The drive signal was increased from 0 to 25 V peak to peak gradually with the signal frequency being in the range of 20–100 kHz. Fig. 7 showed the bidirectional separation processes for viable and non-viable yeast cells in a DEP chip with triangular electrode array. The mixture of viable and non-viable yeast cells was injected into the microchannel (Fig. 7a); non-viable yeast cells experiencing positive DEP move to the highest electric field regions and viable yeast cells experiencing negative DEP move to the lowest electric field regions (Fig. 7b); next, non-viable yeast cells were collected from the other one outlet by flushing a fresh buffer solution (Fig. 7c). Finally the other population is collected at the opposite outlet using same method. In order to test the efficiency of the DEP device yeast viability was counted using the live/dead yeast cell viability kit from molecular probes (Invitrogen) following the method as described in the instruction manual and presented also in [17]. Cell counting was performed using a hemacytometer. The cells were then observed with an Olympus IX71 microscope using the blue fluorescent cube with an excitation wavelength of about 480 nm and an emission wavelength >520 nm for the FUN 1 cell stain. The results show an efficiency of separation of over 95%.
Fig. 7. Optical image with the main steps of the separation technique: (a) insertion of dead and live cell, (b) trapping of the cells by positive and negative DEP and (c) collection of one cell population by flowing a fresh buffer solution.
6. Conclusions This paper proposed a bidirectional separation method in a DEP chip with 3D electrode array, which also functions as microfluidic channels. These electrodes serve a double function. The first function is to generate positive and negative dielectrophoretic force, trapping two populations of cells in different locations. The second function is to produce a gradient of fluid velocity. As a consequence, the resulted hydrodynamic force will drag out the population trapped by positive dielectrophoresis. After the removal of the electric field, the remaining population can then be collected at the outlet. This method has been successfully tested using the mixture of viable and non-viable yeast cells, which should provide a great potential for separation of different bio-particles in biological and medical fields.
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References [1] M.P. Hughes, Strategies for dielectrophoretic separation in laboratory-ona-chip systems, Electrophoresis 23 (16) (2002) 2569–2582. [2] P.R.C. Gascoyne, J. Vykoukal, Particle separation by dielectrophoresis, Electrophoresis 23 (2002) 1973–1983. [3] S. Masuda, M. Washizu, T. Nanba, Novel method for cell fusion in field construction area in fluid integrated circuit, IEEE Trans. Ind. Appl. 25 (4) (1989) 732–737. [4] J. Park, B. Kim, S.K. Choi, H. Su, S.H. Lee, K.-I. Lee, An efficient cell separation system using 3D-asymmetric microelectrodes, Lab Chip 5 (2005) 1264–1270. [5] C. Iliescu, G.L. Xu, V. Samper, F.E.H. Tay, Fabrication of a dielectrophoretic chip with 3D silicon electrodes, J. Micromech. Microeng. 15 (3) (2005) 494–500. [6] C. Iliescu, F.E.H. Tay, G.L. Xu, L. Yu, V. Samper, A dielectrophoretic chip packaged at wafer level, Microsyst. Technol. 12 (10–11) (2006) 987– 992. [7] C. Iliescu, L.M. Yu, G.L. Xu, F.E.H. Tay, A dielectrophoretic chip with a 3D electric field gradient, J. Microelectromech. Syst. 15 (6) (2006) 1506– 1513. [8] R. Pethig, M.S. Talary, R.S. Lee, Enhancing travelling-wave dielectrophoresis with signal superposition, Eng. Med. Biol. Mag. IEEE 22 (6) (2003) 43–50. [9] L. Cui, D. Holmes, H. Morgan, The dielectrophoretic levitation and separation of latex beads in microchips, Electrophoresis 22 (2001) 3893– 3901. [10] B.H. Lapizco-Encinas, B.A. Simmons, E.B. Cummings, Y. Fintschenko, Insulator-based dielectrophoresis for the selective concentration and separation of live bacteria in water, Electrophoresis 25 (10–11) (2004) 1695–1704. [11] C. Iliescu, G.L. Xu, F.C. Loe, P.L. Ong, F.E.H. Tay, A 3 dimensional dielectrophoretic filter chip, Electrophoresis 28 (7) (2007) 1107– 1114. [12] L. Yu, C. Iliescu, G. Xu, F.E.H. Tay, Sequential field-flow cell separation method in a dielectrophoretic chip with 3D electrodes, J. Microelectromech. Syst. 16 (5) (2007) 1120–1129. [13] C. Iliescu, G. Tresset, G.L. Xu, Continuous field-flow separation of particle populations in a dielectrophoretic chip with 3D electrodes, Appl. Phys. Lett. 90 (23) (2007) 234104. [14] F.E.H. Tay, L. Yu, A.J. Pang, C. Iliescu, Electrical and thermal characterization of a dielectrophoretic chip with 3D electrodes for cells manipulation, Electrochim. Acta 52 (8) (2007) 2862–2868. [15] H. Morgan, A.G. Izquierdo, D. Bakewell, N.G. Green, A. Ramos, The dielectrophoretic and traveling wave forces for interdigitated electrode arrays: analytical solution using Fourier series, J. Phys. D: Appl. Phys. 34 (2001) 1553–1561. [16] R. Pethig, J.P.H. Burt, A. Parton, N. Rizvi, M.S. Talary, J.A. Tame, Development of biofactory-on-a-chip technology using excimer laser micromachining, J. Micromech. Microeng. 8 (1998) 57–63. [17] C. Iliescu, G.L. Xu, P.L. Ong, K.J. Leck, Dielectrophoretic separation of biological samples in a 3D filtering-chip, J. Micromech. Microeng. 17 (7) (2007) S128–S136. [18] G.H. Markx, R. Pethig, J. Rousselet, The dielectrophoretic levitation of latex beads, with reference to field-flow fractionation, J. Phys. D: Appl. Phys. 30 (1997) 2470–2477. [19] Y. Huang, X.-B. Wang, J. Tame, R. Pethig, Electrokinetic behaviour of colloidal particles in travelling electric fields: studies using yeast cells, J. Phys. D: Appl. Phys. 26 (1993) 1528–1535. [20] G. Fuhr, S. Fiedler, T. M¨uller, T. Schnelle, H. Glasser, T. Lis, B. Wagner, Particle micromanipulator consisting of two orthogonal channels with travelling-wave electrode structures, Sens. Actuator A 41 (1–3) (1994) 230–239. [21] J. Rousselet, L. Salome, A. Ajdari, J. Prost, Directional motion of Brownian particles induced by a periodic asymmetric potential, Nature 370 (1994) 446–447.
[22] T.B. Jones, Electromechanics of Particles, Cambridge University Press, 1995. [23] C. Iliescu, D.P. Poenar, M. Carp, F.C. Loe, A microfluidic device for impedance spectroscopy analysis of biological samples, Sens. Actuator B 123 (1) (2007) 168–176. [24] C. Iliescu, J. Jing, F.E.H. Tay, J.M. Miao, T.T. Sun, Characterization of masking layers for deep wet etching of glass in an improved HF/HCl solution, Surf. Coat. Technol. 198 (1–3) (2005) 314–318. [25] C. Iliescu, J. Miao, F.E.H. Tay, Optimization of PECVD amorphous silicon process for deep wet etching of Pyrex glass, Surf. Coat. Technol. 192 (1) (2005) 43–47. [26] Y.Y. Lee, L. Yu, F.E.H. Tay, C. Iliescu, Characterization of spray coating photoresist for MEMS applications, Rom. J. Inform. Sci. Technol. 8 (4) (2005) 383–391.
Biographies Ciprian Iliescu was born in Bucharest, Romania in 1965. He received BS degree and PhD degree from school of mechanical engineering, Polytechnic University of Bucharest in 1989 and 1999 respectively. He has more then 17 years working experience in microfabrication. While pursuing his PhD degree he worked at Baneasa S.A. where he was involved in design and fabrication of pressure sensors. Between 1997 and 2000 he collaborated with IMT Bucharest on projects related magnetic sensors. Between 2001 and 2003 he worked as postdoctoral fellow at Nanyang Technological University, Singapore, being involved in projects related microphone, wafer level packaging of MEMS devices and RF microrelay. Currently, he is senior research scientist at the Institute of Bioengineering and Nanotechnology, Singapore. His current research projects are related to dielectrophoresis, electrical characterization of cells by impedance spectroscopy, transdermal drug delivery using microneedles array. Dr. Iliescu is author and co-author of more then 150 papers published in journals and conferences proceedings. Liming Yu, was born in Zhejiang province, China in 1976. He received the BS degree in 1999 and MS degree in 2002 in the Department of Precision Instruments and Mechanology from Tsinghua University, P.R. China. He received his PhD from National University of Singapore, Mechanical Engineering Department in 2007 with a work regarding dielectrophoresis. His research interests include MEMS technologies, Bio-MEMS and Dielectrophoresis (DEP). Francis E.H. Tay received the PhD degree from the Massachusetts Institute of Technology (MIT), Cambridge, in 1995. He is currently an associate professor, with the Department of Mechanical Engineering, Faculty of Engineering, National University of Singapore. He is the Deputy Director (Industry), for the Centre of Intelligent Products and Manufacturing Systems, where he takes charge of research projects involving industry and the Centre. He is also the medical device group leader with the Institute of Bioengineering and Nanotechnology (IBN). Dr. Tay was also the founding director of the Microsystems Technology Initiative (MSTI), and had established the Microsystems Technology Specialization. He had also served as the technical advisor, in the Micro and Nano Systems Laboratory, Institute of Materials Research Engineering (IMRE). His research areas are MEMS, biotechnology, nanotechnology, and wearable devices. He is also the principal investigator for several Agency for Science, Technology and Research (A*STAR) projects. The most recent one is the “MEMSWear: Incorporating MEMS Technology into the Smart Shirt for Geriatric Healthcare”, which was widely published by the local and overseas media and well received by the public. Bangtao Chen received the BS degree in mechanical engineering from Huazhong University of Science and Technology, China, in 2001. He received his PhD degree in mechanical engineering from Nanyang Technological University, Singapore, in 2007. His PhD research was on the development of MEMS microactuators based on silicon-on-glass process. He is currently working as a post-doctoral fellow in Institute of Bioengineering and Nanotechnology, Singapore. His research interests include microfabrication process, sensors and actuators, microfluidic applications in bioengineering, thin film characterization.
Sensors and Actuators B 129 (2008) 491–496
Bidirectional field-flow particle separation method in a dielectrophoretic chip with 3D electrodes夽 Ciprian Iliescu a,∗ , Liming Yu b , Francis E.H. Tay a,b , Bangtao Chen a a
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669, Singapore b National University of Singapore, Singapore Available online 22 November 2007
Abstract This paper proposes a bidirectional field-flow separation method in a dielectrophoretic chip with 3D electrodes. The DEP chip presents a sandwich structure glass/silicon/glass. The top glass layer assures two inlets and two outlets, disposed in cross, for the inlet/outlet of particle suspension and buffer solution, respectively. The silicon layer defines the walls of the microfluidic channels and at the same time the electrodes (rows of pillars with square cross-section) of the DEP device. The bottom glass presents via holes (one for each pillar) and a metallization layer which assures both the connections between pillars (a row of pillar being connected at one electrode) and also the connection of the electrodes with the PCB. The 3D electrodes structure that is used in this device is not only used for generating an uniform DEP force across the microfluidic channel but also for achieving a gradient of the velocity (and in this way a variable hydrodynamic force) in the microfluidic device. DEP and hydrodynamic forces are used in the separation technique of two particle populations. The method consists of four steps. First, the solution with the mixture of two particle population is inserted in the microfluidic chamber between the silicon pillars. Second, by applying an electric field the two populations are separated in different locations according to their electrical properties. In the third step one population is first collected at one outlet by flowing a fresh buffer solution. Finally, the second population is collected at the second outlet by flowing fresh buffer in the perpendicular direction. The device has been tested successfully with live/dead yeast cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Bio-MEMS; Dielectrophoresis; Cell separation; Microfluidic device
1. Introduction Separation of different cell populations is gaining more and more attention for lab-on-a-chip devices due to its potential important applications in biology and medicine. A successful separation of fetal cells from the mother blood can replace the amniocentesis technique used in Down syndrome detection. Also, in terms of cell culture and tissue regeneration, the separation of viable and non-viable cells is one important aspect. Dielectrophoresis (DEP) – the movement of neutral but polarisable particles in a non-uniform electric field – is one of the potential methods that can be used in cells separation [1,2]. An electric field gradient can be generated using three main methods. The first method utilizes microfabrication techniques to create electrodes of different shapes that can induce a change in the electric field magnitude across the particle. These electrodes 夽 This paper is part of the Eurosensors 2006 Special Issue Volume 127 Issue 1, 20 October 2007. ∗ Corresponding author. Tel.: +65 68247137; fax: +65 64789082. E-mail address: [email protected] (C. Iliescu).
0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.11.023
can be thin films [3,4], bulk electrodes that simultaneously define the microfluidic channel [5,6] or even a combination between a thin electrode and a bulk electrode [7]. The second method – travelling wave DEP – involves changing the phase of the applied electric field [8,9]. The last method is called “isolating DEP” or iDEP, as the electric field gradient is generated by a non-homogenous dielectric medium between the electrodes [10,11]. In previous work [12,13], we have proposed two separation methods in DEP devices with 3D electrodes. The important advantages of this structure are the low Joule effect and increased DEP force [14] within the whole volume of the microchannels which makes it very suitable for biological applications. This paper proposes a bidirectional separation method in a DEP chip with 3D electrode array. The DEP chip presents a sandwich structure glass/silicon/glass (Fig. 1). The top glass layer assures two inlets and two outlets, providing bidirectional input/output of fluid. The silicon layer defines the walls of the microfluidic channels and the electrode array (rows of prismatic pillars). The bottom glass presents via holes (one for each pillar) and a metallization layer which assures electrical connections between pillars (a row of silicon pillars being connected at one
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sists of a solution flowing with a particle suspension over an electrode array. When there are two populations that exhibit positive and negative DEP, one population will be trapped near the electrode while the other one will be repelled into the center of the chamber to be subsequentially pushed by the flow towards the outlet. Flow separators using thin electrodes had been reported in [15,16]. We have also reported a sequential field-flow separation technique in [12], separation under continuous flow method in [13] using DEP structure with 3D electrodes (described in [5]) and a field-flow method in an iDEP filtering chip in [17]. Another method uses a fluid velocity gradient (field-flow fraction) to separate particles [18]. Separation method using traveling wave DEP are reported in [19,20]. Early work using ratcheting mechanism (“Christmas tree” electrode) was performed by Rousselet et al. [21]. 3. Bidirectional separation in a DEP chip with 3D electrode array
Fig. 1. Schematic view of the DEP device.
electrode). The 3D electrodes structure that is used in this device is not only used for generating of an uniform dielectric force across the microfluidic channel but also for achieving a gradient of the fluid velocity (and subsequently a variable hydrodynamic force) in the microfluidic channel. 2. Separation methods Hughes in [1] and Gascoyne and Vykoukal in [2] gave a brief description of separation methods using dielectrophoresis. These can be summarized as: flow separation, field-flow fractionation, stepped flow separation, travel wave dielectrophoresis and the ratcheting mechanism. The flow separation method con-
The separation method using DEP chip with 3D electrode array can be described in four steps (Fig. 2). First, the solution with the mixture of two particle populations is injected into the microfluidic chamber. Secondly, by applying an electric field the two populations are separated into different locations according to their electrical properties. In the third step, a fresh buffer solution flows through in one direction, and one of the population is collected at one of the outlets. Finally, the second population is collected at the second outlet by flowing the fresh buffer in the perpendicular direction. As it was previously mentioned, in the second step the populations must be separated in different locations of the chip according to their electrical properties. This was achieved using dielectrophoresis. The DEP force acting on a spherical particle with radius r is given by [22]: F = 2πr 3 εm Re[K(ω)]∇E2
Fig. 2. Main steps of the separation technique.
(1)
C. Iliescu et al. / Sensors and Actuators B 129 (2008) 491–496
493
Besides the DEP force, the particles can also experience hydrodynamic forces due to the movement of fluid. The hydrodynamic force can be given by F = 6πηrv
Fig. 3. The electric field distribution in the DEP structure.
where εm is the absolute permittivity of the suspending medium, E is the electric field strength and Re[K(ω)] is the real part of the polarization factor, defined as [22]: K(ω) =
ε∗p − ε∗m ε∗p + 2ε∗m
(2)
where ε∗p and ε∗m are the complex permittivity of the particle and medium, respectively. Re[K(ω)] can play an important role: if it is positive, the particles will move to the regions with higher electric field (positive DEP). Alternatively, if it presents negative values, the particles will move to regions with lower electric filed (negative DEP). In our particular case, the particles that exhibit positive DEP will be trapped between the tips of the pillars of opposite electrodes (Zone A), while, for negative DEP, the particle will be trapped between the pillar tips of the same electrode (Zone B). This is confirm further by the simulation (using Maxwell software) presented in Fig. 3.
(3)
where η is the viscosity of the fluid, r is the radius of the particle and v is the velocity of the fluid. The hydrodynamic force is directly proportional to the velocity and radius of the particle. Decreasing the velocity by one order decreases the hydrodynamic force by one order. Therefore, the flow profile of the fluid inside the microchannel plays an important role in the movement of particles. Fig. 4 shows an ANSYS simulation of the flow through the electrode-pillars. As a result, these geometries lead to a huge gradient in the flow velocity. The zone with higher fluid velocity experiences larger hydrodynamic forces (Zone A) increases and the particles that are trapped using positive DEP force can be removed. While, in the low velocity regions (Zone B) the hydrodynamic force is too weak to remove the particles that experience negative DEP. 4. Fabrication of the DEP device Some of the major keys of the fabrication processes of the device are shown in Fig. 5. The process is similar with the fabrication of the DEP device presented in [5,7]. Here, we just give a brief description of the fabrication process. Four holes were first etched through in a glass wafer (Corning 7740) with a thickness of 500 m for the inlet–outlet access of fluid using a low-stress amorphous silicon/silicon carbide/photoresist mask (Fig. 5a) technique describe previous in [23]. After that a 4 in. silicon wafer with a thickness of 100 m was wafer-to-wafer anodically bonded to a 4 in. Pyrex glass wafer (Corning 7740) with a thickness of 500 m at 305 ◦ C with an applied voltage of 800 V for 20 min (Fig. 5b). Next, the microchannel walls and electrode array were defined in the silicon wafer using deep
Fig. 4. Simulation of the flow in the microfluidic device.
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Fig. 5. Fabrication processes of DEP chip: (a) wet etching through holes in a Pyrex glass wafer, (b) bonding of a thin heavy doped silicon wafer on glass, (c) DRIE of silicon wafer, (d) second anodic bonding process, (e) thinning of the bottom glass wafer (wet etching), (f) via holes (wet etching process) and (g) metallization.
Fig. 6. Picture (top view and bottom view) with the fabricated DEP chips.
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RIE technology through a silicon oxide mask (Fig. 5c). Next, the top glass wafer with two inlet/outlets was bonded to the patterned silicon wafer using second anodic bonding at 400 ◦ C with an applied voltage of 1200 V and an applied force of 1500 N (Fig. 5d). Subsequently, the bottom glass wafer was thinned from 500 m to around 100 m using an optimized wet etching process in a HF 49%/HCl 37% solution (10/1) (Fig. 5e) [24]. The uniformity of the process was in an acceptable range (5%) and the resulted surface roughness was also acceptable (10 nm). Next, the via holes were wet etched through the thinned bottom glass wafer in the same solution using a low-stress amorphous silicon mask [25], to provide a path to connect the outside metal electrodes and silicon electrodes (Fig. 5f). Finally, a Cr/Au metal layer was deposited on the bottom glass surface, to provide the electrical connection to silicon electrodes (Fig. 5g). The patterning of the metal electrodes in the bottom glass wafer with 100-m deep via holes was transferred from photoresist to metal using an optimized spray-coating process described in [26], with a mixture of positive photoresist AZ4620, MEK and PMGA. The fabricated DEP device is shown in Fig. 6. 5. Experiment The feasibility of the separation mechanism in the DEP chip with 3D electrode array was proved using populations of viable and non-viable yeast cells were used. 100 mg of yeast, 100 mg of sugar and 2 ml DI water were incubated in an Eppendorf tube at 37 ◦ C for 2 h. Next, the cell culture was divided into two with one population being boiled for several minutes in 5 ml boiling DI water (dead cells). Both viable and non-viable populations were mixed and resuspended in the separation buffer, which was a mixture of phosphate buffered saline (PBS) and DI water. The conductivity of the separation buffer was around 20 S cm−1 . The final concentration of the cells was 107 cells/ml. A function generator and a linear amplifier were used for the drive signal generation of the dielectrophoretic chip. The drive signal was increased from 0 to 25 V peak to peak gradually with the signal frequency being in the range of 20–100 kHz. Fig. 7 showed the bidirectional separation processes for viable and non-viable yeast cells in a DEP chip with triangular electrode array. The mixture of viable and non-viable yeast cells was injected into the microchannel (Fig. 7a); non-viable yeast cells experiencing positive DEP move to the highest electric field regions and viable yeast cells experiencing negative DEP move to the lowest electric field regions (Fig. 7b); next, non-viable yeast cells were collected from the other one outlet by flushing a fresh buffer solution (Fig. 7c). Finally the other population is collected at the opposite outlet using same method. In order to test the efficiency of the DEP device yeast viability was counted using the live/dead yeast cell viability kit from molecular probes (Invitrogen) following the method as described in the instruction manual and presented also in [17]. Cell counting was performed using a hemacytometer. The cells were then observed with an Olympus IX71 microscope using the blue fluorescent cube with an excitation wavelength of about 480 nm and an emission wavelength >520 nm for the FUN 1 cell stain. The results show an efficiency of separation of over 95%.
Fig. 7. Optical image with the main steps of the separation technique: (a) insertion of dead and live cell, (b) trapping of the cells by positive and negative DEP and (c) collection of one cell population by flowing a fresh buffer solution.
6. Conclusions This paper proposed a bidirectional separation method in a DEP chip with 3D electrode array, which also functions as microfluidic channels. These electrodes serve a double function. The first function is to generate positive and negative dielectrophoretic force, trapping two populations of cells in different locations. The second function is to produce a gradient of fluid velocity. As a consequence, the resulted hydrodynamic force will drag out the population trapped by positive dielectrophoresis. After the removal of the electric field, the remaining population can then be collected at the outlet. This method has been successfully tested using the mixture of viable and non-viable yeast cells, which should provide a great potential for separation of different bio-particles in biological and medical fields.
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Biographies Ciprian Iliescu was born in Bucharest, Romania in 1965. He received BS degree and PhD degree from school of mechanical engineering, Polytechnic University of Bucharest in 1989 and 1999 respectively. He has more then 17 years working experience in microfabrication. While pursuing his PhD degree he worked at Baneasa S.A. where he was involved in design and fabrication of pressure sensors. Between 1997 and 2000 he collaborated with IMT Bucharest on projects related magnetic sensors. Between 2001 and 2003 he worked as postdoctoral fellow at Nanyang Technological University, Singapore, being involved in projects related microphone, wafer level packaging of MEMS devices and RF microrelay. Currently, he is senior research scientist at the Institute of Bioengineering and Nanotechnology, Singapore. His current research projects are related to dielectrophoresis, electrical characterization of cells by impedance spectroscopy, transdermal drug delivery using microneedles array. Dr. Iliescu is author and co-author of more then 150 papers published in journals and conferences proceedings. Liming Yu, was born in Zhejiang province, China in 1976. He received the BS degree in 1999 and MS degree in 2002 in the Department of Precision Instruments and Mechanology from Tsinghua University, P.R. China. He received his PhD from National University of Singapore, Mechanical Engineering Department in 2007 with a work regarding dielectrophoresis. His research interests include MEMS technologies, Bio-MEMS and Dielectrophoresis (DEP). Francis E.H. Tay received the PhD degree from the Massachusetts Institute of Technology (MIT), Cambridge, in 1995. He is currently an associate professor, with the Department of Mechanical Engineering, Faculty of Engineering, National University of Singapore. He is the Deputy Director (Industry), for the Centre of Intelligent Products and Manufacturing Systems, where he takes charge of research projects involving industry and the Centre. He is also the medical device group leader with the Institute of Bioengineering and Nanotechnology (IBN). Dr. Tay was also the founding director of the Microsystems Technology Initiative (MSTI), and had established the Microsystems Technology Specialization. He had also served as the technical advisor, in the Micro and Nano Systems Laboratory, Institute of Materials Research Engineering (IMRE). His research areas are MEMS, biotechnology, nanotechnology, and wearable devices. He is also the principal investigator for several Agency for Science, Technology and Research (A*STAR) projects. The most recent one is the “MEMSWear: Incorporating MEMS Technology into the Smart Shirt for Geriatric Healthcare”, which was widely published by the local and overseas media and well received by the public. Bangtao Chen received the BS degree in mechanical engineering from Huazhong University of Science and Technology, China, in 2001. He received his PhD degree in mechanical engineering from Nanyang Technological University, Singapore, in 2007. His PhD research was on the development of MEMS microactuators based on silicon-on-glass process. He is currently working as a post-doctoral fellow in Institute of Bioengineering and Nanotechnology, Singapore. His research interests include microfabrication process, sensors and actuators, microfluidic applications in bioengineering, thin film characterization.