Continuous separation of cells by balanced dielectrophoretic forces at multiple frequencies{ Thomas Braschler,{ a Nicolas Demierre,{* a Elisabete Nascimento, b Tiago Silva, b Abel G. Oliva b and Philippe Renaud a Received 6th July 2007, Accepted 25th October 2007 First published as an Advance Article on the web 15th November 2007 DOI: 10.1039/b710303d We present a particle-sorting device based on the opposition of dielectrophoretic forces. The forces are generated by an array of electrode chambers located in both sidewalls of a main flow channel. Particles with different dielectric response perceive different force magnitudes and are therefore continuously focused to different streamlines in the flow channel. We relate the particles’ dielectric response to their output position in the downstream channel. We demonstrate the performance of the device by separating a mixed yeast cell population into pure fractions of viable and nonviable cells. Finally, we use the device to enrich red blood cells infected with Babesia bovis, a major pathogen in cattle and simultaneously confirm the hypothesis that infection with B. bovis causes significant changes in the dielectric response of red blood cells. Introduction BioMEMS are increasingly used for isolation and analysis of cell populations. They offer the potential benefits of portability, low cost, low sample volumes and rapidity of the analysis. There are two approaches for cell sorting in microdevices. One possibility is to first measure the cells and then sort them based on the measurement results. Using this approach fluorescence activated cell sorting 1 (FACS) and dielectric activated cell sorting 2 (DACS) have been imple- mented on chip. The other possibility is to use forces that act directly on the cells, meaning that one sorts directly without prior measurement. We focus here on the second approach because it is potentially simpler, making it more attractive for point-of-care devices. A series of cellular properties have been used for such passive on-chip cell separation: differences in size and density, 3 specific adhesion, 4 magnetic properties, 5 acoustic impedance 6 and dielectric properties. Sorting based on dielectric properties makes use of dielectrophoresis (DEP) and has received much attention mainly because it is a label- free and contactless discrimination of cells. DEP is an electric force that results from the interaction between an electric field and the induced dipole on dielectric particles. The force attracts particles towards regions of high field strength (positive dielectrophoresis, pDEP) or repels them from these regions (negative dielectrophoresis, nDEP) according to the field frequency and the polarisabilities of the particles and the medium. DEP has been used for separating different cell populations, such as viable from nonviable yeast cells 7 , bacteria from nanoparticles 8 and normal mononuclear white blood cells from human promyelocytic leukemia cells. 9 In general, the devices oppose the DEP force to a physical force of different nature: gravity against DEP in free flow fractionation, 9 viscous drag against DEP 8,10–12 and diffusion in ratcheting mechanisms 13 (for a comprehensive review of DEP-based separation strategies, see Hughes 14 ). Most of these separation methods require the devices to be operated in batch mode, but some continuously operating devices have also been reported. 7,10,11 In this paper, we report a novel approach to DEP-based cell sorting. By opposing two DEP force fields driven by multiple frequency signals, we implement an efficient flow cytometer based on the resulting force equilibrium. We have previously shown 15 that by applying opposite negative DEP force fields (nDEP) from electrodes on opposite sides of a channel, particles can be focused towards an arbitrary streamline in a microchannel. Here, we extend the principle to multi- frequency continuous cell sorting. We use a chip consisting of a main channel, with an array of electrodes in dead-end chambers on each side (Fig. 1). The two electrode arrays are driven with AC signals at different frequencies. The particles are focused towards different positions depending on their dielectric response. This gives our device three important advantages: (1) There is no requirement for prior hydrodynamic or other focusing of the particle stream. Indeed, each particle ‘‘finds’’ its own equilibrium line in the channel. The final position does not depend on the position at the beginning of the sorting channel. This greatly facilitates device operation. (2) The device separates particles based on dielectric forces only. Other physical characteristics, such as size or density influence the measurement only as far as they directly influence the dielectric response. Therefore, particles of different sizes and densities can be analysed simultaneously without deteriorating performance. a Laboratory of Microsystems, Ecole Polytechnique Fe ´de ´rale de Lausanne, 1015 Lausanne, Switzerland. E-mail: nicolas.demierre@epfl.ch; Fax: +41 21 693 59 50; Tel: +41 21 693 68 38 b Instituto de Biologia Experimental e Tecnolo ´logical/ITQB, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal { Electronic supplementary information (ESI) available: Video show- ing separation of viable and nonviable cells. See DOI: 10.1039/ b710303d { The authors have equally contributed to the article. PAPER www.rsc.org/loc | Lab on a Chip 280 | Lab Chip, 2008, 8, 280–286 This journal is ß The Royal Society of Chemistry 2008