Dielectrophoretic Characterization and Separation of Antibody-Coated Submicrometer Latex Spheres Michael P. Hughes † and Hywel Morgan* Bioelectronics Research Centre, Department of Electronic Engineering, University of Glasgow, Glasgow G12 8QQ, U.K. The dielectrophoretic behavior of carboxylated 2 1 6 -nm- diameter latex spheres has been characterized as a function of both medium conductivity and applied field frequency. Dielectrophoretic crossover measurements and analysis were used to characterize the dielectric properties of the particles. The particles were function- alized with antibodies using 1 -ethyl-3 -(3 -dimethylamino- propyl)carbodiimide (EDAC)-based coupling. Measure- ments indicated that the surface conductance of the native particles was 1.2 nS and that this reduced to a value of 0.7 nS after EDAC treatment and 0.25 nS after antibody coupling. Changes in the dielectrophoretic spectrum of the particles were exploited to demonstrate the principle of separation of unlabeled and protein-labeled particles. This demonstrates the potential for the development of new affinity separation systems based on ac electrokinetic methods. When a dielectric particle is suspended in a spatially nonuni- form electric field, the interaction of the applied field and the induced dipole moment generates a force on the particle. This force, termed dielectrophoresis (DEP), 1,2 has been demonstrated to be an effective means of manipulating particles in solution. Recent advances in electrode fabrication methods, such as the use of electron beam lithography, means that DEP can now be used to manipulate submicrometer particles such as viruses, 3-5 latex spheres, 6,7 and macromolecules. 8,9 The magnitude and direction of the dielectrophoretic force is governed both by the electric field and by the complex permittivity of the particle and suspending medium. These depend on the frequency of the applied field, so that the magnitude and/ or direction of force exerted on a particle can vary with the frequency. The sign of the force denotes whether the particle is attracted to regions of high electric field or repelled from them, modes of behavior termed positive and negative DEP, respectively. Since the direction of force depends on a particle’s specific dielectric properties, particles with differing properties will experi- ence different forces. It has been demonstrated that, under the appropriate conditions (e.g., medium conductivity and field frequency), particles of different types experience forces in different directionssacting toward or away from regions of high electric field. Thus, dielectrophoretic methods have been used to separate cells such as cancerous and normal blood cells, 10 different strains of bacteria, 11 and different types of viruses. 12 Recent advances in the field of DEP separation have shown that fluid flow (Stoke’s force), gravitational, and DEP forces can be combined to achieve particle separation in a DEP-gravitational field-flow fractionation system. 13-15 As has been shown by Green and Morgan 16,17 the DEP behavior of submicrometer particles is governed largely by surface properties. This means that DEP could be used to detect and measure changes in the composition of a particle’s surface, e.g., changes following binding of an antigen to a surface-immobilized antibody. Such changes are expected to give rise to variations in the dielectric properties of the particles, resulting in differences in the magnitude and/ or direction of the DEP force. Thus, a DEP- based separation technology could be developed for submicrome- ter particles as a new type of affinity fractionation. In this paper, we demonstrate that the dielectrophoretic behavior of 216-nm-diameter fluorescent latex spheres depends on the chemical composition of the surface. The dielectrophoretic properties of the spheres was measured for three cases: (i) beads as supplied by the manufacturer; (ii) after surface activation (for protein attachment) with 1-ethyl-3-(3-dimethylaminopropyl)carbo- diimide (EDAC); and (iii) after immobilization of a monolayer of protein. It is shown that the DEP force on the spheres varies with * Corresponding author: (Tel) +44 141 330 5237; (fax) +44 141 330 4907; (e-mail) h.morgan@ elec.gla.ac.uk. † Present address: European Institute of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 5XH, U.K. (1) Pohl, H. A. Dielectrophoresis; Cambridge University Press: Cambridge, 1978. (2) Jones, T. B. Electromechanics of particles; Cambridge University Press: Cambridge, 1995. (3) Mu ¨ ller, T.; Fiedler, S.; Schnelle, T.; Ludwig, K.; Jung, H.; Fuhr, G. Biotechnol. Tech. 1996 , 10, 221-226. (4) Morgan, H.; Green, N. G. J. Electrostatics 1997 , 42, 279-293. (5) Hughes, M. P.; Morgan, H.; Rixon, F. J.; Burt, J. P. H.; Pethig, R. Biochim. Biophys. Acta 1998 , 1425, 119-126. (6) Mu ¨ ller, T.; Gerardino, A.; Schnelle, T.; Shirley, S. G.; Bordoni, F.; De Gasperis, G.; Leoni, R.; Fuhr, G. J. Phys. D: Appl. Phys. 1996 , 29, 340- 349. (7) Green, N. G.; Morgan, H. J. Phys. D: Appl. Phys. 1997 , 30, 2626-2633. (8) Washizu, M.; Kurosawa, O. IEEE Trans. Ind. Appl. 1990 , 26, 1165-1172. (9) Washizu, M.; Suzuki, S.; Kurosawa, O.; Nishizaka T.; Shinohara T. IEEE Trans. Ind. Appl. 1994 , 30, 835-843. (10) Gascoyne, P. R. C.; Wang, X. B.; Huang, Y.; Becker, F. F. IEEE Trans. Ind. Appl. 1997 , 33, 670-678. (11) Markx, G. H.; Pethig, R. Biotechnol. Bioeng. 1995 , 45, 337-343. (12) Morgan, H.; Hughes, M. P.; Green, N. G. Biophys. J. 1997 , 77, 516-525. (13) Markx, G. H.; Rousselet, J.; Pethig, R. J. Liq. Chromatogr. Rel. Technol. 1997 , 20, 2857-2872. (14) Huang, Y.; Wang, X. B.; Becker, F. F.; Gascoyne, P. R. C. Biophys. J. 1997 , 73, 1118-1129. (15) Wang, X. B.; Vykoukal, J.; Becker, F. F.; Gascoyne, P. R. C. Biophys. J . 1998 , 74, 2689-2701. (16) Green, N. G.; Morgan, H. J. Phys. Chem. 1999 , 103, 41-50. (17) Green, N. G.; Morgan, H. J. Phys. D: Appl. Phys. 1997 , 30, L41-L44. Anal. Chem. 1999, 71, 3441-3445 10.1021/ac990172i CCC: $18.00 © 1999 American Chemical Society Analytical Chemistry, Vol. 71, No. 16, August 15, 1999 3441 Published on Web 06/30/1999