Microparticles have been manipulated in a microfluidic channel by means of negative dielectrophoresis (n-DEP), and the approach applied to a heterogeneous immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 microm diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area when surrounded by intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 6-15 V(peak) and frequencies over 500 kHz were applied to the two facing microelectrodes. A heterogeneous sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing a secondary antibody with a signal source (FITC-labeled anti-mouse IgG antibody), into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 microg/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. Thus, the automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP.