Lanthanide-encoded polystyrene microspheres with methacrylic acid (MAA) as a co-monomer and with diameters on the order of 2 pm and a very narrow size distribution were synthesized by two-stage dispersion polymerization (2-DisP). These microspheres were designed as a platform for mass cytometry-based bioassays. Different lanthanides (Ln) were loaded into these microspheres during the synthesis, through the addition of LnCl(3) salts and excess MAA to the reaction after about 10% conversion of styrene, i.e., well after the microsphere nucleation stage was complete. Different levels of MAA were employed to investigate the relationship between the number of carboxyl group on the particle surface and the amount of MAA used. The reaction remained well controlled with both 2 and 4 wt % MAA. As monitored by inductively coupled plasma (ICP) mass spectrometry, we found high incorporation efficiency (>95%) of Ln ions into the particles when the total amount of LnCl(3) salts in the reaction mixture was sufficiently small. The Ln incorporation efficiency decreased with the increasing amount of LnCl3 salts. Mass cytometry analysis shows that individual microspheres contain ca. 10(5)-10(8) chelated lanthanide ions, either a single element or a mixture of elements. This method of incorporating lanthanide into P(S-MAA) particles through the second stage of two-stage dispersion polymerization yields microspheres suitable for the highly multiplexed detection of biomolecules. (C) 2011 Elsevier Ltd. All rights reserved.