Synthesis and characterization of uniform polyepoxide micrometer sized particles by redox graft polymerization of glycidyl methacylate on oxidized polystyrene and polydivinylbenzene microspheres for enzyme immobilization Melany Omer-Mizrahi, Shlomo Margel * Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel article info Article history: Received 2 October 2009 Received in revised form 7 January 2010 Accepted 7 January 2010 Available online 18 January 2010 Keywords: Polyepoxide microspheres Ozonolysis Conjugated hydroperoxides abstract Polystyrene template microspheres of narrow size distribution were prepared by dispersion polymeri- zation of styrene in 2-methoxyethanol. Uniform polystyrene/poly(divinyl benzene) composite micro- spheres were formed by a single-step swelling process of the polystyrene template microspheres with dibutyl phthalate droplets containing divinyl benzene and benzoyl peroxide, followed by polymerization at 73  C. Uniform poly(divinyl benzene) microspheres of higher surface area were produced by disso- lution of the template polystyrene part of the former composite microspheres with methylene chloride. Hydroperoxide conjugated polystyrene and poly(divinyl benzene) microspheres were produced by controlled ozonolysis of these microspheres. Polyepoxide conjugated microspheres were then formed by redox graft polymerization of glycidyl methacylate on the hydroperoxide-conjugated microspheres. Microspheres with different properties, e.g., size, size distribution, shape, surface morphology, surface area, etc., have been prepared by changing various parameters belonging to the ozonolysis and the grafting polymerization processes, e.g., ozonolysis conditions and monomer volume. Trypsin was then covalently bound to the polyepoxide conjugated microspheres by interacting the epoxide groups of the particles with primary amino groups of the enzyme. A comparison between the enzymatic activity of the conjugated and the free trypsin was also established. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Polymeric microspheres of narrow size distribution have attracted much attention in many applications such as adsorbents for high-pressure liquid chromatography, calibration standards, spacers for liquid crystals, inks, catalysis, and so forth [1–9]. Polymeric microspheres containing functional groups such as aldehydes, chloromethyls, oxiranes, hydroxyls, and thiols have been used for covalent binding, via various activation methods, of bioac- tive reagents (e.g., proteins, enzymes, antibodies and oligonucleo- tides) to the surface of these functional microspheres [1,3,5,10]. The bioactive-conjugated microspheres were then used for various biomedical applications, e.g., specific cell labelling and separation, diagnostics, enzyme immobilization, chromatography, etc. [1,3,5]. Dispersion polymerization is the common method for preparing non-porous microspheres of narrow size distribution in a single step. However, the microspheres formed by this method possess specific properties, e.g., surface morphology and functionality, which can hardly be manipulated [11,12]. Furthermore, uniform microspheres of a diameter larger than approximately 6 mm usually cannot be prepared by dispersion polymerization. These limitations have been overcome by graft polymerization of vinylic monomers on the surface of uniform core microspheres [13–15], and by several swelling methods of PS microspheres with appropriate hydro- phobic monomers and initiators, e.g., multi-step swelling, dynamic swelling [16,17], and a single-step swelling [18–23], followed by polymerization of the monomers within the swollen microspheres. There is much interest among the academic and industrial scientific community in finding new ways to modify the surface of microspheres without changing their bulk properties. The reasons for seeking this kind of modification are many, e.g., changing the surface composition and wettability properties, improving adhe- sion, protein immobilization, blood compatibility, weathering, protection of microspheres, etc. [24–28]. Numerous methods for surface modification of different microspheres, such as high-energy radiation (e.g. gamma, glow discharge, corona discharge or photo irradiation) [29], ozone exposure [26,30], graft polymerization on core microspheres, etc., have been already published [20,31,32]. The present article describes a simple method for surface modification and functionalization of polystyrene (PS) and * Corresponding author. Tel.: þ972 35318861; fax: þ972 37384053. E-mail addresses: ch443@mail.biu.ac.il (M. Omer-Mizrahi), shlomo.margel@ mail.biu.ac.il (S. Margel). Contents lists available at ScienceDirect Polymer journal homepage: www.elsevier.com/locate/polymer 0032-3861/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2010.01.015 Polymer 51 (2010) 1222–1230