Fabrication of Polyglycidyl Methacrylate Multilayer Carboxylated Magnetic Microspheres Immobilized with Affinity Ligands for Diagnostic Allergen Testing

Abstract Magnetic microspheres immobilized with affinity ligands are highly efficient tools for immunoassays. Low non-specific adsorption and high affinity ligand loading capacity are crucial for the sensitivity of immunoassays, especially diagnostic allergen testing. This paper reports a carboxylated magnetic microsphere (PNFC@AA) via a multilayer cross-linked strategy. Monodisperse aminated polyglycidyl methacrylate (PGMA) microspheres were synthesized, followed by the incorporation of Fe3O4 nanoparticles through in situ coprecipitation. Polyacrylic acid (PAA) segments were then grafted onto the microsphere surface via surface radical polymerization using different crosslinkers, ultimately yielding magnetic PNFC@AA microspheres with high carboxyl loading. The prepared PNFC@AA microspheres exhibit uniform particle size, good monodispersity, and typical superparamagnetic properties. It was found that compared to divinylbenzene (DVB) and ethylene glycol dimethacrylate (EGDMA) crosslinkers, the hydrophilic crosslinker polyethylene glycol diacrylate (PEGDA) microspheres exhibited a larger specific surface area (19.17 m2/g), a higher carboxyl loading (655 μmol/g), and a stronger streptavidin (SA)-immobilized biotin binding capacity (2328 pmol/mg). In AMPPD chemiluminescent immunoassay (CLIA), these microspheres effectively suppressed non-specific adsorption, reduced background luminescence, and substantially improved the signal-to-noise ratio. Furthermore, the SA-coupling microspheres were successfully applied to the detection of specific IgE against four common allergens (house dust mite (Dermatophagoides farinae), German cockroach (Blatella germanica), milk, and egg), respectively, which effectively distinguished the sensitization levels of different allergens. This study provides a simple and efficient strategy for the design of high-performance immunodiagnostic carriers.