Trypsin was covalently immobilized by attaching it to porous Fe3O4@SiO2 core/shell nanoparticles with hydrodynamic diameter of about 100-200 nm and specific surface area up to 58 m(2)/g. The particles were prepared by a modified sol-gel method from hydrophilic Fe3O4 nanoparticles. The surface of the Fe3O4@SiO2 particles was then decorated with amino groups by reaction with 3-aminopropyltriethoxysilane (APTES). These amino groups were connected with the carboxylic acid groups of trypsin using EDAC and sulfo-NHS methodology. Core/shell structure was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and powder X-ray diffraction analysis. The specific enzyme activity of the trypsin Fe3O4@SiO2-NH2 nanoparticles was quantified and verified in its recoverability. Kinetic constants K-M and v(max) were measured and compared to those for soluble form of trypsin. Relatively lower value of the apparent Michaelis-Menten constant K-M (0.399-0.658 mM) indicates improvement in affinity between the immobilized enzyme and substrate. The activity and specificity of trypsin Fe3O4@SiO2-NH2 nanoparticles were evaluated using alpha-casein proteolytic digestion (3 h) followed by high-resolution measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer. All measurements confirmed high proteolytic activity and specificity compared to trypsin SiMAG-Amine microparticles and/or to soluble enzyme with 3 h digestion. (C) 2015 Elsevier Ltd. All rights reserved.