A new nanozyme with peroxidase like activity for simultaneous phosphoprotein isolation and detection based on metal oxide affinity chromatography: Monodisperse porous cerium oxide microspheres
Abstract Monodisperse porous cerium oxide (CeO2) microspheres obtained by a new method were used for simultaneous phosphoprotein isolation and detection for the first time. The synthesis method, "staged sol gel templating protocol" provided monodisperse porous CeO2 microspheres 5.0 mm in size, with the specific surface areas and the pore volumes up to 80 m2/g and 0.41 cm3/g, respectively. CeO2 microspheres were directly used as a metal oxide affinity chromatography (MOAC) sorbent without any post functionalization protocol. The isolation/enrichment of phosphoproteins from complex biological samples such as milk and human serum was performed using CeO2 microspheres as the sorbent, with the purities up to 99 In batch MOAC runs, the equilibrium adsorption capacities of 105.3 and 82.4 mg phosphoprotein/g sorbent were obtained for a casein and b casein, respectively. CeO2 microspheres also exhibited peroxidase like activity and were proposed as a new nanozyme for colorimetric determination of phosphoprotein concentration in complex samples. Maximum substrate consumption rate and Km were determined as 384.6 mM/min and 2885.8 mM, respectively, according to Michealis Menten model. The peroxidase like activity of CeO2 microspheres linearly decreased with the increasing phosphoprotein concentration while no appreciable change in this magnitude was observed with the increasing non phosphoprotein concentration. This behavior was explained by the increase in the pseudospecific phosphoprotein adsorption onto CeO2 microspheres with the increasing phosphoprotein concentration. Similar linear tendencies between peroxidase like activity and phosphoprotein concentration observed in the complex samples such as human serum and milk allowed the determination of phosphoprotein concentration up to 300 and 400 mg/mL, respectively, using CeO2 microspheres as the nanozyme. This study provided a new, unique material having a dual function termed as isolation and detection of large phosphorylated molecules without applying any post functionalization protocol.