A new nanozyme with peroxidase-like activity for simultaneous phosphoprotein isolation and detection based on metal oxide affinity chromatography: Monodisperse-porous cerium oxide microspheres

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 μm 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 α-casein and β-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 μM/min and 2885.8 μM, 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 μg/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.