Immobilization of Urokinase onto Magnetically Directed Micromotors

In this presented work, a new micromotor was prepared for urokinase immobilization. A covalent bond was constructed between the urokinase and the carboxyl groups of the graphene oxide, which is located at the outer layer of micromotors by EDC/NHS chemistry. The inner nickel layer gave magnetic properties to the micromotors and enables them to be separated from the reaction medium with the help of a simple magnet. For promising in vivo applications in the future, these micromotors do not require any fuel for their movement. The structures of the synthesized micromotors were illuminated by SEM and EDX analysis, and the movements of the micromotors were observed under an optical microscope with camera equipment. The immobilization yield of urokinase was found to be 68.07% (0.073 mg/100 µL micromotor solution) using the Bradford protein assay. In addition, to compare the activities of the immobilized and free enzymes, Lineweaver-Burk plots were constructed, and the kinetic parameters were calculated. K_m values for free urokinase and immobilized urokinase were 2.0964 mM and 0.5602 mM, respectively. The maximum velocities of free and immobilized urokinase were found to be 25.25 µmol/min and 30.12 µmol/min, respectively. Also, storage stability profiles of the immobilized and free urokinase were monitored for 40-day incubation at + 4 °C, and the immobilized enzyme has 88% of its initial activity, while the free urokinase demonstrated only 30% of its initial activity. As a result, the experiments were carried out in human commercial serum, and specific activity values for free urokinase and immobilized urokinase were found to be 38.06 and 169.84 µmolmg^-1 min^-1, respectively.