Cytochrome P450-Functionalized Spinel Oxide Electrochemical Biosensors for the Enhanced Detection of Cyclophosphamide

Nanostructured metal oxides with spinel crystalline structures are highly attractive for electrochemical sensing due to their ability to enhance the catalytic activity of redox processes, improving sensitivity and lowering limits of detection. At the same time, cytochrome P450 enzymes (P450s) confer substrate specificity, enabling the selective detection of therapeutic compounds. This study investigates the integration of spinel-structured metal oxide nanoparticles—ZnCr $_{\textbf {2}}$ O $_{\textbf {4}}$ , ZnFe $_{\textbf {2}}$ O $_{\textbf {4}}$ , and CuFe $_{\textbf {2}}$ O $_{\textbf {4}}$ —with cytochrome P450 enzymes to enable the selective electrochemical detection of cyclophosphamide, a widely used anticancer drug metabolized by these enzymes. Screen-printed carbon electrodes were functionalized with these spinel oxides and two P450 isoforms: CYP3A4 and CYP2B6. Spinel oxide-modified electrodes showed enhanced catalytic activity, with further sensitivity improvements when combined with P450 enzymes. The ZnCr $_{\textbf {2}}$ O $_{\textbf {4}}$ /CYP2B6 configuration achieved the highest sensitivity of 6.9 nA/ $\upmu$ M, while ZnFe $_{\textbf {2}}$ O $_{\textbf {4}}$ /CYP3A4 improved the limit of detection nearly twentyfold. These findings highlight the synergistic benefits of enzyme-nanoparticle coupling, demonstrating the potential of spinel oxides and P450 enzymes for developing sensitive, selective electrochemical biosensors for drug monitoring.