Europium spinel oxide dual biosensors for cyclophosphamide and ifosfamide detection

Electrochemical sensors convert chemical information into electrical signals, allowing biomolecules to be detected even in complex samples. Nanostructured metal oxides with spinel structures are especially useful due to their high catalytic activity, large surface area, and enhanced electron transfer, which improve sensor sensitivity and selectivity. In this study, europium-based spinel oxide nanoparticles doped with copper, cobalt, and zinc (CuEu₂O₄, CoEu₂O₄, ZnEu₂O₄) were prepared using an autocombustion method in order to exploit the electronic properties of europium which thanks to its f-orbital configuration should facilitate electron exchange. The morphological and crystal structures of the nanoparticles were investigated: Field Emission Scanning Electron Microscope (FESEM) analysis confirmed that the particles were uniform and well dispersed, while X-ray diffraction (XRD) demonstrated the formation of a highly crystalline cubic spinel structure. The electrochemical properties of the different materials were studied using cyclic voltammetry (CV) in ferro/ferricyanide solutions with commercial single sensor screen-printed carbon electrodes (SPCE Metrohm DRP-11L) at various scan rates ( 25 – 100mV/s). The quasi-reversible electron transfer observed allowed the calculation of electron transfer coefficients and diffusion constants by applying Laviron’s model and the Randles-Sevčík equation. Electrochemical impedance spectroscopy (EIS) provided insight into surface properties and charge transfer resistance. Nanoparticles were deposited via drop-casting onto commercial dual screen-printed carbon electrodes (DRP-X1110). Two types of sensors were developed: one with only nanomaterials and another combining nanomaterials with selective enzymes CYP2B6 and CYP3A4, which metabolize cyclophosphamide and ifosfamide, respectively. The sensors without enzymes showed almost no response, in contrast, sensors incorporating enzymes showed significant and stable responses due to the enzymatic reaction required for detecting the prodrugs. The developed biosensors were tested in phosphate-buffered saline (PBS) at drug concentrations of 5–100 µM (cyclophosphamide) and 10–120 µM (ifosfamide) revealing copper-doped europium oxide (CuEu₂O₄) as the most reliable sensor, showing good sensitivity and a linear detection range. The sensitivity values were 1.35 ± 0.19 nA/μM·mm² for CuEu₂O₄ combined with CYP2B6 for cyclophosphamide detection, and 1.50 ± 0.14 nA/μM·mm² for CuEu₂O₄ combined with CYP3A4 for ifosfamide detection. Although cobalt europium oxide exhibited slightly higher sensitivity for cyclophosphamide, its response lost linearity above 50 µM. For this reason, CuEu₂O₄ was chosen for further evaluation in human serum to simulate biological conditions. In conclusion, europium-based spinel oxides, especially CuEu₂O₄, show promising results for developing dual electrochemical biosensors for key drug targets. Future studies will aim to expand detection capabilities to other drugs, supporting the advancement of point-of-care technology for personalized medicine.