A ratiometric electrochemical biosensor for glycated albumin detection based on enhanced nanozyme catalysis of cuprous oxide-modified reduced graphene oxide nanocomposites

Nanozymes have enzyme-like characteristics and nanozyme-based electrochemical sensors have been widely studied for biomarker detection. In this work, cuprous oxide-modified reduced graphene oxide (Cu₂O-rGO) nanozyme was prepared by simultaneous reduction of copper chloride and graphene oxide. This Cu₂O-rGO nanozyme displayed an outstanding electrocatalytic activity to glucose oxidation and was used as the modified material of a glassy carbon electrode to fabricate an electrochemical ratiometric biosensor for glycated albumin (GA) detection. In this ratiometric biosensor, methylene blue-labeled DNA tripods (MB-tDNA) were adsorbed on the Cu₂O-rGO/GCE surface to form a bioinspired electrode (MB-tDNA/Cu₂O-rGO/GCE), in which the catalytic sites of Cu₂O-rGO were covered by MB-tDNA. In the presence of target GA, GA could be identified by the aptamer sequence contained in MB-tDNA, and a MB-tDNA/GA complex was formed and released into the solution, so the reduced current of MB-tDNA was decreased. Simultaneously, the oxidized current of the outer added glucose was increased since more catalytic sites of Cu₂O-rGO nanozyme on the substrate electrode surface were exposed. The ratio of the peak currents of glucose oxidation and methylene blue reduction (I_Glu/I_MB) was used to monitor the GA level and ultimately improve the accuracy of the method. The electrochemical sensor showed a low detection limit of 0.007 μg mL⁻¹and a wide linear range from 0.02 to 1500 μg mL⁻¹. The proposed sensor was also successfully used to measure the GA expression level in the blood serum of a diabetic mouse model.