Bioelectrocatalysis of Hemoglobin on Electrodeposited Ag Nanoflowers toward H₂O₂ Detection

Hydrogen peroxide (H₂O₂) is a partially reduced metabolite of oxygen that exerts a diverse array of physiological and pathological activities in living organisms. Therefore, the accurate quantitative determination of H₂O₂ is crucial in clinical diagnostics, the food industry, and environmental monitoring. Herein we report the electrosynthesis of silver nanoflowers (AgNFs) on indium tin oxide (ITO) electrodes for direct electron transfer of hemoglobin (Hb) toward the selective quantification of H₂O₂. After well-ordered and fully-grown AgNFs were created on an ITO substrate by electrodeposition, their morphological and optical properties were analyzed with scanning electron microscopy and UV-Vis spectroscopy. Hb was immobilized on 3-mercaptopropionic acid-coated AgNFs through carbodiimide cross-linking to form an Hb/AgNF/ITO biosensor. Electrochemical measurement and analysis demonstrated that Hb retained its direct electron transfer and electrocatalytic properties and acted as a H₂O₂ sensor with a detection limit of 0.12 µM and a linear detection range of 0.2 to 3.4 mM in phosphate-buffered saline (PBS). The sensitivity, detection limit, and detection range of the Hb/AgNF/ITO biosensor toward detection H₂O₂ in human serum was also found to be 0.730 mA mM^-1 cm^-2, 90 µM, and 0.2 to 2.6 mM, indicating the clinical application for the H₂O₂ detection of the Hb/AgNF/ITO biosensor. Moreover, interference experiments revealed that the Hb/AgNF/ITO sensor displayed excellent selectivity for H₂O₂.