Hydrogen peroxide (H2O2) is a vital messenger molecule facilitating signal transmission within cellular systems. The accurate quantification of H2O2 in complex environments, such as human serum, is instrumental in elucidating its diverse biological functions. In this study, we present the development of a robust electrode platform by integrating hemoglobin onto iron oxide (Fe3O4) nanoparticles and reduced graphene oxide (rGO) as a conducting support for the sensitive detection of H2O2 in human serum. The Fe3O4/rGO nanocomposite was synthesized via a facile single-step thermolysis reaction in the presence of oleylamine. Transmission electron microscopy (TEM) analysis revealed the formation of highly monodisperse Fe3O4 nanoparticles that were uniformly dispersed over the reduced graphene surface. The rGO surface provided abundant active sites (Fe3O4) for H2O2 sensing. Consequently, the resulting Hb/Fe3O4@rGO modified electrode exhibited a wide linear range of H2O2 concentrations from 1.5 to 2684 µM, demonstrating high sensitivity and low detection limit. Further characterization of the Hb/Fe3O4@rGO/GC electrode revealed a high surface coverage (τ = 3.0 × 10−9 mol cm−2), charge transfer coefficient (α = 0.83), and electron transfer rate constant (ks = 1.33 s−1). Subsequently, the Hb/Fe3O4@rGO/GC sensor probe was employed to analyze H2O2 in real human serum samples, demonstrating excellent recovery rates ranging from 99.8 % to 104.0 %. The nanocomposite material, designed through the innovative combination of graphene oxide reduction, Fe3O4 nanoparticle decoration, and oleylamine amide/amine bonding, serves as an excellent electrode material for H2O2 concentration sensing. Thus, this study presents a promising platform for the future determination of H2O2 biomarkers in clinical settings.
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