Hemoglobin–metal2+ phosphate nanoflowers with enhanced peroxidase-like activities and their performance in the visual detection of hydrogen peroxide

Abstract

Hemoglobin (Hgb)–metal²⁺ phosphate nanoflowers (Hgb–X²⁺-Nfs) were synthesized using Co²⁺, Zn²⁺, Ca²⁺, and Fe²⁺ separately as inorganic components, to generate a visual hydrogen peroxide (H₂O₂) biosensor for the first time. While there was no flower-like structure using Ca²⁺ and Fe²⁺ ions, the average diameters of nanoflowers obtained using Co²⁺ and Zn²⁺ were about 7–8 μm and 1.2 μm, respectively. Their encapsulation efficiencies were 85% and 90%, respectively. The peroxidase-like activities of Hgb–X²⁺-Nfs were evaluated against the ABTS substrate, compared to free hemoglobin. The obtained Hgb–X²⁺-Nfs were used as a biocatalyst to generate visual and spectrometric biosensors. The working range that can be detected visually was 0.005–0.667 mM in both hybrid nanoflowers, while the working range that can be detected spectrometrically was 0.00017–0.667 mM for Hgb–Co²⁺-Nfs and 0.0017–0.667 mM for Hgb–Zn²⁺-Nfs. The proposed biosensing methods were successfully applied for the determination of hydrogen peroxide in tap water and milk samples, with recoveries ranging from 95.23% to 107.01%. The findings of this study can be used for the production of new hybrid nanoflowers incorporating hemoglobin and various metal ions for use as potential biosensors. In addition, the proposed biosensors have the potential to provide a successful analytical tool in many areas of analysis, such as pharmaceutical, clinical, food and environmental.