In Situ Synthesized Gold-Conjugated Hemoglobin-Cu₃ (PO₄)₂ Hybrid Nanopetals for Enhanced Electrochemical Detection of H₂O₂

In situ synthesis of novel hybrid organic–inorganic nanopetals (HNPs) of Copper (Cu²⁺) and gold-conjugated hemoglobin (Au@Hb) is reported. The presence of Au within the protein matrix prevents the formation of a flower-like assembly of the formed nanopetals of Au@Hb and Cu²⁺ via the co-precipitation method. Morphological, chemical, and electrocatalytic activities of in situ synthesized Au@Hb-Cu HNPs were examined systematically. The hybrid nanopetal (Au@Hb-Cu HNP)-modified screen-printed PET electrodes show enhanced electrocatalytic activity toward the oxidation of H₂O₂ compared to electrodes modified with Hb-copper hybrid nanoflowers (Hb-Cu HNFs) without Au conjugation. The proposed biosensor exhibits excellent electrochemical performance with broad linear responses over a H₂O₂ concentration ranging from 5 to 1000 µM (R² = 0.99) and showed a lower detection limit of 1.46 µM at 0.30 V vs. pseudo Ag/AgCl. Enhanced electrochemical performance is attributed to heterogeneous active sites over hybrid nanopetal surfaces. Moreover, the hybrid nanopetal–modified electrodes showed excellent stability and anti-interference performance in the presence of ascorbic acid, uric acid, fructose, and glucose. These results demonstrate that Au@Hb-Cu HNPs offer a better and more promising alternative for the electrochemical detection of H₂O₂ sensitively.