Immobilization of hemoglobin on functionalized multi-walled carbon nanotubes-poly-l-histidine-zinc oxide nanocomposites toward the detection of bromate and H2O2

• Simple and eco-friendly strategy for the preparation of f-MWCNT–P-l-His–ZnO and Hb/f-MWCNT–P-l-His–ZnO composite.
• It offered good selectivity, stability, reproducibility with a wide linear range and low LOD.
• The proposed biosensor was applied to detect H2O2 and bromate in real samples.

A novel biocompatible sensing strategy has been developed based on functionalized multi-walled carbon nanotube (f-MWCNT), poly-l-histine (P-l-His), and ZnO nanocomposite film for the immobilization of hemoglobin (Hb). The direct electron transfer properties and bioelectrocatalytic activity of the Hb in f-MWCNT–P-l-His–ZnO composite film is further investigated. The apparent heterogeneous electron transfer rate constant (ks) of Hb confined to f-MWCNT–P-l-His–ZnO nanocomposite is found to be 5.16 s−1 using Laviron's equation. Moreover, the surface coverage concentration (Γ) of the electroactive Hb in the f-MWCNT–P-l-His–ZnO film is estimated to be 1.88 × 10−9 mol cm−2. The fabricated electrochemical biosensor based on the immobilized Hb revealed a fast response time (<3 s) with a wide linear range (4–18,000 μM and 2–15,000 μM) and detection limit (as low as 0.01 μM and 0.30 μM) for the electrocatalytic determination of a mediator-free H2O2 and bromate under optimal experimental conditions. The ca. apparent Michaelis–Menten constant is 0.14 mM, which indicates that the Hb has a high affinity to H2O2. The high sensitivity, good reproducibility, and long-term stability of the proposed nanocomposite film indicates that it can serve as an electrode for the development of an amperometric H2O2 and bromate-based biosensor. The proposed third-generation biosensor was successfully applied to milk and urine samples for the detection of H2O2 and bromate.

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