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

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 (k_s) 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 (F) 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.