Glassy carbon (GC) electrode modified with covalently attached amine-terminated ionic liquid (NH2-IL) and titanium nitrite nanoparticles (TiNnp) was used as support for immobilization of hemoglobin (Hb) protein. The presence of well-defined and nearly reversible CV peaks with E°′ of −0.03 V (vs. Ag/AgCl) indicated the successful immobilization of Hb on GC/TiNnp/NH2-IL surface. The surface coverage of Hb immobilized on nanocomposite film (Γ), and heterogeneous electron transfer rate constant (ks) were 5.12 (±0.05) × 10−11 mol cm−2 and 5.10 ± 0.10 s−1 respectively, indicating high loading capacity of nanocomposite for direct electron transfer of Hb on the electrode surface. Moreover, the GC/TiNnp/NH2-IL/Hb electrode revealed excellent electrocatalytic activity toward nitrite reduction. This sensor exhibited fast amperometric response with sensitivity of 0.027 ± 0.009 μA/μM and limit of detection of 0.10 ± 0.010 μM at the concentration range up to 2 mM. The calculated Michaelis–Menten constant (KM) of 0.065 (±0.030) mM indicated that Hb immobilized onto the nanocomposite film retained its native activity. The sensor also showed good stability, reproducibility and long lifetime and it was successfully applied for nitrite detection in real samples with obtained satisfactory results.
