Direct electrochemistry of covalently immobilized hemoglobin on a naphthylimidazolium butyric acid ionic liquid/MWCNT matrix

Monitoring the concentration levels of hydrogen peroxide (H₂O₂) is significant in both clinical and industrial applications. Herein, we develop a facile biosensor for the detection of H₂O₂ based on direct electron transfer of hemoglobin (Hb), which was covalently immobilized on a hydrophobic naphthylimidazolium butyric acid ionic liquid (NIBA-IL) over a multiwalled carbon nanotube (MWCNT) modified glassy carbon electrode (GCE) to obtain an Hb/NIBA-IL/MWCNT/GCE. Highly water-soluble Hb protein was firmly immobilized on NIBA-IL via stable amide bonding between the free NH₂ groups of Hb and COOH groups of NIBA-IL via EDC/NHS coupling. Thus fabricated biosensor showed a well resolved redox peak with a cathodic peak potential (E_pc) at -0.35 V and anodic peak potential (E_pa) at -0.29 V with a formal potential (E°') of -0.32 V, which corresponds to the deeply buried Fe^III/Fe^II redox centre of Hb, thereby direct electrochemistry of Hb was established. Further, the modified electrode demonstrated very good electrocatalytic activity towards H₂O₂ reduction and showed a wide linear range of detection from 0.01 to 6.3 mM with a limit of detection and sensitivity of 3.2 μM and 111 μA mM^-1 cm^-2, respectively. Moreover, the developed biosensor displayed high operational stability under dynamic conditions as well as during continuous potential cycles and showed reliable reproducibility. The superior performance of the fabricated biosensor is attributed to the effective covalent immobilization of Hb on the newly developed highly conducting and biocompatible NIBA-IL/MWCNT/GCE platform.