High‐Quality Covalently Grafting Hemoglobin on Gold Electrodes: Characterization,Redox Thermodynamics and Bio‐electrocatalysis

Herein, we report a versatile surface chemistry methodology to covalently immobilize ligands and proteins to self‐assembled monolayers (SAMs) on gold electrode. The strategy is based on two steps: 1) the coupling of soluble azido‐PEG‐amimo ligand with an alkynyl‐terminated monolayer via click reaction and 2) covalent immobilization hemoglobin (Hb) to the amine‐terminated ligand via carbodiimide reaction. Surface‐enhanced Raman scattering spectroscopy (SERS), atomic force microscopy (AFM), reflection absorption infrared spectroscopy (RAIR) and cyclic voltammetry are used to characterize the model interfacial reactions. We also demonstrate the excellent biocompatibility of the interface for Hb immobilization and reliable application of the proposed method for H₂O₂ biosensing. Moreover, the redox thermodynamics of the Fe³⁺/Fe²⁺ couple in Hb is also investigated.