Direct electron transfer and characterization of hemoglobin immobilized on a Au colloid–cysteamine-modified gold electrode

Hemoglobin (Hb) was immobilized successfully on nanometer-sized gold colloid particles associated with a cysteamine monolayer on a gold electrode surface, and was characterized by atomic force microscopy (AFM), UV–vis spectroscopy (UV–vis), surface enhanced Raman spectroscopy (SERS) and electrochemical impedance spectroscopy (EIS). The immobilized Hb was shown to keep its biological activity well. Direct electron transfer (eT) between Hb and the modified electrode was achieved without the aid of any electron mediator. In pH 7.2 phosphate buffer solution, the formal potential (E°′) of Hb was −0.051 V (vs. SCE) and the eT rate constant was 0.49 s−1. The average surface coverage of Hb immobilized on the gold colloid was about 6.71×10−11 mol cm−2. The immobilized Hb displayed the features of a peroxidase and gave an excellent electrocatalytic response to the reduction of H2O2. The Michaelis–Menten constant (Km) was 1.2×10−4 M. The currents were proportional to the H2O2 concentration from 3.6×10−7 to 8.6×10−4 M and the detection limit was as low as 1.2×10−7 M (S/N=3).