• hemoglobin;
  • room temperature ionic liquid;
  • multi-walled carbon nanotube;
  • electrochemistry;
  • electrocatalysis


A robust and effective composite film combined the benefits of Nafion, room temperature ionic liquid (RTIL) and multi-wall carbon nanotubes (MWNTs) was prepared. Hemoglobin (Hb) was successfully immobilized on glassy carbon electrode surface by entrapping in the composite film. Direct electrochemistry and electrocatalysis of immobilized Hb were investigated in detail. A pair of well-defined and quasi-reversible redox peaks of Hb was obtained in 0.10 mol·L−1 pH 7.0 phosphate buffer solution (PBS), indicating that the Nafion-RTIL-MWNTs film showed an obvious promotion for the direct electron transfer between Hb and the underlying electrode. The immobilized Hb exhibited an excellent electrocatalytic activity towards the reduction of H2O2. The catalysis current was linear to H2O2 concentration in the range of 2.0×10−6 to 2.5×10−4 mol·L−1, with a detection limit of 8.0×10−7 mol·L−1 (S/N=3). The apparent Michaelis-Menten constant (Kmapp) was calculated to be 0.34 mmol·L−1. Moreover, the modified electrode displayed a good stability and reproducibility. Based on the composite film, a third-generation reagentless biosensor could be constructed for the determination of H2O2.