Novel magnetic Fe(3)O(4)/chitosan (CS) microspheres were prepared using magnetic Fe(3)O(4) nanoparticles and the natural macromolecule chitosan. Then, using an easy and effective hemoglobin (Hb) immobilization method, an innovative biosensor with a Fe(3)O(4)/CS-Hb-Fe(3)O(4)/CS "sandwich" configuration was constructed. This biosensor had a fast (less than 10 s) response to H(2)O(2) and excellent linear relationships were obtained in the concentration range of 5.0 × 10(-5) to 1.8 × 10(-3) M and 1.8 × 10(-3) to 6.8 × 10(-3) M with a detection limit of 4.0 × 10(-6) M (s/n = 3) under the optimum conditions. The apparent Michaelis-Menten constant K(m) was 0.29 mM and it showed the excellent biological activity of the fixed Hb. Moreover, the biosensor had long-time stability and good reproducibility. The method was used to determine H(2)O(2) concentration in real samples.
Keywords: biosensor; hemoglobin; hydrogen peroxide; hydroquinone; magnetic microspheres.