An amperometric H2O2 biosensor based on hemoglobin nanoparticles immobilized on to a gold electrode

The nanoparticles (NPs) of hemoglobin (Hb) were prepared by desolvation method and characterized by transmission electron microscopy (TEM), UV spectroscopy and Fourier-transform IR (FTIR) spectroscopy. An amperometric H2O2 biosensor was constructed by immobilizing HbNPs covalently on to a polycrystalline Au electrode (AuE). HbNPs/ AuE were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) before and after immobilization of HbNPs. The HbNPs/AuE showed optimum response within 2.5 s at pH 6.5 in 0.1 M sodium phosphate buffer (PB) containing 100 mu M H2O2 at 30 degrees C, when operated at -0.2 V against Ag/AgCl. The HbNPs/AuE exhibited V-max of 5.161 +/- 0.1 mu A cm(-2) with apparent Michaelis-Menten constant (K-m) of 0.1 +/- 0.01 mM. The biosensor showed lower detection limit (1.0 mu M), high sensitivity (129 +/- 0.25 mu A cm(-2) mM(-1)) and wider linear range (1.0-1200 mu M) for H2O2 as compared with earlier biosensors. The analytical recoveries of added H2O2 in serum (0.5 and 1.0 mu M) were 97.77 and 98.01% respectively, within and between batch coefficients of variation (CV) were 3.16 and 3.36% respectively. There was a good correlation between sera H2O2 values obtained by standard enzymic colorimetric method and the present biosensor (correlation coefficient, R-2 = 0.99). The biosensor measured H2O2 level in sera of apparently healthy subjects and persons suffering from diabetes type II. The HbNPs/AuE lost 10% of its initial activity after 90 days of regular use, when stored dry at 4 degrees C.