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 μM H₂O₂ at 30°C, when operated at -0.2 V against Ag/AgCl. The HbNPs/AuE exhibited V_max of 5.161 ± 0.1 μA cm^-2 with apparent Michaelis-Menten constant (K_m) of 0.1 ± 0.01 mM. The biosensor showed lower detection limit (1.0 μM), high sensitivity (129 ± 0.25 μA cm^-2 mM^-1) and wider linear range (1.0-1200 μM) for H₂O₂ as compared with earlier biosensors. The analytical recoveries of added H₂O₂ in serum (0.5 and 1.0 μM) 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 =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°C.