H2O2 determination by a biosensor based on hemoglobin

The detection of hydrogen peroxide, H2O2, plays an important role in many fields including industry, environmental protection, and clinical control. Hydrogen peroxide can be toxic if ingested, inhaled, or by contact with the skin or eyes. Hemoglobin is a molecule with four electroactive iron hemes which can be used as an ideal model molecule for the study of electron transfer reactions of heme proteins and also for biosensing and electrocatalysis. The present study describes the immobilization of hemoglobin on a Clark electrode surface to develop a novel electrochemical biosensor for the detection of hydrogen peroxide. The principle of the measurements was based on the electrocatalytic activity of the immobilized hemoglobin to the reduction of hydrogen peroxide. Hemoglobin was crosslinked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The optimum conditions for the biosensor were established. The most suitable hemoglobin and gelatin amounts and glutaraldehyde ratio were determined. Characterization studies of the biosensor, such as optimum pH and optimum temperature, were carried out. The repeatability experiments were done and the average value (x), standard deviation (S.D.), and variation coefficient (C.V.) were calculated. After the optimization and characterization studies the proposed biosensor was applied to determination of H2O2 in real samples.