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

Abstract

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 Vmax of 5.161 ± 0.1 μA cm−2 with apparent Michaelis-Menten constant (Km) 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, R2 =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.

Direct Electrochemistry of Catalase Immobilized at Polymerized-SnO2 Multiporous Modified Electrode for an Amperometric H2O2 Biosensor

“…However, a decline of reduction current is obtained when the pH is further increased from 8.0 to 10.0. The current response is very low at the lower pH values (<4.0) of electrolytes which can be caused by the denaturation of bio molecules [35][36][37]. Therefore, the pH 7.0 was determined as the optimal pH value of electrolyte to obtain highest current response and it corroborates the concept that the pH range of 6.0-7.5 provides the optimum environment for enzymatic electrochemical reactions [35].…”

Novel manufacturing method for producing apohemoglobin and its biophysical properties

“…Additionally, when Hb solutions with concentrations >50 mg/ml were used as the initial basis for the process, the protein rapidly denatured into a red precipitate when contacted with the acidic 80% ethanol solution. This event likely occurred due to desolvation of the protein, leading to particle formation (Narwal, Yadav, Thakur, & Pundir, ; Yen, ). Thus, to minimize this effect, Hb solutions with concentrations of 20–25 mg/ml were used when adding Hb to the acidic ethanol solution.…”

Peroxidase-like activity of hemoglobin-based hybrid materials against different substrates and their enhanced application for H2O2 detection

“…To date, various researches have been carried out using such materials as carbon, metal, metal oxide-based nanoparticles, and metal complexes to design functional synthetic materials with peroxidase and catalase-like activities [13][14][15]. Heme groupcontaining proteins such as hemoglobin (Hb), myoglobin and cytochrome C exhibit peroxidaselike activity due to their electroactive heme groups and can be used to reduce hydrogen peroxide and as H2O2 sensors [16,17]. Among these, hemoglobin is widely used in the analysis because of its well-known molecular structure, its cheapness, commercial usable and high stability [10,18].…”

“…Among these, hemoglobin is widely used in the analysis because of its well-known molecular structure, its cheapness, commercial usable and high stability [10,18]. Hemoglobin is a 5.5 nm sized protein with tetrameric, globuler and oxygen carrier, each monomer weighing 17 kDa [11,16,19,20].…”