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

Vinay Narwal,Chandra S Pundir,Manisha Thakur,Neelam Yadav

doi:10.1042/bsr20170194

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.

Highlights

Hydrogen peroxide (H2O2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells, depending on the extent, timing and location of its production
The various supports used for the H2O2 biosensors are: platinum nanoparticles (NPs) (PtNPs)/reduced graphene oxide (RGO)/chitosan (CS)/ferrocene (Fc) [28], self-assembled dipeptide (DP)-gold NPs (AuNPs)/horseradish peroxidase (HRP)/glassy carbon electrode (GCE) [29], graphene capsules (GRCAPS)/HRP/indium tin oxide (ITO) [30], HRP/poly(aniline-co-N-methyllanthionine) (PAN-PNMThH) [31], electrochemically reduced graphene oxide (ERGO)/GCE [32], 3,3,5,5 -teramethylbencidine (TMB)/HRP/polydimethylsiloxane (PDMS)/tetraethylorthosilicate (TEOS)/silicon oxide NPs (SiO2NPs) [33], turnip tissue paper (TTP)/Screen printed carbon electrode (SPCE) [34], Platinum nanostructure on reduced graphene (GPtNP) [35], Platinum-Ruthenium bimetallic catalyst (PtRu)/Three dimensional graphene foam (3DGF) [36], cytochrome c (Cytc)/nickel oxide nanaoparticles (NiONPs)/collagen-multiwall carbon nanotubes (c-MWCNT)/polyaniline (PANI)/Au [37]
In comparison with the Fourier-transform IR (FTIR) spectrum of the Hb and HbNPs, the major shift was observed in the carbonyl, hydroxyl, and amino groups of protein, which is due to ethanol, glutaraldehyde, and cysteamine treatment of HbNPs during their preparation

Summary

Introduction

Hydrogen peroxide (H2O2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells, depending on the extent, timing and location of its production. The various supports used for the H2O2 biosensors are: platinum nanoparticles (NPs) (PtNPs)/reduced graphene oxide (RGO)/chitosan (CS)/ferrocene (Fc) [28], self-assembled dipeptide (DP)-gold NPs (AuNPs)/HRP/glassy carbon electrode (GCE) [29], graphene capsules (GRCAPS)/HRP/indium tin oxide (ITO) [30], HRP/poly(aniline-co-N-methyllanthionine) (PAN-PNMThH) [31], electrochemically reduced graphene oxide (ERGO)/GCE [32], 3,3 ,5,5 -teramethylbencidine (TMB)/HRP/polydimethylsiloxane (PDMS)/tetraethylorthosilicate (TEOS)/silicon oxide NPs (SiO2NPs) [33], turnip tissue paper (TTP)/SPCE [34], GPtNPs [35], PtRu/3DGF [36], cytochrome c (Cytc)/nickel oxide nanaoparticles (NiONPs)/c-MWCNT/polyaniline (PANI)/Au [37] These methods involve the complex strategies for construction of working electrode and have poor detection limit and narrow linear range. The present work describes the construction of an H2O2 biosensor based on covalent immobilization of HbNPs on to AuE, its characterization, evaluation, and application for amperometric determination of H2O2 in sera of diabetic patients

Experimental procedures

Results and discussion

Conclusion