H2O2 biosensor consisted of hemoglobin-DNA conjugate on nanoporous gold thin film electrode with electrochemical signal enhancement

Abstract

In this research, we developed electrochemical biosensor which was composed of hemoglobin (Hb)-DNA conjugate on nanoporous gold thin film (NPGF) for hydrogen peroxide (H₂O₂) detection. For the first time, Hb and DNA was conjugated as a sensing platform for uniform orientation of Hb on electrode. The newly developed Hb-DNA conjugate was designed to prevent Hb from aggregation on electrode. DNA hybridization of Hb-DNA conjugate and complementary DNA (cDNA) on NPGF electrode induced uniformly assembled biosensor. Furthermore, NPGF electrode fabrication method was introduced to the increment of the surface area. To confirm the conjugation of Hb-DNA conjugate, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ultraviolet-visible spectroscopy (UV-VIS) were used. Formation of the NPGF electrode was verified by scanning electron microscope (SEM). Atomic force microscopy (AFM) was operated for the confirmation of Hb-DNA immobilization on electrode. The electrochemical property of fabricated electrode was investigated by cyclic voltammetry (CV). Also, H₂O₂ sensing performance of fabricated electrode was investigated by amperometric i-t curve technique. This sensor showed a wide linear range from 0.00025 to 5.00 mM and a correlation coefficient of R² = 0.9986. The detection limit was 250 nM. Proposed biosensor can be utilized as a sensing platform for development of biosensor.

Keywords: Biosensor; DNA hybridization; Hemoglobin; Hydrogen peroxide; Nanoporous gold thin film.

Fig. 1

The schematic diagram of Hb-DNA conjugate on the NPGF electrode

Fig. 2

SEM images of a bare gold; b NPGF electrode; c SDS-PAGE (lane 1 for protein ladder, lane 2 for Hb, lane 3 for Hb-DNA conjugate); d UV–VIS results of Hb-DNA conjugate

Fig. 3

AFM results of a aggregated Hb/6-MHA/gold electrode; b Hb/DNA hybridization/gold electrode

Fig. 4

a Cyclic voltammograms of Bare gold, NPGF electrode, NPGF/6-MHA/Hb, NPGF/DNA/Hb in PBS; amperometric i-t curves of b addition of 10 μL of 100 μM H₂O₂ solution; c Successive addition of 1 µM UA, AA, NaNO₂, NaHCO₃, and 1 µM H₂O₂ solution; d successive addition of serum without H₂O₂ once and serum with 1 µM of H₂O₂ twice

Fig. 5

a Calibration curve of 0.5 mM concentration of H₂O₂ versus catalytic peak current and error bar with trend line equation and linear range (0.00025–5.0 mM) with a correlation coefficient of 0.9986; b amperometric response curves of Hb/DNA/NPGF addition of 30 nM, 62.5 nM, 125 nM, 250 nM and 500 nM H₂O₂ solution