Direct electrochemistry of hemoglobin immobilized on a functionalized multi-walled carbon nanotubes and gold nanoparticles nanocomplex-modified glassy carbon electrode

Abstract

Direct electron transfer of hemoglobin (Hb) was realized by immobilizing Hb on a carboxyl functionalized multi-walled carbon nanotubes (FMWCNTs) and gold nanoparticles (AuNPs) nanocomplex-modified glassy carbon electrode. The ultraviolet-visible absorption spectrometry (UV-Vis), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) methods were utilized for additional characterization of the AuNPs and FMWCNTs. The cyclic voltammogram of the modified electrode has a pair of well-defined quasi-reversible redox peaks with a formal potential of -0.270 ± 0.002 V (vs. Ag/AgCl) at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was evaluated to be 4.0 ± 0.2 s(-1). The average surface concentration of electro-active Hb on the surface of the modified glassy carbon electrode was calculated to be 6.8 ± 0.3 × 10(-10) mol cm(-2). The cathodic peak current of the modified electrode increased linearly with increasing concentration of hydrogen peroxide (from 0.05 nM to 1 nM) with a detection limit of 0.05 ± 0.01 nM. The apparent Michaelis-Menten constant (K(m)(app)) was calculated to be 0.85 ± 0.1 nM. Thus, the modified electrode could be applied as a third generation biosensor with high sensitivity, long-term stability and low detection limit.

Figure 1.

Preparation process of functional membrane modified glassy carbon (GC) electrode.

Figure 2.

(A) CVs of different modified electrodes (from inner to outer): (a) Bare GC electrode; (b)NF/Hb/GC; (c) NF/FMWCNTs/Cys/AuNPs/GC; (d) NF/Hb/FMWCNTs/GC; (e) NF/Hb/FMWCNTs/Cys/AuNPs/GC. The experiments were carried out in 0.05 M PBS (pH7.0) at a scan rate of 0.05 V/s. (B) CVs of NF/Hb/FMWCNTs/Cys/AuNPs/GC electrode in 0.05 M PBS (pH 7.0) at various scan rates (from inner to outer): 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18… 4 V/s, respectively; (C) Plot of peck current I_p vs. scan rate ν; (D) Plot of peak potential E_p vs. ln (ν).

Figure 3.

(A) CVs of NF/Hb/FMWCNTs/Cys/AuNPs/GC electrode in 0.05 M PBS at different pH values: (a) 5.0, (b) 6.0, (c) 7.0, and (d) 8.0, respectively; (B) plot of I_pc vs. pH value; (C) Plot of E ° ′ vs. pH value.

Figure 4.

LSVs of (A) NF/Hb/FMWCNTs/Cys/AuNPs/GC electrode in the absence or presence of different concentrations of H₂O₂ (from curve a to curve e): 0, 0.1, 0.13, 0.16, 0.20 mM, respectively. LSVs of (B) Cys/AuNPs/GC; (C) NF/Cys/AuNPs/GC and (D) NF/Hb/Cys/AuNPs/GC electrodes, respectively in the presence of different concentrations of H₂O₂ (from a to curve d): 0.1, 0.3, 0.5 and 0.7 mM, respectively.

Figure 5.

Amperometirc response of the modified electrode to successive additions of 5 μL of 1 nM (a), 10 nM(b) or 20 nM(c) H₂O₂ in 5 mL of 0.05 M PBS, pH 7.0, at the applied potential of −0.35 V (vs. Ag/AgCl). Inset shows the typical current response for each addition process: (1) previous steady state current; (2) maximum response current; (3) steady state current; (4). next maximum response current.

Figure 6.

(A) The typical steady current vs. [H₂O₂] in the process of successive additions of 5 μL of 10 nM (●) and 20 nM (▲; H₂O₂ in 5 mL of 50 mM PBS (pH 7.0) at the applied potential of −0.35 V (vs. Ag/AgCl). (B) The determination of the H₂O₂ detection limit for NF/Hb/FMWCNTs/Cys/AuNPs/GC electrode. The detection limit was determined from the cross point of the lines fitted to the linear segments of the steady current Is vs. [H₂O₂] in the process of successive additions of 5 μL of 10 nM H₂O₂ in 5 mL of 50 mM PBS (pH7.0). (C) Lineweaver-Burk plot for K_m^app determination.