Ultrasensitive Label-free Electrochemical Immunosensor based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein

Abstract

In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of alpha fetoprotein (AFP). Multifunctionalized graphene nanocomposites (TB-Au-Fe₃O₄-rGO) were applied to modify the electrode to achieve the amplification of electrochemical signal. TB-Au-Fe₃O₄-rGO includes the advantages of graphene, ferroferric oxide nanoparticles (Fe₃O₄ NPs), gold nanoparticles (Au NPs) and toluidine blue (TB). As a kind of redox probe, TB can produce the electrochemical signal. Graphene owns large specific surface area, high electrical conductivity and good adsorption property to load a large number of TB. Fe₃O₄ NPs have good electrocatalytic performance towards the redox of TB. Au NPs have good biocompatibility to capture the antibodies. Due to the good electrochemical performance of TB-Au-Fe₃O₄-rGO, the effective and sensitive detection of AFP was achieved by the designed electrochemical immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 1.0 × 10^-5 ng/mL to 10.0 ng/mL with a low detection limit of 2.7 fg/mL for AFP. It also displayed good electrochemical performance including good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers.

Figure 1. The synthesis procedure of the TB-Au-Fe₃O₄-rGO and SEM image of the Au-Fe₃O₄-rGO (inset).

Figure 2. The schematic diagram of the label-free electrochemical immunosensor fabricated on the GCE.

Figure 3

SEM images of Au-Fe₃O₄-rGO (A), Au-rGO (C) and Fe₃O₄-rGO (D); EDX spectrum of Au-Fe₃O₄-rGO (B).

Figure 4

Effect of pH (A) and the concentration of TB-Au-Fe₃O₄-rGO (B) on the electrochemical current responses of the immunosensor for the detection of 10.0 ng/mL of AFP. Error bar = RSD (n = 5).

Figure 5

Electrochemical current responses recorded from −0.6 V to 0 V in PBS at pH 6.8 (A): TB-Au-Fe₃O₄-rGO/GCE (a), TB-Au-rGO/GCE (b), TB-Fe₃O₄-rGO/GCE (c), anti-AFP/TB-Fe₃O₄-rGO/GCE (d) and Au-Fe₃O₄-rGO/GCE (e); Electrochemical current responses recorded from −0.6 V to 0 V in PBS at pH 6.8 (B) and Nyquist plots of the A.C. impedance method (C): bare GCE (a), TB-Au-Fe₃O₄-rGO/GCE (b), anti-AFP/TB-Au-Fe₃O₄-rGO/GCE (c), BSA/anti-AFP/TB-Au-Fe₃O₄-rGO/GCE (d) and AFP/BSA/anti-AFP/TB-Au-Fe₃O₄-rGO/GCE (e); Inset shows the Randles model for the equivalent circuit, which represents each component at the working electrode interface and in the solution during the electrochemical reaction in the presence of Fe(CN)₆³⁻/Fe(CN)₆⁴⁻: solution resistance (R_s), electron transfer resistance (R_ct), capacitance of double layer (C_dl), Warburg impedance (Z_w).

Figure 6

(A) Electrocatalytic current responses of the immunosensor for the detection of different concentrations of AFP: 1.0 × 10⁻⁵ ng/mL (a), 1.0 × 10⁻⁴ ng/mL (b), 1.0 × 10⁻³ ng/mL (c), 1.0 × 10⁻² ng/mL (d), 0.1 ng/mL (e), 1.0 ng/mL (f) and 10.0 ng/mL (g); (B) Calibration curve of the immunosensor for the detection of different concentrations of AFP. Error bar = RSD (n = 5).

Figure 7

(A) Electrochemical signal responses of the immunosensor fabricated on five different electrodes for the detection of 1.0 ng/mL AFP; (B) Electrochemical signal responses of the immunosensor to 1.0 ng/mL AFP, 1.0 ng/mL AFP + 100 ng/mL CEA, 1.0 ng/mL AFP + 100.0 ng/mL PSA, 1.0 ng/mL AFP + 100.0 ng/mL IgG and 1.0 ng/mL AFP + 100.0 ng/mL BSA. Error bar = RSD (n = 5).