Fe₃O₄/Au magnetic nanoparticle amplification strategies for ultrasensitive electrochemical immunoassay of alfa-fetoprotein

Figures & data

Figure 1 Schematic representation of the preparation of (A) Fe₃O₄/Au, (B) Fe₃O₄/Au-HRP-Ab₂, and (C) immunosensor.

Abbreviations: AFP, alfa-fetoprotein; BSA, bovine serum albumin; HRP, horseradish peroxidase; Ab₂, secondary antibody.

Figure 2 Transmission electron microscopic images of (A) Fe₃O₄ and (B) Fe₃O₄/Au. Diameter distribution of (C) Fe₃O₄/Au nanoparticles.

Figure 3 (A) X-ray powder diffraction patterns for (a) Fe₃O₄ nanoparticles and (b) Fe₃O₄/Au nanoparticles. (B) X-ray photoelectron spectra binding-energy spectra for a sample of Fe₃O₄/Au.

Figure 4 Magnetization hysteresis for (a) Fe₃O₄ and (b) Fe₃O₄/Au nanoparticles recorded at room temperature.

Figure 5 Ultraviolet-visible spectra for (a) Fe₃O₄, (b) Au, (c) Fe₃O₄/Au, (d) Ab₂, (e) HRP, and (f) Fe₃O₄/Au-HRP-Ab₂.

Abbreviations: HRP, horseradish peroxidase; Ab₂, secondary antibody.

Figure 6 (A) Energy dispersive spectroscopy pattern of Au/GCE electrode. Scanning electron microscopic images of (B) Au/GCE and (C) Ab₁/Au/GCE.

Abbreviation: GCE, glass carbon electrode.

Figure 7 Cyclic voltammograms obtained at (a) Ab₁/Au/GCE in pH 6.5 phosphate-buffered solution, (b) Ab₁/Au/GCE, (c) Fe₃O₄/Au-HRP-Ab₂/AFP/Ab₁/Au/GCE, in pH 6.5 phosphate-buffered solution containing 2 mM hydroquinone and 5 mM H₂O₂.

Abbreviations: HRP, horseradish peroxidase; GCE, glass carbon electrode.

Figure 8 Cyclic voltammograms of the electrochemical sandwich immunosensors toward 5 ng/mL alfa-fetoprotein in the absence (A) and presence (B) of 5 mM H₂O₂ in an electrolytic cell in phosphate-buffered solution containing 2 mM hydroquinone by using various detection antibodies (a) HRP-anti-Ab₂, (b) Au-HRP-Ab₂, and (C) Fe₃O₄/Au-HRP-Ab₂.

Abbreviations: HRP, horseradish peroxidase; Ab₂, secondary antibody.

Figure 9 Effects of (A) HRP/Ab₂ ratio, (B) pH of phosphate-buffered solution, and (C) incubation time on electrochemical responses. A 1 ng/mL AFP concentration was used during the incubation process.

Abbreviations: Ab₂, secondary antibody; AFP, alfa-fetoprotein; HRP, horseradish peroxidase.

Figure 10 Calibration curves for the electrochemical immunosensor towards the AFP standard using various detection antibodies: (a) Fe₃O₄/Au-HRP-Ab₂, (b) Au-HRP-Ab₂, and (c) HRP-anti-AFP (inset: cyclic voltammograms of the immunosensor using Fe₃O₄/Au-HRP-Ab₂ towards AFP standard with various concentrations from bottom to top: 0, 0.005, 0.01, 0.05, 0.5, 1, 5 and 10 ng/mL in pH 6.5 phosphate-buffered solution containing 5 mM H₂O₂ and 2 mM hydroquinone).

Abbreviations: Ab₂, secondary antibody; AFP, alfa-fetoprotein; HRP, horseradish peroxidase.

Table 1 Comparison of serum AFP levels determined using two methods

Serum samples	1	2	3	4	5	6
Immunosensor (ng/mL)a	20.9	12.1	53.9	96.3	33.6	3.2
ELISAa	19.6	11.5	51.5	93.5	31.9	_b
Relative deviation (%)c	6.6	5.2	4.7	3.0	5.3

a Notes: Average value of five successive determinations;

b not detected;

c relative deviation was calculated by comparing results obtained using the present method with reference values obtained by the commercial ELISA method.

Abbreviations: AFP, alfa-fetoprotein; ELISA, enzyme-linked immunosorbent assay.