An Ultrasensitive Electrochemical Immunosensor for HIV p24 Based on Fe₃O₄@SiO₂ Nanomagnetic Probes and Nanogold Colloid-Labeled Enzyme-Antibody Copolymer as Signal Tag

Abstract

An ultrasensitive portable electrochemical immunosensor for human immunodeficiency virus p24 (HIV p24) antigen detection has been developed, whereby the detection sensitivity was 1000 times higher than that of the ELISA method. Firstly, a novel HRP enzyme-antibody copolymer (EV-p24 Ab2) was synthesized through an EnVision regent (EV, a dextrin amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of anti IgG), then incubated in the secondary antibody of p24. Secondly, the copolymer was immobilized on the gold nanocolloids (AuNPs) to fabricate a novel signal tag (AuNPs/EV-p24 Ab2). Subsequently, a sandwich-type immunoreaction would take place between the capture probe (silicon dioxide-coated magnetic Fe₃O₄ nanoparticles (MNPs) labeled with the primary p24 antibody (MNPs-p24 Ab1)), p24 (different concentrations) and the signal tag [AuNPs/EV-p24 Ab2)] to form the immunocomplex. Finally, the immunocomplex was absorbed on the surface of screen printed carbon electrode (SPCE) by a magnet and immersed in the o-hydroxyl phenol (HQ) and H₂O₂. The large amounts of HRP on the signal tag can catalyze the oxidation of HQ by H₂O₂, which can induce an amplified reductive current. Moreover, the capture probe could improve the accumulation ability of p24 and facilitate its separation from the substrate through the magnet. Under optimal conditions, the proposed immunoassay exhibited good sensitivity to p24 within a certain concentration range from 0.001 to 10.00 ng/mL, with a detection limit of 0.5 pg/mL (S/N = 3). The proposed method can be used for real-time and early detection of HIV-infected people.

Keywords: Fe3O4@SiO2 magnetic beads; HIV p24; electrochemical immunosensor; nanogold-labeled EnVision antibody enzyme complex; signal amplification.

Figure 1

Scanning electron microscopy (SEM) images of (a) EnVision reagent (EV); (b) EV-p24 Ab2 and (c) Au NPs/EV-p24 Ab2.

Figure 2

Transmission electron microscopy (TEM) images of (a) Fe₃O₄@SiO₂ (MNPs) and (b) MNPs-p24 Ab1 and (c) MNPs-p24 Ab1 capture probes in the absence and (d) presence of an external magnetic field (d, right).

Figure 3

The X-ray fluorescent spectroscopic (XRF) spectrum of MNPs.

Figure 4

The ultraviolet-visible absorption spectrometry of (a) EV; (b) EV-p24 Ab2 and (c) Au/EV-p24 Ab2 signal tag, respectively.

Figure 5

Cyclic voltammograms obtained at (a) SPCE/MNPs-p24 Ab1 electrode in pH 7.0 PBS; (b) SPCE/MNPs-p24 Ab1; (c) SPCE/MNPs-p24 Ab1 incubated in 5 ng/mL p24; and (d) c was further sandwich immunoreacted in Au NPs/EV-p24 Ab₂ in pH 7.0 PBS containing 5 mmol/L HQ and H₂O₂.

Figure 6

The effect of different (a) concentration of HQ; (b) Concentration of H₂O₂; (c) pH to the anodic peak currents responses for the immunosensor; (d) Incubation time after the immunosensor was incubated with 0.5 ng/mL p24 solution.

Figure 7

Cyclic voltammograms of the electrochemical sandwich immunosensor toward 5 ng/mL p24 in the absence (a) and in the presence of 5 mmol/L H₂O₂ in electrolytic cell in pH 7.0 PBS containing 5 mmol/L HQ by using various signal tags: (b) HRP-p24 Ab2; (c) EV-p24 Ab2; and (d) AuNPs/EV-p24 Ab2.

Figure 8

Voltammogram of the modified electrode using different concentrations of antigen (0~10 ng/mL, respectively) (a) DPV curves; (b) plot of ΔI vs. the concentrations of p24. All experiments were carried out in 0.01 mol/L PBS, 0.1 mol/L KCl (pH 7.4) and added 5 mmol/L HQ and H₂O₂, at a potential range of 0.2 V to −0.5 V, vs. SCE. Scan rate 50 mV/s.

Scheme 1

Illustration of the fabrication and detection procedure of the immunosensor.