Effects on molecular interactions of hollow gold nanoparticles and antibody for sensitizing P24 antigen determination

Abstract

In recent years, with the rapid development of point-of-care testing, the application of lateral flow immunochromatography assay (LFIA) has become increasingly widespread. The key to the success of these detection technologies is the effective binding with diagnostic materials and detection antibody proteins. Although many researchers have tried to optimize antibody binding, a universally accepted strategy that can provide maximum performance has not been determined. In this study, the HIV infection P24 antigen was selected as the detection biomarker. Then the binding mechanism between hollow gold nanoparticles as diagnostic materials and detection antibodies was explored through dynamic light scattering, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and other methods. It was found that the binding efficiency is related to the change in protein secondary conformation during binding, hydrogen bonding, and van der Waals force maintain the binding mechanism between antibodies and nanoparticles. The main forces of particle complexation and the main binding site of the antibody were discussed and analyzed. Finally, an immunochromatographic system was constructed to evaluate the significant advantages of this platform compared to the common colloidal gold immunochromatographic system.

Fig. 1. Fundamental principle of the HGNPs–LFIAs. The double antibody sandwich structure formed by HGNPs–McAb and p24 coating antibody is shown in the black dashed rectangular box.

Fig. 2. Characterization of PVP@HGNPs. Appearance (A) and TEM image (B) of PVP@HGNPs. (Scale bar = 50 nm); (C) the diameter of PVP@HGNPs; (D) the absorbance spectra of PVP@HGNPs.

Fig. 3. Different absorbance (A) and particle size (B) during the binding process between HGNPs and McAb under different pH conditions. (C) TEM images of HGNPs–McAb; (scale bar = 50 nm) (D) zeta potential of HGNPs and HGNPs–McAb at optimal pH. Mean ± SD, n = 3.

Fig. 4. Effect of HGNPs on the fluorescence spectrum of McAb at 298 K (A) and 310 K (B); (C) the Stern–Volmer fitting of HGNPs effect on McAb fluorescence intensity at 298 K and 310 K; (D) double-logarithm plot for the quenching of McAb protein by HGNPs at different temperatures.

Fig. 5. Conformational studies of McAb. (A) Infrared scanning spectra of HGNPs, McAb and HGNPs–McAb (pH = 6.5); (B) CD spectra of interaction between HGNPs and McAb under different pH conditions.

Fig. 6. Representative images of Alere HIV Combo (A) and HGNPs–LFIA (B) for p24 antigen detection in serum. *represents the visual detection limit of the p24 antigen. (C) Comparison OD_T of HGNPs–LFIAs and Alere HIV combo for p24 antigen detection in serum. Mean ± SD, n = 3.

Fig. 7. Representative images of Alere HIV Combo(A) and HGNPs–LFIA (B) for p24 antigen for preclinical samples. (C) Comparison OD_T of HGNPs–LFIAs and Alere HIV combo for p24 antigen detection for preclinical samples. Mean ± SD, n = 3.