Impact of conjugation strategies for targeting of antibodies in gold nanoparticles for ultrasensitive detection of 17β-estradiol

Abstract

Antibody-coated nanoparticles have recently attracted considerable attention, with the focus falling on diagnostics. Nevertheless, controlled antibody bioconjugation remains a challenge. Here, we present two strategies of bioconjugation with the aim of evaluating the best approach for the coupling of antibodies on the surface of nanomaterials in an oriented way. We employed electrostatic interaction (physical adsorption) and covalent conjugation in the orientation of antibodies on the metallic surface as coupling methods, and their influence on the detection of 17β-estradiol was addressed with localized surface plasmon resonance. The understanding of these mechanisms is fundamental for the development of reproducible inorganic bioconjugates with oriented surface as well sensibility of immunoassays.

Figure 1

UV-Visible absorption spectroscopy for synthesized gold colloid showing the characteristic plasmony peak at 522 nm (A) X-ray diffraction pattern depicting the characteristic peaks of nanocrystals. (B) Images obtained by transmission electron microscopy at different magnifications showing the size and distribution of the synthesized nanoparticles. Scale bar 100 nm (C,D) and 10 nm (E,F).

Figure 2

Normalized UV-Vis absorption spectroscopy of AuNPs in black and AuNPs functionalized with mercaptoundecanoic acid (MUA) in blue. (A) The inset shows the band displacement caused by the MUA connection with the metal surface; Infrared spectrum of the binder used (MUA) and the functionalized AuNPs showing the overlapping characteristic peaks (B). Raman spectra of AuNPs (black) and AuNPs-MUA (blue) (C).

Figure 3

Comparison between the amounts of bound anti-17β-estradiol antibodies and antibodies targeted on the AuNPs metal surface by the conjugation strategies evaluated (Physical adsorption and covalent attachment). The rate of bound antibodies was determined by the intrinsic fluorescence of the amino acids present in the IgG-like antibody structure (Excitation 280/emission 350 nm) and the rate of targeted antibodies was determined by the signal emitted by the anti-Fab Alexa Fluor 750 secondary antibodies.

Figure 4

Images obtained by transmission electron microscopy presenting several possible combinations according to the orientation of the primary antibody anchored on the metal surface. Superstructures are formed by recognition of Fab sites by immunogold (5 nm). Immunoconjugates prepared by physical adsorption (A–E) and immunoconjugates prepared by covalent conjugation through the mercaptoundecanoic acid linker (F–J). Scale bar 20 nm.

Figure 5

Absorption spectra of the AuNPs conjugated solutions by physical adsorption with anti-17β-estradiol antibodies under different concentrations of 17β-estradiol (A); Typical calibration curve for detection of 17β-estradiol with immunoconjugates prepared by physical adsorption. (B) Absorption spectra of the AuNPs conjugated by covalent binding with anti-17β-estradiol antibodies under different concentrations of 17β-estradiol (C); Typical calibration curve for detection of 17β-estradiol with immunoconjugates prepared by covalent conjugation (D).

Figure 6

Comparison of the maximum lambda displacement (nm) evolution as a function of increasing concentrations of 17β-estradiol (ng.mL⁻¹) for both conjugation strategies evaluated (physical adsorption and covalent attachment).