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.
Highlights
Due to their high specificity and great diversity, antibodies (Ab) are widely used to provide specificity and bioactivity to nanoparticles (NPs), mainly for biosensor applications, drug delivery and imaging marking[1,2,3,4,5,6]
There is evidence that the plane of interaction of the antibodies with the metal surface does not affect the antigen binding portion (Fab), due to the net charge distribution and the asymmetry of the antibody. Another method of bioconjugation involves the modification of the surface of nanoparticles with reactive groups such as carboxyl and amine groups, which can be covalently coupled to amino acid side chains on the surface of the antibody using standardized bioconjugation methods such as ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)/NHS27
Ligated binding was determined by fluorescence using a secondary antibody anti-Fab Alexa Fluor 750 and by transmission electron microscopy using immunogold anti-Fab
Summary
The physical adsorption conjugation method had a higher rate of antibodies bound in AuNPs, the orientation rate was only 38.2% (Fig. 3) This result confirms that the Fab regions of many molecules are involved during immobilization, thereby reducing their antigen recognition efficiency. In covalent conjugation (Fig. 4F–J), a greater number of antibodies available for recognition – approximately 6.5 per NP – was found, confirming the results obtained by fluorescence labeling discussed above The characterization of these systems by MET revealed the successful formation of superstructures, confirming the recognition between the anti-IgG Fab (Immunogold) and the anti-17β estradiol, as well as proving that the bioconjugates retain their biological activity. Due to its simplicity, it becomes a potential candidate for the development of miniaturized and portable systems capable of quick and accurate detection of biomolecules
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
