Design and Simple Assembly of Gold Nanostar Bioconjugates for Surface-Enhanced Raman Spectroscopy Immunoassays

Abstract

Immunoassays using Surface-Enhanced Raman Spectroscopy are especially interesting on account not only of their increased sensitivity, but also due to its easy translation to point-of-care formats. The bases for these assays are bioconjugates of polyclonal antibodies and anisotropic gold nanoparticles functionalized with a Raman reporter. These bioconjugates, once loaded with the antigen analyte, can react on a sandwich format with the same antibodies immobilized on a surface. This surface can then be used for detection, on a microfluidics or immunochromatographic platform. Here, we have assembled bioconjugates of gold nanostars functionalized with 4-mercaptobenzoic acid, and anti-horseradish peroxidase antibodies. The assembly was by simple incubation, and agarose gel electrophoresis determined a high gold nanostar to antibody binding constant. The functionality of the bioconjugates is easy to determine since the respective antigen presents peroxidase enzymatic activity. Furthermore, the chosen antibody is a generic immunoglobulin G (IgG) antibody, opening the application of these principles to other antibody-antigen systems. Surface-Enhanced Raman Spectroscopy analysis of these bioconjugates indicated antigen detection down to 50 µU of peroxidase activity. All steps of conjugation were fully characterized by ultraviolet-visible spectroscopy, dynamic light scattering, ζ-Potential, scanning electron microscopy, and agarose gel electrophoresis. Based on the latter technique, a proof-of-concept was established for the proposed immunoassay.

Lateral Flow Immunoassay of SARS-CoV-2 Antigen with SERS-Based Registration: Development and Comparison with Traditional Immunoassays

“…In this study, non-covalent conjugation was preferable since this method is gentle and straightforward and allows antibodies to be immobilized on the surface of nanoparticles modified by reporter molecules with minimal conformational changes. Since 4-mercaptobenzoic acid molecules are adsorbed on the surface of AuNPs through the S-atom, and the binding of antibodies to AuNPs is dominated by electrostatic interactions between the negatively charged surface of nanoparticles promoted by carboxylic groups of MBA and antibodies containing a positive charge, it is assumed that the reporter does not significantly affect the adsorption of antibodies [ 31 , 32 ]. For adsorption of MBA on the surface of AuNP through thiol groups, different amounts of MBA were added to 10 mL of the colloidal solution so that its final concentration was from 1 to 10 μM.…”

Gold Nanostars Bioconjugation for Selective Targeting and SERS Detection of Biofluids

“…At first, different concentration ratios were exploited for ligand exchange and the quantity of SH-PEG-COOH added to the solution of NPs was optimized to achieve the 100% coverage. The optimized amount of SH-PEG-COOH necessary to fully cover the NPs was tested by running agarose gel electrophoresis (AGE), a technique which has found application also in the field of colloid and NPs characterization to find out the modifications in charge and dimensions and consequently to demonstrate functionalization and conjugation processes [ 14 ]. NPs are differentiated according to variances in size and surface charge and bands can be discovered without any dye molecules, since NPs solutions are coloured (red and blue for NSps and NSts respectively) due to their plasmonic activities.…”

“…Other works based on physical adsorption on AuNSts were reported in the literature [ 12 , 13 ]. In this context, Oliveira M. J. et al published a really interesting and extensive work on the characterization of bioconjugation process through non-covalent interactions of antibodies with bifunctional ligands grafted onto the surface of AuNSts [ 14 ]. For what concern the chemisorption strategy instead, some articles can be found in literature.…”

Raman Scattering-Based Biosensing: New Prospects and Opportunities

“…The traditional methods of the synthesis of colloidal metal substrates include the chemical reduction of metal salts or laser ablation. Currently, methods of synthesis and the application of various nanostructures, including nanospheres [ 35 ], nanovoids [ 90 ], nanostars [ 91 , 92 ], and nanoflowers [ 93 ] in SERS-based techniques, are described. The signal enhancement efficiency is influenced by the size and shape of the metal nanoparticles.…”