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, -Potential, scanning electron microscopy, and agarose gel electrophoresis. Based on the latter technique, a proof-of-concept was established for the proposed immunoassay.
Highlights
Raman spectroscopy has been intensively studied as a non-destructive and sensitive technique for in situ biological applications, due to the low background signal of water [1,2,3]
We demonstrate a simple method for optimizing and testing bioconjugates for Surface Enhanced RamanScattering (SERS)-based immunoassays
Agarose gel electrophoresis was employed to determine the variations in charge and size, as previously reported for gold nanoparticles of different functionalities and, used as a tool to demonstrate the formation of the bioconjugates [12,15,34,35,36,37]
Summary
Raman spectroscopy has been intensively studied as a non-destructive and sensitive technique for in situ biological applications, due to the low background signal of water [1,2,3]. For protein antigen detection applications, the most useful strategy for improved sensitivity is a Raman reporter molecule coupled to a silver or gold NP and conjugated with a specific antibody [5,6]. We present the design, simple assembly process, and testing of bioconjugates based on gold nanostars (AuNSs) and antibodies, for immuno-detection SERS applications. Antibodies are conjugated with functionalized AuNSs by a simple incubation method, which relies on establishing non-covalent interactions with the bifunctional ligand that is a Raman reporter. Our bioconjugates consisted of AuNSs with a monolayer of the thiol and Raman reporter 4-mercaptobenzoic acid (MBA) In this way, MBA confers a distinct spectral signature and, simultaneously, a stabilizing coating to the nanoparticles and a surface for conjugation with a molecular recognition element, the antibody. The bioconjugates formed in aqueous solution were characterized by ultraviolet-visible spectroscopy (UV-Vis), DLS, and ζ-potential measurements
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