Antibody-conjugated nanoparticles for SERS-based lateral flow immunoassay

  • Pietro Gancitano

Student thesis: Doctoral Thesis

Abstract

The success to obtain a sensitive analytical tool such as a nanosensor, starts from its design and fabrication. The investigation of the chemistry behind metallic nanostructures and antibody interactions was a fundamental part of the research described this thesis. In addition, the development of surface enhanced Raman scattering (SERS) based lateral flow immunoassays (LFIA) for the highly sensitive detection of specific antigens was carried out.Gold nanoparticles (AuNPs) were used in the development of a universal method for histidine tagged antibodies conjugation. To achieve this, AuNPs were functionalised with a linker that contained a thiol group, which has affinity to the gold surface and a nitrilotriacetic acid (NTA) metal ion complex, which is known to have an affinity towards histidine. A fragment antigen-binding (Fab) antibody that contains 7-histidine residues was conjugated to the functionalised AuNPs in a rapid and straightforward manner. Different metal ions were investigated at various concentrations.Results demonstrated that the antibody uptake was controlled by metal ion concentration and that nickel (II) gave the stronger antibody binding. The Fab conjugated NTA-Ni AuNPs were also evaluated for use in SERS-based LFIA, showing high stability and compatibility compared to passive adsorption conjugates. A sandwich-like format SERS-based LFIA for the detection of C-reactive protein (CRP) was developed. Results demonstrated that, by using a Raman reporter molecule (RRM) that was in resonance with the excitation laser, a 25-fold improvement in sensitivity was achieved, compared to visual detection. A competitive format SERS-based LFIA was also developed, showing the possibility of detecting a wider range of CRP concentrations, compared to the sandwich format. Anisotropic nanoparticles such as gold nanostars (AuNSs) were used for the detection of human chorionic gonadotropin (hCG) by LFIA. AuNSs were conjugated to anti-hCG antibodies and labelled with RRMs in order to allow a SERS detection. AuNSs showed better assay performance than spherical nanoparticles, either in SERS and visual detection. AuNSs morphology and shape consented the generation of “hot spots” and the possibility to allocate antibodies in an orientated manner on the AuNSs surface, which resulted in highly sensitive signal response. The optimisation of the SERS labelled AuNSs-antibody conjugates along with the choice of an appropriate RRM led to the detection in the picogram range of hCG concentration in a pseudo-biological matrix.
Date of Award1 Jul 2017
LanguageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorDuncan Graham (Supervisor) & Karen Faulds (Supervisor)

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