The aim of this investigation was to define the optimum method of binding antibodies to the surface of gold nanoparticles (AuNPs) and then to apply the optimised antibody-functionalised AuNPs for the detection of a target antigen. A detailed investigation of three different techniques for the functionalisation of AuNPs with anti-cocaine antibody and methods for the subsequent characterisation of the antibody-functionalised AuNP are reported. The addition of anti-cocaine antibody onto the AuNP surface was facilitated by either: a polyethylene glycol (PEG) linker with a –COOH terminal functional group; an aminated PEG ligand; or an N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-Protein A/G intermediate. Characterisation of the functionalised particles was performed using transmission electron microscopy, UV-Visible spectrophotometry and by agarose gel electrophoresis. In addition, the cocaine binding efficacy of the resultant AuNPs and their cocaine-binding capacity was determined using a cocaine-horseradish peroxidase conjugate, and by the application of a microtiter plate-based immunoassay. The results showed that the number of antibody per particle was the highest when the AuNP were functionalised with the Protein A/G intermediate. As compared to free antibody, the cocaine binding efficacy was significantly enhanced using the AuNP-Protein A/G-antibody complex. This optimal antibody-antigen binding efficacy is thought to be the result of the large number of antibody per particle and the oriented binding of the antibody to the Protein A/G on the AuNP surface. These results highlight the ideal immuno-gold nanoparticle characteristics for the detection of target antigens such as cocaine.
- Gold nanoparticles