title: Optimisation of immuno-gold nanoparticle complexes for antigen detection.
abstract: 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.
authors.affiliation: School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, Norfolk NR4 7TJ, UK. Electronic address: sv377@cam.ac.uk.
authors.forename: Susan
authors.initials: S
authors.lastname: van der Heide
authors.affiliation: School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, Norfolk NR4 7TJ, UK. Electronic address: d.russell@uea.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Russell
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000941:Antigens / Q000032:analysis*; D001426:Bacterial Proteins / Q000737:chemistry; D003042:Cocaine / Q000032:analysis*; D006046:Gold / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry; D007124:Immunoenzyme Techniques / Q000379:methods; D053768:Metal Nanoparticles / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000737:chemistry
chemical_list: D000906:Antibodies; D000941:Antigens; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D013205:Staphylococcal Protein A; D011092:Polyethylene Glycols; D006046:Gold; D006735:Horseradish Peroxidase; D003042:Cocaine; C000595209:polyethylene glycol 1000
keywords: Antibodies; Cocaine; Conjugation; Gold nanoparticles