Measuring antibody coatings on gold nanoparticles by optical spectroscopy

Abstract

The adsorption of antibodies onto gold nanoparticles to make gold–antibody conjugates is finding application in multiple areas. Gold–antibody conjugates for use in malaria diagnostics were prepared and a method of characterisation that can be applied to any gold–protein conjugate was developed. When protein adsorbs onto a gold nanoparticle, it changes the local refractive index and so changes the surface plasmon resonance of the gold particle. Changes to the surface plasmon resonance manifest in the absorbance spectrum of the conjugates. This was measured by optical spectroscopy and relatively simple equations to convert spectral shifts to predictions of the protein layer thickness and mass coverage are presented. As with most protein adsorption reported in the literature, the results showed the protein adsorption to depend on antibody concentration, reaching a plateau at around 1 μg ml⁻¹. The coverage was estimated to be approximately 2–3 mg m⁻² and the coating thickness estimates were approximately 10 nm, which is consistent with active antibody. The results suggested more antibody was used in conjugate preparation than was necessary for complete coverage of the gold. This excess antibody could bind to the target antigen to reduce malaria test sensitivity. A key advantage of this characterisation method is that it is sufficiently simple to be used for quality control of conjugate production and the equations presented can be applied to other coatings on gold nanoparticles.