Bioconjugation and characterisation of gold colloid-labelled proteins

Abstract

Colloidal metal particles, in particular gold, have found many biological applications often as probes in light and electron microscopy, and more recently since the 1980s in membrane-based rapid immunoaffinity tests. The surface plasmon resonance absorbance properties in the visible spectroscopy region of gold colloids make them useful tools in medical devices, as the colloids are directly visible to the naked eye. Despite the relative ease with which gold-protein conjugates can be prepared a major issue is the manufacture of poor-quality and poorly characterised bioconjugates that can result in the under performance of subsequent diagnostic tests. This paper describes the preparation of good-quality conjugates for use in immunoassays by optimising the adsorption of antibodies onto the surface of gold colloids, followed by their subsequent characterisation. The conjugates were characterized for size, aggregation and quality using a range of techniques: UV-visible (UV/Vis) absorption spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The biological activities of the conjugated products were also assessed using an immunoassay format and electrochemical measurements. By utilising a number of measurement techniques we aimed to gain a better understanding of the extent of particle aggregation, and the resulting stability and activity of the biological molecule on the surfaces of nanoparticles. The tools developed will enable researchers and companies to ensure the sensitivity, quality and reproducibility of batches of nanoparticle bio-conjugates.