Sensitive Protein Assay with Distinction of Conformations Based on Visible Absorption Changes of Citrate-Stabilized Gold Nanoparticles

In this work, we introduce a new and potentially general method of assay of proteins in solution. In addition, the method could distinguish conformations of protein (native and denatured forms). The method is based on the changes in visible absorption spectra of citrate-stabilized Au nanoparticles (NPs) upon addition of a measured amount of protein. The behavior of four proteins, namely alpha-amylase, green fluorescent protein (GFP), amyloglucosidase (AMG) and bovine serum albumin (BSA) have been found to be different with respect to changes in the spectra of Au NPs. The spectral behaviors were also different between the native and denatured forms of the same protein. Interestingly, spectral changes in the presence of thiol-containing proteins ((x-amylase and GFP) were different from those that either did not contain thiol at all or contained thiol that was not exposed to the solution (AMG and BSA). Transmission electron microscopic investigations revealed agglomeration of Au NPs in the presence of protein as the reason behind the change in absorption spectra. This has been further supported by dynamic light scattering based particle size analysis experiments. We have proposed a model based on the agglomeration of Au NPs in the presence of enzyme to account for the changes in the optical spectra. In addition, deconvolution analyses of the absorption spectra clearly indicated different levels of agglomeration for different proteins making the distinction possible.