Au Nanoparticles as Substrates in Developing Simple and Sensitive Immunoassay for PSA

Abstract: The current project work aims to develop a simple and sensitive immunoassay for prostate specific antigen (PSA) by using changes of fluorescence of gold nanoparticles with varying coverage of proteins. In this work we attempted to investigate the changes in optical properties caused by coating the nanoparticles with antibody-antigen complex. We wanted to construct biosensor that utilizes these changes to monitor biological bindings without fluorescent marker.The underlying idea of the project has been to use label free immunoassay to monitor not only presence or absence of antigens in sample solution but also to quantify the antigen concentration, we have tried to develop a simple and time and labour saving method based on a non competitive heterogeneous but label free immunoassay.There are many instrumentation techniques for analysis of changes of optical properties of nanoparticles used in an immunoassay, like absorption spectroscopy, surface plasmon resonance spectroscopy, Raman spectroscopy, time resolved fluorescence and electrochemical techniques [1]. In present work we have investigated whether the adsorption of antibodies onto Au (gold) nanoparticles would change the optical properties of antibodies to an extent sufficient to differentiate them from the free antibodies. We have furthermore investigated whether the subsequent antigen binding to antibodies also induces changes of optical properties sufficient for quantitative analysis. We have chosen to monitor optical properties via measurement of fluorescence because of its sensitivity and selectivity.Our objective here has been not only to investigate spectral changes but also to develop a robust assay protocol, for example with respect to the antibody binding and nanoparticles separation techniques. Two critical steps of the experimental procedure developed here have been (i) the separation of excess prostate specific monoclonal antibodies (PSA10) from the solution containing nanoparticles with adsorbed PSA10 and free PSA10, and the stability of Au-PSA10 conjugates, and (ii) quantification of the binding of PSA to PSA10 covered nanoparticles.We have encountered problems with agglomeration of gold nanoparticles, both naked and with PSA10 conjugates. The naked nanoparticles were “sticky” and bound easily with other materials, for example with agarose beads from the separation columns. Fortunately the coated nanoparticles turned out to be much more inert. This allowed the separation and, simultaneously, acted as a test for antibody coverage.