Preparation and quality control of silver nanoparticle-antibody conjugate for use in electrochemical immunoassays

Metal nanoparticle-antibody conjugates are often used as optical or electrochemical markers in applications like immunohistochemistry, lateral flow tests, biosensors and immunoassays. In order to serve that role, an antibody needs to be immobilized on the surface of the nanoparticle. This is easily done, as proteins bind to gold and silver nanoparticles spontaneously. However, this immobilization process might result in nanoparticle aggregation or the loss of the bioactivity of the conjugated antibodies. In this work the optimization of antibody immobilization on silver colloid in order to obtain conjugates with the best possible activity is investigated. The parameters investigated were the type of immobilization buffer, its molarity and pH, the nanoparticle/antibody ratio and also blocking and washing protocols to reduce non-specific binding. The functionality of the obtained conjugates was tested with electrochemical immunoassay. It was found out that the optimum environment for immobilization of an anti-myoglobin antibody on silver nanoparticles was 0.2 M boric acid pH 6.5 with 10 μg of antibody loading per 1 mL of silver colloid. For an anti-troponin antibody it was 0.1 M boric acid pH 7.5 also with 10 μg/mL of antibody loading. The main problem for silver conjugation was the tendency of silver nanoparticles to aggregate during the immobilization process, but by choosing the optimum conditions the aggregation problem was completely removed. Here it is demonstrated that by using the conjugates prepared with an optimized protocol an increase in the sensitivity of the assay 10 times can be achieved. The electrochemical immunoassay described here can be used as a test for quality control of conjugates and for the estimation of batch-to-batch variability.