Evaluating gold nanoparticles parameters in competitive Immunochromatographich Assay via Dot Blot and Bradford Assay as new approaches

Abstract Immunochromatographic assay (ICA) is the applicable commercialized assay that is used gold nanoparticles (GNPs) as label for detection of different molecules. Various parameters like size of GNPs, pH of GNPs solution and antibody concentration which affect the performance of GNPs as label were evaluated in this study. GNPs in three different sizes of 12 ± 1.08, 32 ± 1.46 and 64 ± 2.24 nm synthesized via reversed Turkevich method at 80 °C. GNPs samples were characterized by UV–vis spectroscopy, DLS and TEM. Optimum conjugation pH and optimum concentration of conjugating antibody were obtained as pH 8 and 10.0 µg/mL, respectively. The adsorption efficiency of antibody molecules on the surface of GNPs was determined by Bradford protein assay. The results indicate that, increasing the size of GNPs leads to increasing the adsorption efficiency of antibody. Function of antibody-GNPs against morphine and structure accuracy of Morphine-BSA (MOR-BSA) was confirmed thorough dot-blot assay. Finally, the effect of size of GNPs in the LOD of ICA was evaluated. The results indicated ICA that constructed by the lowest size of GNPs have the highest LOD about 100 ng/mL and by increasing the size of GNPs up to 32 ± 1.46 the LOD decreased two folds (50 ng/mL). After that, increasing the size of GNPs was not effective in the LOD of the ICA and it was still remained at 50 ng/mL.