Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation

In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis. Experimentally measured PG-fluorescence intensity vs DNA concentration functions were fitted by a derived analytical expression, in which parameters of PG binding to DNA and chromophore fluorescence properties were included. We show that silver nanoparticles significantly increase the ratio between the fluorescence of PG bound to DNA and free PG, due to the metal-enhanced fluorescence effect (MEF), which enhances the lower limit of detectability of DNA concentrations by several orders of magnitude. An additional order of magnitude increase of PG/DNA assay sensitivity (~1 pg/ml) can be achieved by decreasing the PG concentration. We show herein that the use of MEF substrates in surface assays has a profound effect on assay sensitivity.