Synthesis and characterisation of far-red fluorescent cyanine dye doped silica nanoparticles using a modified microemulsion method for application in bioassays

Silica nanoparticles (NPs) doped with far-red fluorescent cyanine dyes, Cy5 and FR670, were prepared using the microemulsion method. The effects of nucleation pathway on dye loading and NP morphology were investigated using UV-Vis spectroscopy and transmission electron microscopy for different combinations of three surfactants, Triton® X-100, AOT and NP-5. Successful synthesis of monodispersed NPs with efficient dye loading was achieved using an intramicellar nucleation pathway combined with a negatively charged surfactant. For effective bioconjugation NPs were coated with a silica shell using either the microemulsion or the Stöber method. To maximise fluorescence intensity a series of NPs were doped with different amounts of dye ranging from 0.5 to 6% (w/w). At dye loadings of 0.5 and 0.25% (w/w) for Cy5 and FR670, respectively, NPs were 72 and 87 times more fluorescent than free dye labels. For Cy5 the change in fluorescence intensity with dye loading matched closely with a standard model for homo Förster resonance energy transfer (hFRET). A significant drop in fluorescence lifetime inside the NPs with increased dye loading was also observed and correlated with changes in energy transfer and quantum efficiency. Cy5 dye doped NPs were tested as biolabels in a fluorescence immunoassay to detect C-reactive protein, which is a recommended biomarker for cardiovascular disease. The NP label assay showed approximately an order of magnitude improvement in limit-of-detection when compared to a free dye assay.