A new type of phosphorescent nanospheres for use in advanced time-resolved multiplexed bioassays

A new concept to design phosphorescent nanospheres is presented. The spheres are distinguishable by their individual decay time and spectral distribution of their emission spectra. They are composed of a phosphorescent ruthenium metal-ligand complex (MLC) dissolved, along with certain strongly fluorescent cyanine dyes, in modified polyacrylonitrile-based nanospheres. Since the emission spectrum of the MLC overlaps the absorption spectrum of the cyanine and both the MLC (the donor) and the cyanine (the acceptor) are in close spatial proximity, efficient resonance energy transfer (RET) does occur. Thus, the nanospheres emit dual luminescence, one from the acceptor dye and the other from the donor MLC. Variation of the concentrations of the acceptor dye results in a varying efficiency of RET, thus making the spheres distinguishable. Hence, a set of multiplexable sphere labels is obtained by using one MLC (acting as the phosphorescent donor and present in constant concentration) and one acceptor dye (which varies in terms of both spectral properties and concentration). The nanospheres can be identified by the emission maximum (reflecting the kind of acceptor dye) and by decay time (reflecting its concentration). Since the same donor MLC is used throughout, all nanospheres can be excited with the same light source.