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

A new concept to design phosphorescent nanospheres is presented. The sphere s are distinguishable by their individual decay time and spectral distribut ion of their emission spectra. They are composed of a phosphorescent ruthen ium metal-ligand complex (MLC dissolved, along with certain strongly fluore scent cyanine dyes, in modified polyacrylonitrile-based nanospheres. Since the emission spectrum of the MIC overlaps the absorption spectrum of the cy anine and both the NMC (the donor) and the cyanine (the acceptor) are in cl ose spatial proximity, efficient resonance energy transfer (RET) does occur . Thus, the nanospheres emit dual luminescence, one from the acceptor dye a nd the other from the donor MLC. Variation of the concentrations of the acc eptor dye results in a varying efficiency of RET, thus making the spheres d istinguishable. Hence, a set of multiplexable sphere labels is obtained by using one MLC (acting as the phosphorescent donor and present in constant c oncentration) and one acceptor dye (which varies in terms of both spectral properties and concentration). The nanospheres can be identified by the emi ssion maximum (reflecting the kind of acceptor dye) and by decay time (refl ecting its concentration). Since the same donor MLC is used throughout, all nanospheres can be excited with the same light source. (C) 2001 Academic P ress.