Luminescence decay time encoding of magnetic micro spheres for multiplexed analysis

Magnetic microspheres are optically encoded by doping with three luminescent dyes. The combination of a fluorophore with a nanosecond decay profile and two phosphorescent Ruthenium metal ligand complexes with a microsecond decay profile generates a characteristic signature described by three features: bead brightness, luminescent decay time and dual lifetime referencing (DLR). The beads are identified by time resolved imaging in the microsecond range. A series of fluorophores is tested and the interference of the resulting luminescent code in the red and green label detection channels is investigated. A detailed staining procedure is worked out to increase the staining efficiency of the dyes with hydrophilic character into the lipophilic polystyrene microspheres. A mathematical model is established to calculate the dye amounts that are needed for staining a bead family with a specific feature set. Nineteen bead families were prepared representing the grid points in the three planes of a cube referring to the three features. The coefficient of variation over all bead families is 7%, 1.4% and 1.6% for bead brightness, luminescence decay time and DLR, respectively. The combination of these features and the bead size as additional feature enables the creation of 840 distinguishable bead families.