Lifetime multiplexing with lanthanide complexes for luminescence in situ hybridisation

Long-lived luminescence probes with engineered lifetimes offer great potential for multiplexed biodetection and imaging at high sensitivity and contrast. The overwhelming majority of lifetime-multiplexing probes are based on luminescent nanoparticles; however, these typically suffer practical limitations compared to molecular probes when applied to biological systems. By surveying commercially available ligands for lanthanide coordination, we identify three europium complexes with distinguishable lifetime from 100 μs to 1 ms. Spectroscopic analysis reveals this to be due to intrinsic radiative lifetime and non-radiative relaxation. These europium complexes are developed as in situ hybridisation probes for luminescence lifetime imaging of bacterial biofilms. Utilising time-resolved detection we achieve sensitive and robust detection and identification of the target bacteria species, thus demonstrating the practicality of lanthanide-based molecular probes for lifetime multiplexing.