Design of a Room-Temperature Phosphorescence-Based Molecular Beacon for Highly Sensitive Detection of Nucleic Acids in Biological Fluids

Ultrasensitive fluorescent analysis or monitoring of significant molecules in complex samples is important for many biological studies, clinical diagnosis, and forensic investigations, the major obstacle for which is the background signals from ubiquitous endogenous fluorescent components of the environments. Herein, a room-temperature phosphorescence (RTP)-based molecular beacon (MB), employing a Eu3+ complex of chlorosulfonylated tetradentate β-diketone (L) and the quencher BHQ-2, was engineered for highly sensitive detection of DNA sequences in biological fluids. Complexation of Eu3+ with the ligand L formed a strongly luminescent complex EuL2. But when EuL2 and BHQ-2 were labeled to two ends of a DNA molecule with hairpin structure, the luminescence of EuL2 was quenched by BHQ-2 due to the stem-closed conformation of the beacon. Due to very low background luminescence from the probe molecule, >200-fold signal enhancement was achieved when nanomolar target sequence was introduced. This sensitivity is about 20-fold higher than the level achieved with conventional fluorescence-based molecular beacons. Furthermore, because the Eu3+ complex has a much longer luminescence lifetime (≈0.8 ms) than that of the background (<10 ns), RTP measurements were used to directly detect as low as 500 pM DNA in cell media quantitatively without any sample pretreatment.