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

摘要

ABSTRACT: Ultrasensitive fluorescent analysis or monitoringnof significantmolecules in complex samples is important formanynbiological studies, clinical diagnosis, and forensic investigations,nthe major obstacle for which is the background signals fromnubiquitous endogenous fluorescent components of the environ-nments. Herein, a room-temperature phosphorescence (RTP)-nbased molecular beacon (MB), employing a Eu3þ complex ofnchlorosulfonylated tetradentate β-diketone (L) and the quenchernBHQ-2, was engineered for highly sensitive detection of DNAnsequences in biological fluids. Complexation of Eu3þ with thenligand L formed a strongly luminescent complex EuL2.ButwhenEuL2 and BHQ-2 were labeled to two ends of a DNA molecule withnhairpin structure, the luminescence of EuL2 was quenched by BHQ-2 due to the stem-closed conformation of the beacon. Due to very lownbackground luminescence from the probe molecule, >200-fold signal enhancement was achieved when nanomolar target sequence wasnintroduced. This sensitivity is about 20-fold higher than the level achieved with conventional fluorescence-based molecular beacons.nFurthermore, because the Eu3þ complex has a much longer luminescence lifetime (≈0.8 ms) than that of the background (<10 ns), RTPnmeasurements were used to directly detect as low as 500 pM DNA in cell media quantitatively without any sample pretreatment.