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The accurate detection of biological substances is highly desirable to study various biological processes and evaluate disease progression. Herein, we report a self-validated fluorescent probe which is composed of a coumarin fluorophore as the energy donor and a fluorogen with aggregation-induced emission characteristics (AIEgen) as the energy quencher linked through a caspase-3 specific peptide substrate. Unlike the traditionally widely studied fluorescence resonance energy transfer (FRET) probes, our new generation of FRET probe is non-fluorescent itself due to the energy transfer as well as the dissipation of the acceptor energy through the free molecular motion of AIEgen. Upon interaction with caspase-3, the probe displays strong green and red fluorescent signals synchronously due to the separation of the donor-quencher and aggregation of the released AIEgen. The fluorescence turn-on with dual signal amplification allows real-time and self-validated enzyme detection with a high signal-to-background ratio, providing a good opportunity to accurately monitor various biological processes in a real-time manner.

A FRET probe with AIEgen as the energy quencher: dual signal turn-on for self-validated caspase detection