Bispyrene-fluorescein hybrid based FRET cassette: a convenient platform toward ratiometric time-resolved probe for bioanalytical applications.

Pyrene excimer possesses a large Stokes shift and long fluorescence lifetime and has been widely applied in developing time-resolved biosensing systems to solve the autofluorescence interference problems in biological samples. However, only a few of pyrene excimer-based small molecular probes have been reported so far. Ratiometric probes, on the other hand, can eliminate interferences from environmental factors such as instrumental efficiency and environmental conditions by a built-in correction of the dual emission bands but are ineffective for endogenous autofluorescence in biosystems. In this work, by combining the advantages of time-resolved fluorescence technique with ratiometric probe, we reported a bispyrene-fluorescein hybrid FRET cassette (PF) as a novel ratiometric time-resolved sensing platform for bioanalytical applications, with pH chosen as a biorelated target. The probe PF showed a fast, highly selective, and reversible ratiometric fluorescence response to pH in a wide range from 3.0 to 10.0 in buffered solution. By employing time-resolved fluorescence technique, the pH-induced fluorescence signal of probe PF can be well-discriminated from biological autofluorescence background, which enables us to detect pH in a range of 4.0-8.0 in cell media within a few seconds. It has also been preliminarily applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. Since many fluorescein-based fluorescence probes have been developed, our strategy might find wide applications in design ratiometric time-resolved probes for detection of various biorelated targets.