Next, we examined the optical sensing behavior of RhQ towards VDPs (with rBSA as the model VDPs) in HEPES (10 mM HEPES, 5 mM EDTA, pH 7.4, 1% DMSO) buffer solution at 37 o C. Firstly, the free probe RhQ exhibited a weak fluorescence emission (Figure ) and the quantum yield of RhQ was quite low (φF = 0.014 in ethanol, using rhodamine B as reference) due to the quenching effect of a naphthalimide-based azo group in RhQ. Upon addition of rBSA (1 equiv.), the fluorescence intensity increased as a result of the FRET being interrupted (Figure ). A small enhancement of the shoulder peak at 570 nm in the absorption spectra which was assigned to the rhodamine fluorophore further verified the interaction between the rhodamine part of RhQ and rBSA (Figure ). To explore the stability of the carbamate bond in RhQ under physiological conditions, time-dependent fluorescence changes of RhQ and control probe HC were monitored in HEPES buffer. It was found that both RhQ and HC were stable for at least 4 hours without significant fluorescence changes in HEPES buffer (Figure ). Combining this information with the results that the azo bond in Q-1 could not be reduced by rBSA (Figure ), we concluded that RhQ exhibits stable sensing behavior for rBSA. Then, the time-dependent fluorescence response of RhQ towards rBSA (150 equiv.) was evaluated. The addition of rBSA resulted in a significant fluorescence change. With a gradual increase in fluorescence intensity, which reached a plateau after 90 min (Figure and). Therefore, RhQ exhibited an obvious off-on fluorescence response to rBSA, and 90 min was sufficient for the interaction between RhQ and rBSA. Compared to our previous probes that provide a relatively fast response to VDPs, we envisioned that a 90 min response might be much more suitable for recording dynamic changes of VDPs in live cells since the interaction between RhQ and VDPs could be gradual visualized by the release of fluorescence signal from the rhodamine part of RhQ. And the gradually evolving labelled fluorescence signal could be successively recorded by fluorescence microscopy using live cell imaging.