Recently, a new fluorescent probe AE-Phoswas reported to detect the activity of alkaline phosphatases (ALP) in different living cell lines. Here, we present an in-depth computational analysis of the mechanism and source of the fluorescence of the AE-Phos probe. There is an intermediate product (AE-OH-Phos) in the experiment as well as a different configuration of products that may emit fluorescence. It is essential to investigate the origin of fluorescence and the detection mechanism of the probe, which could help us eliminate the interference of other substances (including an intermediate product and possible isomers) on fluorescence during the experiment. According to the change of geometric parameters and Infrared spectra, we deduce that the dual intramolecular hydrogen bonds of salicylaldehyde azine (SA) were enhanced at the excited state, while AE-OH-Phos was attenuated. Considering the complex ESIPT behavior of the dual proton-type probe, the potential energy surfaces were further discussed. It can be concluded that the single proton transfer structure of SA (SA-SPT) is the most stable form. Both the concerted double proton transfer process and stepwise single proton transfer process of SA were forbidden. The fluorescence for SA was 438 nm, while that of SA-SPT was 521 nm, which agrees with the experimentally measured fluorescence wavelength (536 nm). The conclusion that single proton transfer occurs in SA is once again verified. In addition, the distribution of electron-hole and relative index was analyzed to investigate the intrinsic mechanism for the fluorescence quenching of the probe and the intermediate product. The identification of the origin of fluorescence sheds light on the design and use of dual-proton type fluorescent probes in the future.
Keywords: alkaline phosphatase; double proton transfer; fluorescence probe; time-dependent density functional theory.