Study on the pH Dependence of the Photophysical Properties of a Functionalized Perylene Bisimide and Its Potential Applications as a Fluorescence Lifetime Based pH Probe

In this work, we have investigated the dependence on the pH of the photophysical properties of a functionalized perylene bisimide (PBI) and its potential pH sensing applications. Observed was the presence of aggregates which diminishes at acid pH values and low concentrations, without totally disappearing until a temperature of 80 °C was reached. At basic pH, significant changes in the absorption spectrum were observed, which were associated with more strongly coupled aggregates. The 1H NMR spectra of the PBI dye in D2O/TFA also showed the dependence of aggregation on concentration and temperature. PBI fluorescence intensity and lifetime were also sensitive to pH values. The maximum fluorescence intensity and lifetime were observed in acid medium, in which protonation of the secondary amines on the PBI side chains likely hinders the formation of strongly coupled aggregates. On the contrary, the fluorescence intensity significantly decreased in basic medium, due to deprotonation of the amine groups and the formation of stronger aggregates. Density functional theory calculations corroborated that π-stacked aggregates of PBI derivatives are stable in the protonated state, but their supramolecular structure changes. In the aggregate, monomeric units slide over their adjacent ones and increase the intermolecular distance upon the protonation. Intermolecular hydrogen bonds can help maintain the stability of the protonated aggregate. Fluorescence lifetime showed a sigmoidal dependence on pH, with a linear response range between pH 6 and 8, both in Tris·HCl buffered solutions and in a synthetic buffer mimicking the intracellular environment. The biocompatibility of the PBI dye was tested in C3H10T1/2 mesenchymal cells. The cellular uptake was confirmed by confocal fluorescence microscopy. No significant effects on cellular viability and morphology were observed at the conditions in which compound 1 can be used as a fluorescent probe. This work supports the idea that PBI derivatives can be suitable dyes for fluorescence lifetime sensing applications.