8-hydroxyquinoline derivatives as powerful, versatile, and sensitive fluorescent probes to assay phospholipase D

Due to their potential as efficient fluorescent chemosensors, the development of novel complexes using 8-hydroxyquinoline (8HQ) derivatives has garnered significant attention. In this study, the properties of forming a specific complex with phosphatidic acid (PA) through calcium ion binding were evaluated using commercial and newly synthesized 8HQ derivatives. PA is the invariant product that phospholipases D (PLDs) liberate from their various phospholipidic substrates, particularly phosphatidylcholine (PC). In order to enhance direct and sensitive PLD assays, 8HQ derivatives were subsequently tested as fluorescent probes for their capacity to distinguish PA from PC. 8HQ derivatives with chloro groups outperformed all other simple functional groups, providing the strongest discriminating signal, a broad range of excitation and emission wavelength ratio (λ_ex/λ_em) and a detection limit (LOD) of 0.1 nmol of PA, lower than previously reported. To prevent nonspecific hydrophilic interactions and to increase the proximity between PA and the probe, we grafted a hydrophobic alkyl chain to anchor 8HQ to phospholipidic micelles. These conditions did increase both the fluorescence signal ratio PA/PC and the sensibility of PA detection. The ¹H-RMN analyses of the complex PA-Ca²⁺-probe enabled a more comprehensive characterization of the entire interaction, demonstrating that the spectral changes of PA were enhanced in the presence of 8HQ and calcium ions. In conclusion, this novel collection of 8HQ derivatives, which exhibited enhanced fluorescence signals and sensitivity, a broad range of λ_ex/λ_em, varying λ_max, or efficient membrane localization, enabled their application in a wider range of conditions and the development of more sensitive, powerful, and specific PLD assays.