A Cationic 3-hydroxyflavone-Based Fluorescent Probe for Wide Range of pH and Adenosine Triphosphate Determination

Cellular ATP concentration is a key marker of viability and metabolic activity, as well as pathological conditions such as cancer, hypoxia, and neurodegenerative diseases. Therefore, its determination is of great importance. Fluorescent probes used for this purpose often lack sufficient selectivity over other nucleotides. In this work, the ATP-sensing properties of the synthetic wide-range pH indicator FAM345 (bis-(dimethylaminomethyl)flavone) were investigated. At physiological pH, FAM345 exists in a protonated form, and its association with ATP yields a fourfold fluorescence enhancement and a 35 nm redshift in the excitation spectrum. Maximum fluorescence is achieved at pH 6.7-7.2, where the FAM345⁺ and ATP^4- species dominate. The formation of a 1:1 associate with an apparent association constant of 5.8×10³ M^-1 has been confirmed. The limit of quantification is 5.87 µM within a linear range of 1.76-50 µM. ADP, AMP, GTP, CTP, UTP, and NAD⁺ exhibit negligible interference, with spectral responses 2- to 5-fold lower than that of ATP. FAM345 was successfully applied for precise and accurate ATP determination in the pharmaceutical formulation "Cocarnit," demonstrating its suitability for analyzing sample with complex matrices. Thus, we have developed a highly selective ATP probe capable of simultaneously monitoring the pH of the medium.