Overlooking pH Decrease Induced by ATP (or ATP-2Na) Results in False Positive Response of Rhodamine Spirolactam Derivatives to Millimolar ATP

An outstanding fluorescent probe for adenosine 5′-triphosphate (ATP) in the millimolar range will shed deep light on the physiological role of ATP. Many fluorescent probes based on rhodamine spirolactam derivatives have been developed for this purpose and claimed to detect millimolar ATP excellently, where ATP or adenosine 5′-triphosphate disodium salt (ATP-2Na) dissolved in a buffer solution is employed to examine their response to ATP. However, in this work, it was found that both millimolar ATP and ATP-2Na could significantly decrease the pH of the common buffer solution even for several units. Considering the dependence of the rhodamine spirolactam fluorescence on pH, a series of rhodamine spirolactam-based probes was synthesized to investigate their fluorescence response to pH and ATP-2Na. The results demonstrated that the fluorescence response of the probes to millimolar ATP-2Na indeed resulted from both ATP-2Na itself and the pH decrease induced by ATP-2Na. Furthermore, it was evidenced that acidic pH was crucial for the probes to produce fluorescence response to millimolar ATP-2Na itself, and the rhodamine spirolactam-based probes would show poor sensitivity to millimolar ATP-2Na at near-natural pH. Accordingly, the previous conclusion that the rhodamine spirolactam-based probes could detect millimolar ATP excellently was almost erroneous, and might mislead the design and practice of intracellular ATP probe in the future. This work notes that attention should be paid to the physiochemical properties of the analyte and the environmental changes produced by the target analyte.