An outstanding fluorescent probe for adenosine 5′-triphosphate (ATP) in the millimolar range will shed deep light on the physiological role of ATP. Many fluorescentprobes 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) 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.

中文翻译：

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忽视 ATP（或 ATP-2Na）引起的 pH 降低会导致罗丹明螺内酰胺衍生物对毫摩尔 ATP 产生假阳性反应

毫摩尔范围内的 5'-三磷酸腺苷 (ATP) 出色的荧光探针将深入揭示 ATP 的生理作用。为此目的，开发了许多基于罗丹明螺内酰胺衍生物的荧光探针，并声称能够出色地检测毫摩尔 ATP，其中使用 ATP 或腺苷 5'-三磷酸二钠盐 (ATP-2Na) 来检查它们对 ATP 的响应。然而，在这项工作中，发现毫摩尔ATP和ATP-2Na都可以显着降低普通缓冲溶液的pH值，甚至可以降低几个单位。考虑到罗丹明螺内酰胺荧光对pH的依赖性，合成了一系列基于罗丹明螺内酰胺的探针来研究它们对pH和ATP-2Na的荧光响应。结果表明，探针对毫摩尔ATP-2Na的荧光响应确实是由ATP-2Na本身和ATP-2Na引起的pH降低共同引起的。此外，事实证明，酸性pH对于探针对毫摩尔ATP-2Na本身产生荧光响应至关重要，并且基于罗丹明螺内酰胺的探针在接近自然pH下对毫摩尔ATP-2Na表现出较差的灵敏度。因此，之前关于基于罗丹明螺内酰胺的探针可以很好地检测毫摩尔ATP的结论几乎是错误的，并且可能会误导未来细胞内ATP探针的设计和实践。这项工作指出，应关注分析物的理化性质以及目标分析物产生的环境变化。