Unraveling Ratiometric Chemiluminescence Probe: Theoretical Insights into pH Modulation, Luminescence Dynamics, and Energy Transfer Mechanisms

Maintaining a stable physiological pH is essential for the normal functioning of both whole organisms and individual cells. Ratiometric chemiluminescence probes have been widely employed to monitor pH in cells and living organisms due to their high sensitivity, resistance to external interferences, and noninvasiveness. In this study, the working mechanism of a specific ratiometric chemiluminescent probe, Ratio‐pHCL‐1, is investigated using (time‐dependent) density functional theory. The mechanism can be divided into three stages. At first, pH influences the protonation state of Ratio‐pHCL‐1 in physiological pH range of 6.8–8.4. Subsequently, Ratio‐pHCL‐1 decomposes to generate the light emitter in the first excited state (S 1 ) via a gradually reversible charge‐transfer initiated luminescence mechanism. Finally, at higher pH values, the intramolecular energy transfer (ET) occurs, resulting in a redshift of the emission wavelength. The redshift of the emission wavelength effectively enhances the luminescence intensity and improves the imaging ability. While at lower pH values, the ET process does not occur. This is the first systematic study on the working mechanism of ratiometric chemiluminescent probes at the molecular and electronic‐state levels. The findings can also be extended to understand the mechanism of a class of ratiometric chemiluminescent probes.