Near-Infrared Fluorescent Probe for H₂S Detection: Will pH Affect the Intracellular Sensing?

Near-infrared (NIR) fluorescent probe has exhibited unique advantages for in vitro and in vivo detection of hydrogen sulfide (H₂S), an important endogenous gasotransmitter in redox homeostasis and multiple life processes. However, both the pH-dependent emission of NIR probes and H₂S conversions would normally affect the accurate detection in cellular environments in different acidic conditions. Herein, both experiments and theoretical calculations were carried out to examine the effect of pH on intracellular sensing of H₂S by the NIR probe. Selecting a NIR probe of R1 with dual-excited NIR responses to H₂S as the model, the pH-dependent R1 emission was confirmed by optical measurements, whose structural changes were further examined by mass spectrometry (MS). Significantly, the dynamic changes versus pH increase were employed for the online monitoring of ambient MS (AMS), observing important intermediate species without sample pretreatments. Thereby, intermediates and transition states were confirmed by theoretical calculations, which proposed the mechanism of nucleophilic substitution, followed by the hydrolysis process with increasing pH. As examined, R1 exhibited a relatively stable NIR emission at pH 4-8, while a dramatic change in signals occurred at higher-pH conditions. Therefore, R1 was demonstrated to be reliable for intracellular sensing of H₂S and had been confirmed by cell imaging. This work has initiated a comprehensive strategy for evaluating fluorescence (FL) probes, showing potential for the development of fluorescent probes.