A phenyl-substituted hemicyanine with maintained pKa as ratiometric near-infrared fluorescent probe for in vitro and in vivo pH determinations

In this study a new ratiometric pH probe, PHE-C, was designed and synthesized with the aim of enlarging the wavelength difference (& UDelta;?fl) between the two peaks of the typical hemicyanine fluorophore without significantly affecting its pKa value. The two emission maxima at 682 and 730 nm, associated with the respective protonation states of the hydroxyl group in PHE-C, have a larger & UDelta;?fl (48 nm) than that of unsubstituted hemicyanine. Importantly, the pKa of PHE-C (6.55) is the same as that of the unsubstituted counterpart. Consequently, the ratio of the intensities of the 682 and 730 nm emission bands is linearly related to pH in the range of 5.5-7.5, which is appropriate for monitoring small changes in pH of weakly acidic physiological environments. Moreover, PHE-C possesses good photostability, sensitivity and reversibility, and it displays the same response as pH meter when determining the pH in bovine serum solutions, indicating that it can be utilized in complex biological environments. Finally, the results of studies aimed at assessing the applicability to monitoring pH changes in living cells and mice demonstrate that the new probe is a highly sensitive ratiometric probe for in vivo pH determinations.

Automated summary

In this study, a new ratiometric pH probe, PHE-C, was developed to enhance the wavelength difference between the two peaks of the typical hemicyanine fluorophore without affecting its pKa value. The probe exhibited two emission maxima at 682 and 730 nm, with a larger wavelength difference compared to the unsubstituted hemicyanine. It demonstrated linear response to pH changes in the range of 5.5-7.5 and showed good performance in complex biological environments such as bovine serum solutions, making it a highly sensitive ratiometric probe for in vivo pH determinations.