A phenyl-substituted hemicyanine with maintained pK_a 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 (Δλ_fl) between the two peaks of the typical hemicyanine fluorophore without significantly affecting its pK_a 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 Δλ_fl (48 nm) than that of unsubstituted hemicyanine. Importantly, the pK_a 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.