Near-infrared, dual-ratiometric fluorescent label for measurement of pH

We describe the spectral properties of an amine-reactive, pH-sensitive, long-wavelength ratiometric fluorescent label having a pK_a in the physiological pH range. The label exhibits its main absorption and emission in the near-infrared (NIR) region. On deprotonation, a blue shift of the excitation maximum is observed. Importantly, both the protonated and deprotonated forms of the label are fluorescent, with the deprotonated form having an extremely large Stokes shift of more than 100 nm. The spectral and photophysical properties of this pH label are compared with the properties of the protein-conjugated forms. Due to the observed pK_a shift to the acidic pH range upon conjugation to proteins, such labels are ideal for studying phagocytic events and their regulation by drugs and/or environmental factors.