Amino-coumarin based fluorescence ratiometric sensors for acidic pH and their application for living cells imaging

Two novel ratiometric sensors, 1a and 1b, for acidic pH have been rationally developed with 7-diethylamino-coumarin as the fluorophore and pyridine as the receptor. Both of the sensors exhibited a fluorescence ratiometric response to acidic pH. For sensor 1a, upon decreasing the pH from 8.35 to 2.36, the fluorescence emission spectra exhibited a large red shift from 529 to 616 nm, the emission ratio (I-529 : I-616) changed dramatically from 8.58 to 0.09, and the pK(a) value was calculated to be 5.36. The emission ratio also displayed a good linearity with the pH in the range of 4.0 to 6.5, which is valuable for quantitative determination of pH in this acidic pH window. Similar behaviour was observed for sensor 1b. In addition, NMR experiments and theoretical calculations demonstrated that the ratiometric response of the sensors to acidic pH was due to H+ binding with the N of pyridine and the induced enhancement of the intramolecular charger transfer (ICT) process. The sensors have been successfully applied to quantitatively detect pH in biological fluids. The intracellular pH imaging experiments also proved that the sensors are suitable for detecting acidic pH fluctuations in living cells.