Two‐Color Emitting Colloidal Nanocrystals as Single‐Particle Ratiometric Probes of Intracellular pH

Intracellular pH is a key parameter in many biological mechanisms and cell metabolism and is used to detect and monitor cancer formation and brain or heart diseases. pH‐sensing is typically performed by fluorescence microscopy using pH‐responsive dyes. Accuracy is limited by the need for quantifying the absolute emission intensity in living biological samples. An alternative with a higher sensitivity and precision uses probes with a ratiometric response arising from the different pH‐sensitivity of two emission channels of a single emitter. Current ratiometric probes are complex constructs suffering from instability and cross‐readout due to their broad emission spectra. Here, we overcome such limitations using a single‐particle ratiometric pH probe based on dot‐in‐bulk CdSe/CdS nanocrystals (NCs). These nanostructures feature two fully‐separated narrow emissions with different pH sensitivity arising from radiative recombination of core‐ and shell‐localized excitons. The core emission is nearly independent of the pH, whereas the shell luminescence increases in the 3–11 pH range, resulting in a cross‐readout‐free ratiometric response as strong as 600%. In vitro microscopy demonstrates that the ratiometric response in biologic media resembles the precalibralation curve obtained through far‐field titration experiments. The NCs show good biocompatibility, enabling us to monitor in real‐time the pH in living cells.