Reliable Quantification of pH Variation in Live Cells Using Prussian Blue-Caged Surface-Enhanced Raman Scattering Probes

Intracellular pH is an important parameter that is highly associated with diverse physiological processes. The reliable measurement of pH values inside cells remains a formidable challenge because of the complexity of cytoplasm. Herein, we report a robust Prussian blue (PB)-caged pH-responsive surface-enhanced Raman scattering (SERS) probe for precisely mapping the dynamic pH values in live cells. The PB shell has a subnanoscale porous structure that allows only very small biospecies such as H⁺ or OH^- to pass freely through the shell and react with the encased pH-responsive SERS probe, while physically resisting the entry of large biomolecules. This probe achieved unmatched detection linearity (<i>R</i>² > 0.999) for pH measurements in diverse complex biological samples. Moreover, the nitrile (C≡N) in PB shows a sharp band in the cellular Raman-silent region, which serves as a background-free internal standard for accurate profiling of the probe distribution inside the cells. We applied the proposed probe to monitor the dynamic pH changes during cellular autophagy induced by different stimuli and thereby demonstrated that the PB-caged probe can reliably quantify subtle intracellular pH variations, providing an effective tool for revealing the relationship between abnormal intracellular pH and cellular functions.