A Tetrazine-Based Ratiometric Sensor Quantifying pH Gradient in Tumorspheres through Bio-Orthogonal Labeling

, we tracked and visualized the glycolysis-associated pH fluctuations in tumor cells and spheres. Specifically, we quantified the pH gradient throughout the tumorspheres post various glycolysis states, e.g., the average pH values of the tumorsphere were measured to be 7.74 ± 0.07, 7.48 ± 0.09, and 5.69 ± 0.06 at 12, 24, and 48 h post glycolysis, respectively; and the pH values were measured to be 5.72 ± 0.05, 6.47 ± 0.06, and 7.04 ± 0.08 from the internal to the surface layer of tumorspheres, respectively. This tetrazine-based structural feature and bio-orthogonal labeling strategy enabled spatial and temporal quantitation of pH within tumorspheres, providing a universal approach for quantifying disease-related factors.