Two‐Dimensional Design Strategy to Construct Smart Fluorescent Probes for the Precise Tracking of Senescence

The tracking of cellular senescence usually depends on the detection of senescence‐associated β‐galactosidase (SA‐β‐gal). Previous probes for SA‐β‐gal with this purpose only cover a single dimension: the accumulation of this enzyme in lysosomes. However, this is insufficient to determine the destiny of senescence because endogenous β‐gal enriched in lysosomes is not only related to senescence, but also to some other physiological processes. To address this issue, we introduce our fluorescent probes including a second dimension: lysosomal pH, since de‐acidification is a unique feature of the lysosomes in senescent cells. With this novel design, our probes achieved excellent discrimination of SA‐β‐gal from cancer‐associated β‐gal, which enables them to track cellular senescence as well as tissue aging more precisely. Our crystal structures of a model enzyme E. coli β‐gal mutant (E537Q) complexed with each probe further revealed the structural basis for probe recognition. We report a two‐dimensional design strategy to construct smart fluorescent probes for the precise tracking of senescence. Our approach relies on the detection of senescence‐associated β‐galactosidase activity in combination with the monitoring of lysosomal microenvironmental pH since de‐acidification is a unique feature of the lysosomes in senescent cells.