Fan_2026_Anal.Methods__

Esterases constitute vital hydrolases participating in lipid metabolism, prodrug activation, and tumor progression. Due to the significant heterogeneity in their expression levels across different types of tumor microenvironments, esterases have emerged as promising biomarkers for cancer diagnosis. This probe achieves fluorescence quenching through an intramolecular photoinduced electron transfer (PET) mechanism. Under the catalysis of the esterase, the amide bond is hydrolyzed and the heptafluorobutyramide group is cleaved, restoring the electron-donating capacity of the amino group, blocking the PET effect, and restoring the fluorescence signal. CYF exhibits high sensitivity (limit of detection as low as 0.04749 U mL(-1)), excellent selectivity, and good photostability. Molecular docking and theoretical simulations show that CYF can stably bind to the esterase's active pocket through multiple molecular forces, with a binding affinity of -9.3 kcal mol(-1). The HOMO-LUMO band gap clearly elucidates the probe's fluorescence response mechanism. CYF demonstrates good biocompatibility and low cytotoxicity, effectively distinguishing disparities in esterase activity between normal thyroid cells (Nthy-ori3-1) and thyroid cancer cells (TPC-1), with significantly upregulated esterase activity observed in TPC-1 cells. In 4T1 tumor-bearing mice, the fluorescence intensity of probe CYF in the tumor region was significantly lower than in normal tissue; this signal was further suppressed following pretreatment with the inhibitor AEBSF. These results confirm the low expression of esterase activity in the breast cancer intratumoral microenvironment and demonstrate CYF's capability for the time-dependent, in situ dynamic tracking of esterase activity in vivo.