A cascade-driven fluorescent probe for serum albumin-mediated highly selective detection of biothiols in living cells

Precisely detecting biothiols in cancer cells holds great significance as it provides insight into the redox state of tumors and enables early cancer detection by identifying abnormal biothiol levels. Despite the elevated biothiol levels often observed in cancer cells compared to normal cells, the high concentration of biothiols in normal tissues, particularly glutathione (GSH) at millimolar levels, makes it challenging to distinguish cancer cells from normal ones using a general biothiol probe. Herein, a cascade-driven fluorescent probe (TNY) was developed for the serum albumin-mediated detection of biothiols. Leveraging an intramolecular charge transfer (ICT) mechanism, TNY was intrinsically non-fluorescent, exhibited no fluorescence change upon direct reaction with GSH alone, and showed a negligible response to serum albumin. However, the fluorescence response to GSH (LOD = 0.43 μM) was turned on in the presence of serum albumin. This process is driven by the specific interaction between the generated TNY-OH and the protein, as well as the robust responsiveness of TNY-OH to albumin (LOD = 0.71 μM). Benefiting from synergistic non-covalent interactions, both TNY (K_TNY = (0.41 ± 0.01) × 10⁵ M^-1) and TNY-OH (K_TNY-OH = (2.29 ± 0.08) × 10⁵ M^-1) exhibited strong binding affinities for serum albumin. This characteristic effectively shielded the albumin responsiveness of TNY-OH from interference by common drugs. In vitro cellular studies demonstrated that TNY exhibited weak fluorescence in cells with low levels of either GSH or serum albumin but showed a strong fluorescent signal specifically in cells co-enriched with both analytes. TNY stands as a reliable cascade-driven tool for serum albumin-mediated detection of biothiols, offering great promise for precise cancer diagnostics.