A Meso-Quinolinium-Anionic Cyanine 5 Probe with Charge-Switch-Gated PET for Equivalent Total Biothiol Sensing and High-Contrast Imaging for Liver Injury Auxiliary Diagnosis.

Biothiols are central to redox homeostasis, yet their different reactivities prevent existing fluorescent probes from sensing them equally, making accurate total biothiol (T-SH) quantification in biofluids difficult. Moreover, most biothiol probes suffer from signal drift and cannot support long-term, high-contrast imaging. To address these issues, we developed ACy5-Ql-RSH, a meso-quinolinium-anionic Cy5 probe based on a charge-switching gated PET sensing mechanism. Installing the quinolinium at the meso-position creates an optimal electron donor-acceptor pair with twisted orthogonal geometry, minimizing charge-transfer distance for efficient d-PET (near-zero background). This design also neutralizes the anionic backbone, conferring lipophilicity and cell permeability. Critically, a chromene-mediated biothiol "click" reaction triggers quinolinium-to-quinoline rearrangement, shifting the Michael addition equilibrium and accelerating further biothiol additions. This enforces equivalent reactivity across Cys, Hcy, and GSH, enabling direct and accurate T-SH quantification. The quinolinium-to-quinoline conversion shuts off PET and restores bright NIR emission (SBR ≈ 60-fold). Using only 1 μL of plasma, T-SH quantification revealed 204.3 μM in healthy mice versus 131.9 μM post-APAP injury─aligned with commercial kit validations. The released anionic ACy5 is retained in lysosomes, allowing sustained high-contrast liver imaging for 5 days. This probe thus enables equivalent biothiol sensing with nearly identical kinetics and supports prolonged monitoring of APAP-induced liver injury─showing promise for plasma diagnostics and long-term bioimaging surgery.