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

Abstract 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.