Photoactivated fluorescent nanozymes from mulberry leaf-derived carbon dots for dual-mode antioxidant determination and biothiol contribution estimation.

Contribution-resolved antioxidant determination in complex food matrices remains challenging because the total antioxidant response usually arises from multiple overlapping reducing species. Herein, red-emissive carbon dots (CDs) were prepared from mulberry leaves via a solvothermal method and developed as highly efficient photoactivated fluorescent nanozymes for dual-mode antioxidant determination. The CDs exhibited a large Stokes shift (Δλ = 272 nm) and were stably dispersed in aqueous media with the assistance of Tween-20. Under 365 nm irradiation, the CDs showed oxidase-like activity toward 3,3',5,5'-tetramethylbenzidine (TMB), producing oxidized TMB (oxTMB) and simultaneously inducing fluorescence quenching through an inner-filter-effect-dominated process. In the presence of antioxidants, oxTMB was reduced, accompanied by color fading and fluorescence recovery, thereby establishing a light-gated "oxidation-quenching/reduction-recovery" dual-mode sensing platform. Using biothiols as model reductive analytes, the limits of detection for Cys and GSH were 0.29 and 0.16 µM in the colorimetric mode, and 0.34 and 0.26 µM in the fluorescent mode, respectively. More importantly, by integrating N-ethylmaleimide-mediated thiol blocking with total antioxidant measurement, the platform enabled differential estimation of the biothiol-associated reducing contribution in complex soy sauce samples. The total antioxidant capacity was determined to be 1392.3-2643.5 µM Cys-equivalent, while the biothiol-associated contribution was estimated to be 102.4-384.8 µM Cys-equivalent. This work provides a light-gated dual-mode nanozyme platform and a chemically selective strategy for contribution-resolved antioxidant determination in complex fermented food matrices.