Enzymatic superoxide generation coupled with dual-signal transduction for gallic acid determination in gallnuts

Gallic acid (GA) serves as the primary quality marker for gallnuts in traditional medicine, with its quantification essential for pharmacopoeial standardization and herbal product authentication. This work presents a dual-mode sensing platform for GA determination based on analyte-mediated suppression of superoxide radicals (O₂•^-) generated from a xanthine/xanthine oxidase (X/XO) enzymatic system. The detection exploits GA's potent superoxide scavenging to modulate two independent signals: (1) colorimetric detection via reduced conversion of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) to colored formazan, where GA scavenging of O₂•^- produces dose-dependent absorbance decrease, and (2) fluorometric detection via carbon dot (CD) fluorescence recovery, where GA suppression of formazan formation reduces optical attenuation, causing proportional fluorescence increase. Following acid hydrolysis to convert gallotannins into GA monomers and appropriate sample dilution to reduce matrix interference, the platform provided two independent calibration regimes: the fluorometric mode exhibited a linear response over 0.1-13.0 μM with an LOD of 0.038 μM, while the colorimetric mode showed linearity over 1.0-40.0 μM with an LOD of 0.41 μM. Extraction recoveries ranged from 96.5 to 97.8% (fluorometric, RSD 1.83-3.52%) and 97.6-101.0% (colorimetric, RSD 1.68-2.89%). The work is novel in coupling enzymatic superoxide generation with dual optical readouts in one platform, enabling internal cross-validation, and a practical method for routine gallnut quality control and traditional medicine standardization.