Light-regulated, cofactor-free amperometric biosensor for ultrasensitive TNT detection based on XenB-modified...

Nitroaromatic explosives such as 2,4,6-trinitrotoluene (TNT) are persistent environmental contaminants that require rapid, sensitive, and field-deployable detection strategies. In this study, we report a light-regulated, cofactor-free electrochemical biosensor based on xenobiotic reductase (XenB)-modified electrodes for ultrasensitive TNT detection. The immobilized flavoenzyme operates in two complementary modes: 1) a classical cofactor-assisted regime in the dark using NADPH as electron donor, and 2) a cofactor-free, light-regulated regime under metal halide lamp irradiation, where the polarized electrode supplies electrons while light enhances flavin redox cycling ("light-assisted flavin charging"). Under illumination in the absence of soluble cofactors, the bioelectrode exhibits a pronounced increase in catalytic current and significantly improved substrate affinity, enabling TNT detection down to 0.3 nM with a high sensitivity of 56000 ± 140 A M^-1 m^-2. The sensor shows high selectivity toward TNT over structurally related compounds and good reproducibility. Mechanistic studies suggest that the light-induced signal amplification originates from flavin-mediated redox processes influenced by the protein microenvironment. The analytical performance was further validated in real post-explosion soil samples, demonstrating practical applicability in complex matrices.