Molecular Network-Bearing Fe-N-C Single-Atom Nanozymes for Monitoring Intracellular Glutathione Fluctuations

Nanozymes have drawn much attention owing to their low cost and high stability compared with natural enzymes. However, limited specificity intrinsically presents a challenge to their efficiency in sensing applications. Although specific biorecognition units and molecularly imprinted polymers have been introduced to enhance the specificity of nanozymes, they have some shortcomings like poor stability, high cost, complicated operation, and weakened activity. To address these issues, we developed here a simple and efficient surface engineering strategy to build the target-responsive sensing interface by weaving a molecular network on nanozymes to afford a pocket-like structure. Glutathione (GSH), an important intracellular biothiol, was used as the model target, considering the difficulty in distinguising GSH from other biothiols, especially cysteine (Cys) and homocysteine (Hcy). With high-performance single-atom nanozyme (SAzyme) Fe-N-C as the parent nanozyme, a meshy SAzyme Fe/PSAs-DTSSP containing GSH-cleavable disulfide bonds was fabricated via facile surface modification. Interestingly, the molecular network adhered onto the Fe-N-C surface modulated the peroxidase-like properties, including substrate affinity and catalytic stability under harsh conditions. Moreover, the molecular network could specifically respond to GSH over Cys, Hcy, and other interferents, which suppressed the oxidation of the chromogenic substrate 3,3 ',5,5 '-tetramethylbenzidine (TMB) due to the reversion of the surface charge property of Fe/PSAs-DTSSP, resulting in a sensitive response identified by the naked eye. The usefulness of this assay was validated by monitoring the fluctuation of the GSH level in cancer cells treated with glucose deprivation or incubated with doxorubicin, showing good accuracy and reliability.