Reactive oxygen species (ROS) are a class of small-molecule species with unpaired electrons that are directly or indirectly transformed from molecular oxygen. Meanwhile, they are highly active chemical substances produced during aerobic metabolism. As a key member of the ROS family, endogenous H₂O₂ plays crucial roles in physiological processes, such as cellular signal transduction and immune defense. However, abnormal accumulation of H₂O₂ is closely associated with various pathological conditions, including cancer, neurodegenerative diseases, and cardiovascular disorders. Therefore, real-time and sensitive monitoring of the differences in H₂O₂ content changes can provide new insights and strategies for disease biology research and disease diagnosis. The existing H2O2 detection techniques include colorimetry, fluorescent probe, gene-encoded, electrochemical sensing, and chemiluminescence. The research suggested that fluorescent probes with halide, naphthylimide, and other fluorescent reporter groups have high detection sensitivity and selectivity for H₂O₂.

In recent years, advanced materials, including natural enzyme composites and biomimetic nanoenzyme catalytic materials, have attracted broad research interest. Enzymes are a class of natural biological catalysts that participate in regulating important biochemical reactions in living systems. However, the inherent fragility of enzymes leads to their unstable activity in complex environments. Immobilizing enzymes within nanomaterial frameworks is an effective strategy to alleviate the structural instability. Metal-organic frameworks (MOFs) materials can enhance the activity, stability, and sensitivity of encapsulated enzymes through structural restrictions. Zeolitic imidazolate frameworks (ZIFs) materials, composed of zinc ions and imidazole ligands, are the most widely studied type of MOFs, which have advantages such as simple synthesis conditions and good biocompatibility. In-situ encapsulation of enzymes within MOFs has always been a research hotspot in this field. This method has been widely applied in the in-situ encapsulation and performance improvement of biological macromolecules, such as enzymes, antibodies, and nucleic acids. Moreover, encapsulating enzymes in MOFs for catalytic signal generation to achieve sensitive detection of H₂O₂ has become an important research direction. Therefore, the construction of functional composite systems that can stably enhance the catalytic activity of enzymes is a cutting-edge research hotspot at the intersection of biomedicine and advanced materials. MOFs are widely used in the modification and sensitization of various natural enzymes due to their simple preparation and adjustable structure.

In this study, MOFs were used as carriers for the in-situ encapsulation of horseradish peroxidase (HRP) in aqueous solvents in the presence of flexible linkers Bis-COOH-PEG_n (n = 2, 4, 8) to construct new MOF-based sensing materials for H₂O₂. The loading capacity of metal ions, organic ligands, and flexible linkers was optimized. Using the fluorescence generation of a known fluorogenic substrate of HRP, Amplex Red, the optimal MOF sensing material was obtained, which exhibited approximately 5.7-fold higher catalytic activity than that of unencapsulated HRP. Importantly, we found that the presence of the flexible linker significantly improved the sensitivity of HRP-encapsulated MOFs. The optimal MOF material was applied to the detection of endogenous H₂O₂, including in lysate from six cell lines and the solution of eight bacterial strains.

In conclusion, this work constructs a MOF-based biomaterial for the sensitive detection of H₂O₂. It innovatively introduces PEG flexible linkers with dicarboxylic acid arms of varying chain lengths. This strategy not only provides new insights into the construction of MOFs with flexible chains and their biosensing applications, but also offers a novel research tool for the sensitive detection of endogenous H₂O₂.