Construction of genetically encoded biosensors for monitoring cytosolic and mitochondrial H<sub>2</sub>O<sub>2</sub> in response to nanozymes in THP-1 cells

Intracellular H₂O₂ levels are tightly regulated and can be modulated by various stimuli. A variety of nanozymes have been revealed with the ability to catalyze substrates of oxidoreductases, mostly including peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), and some of them display multienzyme-like properties, which make them highly attractive for biomedical applications. However, the specific manifestations of nanozyemes within cells remain challenging to predict and detect. In this study, we developed a real-time, dynamic, and highly sensitive live-cell biosensor by expressing HyPer7 probe in the cytosol and mitochondria to monitor the cytosolic and mitochondrial H₂O₂ dynamics in a leukemia cell line THP-1. The successful expression of the probes in the cytosol and mitochondria was confirmed using confocal fluorescence microscopy. When the THP-1 cells were exposed to exogenous H₂O₂, the fluorescence intensity at 525 nm upon excitation with 405 nm lasers (referred to as F405) decreased, while that upon excitation with 488 nm lasers (referred to as F488) increased. Using this biosensor, we examined the dynamics of cytosolic and mitochondrial H₂O₂ in response to Daunorubicin, Fe₃O₄ nanozyme with Polyetherimide (PEI)- or Dextran (Dex)-modification, and Prussian blue nanozyme with different diameters. Results indicated that the particle size of PBNPs and surface modification of Fe₃O₄ play critical roles in their intracellular effects on the aspect of H₂O₂ modulation. The live-cell biosensors thus provide a powerful tool for detecting the variations of cytosolic and mitochondrial H₂O₂ in response to nanozymes, thereby facilitating a better understanding of the biological effects of nanozymes and their potential biomedical applications.