Construction of genetically encoded biosensors for monitoring cytosolic and mitochondrial H

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.