Construction of an Oxidative Cleavage-Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity

Myeloperoxidase (MPO) is a mammalian pro-oxidant protease and it is closely related to severe infections and diverse inflammatory diseases. Rapid and sensitive measurement of MPO activity is essential for anticancer drug discovery and inflammatory research. Herein, we demonstrate the construction of an oxidative cleavage-activated deoxyribozymes (DNAzyme) biosensor for rapid detection and cellular imaging of the MPO activity. When target MPO is present, the phosphorothioate (PS)-modified hairpin probe is site-specifically cleaved by MPO, releasing the intact Mg²⁺-dependent DNAzyme sequences. Subsequently, the activated DNAzyme initiates the cyclic cleavage of the signal probe with the assistance of cofactor Mg²⁺, liberating large numbers of Cy5 molecules. This assay possesses the characteristics of easy operation, low sample consumption, without the requirements of expensive radiolabeling, antibodies, and nanomaterials. Especially, this assay can be performed in one pot under isothermal conditions (37°C) within 60 min. Due to the high efficiency of DNAzyme-based cyclic cleavage reaction and the intrinsic advantages of single-molecule detection, this assay achieves high sensitivity with a limit of detection (LOD) of 2.74×10⁻³ ng μL⁻¹. It can be applied to screen MPO inhibitors, measure cellular MPO activity at the single-cell level, and image intracellular MPO in living cells, providing a powerful platform for early clinical diagnosis and drug discovery.