Caiyun Liu

Peroxynitrite (ONOO−) is a reactive oxygen species predominantly generated in mitochondria. Its concentration-dependent dynamics exhibit complex bidirectional effects, making quantitative measurement essential for understanding its pathological roles. Despite advances in fluorescent probes for ONOO− imaging, design limitations and interference from complex cellular environments still compromise detection reliability. Herein, we report the first reliable ratiometric fluorescent probe RDC for quantitative detection of ONOO− in mitochondria. Under single-wavelength excitation, the probe exhibits two emission peaks at 563 nm and 690 nm. Upon reaction with ONOO−, the 690 nm signal remains constant as an internal reference, while the 563 nm signal increases significantly, allowing the ratio (F563/F690) to achieve quantitative detection. The probe exhibits excellent selectivity and high sensitivity (LOD = 96 nM). Cell imaging reveals a linear relationship between the ratiometric signal and exogenous ONOO− concentration, validating its quantitative capability in living cells. Moreover, during pyroptosis, the probe reveals a marked increase in mitochondrial ONOO− levels, which is reversed by a pyroptosis inhibitor, indicating ONOO− as a key effector in pyroptosis. The probe also enables ONOO− detection in tissue samples, demonstrating its potential in complex biological systems. This probe serves as a reliable tool for quantifying mitochondrial ONOO− in cells and tissues and for studying its roles in pyroptosis and related diseases.

Precise visualization of hypochlorous acid (HOCl) in vivo is limited by tissue autofluorescence and nonspecific activation. Addressing these limitations requires dual-locked logic-driven molecular tools that integrate near-infrared (NIR) emission with stringent signal gating. As a key indicator of both physiological regulation and pathological dysfunction, pH provides an important cue for disease detection. Herein, we reported a pH-gated dual-locked NIR fluorescent probe, Ac-HEP, constructed on an acridine scaffold and functionalized with pH-responsive piperazine moieties for the selective detection of HOCl through the dual-locked fluorescence-activation strategy. Unlike most tandem dual-locked probes, Ac-HEP emitted fluorescence immediately upon activation in an acidic environment, which significantly enhanced the specificity of HOCl recognition. The ability of Ac-HEP to detect pathological HOCl was verified by imaging a pyroptosis model and real-time monitoring of HOCl in an inflammation zebrafish model. Notably, Ac-HEP achieved high contrast tumor diagnosis in live mice, offering a robust tool for spatiotemporal analysis of oxidative stress. Therefore, this pH-gated dual-locked fluorescent probe capable of detecting HOCl in different pathological conditions provides a new strategy for disease diagnosis.

A simple methoxyquinoline‐based ratiometric fluorescent probe was synthesized for detecting hydrogen peroxide (H₂O₂). After the probe reacted with H₂O₂, 6‐methoxyquinoline fluorescence dye was released, and the fluorescence intensity increased at 366 nm and decreased at 450 nm. And the fluorescence ratio of the probe at 366 and 450 nm is linearly related to the concentration of H₂O₂ in the range of 0–100 μM; it shows a good ratiometric fluorescence signal. In addition, the probe has good characteristics such as rapid response speed and high sensitivity and selectivity. This fluorescent probe with low biotoxicity and excellent cell membrane permeability was further used to image H₂O₂ in HeLa cells and zebrafish.