MnO₂ nanozyme-based ratiometric fluorescent nanoplatform for glutathione detection and intracellular imaging

Ratiometric fluorescent nanoplatforms with self-calibrating capabilities offer enhanced detection accuracy by minimizing environmental interference. However, challenges such as signal cross-interference and nanomaterial instability persist in complex biological systems. To address these issues, we developed a glutathione (GSH)-responsive ratiometric fluorescent nanoprobe by integrating a porphyrin-based metal-organic framework (PCN-224) with MnO₂ nanosheets (NSs). The PCN-224 serves as both a nanocarrier and luminescent moiety, while MnO₂ acts as oxidase (OXD) mimetic nanozyme and the reactive site for GSH. The ratiometric detection mechanism relies on the intrinsic fluorescence of PCN-224 (emission at 655 nm) and the OXD-like activity of PCN-224@MnO₂, which catalyzes the oxidation of o-phenylenediamine (OPD) to generate fluorescent 2,3-diaminophenazine (DAP, emission at 570 nm). Upon GSH exposure, the redox reaction between GSH and MnO₂ produces Mn²⁺, quenching the OXD-like activity and diminishing the DAP fluorescence, while the PCN-224 fluorescence recovers due to MnO₂ decomposition. The nanoplatform exhibits a wide detection range spanning from 0.1 to 60 μM with a low detection limit of 15 nM and high selectivity. Furthermore, it enables accurate GSH quantification in complex biological matrices, including human serum and cell lysates, and facilitates real-time monitoring of endogenous and exogenous GSH in living cells via fluorescence imaging. This study not only presents sensitive and reliable platform for GSH detection, but also overcomes key limitations in ratiometric sensing, such as signal interference and nanomaterial instability, by utilizing a stimuli-responsive fluorescent nanoprobe design.