Glutathione (GSH) is a critical intracellular antioxidant that regulates redox homeostasis and protects cells from oxidative damage. Altered GSH levels are implicated in numerous diseases, including cancer, Parkinson’s disease, and metabolic disorders. The ability to monitor GSH dynamics in real time within living systems is essential for understanding cellular function and pathological processes. In this study, we report the development of a ratiometric fluorescent probe based on Förster resonance energy transfer (FRET) between a coumarin donor and a near-infrared rhodamine acceptor, connected by a reducible disulfide linker.
The probe operates through a reversible mechanism: in its intact form, efficient FRET occurs from the coumarin donor (excited at 405 nm) to the rhodamine acceptor, resulting in strong emission at 652 nm and weak emission at 470 nm. Upon reduction by glutathione, the disulfide bond is cleaved, leading to spatial separation of the fluorophores and disruption of FRET. This results in a significant increase in coumarin fluorescence and a concomitant decrease in rhodamine emission, generating a ratiometric signal that is self-calibrating and resistant to environmental fluctuations.
Spectroscopic analysis confirmed a rapid and concentration-dependent response to GSH. In a 20 mM PBS buffer (pH 7.4) containing 30% ethanol, the probe exhibited a linear increase in the coumarin-to-rhodamine fluorescence ratio with increasing GSH concentrations over the range of 1–10 μM, with a detection limit of 0.8 μM. Complete response was achieved within 60 minutes, indicating favorable kinetics.219989-84-1 IUPAC Name UV-Vis absorption spectra showed two distinct peaks at 430 nm (coumarin) and 625 nm (rhodamine), both increasing upon GSH addition, consistent with the formation of cleavage products.
The probe demonstrated high selectivity for GSH over other amino acids, including cysteine, homocysteine, serine, leucine, and tryptophan. While cysteine induced a partial response due to its thiol reactivity, the effect was significantly weaker than that observed with GSH. Importantly, the probe remained stable across physiological pH values (5.0–8.0), ensuring reliable performance under biologically relevant conditions.
Cellular imaging using HeLa cells revealed excellent membrane permeability and low cytotoxicity. MTT assays showed over 85% cell viability even at 50 μM probe concentration. Confocal microscopy indicated mitochondrial localization, likely driven by electrostatic interactions between the positively charged rhodamine moiety and the negatively charged mitochondrial membrane. In untreated cells, a moderate ratiometric signal indicated baseline GSH levels. After treatment with N-ethylmaleimide (NEM), which depletes intracellular GSH, a dramatic shift toward higher coumarin emission was observed, confirming effective GSH removal. Conversely, exogenous GSH addition reversed this trend, demonstrating dynamic responsiveness.69075-42-9 InChIKey
In vivo applications were validated using first-instar Drosophila melanogaster larvae.PMID:31424814 Following incubation with the probe and varying GSH concentrations, larvae displayed clear, dose-dependent changes in fluorescence ratios. Control experiments confirmed no background signal from the probe alone or endogenous thiols. Imaging under both 405 nm and 559 nm excitation yielded consistent results, reinforcing the reliability of the ratiometric readout.
Theoretical calculations using density functional theory (DFT) supported the experimental observations. Calculations showed that the excited states of the coumarin and rhodamine moieties are localized independently in the intact probe, with minimal electronic coupling. Upon disulfide cleavage, increased molar absorptivity and oscillator strength were predicted for both fragments, explaining the enhanced fluorescence signals observed experimentally.
This work presents a robust, ratiometric FRET-based probe capable of real-time, non-invasive monitoring of glutathione in live cells and whole organisms. Its combination of sensitivity, selectivity, photostability, and self-calibration makes it an ideal tool for studying redox biology, evaluating oxidative stress, and screening potential therapeutics targeting GSH metabolism.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com