Fluorescent Probes for Thiol Detection in Biological Systems

Thiol-containing biomolecules such as glutathione, cysteine and homocysteine play central roles in cellular redox balance, signal transduction and metabolic regulation. The real-time mapping of these analytes in living systems has driven the development of fluorescent probes that combine high sensitivity, selectivity and minimal perturbation of native processes. Modern designs exploit reaction-based mechanisms—such as disulfide bond cleavage, Michael addition or nucleophilic displacement—to trigger a fluorescence “turn-on” or a ratiometric response in the presence of target thiols. Recent advances have introduced sensors that are targetable to specific organelles, operate in the near-infrared window for deep-tissue imaging and enable quantitative live-cell measurements. Two-photon excitation, chemogenetic fusion and reversible reaction chemistry have further extended temporal and spatial resolution. Collectively, these tools are enhancing our understanding of thiol dynamics in health and disease and opening new paths for diagnostic imaging, drug screening and the study of oxidative stress in complex biological environments.

Research from Nature Portfolio

Recent studies have introduced a chemogenetic ratiometric sensor that remains non-fluorescent until activated by glutathione in a predefined cellular compartment. By fusing this small-molecule probe to a fluorescent protein reporter, researchers achieved quantitative live-cell imaging of nuclear and cytosolic glutathione pools and simultaneous measurement of redox potential in the endoplasmic reticulum. The modular design allows exchange of fluorescent proteins to generate near-infrared sensors for deep-tissue visualisation.

Earlier foundational work reported a reversible reaction-based probe that quantifies real-time glutathione dynamics in single cells. This reagent supports both confocal microscopy and flow cytometry for high-throughput measurement of antioxidant responses, revealing transient changes during neuronal activation and ferroptotic cell death. Its reversible chemistry affords continuous monitoring of intracellular thiol fluctuations under physiological and pathological stimuli.

Fluorescent Probes for Thiol Detection in Biological Systems publication trend

The graph below shows the total number of articles in fluorescent probes for thiol detection in biological systems across all publications each year (not limited to Nature Index journals).

Technical terms

Thiol: An organic compound containing a sulphur–hydrogen (–SH) group, critical in redox chemistry and protein function.

Ratiometric fluorescent probe: A sensor that produces two emission wavelengths to provide a built-in reference for quantitative analysis.

Chemogenetic sensor: A hybrid tool combining a small-molecule probe with a genetic reporter to achieve orthogonal control and targeting in live cells.

Two-photon microscopy: An imaging technique using simultaneous absorption of two low-energy photons to excite fluorescence, allowing deep-tissue visualisation with reduced phototoxicity.

Disulfide bond cleavage: A reaction mechanism whereby a probe’s disulfide linkage is broken by thiolate nucleophiles, triggering a fluorescence response.

References

1. A locally activatable sensor for robust quantification of organellar glutathione. Nature Chemistry (2023).
2. Quantitative real-time imaging of glutathione. Nature Communications (2017).
3. A dual-response BODIPY-based fluorescent probe for the discrimination of glutathione from cystein and homocystein. Chemical Science (2015).
4. A minimalist fluorescent probe for differentiating Cys, Hcy and GSH in live cells. Chemical Science (2016).
5. Exploring cysteine regulation in cancer cell survival with a highly specific “Lock and Key” fluorescent probe for cysteine. Chemical Science (2019).