Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Imaging

Background:

• Thiols, including glutathione and cysteine, are vital biomolecules for maintaining intracellular redox homeostasis.
• Sensitive and selective detection methods for thiols are essential for understanding cellular processes.
• Existing methods may lack the sensitivity or specificity required for complex biological systems.

Purpose of the Study:

• To design and synthesize a novel chemoselective probe, SEluc-1 (sulfinate ester luciferin), for thiol detection.
• To achieve a ratiometric and turn-on response for thiols using both fluorescence and bioluminescence.
• To validate the probe's performance in biological imaging and assays.

Main Methods:

• Design and synthesis of SEluc-1 based on a "caged" luciferin strategy.
• Evaluation of probe's selectivity, signal-to-noise ratio, and limit of detection (LOD) for thiols.
• Application of SEluc-1 for fluorescence imaging of thiol activity in HeLa cells.
• Bioluminescent assays using SEluc-1 to detect thiol fluctuations in COS-7 and MCF-7 cells under oxidative stress.

Main Results:

• SEluc-1 exhibits a chemoselective, ratiometric fluorescence, and turn-on bioluminescence response to thiols.
• The probe demonstrates excellent selectivity, a high signal-to-noise ratio (>240 in bioluminescence), and a biologically relevant LOD (80 nM for cysteine).
• Successful application in fluorescence imaging of thiol activity in living HeLa cells and bioluminescent detection of thiol changes in COS-7 and MCF-7 cells.

Conclusions:

• SEluc-1 is a highly sensitive and selective probe for thiol detection, suitable for both fluorescence and bioluminescence applications.
• The probe's ability to detect thiol fluctuations induced by oxidative stress highlights its utility in studying cellular redox dynamics.
• SEluc-1 represents a valuable tool for advancing research in cellular biology and disease mechanisms involving thiol metabolism.