Visualizing Thiol Stress Responses in Cells with a Water-Soluble Raman Sensor.

Abstract

Accurate quantification of intracellular thiols in live cells remains challenging owing to the inherent properties of fluorescent probes, such as signal saturation, photobleaching, and dye aggregation, despite the development of numerous thiol-detecting probes. Here, we present (<i>E</i>)-2-cyano-3-isopropylacrylamide (iPrCAA) as a ratiometric Raman probe for quantifying and visualizing intracellular thiol concentrations. iPrCAA is the smallest thiol-detecting probe currently available for live-cell detection (molecular weight 138 g/mol). It exhibits high reactivity toward thiol-adduct formation and solubility in water, which enhance its sensitivity in relation to that of existing Raman probes. Thus, iPrCAA enables the real-time monitoring of thiol depletion in live cells, demonstrating that a thiol-free medium reduces thiol levels by approximately 3-fold. Through its ability to visualize endogenous molecules, iPrCAA revealed that the thiol distribution closely resembles that of proteins. This Raman probe provides a practical means for efficiently quantifying endogenous thiols and dynamically tracking thiol fluctuations under stress conditions.