Two-mode ratiometric electrochemical and turn-on fluorescence strategy for detecting biothiols in serum with all-in-one molecular probe

Reactive sulfur species (RSS) play crucial roles in human physiology, with biothiols like hydrogen sulfide (H 2 S), cysteine (Cys), and glutathione (GSH) being key regulators of oxidative stress and signal transduction. Here, a dual-mode resorufin-based (Re) small-molecule probe (Re-RSS) with electrochemical and fluorescent properties was developed for biothiol detection. Using a ratiometric electrochemical strategy and a “turn-on” fluorescence method, Re-RSS enabled sensitive quantification of H 2 S, Cys, and GSH. The electrochemical approach showed linear ranges of 0–0.3 mM for H 2 S, 0–0.1 mM for Cys, and 0–0.08 mM for GSH. Fluorescence spectrometry revealed linear relationships in the ranges of 1 μM–0.3 mM (H 2 S), 2 μM–0.1 mM (Cys), and 5 μM–0.08 mM (GSH), with detection limits as low as 0.02, 0.01, and 0.08 μM (S/N = 3), respectively. This dual-mode sensor offers three major advantages: (1) dual-readout correction of system/background errors for improved accuracy; (2) independent signal transduction from distinct mechanisms, eliminating cross-interference; (3) negative-potential electrochemical detection free from biological interference. The direct dual-mode quantification highlights its significance and reliability for biothiol analysis in biological systems.