Biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are crucial in maintaining the redox balance in the body, and the metabolism and transportation of biothiols rely on the coreaction of diverse proteins and enzymes. The abnormal concentrations and metabolism of biothiols are closely associated with many diseases. However, due to the same active reaction site of the sulfydryl group in biothiols, it is inevitable to bear a confused signal of mutual influence on both nonselective detection and discriminate detection, which presents a serious challenge of accurately sensing or imaging the three biothiols. By assigning an alpha,beta-unsaturated ketone moiety as a Michael acceptor to trigger thiols to complete the irreversible equivalent domino response processes of nucleophilic addition, olefinic bond migration, and self-immolation, a targeted strategy was rationally pointed out, and herein, a hemicyanine-based probe CyOCy was prepared as a proof of strategy demonstration. The new probe could be equivalently lit up by Cys, Hcy, GSH, and even biothiol combinations (Cys/Hcy, Cys/GSH, Hcy/GSH, or Cys/Hcy/GSH) with unified linear ranges, detection limits, and response times. The probe CyOCy has been successfully used for the accurate quantification of total biothiols in the serum samples of healthy persons and coronary heart disease patients. In addition, the probe has been applied for cell screening, exogenous biothiol imaging, and monitoring drug-induced biothiol fluctuations. The purposive thinking of this work may provide an effective avenue for the accurate sensing of multicomponent samples.
