Equivalent Response Strategy for Sensing Total Biothiols in Human Serums and Living Cells Using a Hemicyanine-Based...

Biothiols play a central role in maintaining redox homeostasis, yet their detection remains challenging due to overlapping reactivity sites. Prior research has shown that cysteine, homocysteine, and glutathione share a sulfydryl group, which complicates selective sensing. This overlap leads to cross-reactivity in detection methods, making it difficult to distinguish individual biothiols. While existing techniques offer some detection capabilities, they often lack precision in mixed biothiol environments. No prior work had resolved the issue of equivalent response across multiple biothiols. This gap motivated the development of a new detection strategy. The need for accurate quantification in clinical and cellular settings remains unmet. Researchers have proposed alternative methods, but none have achieved uniform response across all three biothiols. This paper introduces a novel approach to address these limitations.

The aim of this study was to develop a detection method that could equally respond to multiple biothiols without interference. The specific problem addressed is the inability of current methods to distinguish or quantify biothiols in complex mixtures. The motivation comes from the clinical relevance of biothiol imbalances in diseases like coronary heart disease. The authors sought to design a probe that could detect all three biothiols simultaneously. The study aimed to create a unified detection platform for biothiols in serum and cells. The goal was to achieve equivalent response across all biothiols, including combinations. The probe needed to maintain consistent detection limits and response times. This approach could improve accuracy in both clinical diagnostics and cellular imaging.

The study employed a hemicyanine-based self-immolative probe named CyOCy. The probe was designed with an α,β-unsaturated ketone as a Michael acceptor. This structure enabled a domino response involving nucleophilic addition and self-immolation. The probe was tested in human serum and living cells to assess its performance. Detection limits and linear ranges were measured for individual and combined biothiols. The probe's response time was evaluated under various conditions. The method focused on achieving equivalent fluorescence activation across all biothiols. The probe was validated for use in clinical and cellular applications.

The probe CyOCy demonstrated equivalent fluorescence activation when exposed to Cys, Hcy, GSH, and their combinations. The detection limits were consistent across all tested biothiols. Linear ranges remained uniform regardless of the biothiol type or combination. Response times were within 5 minutes for all tested scenarios. The probe successfully quantified total biothiols in healthy and coronary heart disease patient sera. Cellular imaging experiments showed clear biothiol localization in living cells. The probe detected exogenous biothiols with high specificity. Drug-induced biothiol fluctuations were monitored effectively using CyOCy.

The authors propose that the equivalent response strategy offers a reliable method for detecting multiple biothiols. The probe CyOCy was shown to activate uniformly across all tested biothiols. This approach avoids the confusion caused by overlapping sulfydryl reactivity. The probe's performance in serum and cells supports its practical application. The method provides consistent detection limits and linear ranges. The study suggests that this strategy could be extended to other multicomponent systems. The authors propose that this work may guide future efforts in multianalyte sensing. The probe's success in clinical and cellular settings supports its potential use in diagnostics.