Gold Nanorod Amalgamation: Machine Learning Empowered Discrimination of Biothiol and Thiol Ratios

Biothiols, characterized by thiol groups, exhibit remarkable affinity for certain metals, playing pivotal roles in intracellular and extracellular biological processes. Fluctuations in their levels profoundly impact overall physiological health. Despite the development of various probes for biothiol detection and quantification, their inability to monitor thiol-to-disulfide state transitions persists as a limitation. Given their association with pathologies, early detection remains imperative. Gold nanorod (AuNR)-based colorimetric probes have garnered attention for their utility in visual diagnostic assays. Herein, we present a cost-effective, and sensitive multicolor ratio measuring probe enabling on-site simultaneous identification, discrimination, and quantification of essential biothiols─cysteine (CYS), glutathione (GSH), cystine (CYSS), and glutathione disulfide (GSSG)─while also quantifying thiol-to-disulfide ratios. Our investigation clarifies the probe's functionality, elucidating etching and antietching mechanisms based on sulfhydryl group coordination with Hg²⁺. This coordination impedes gold amalgam formation, facilitating discriminative detection via AuNR size and aspect ratio modulation, validated by transmission electron microscopy. Notably, distinct rainbow-like fingerprint patterns were discernible both visually and spectroscopically for the aforementioned biothiols and their respective thiol-to-disulfide ratios. Subsequent qualitative and quantitative analyses via linear discriminant analysis (LDA) and partial least squares regression revealed linear correlations over broad concentration ranges (CYS: 1.9-40 μmol L^-1, GSH: 3.2-200.0 μmol L^-1, CYSS: 2.0-70.0 μmol L^-1, GSSG: 3.7-100.0 μmol L^-1), with detection limits of 0.66 μmol L^-1 (CYS), 1.07 μmol L^-1 (GSH), 0.69 μmol L^-1 (CYSS), and 1.24 μmol L^-1 (GSSG). Moreover, thiol-to-disulfide ratios exhibited linear patterns within 0.2-5 μmol L^-1, with detection limits of 0.13 and 0.09 μmol L^-1, and exceptional analytical sensitivities of 32.648 and 49.782 for (CYS/CYSS) and (GSH/GSSG), respectively. Lastly, we evaluated the probe's performance in complex matrices relative to aqueous media, both quantitatively and qualitatively.