Gold Nanorod Amalgamation:\nMachine Learning Empowered\nDiscrimination of Biothiol and Thiol Ratios

Biothiols, characterized by thiol groups, exhibit remarkable\naffinity\nfor certain metals, playing pivotal roles in intracellular and extracellular\nbiological processes. Fluctuations in their levels profoundly impact\noverall physiological health. Despite the development of various probes\nfor biothiol detection and quantification, their inability to monitor\nthiol-to-disulfide state transitions persists as a limitation. Given\ntheir association with pathologies, early detection remains imperative.\nGold nanorod (AuNR)-based colorimetric probes have garnered attention\nfor their utility in visual diagnostic assays. Herein, we present\na cost-effective, and sensitive multicolor ratio measuring probe enabling\non-site simultaneous identification, discrimination, and quantification\nof essential biothiolscysteine (CYS), glutathione (GSH), cystine\n(CYSS), and glutathione disulfide (GSSG)while also quantifying\nthiol-to-disulfide ratios. Our investigation clarifies the probe’s\nfunctionality, elucidating etching and antietching mechanisms based\non sulfhydryl group coordination with Hg<sup>2+</sup>. This coordination\nimpedes gold amalgam formation, facilitating discriminative detection\nvia AuNR size and aspect ratio modulation, validated by transmission\nelectron microscopy. Notably, distinct rainbow-like fingerprint patterns\nwere discernible both visually and spectroscopically for the aforementioned\nbiothiols and their respective thiol-to-disulfide ratios. Subsequent\nqualitative and quantitative analyses via linear discriminant analysis\n(LDA) and partial least squares regression revealed linear correlations\nover broad concentration ranges (CYS: 1.9–40 μmol L<sup>–1</sup>, GSH: 3.2–200.0 μmol L<sup>–1</sup>, CYSS: 2.0–70.0 μmol L<sup>–1</sup>, GSSG: 3.7–100.0\nμmol L<sup>–1</sup>), with detection limits of 0.66 μmol\nL<sup>–1</sup> (CYS), 1.07 μmol L<sup>–1</sup> (GSH), 0.69 μmol L<sup>–1</sup> (CYSS), and 1.24 μmol\nL<sup>–1</sup> (GSSG). Moreover, thiol-to-disulfide ratios\nexhibited linear patterns within 0.2–5 μmol L<sup>–1</sup>, with detection limits of 0.13 and 0.09 μmol L<sup>–1</sup>, and exceptional analytical sensitivities of 32.648 and 49.782 for\n(CYS/CYSS) and (GSH/GSSG), respectively. Lastly, we evaluated the\nprobe’s performance in complex matrices relative to aqueous\nmedia, both quantitatively and qualitatively.