Several spectroscopic and electron microscopic techniques can monitor sulfur defects in molybdenum disulfide (MoS2)-based nanomaterials, but they often require costly instrumentation. Herein, we introduce a straightforward, fast, colorimetric method to evaluate sulfur vacancy degree in MoS2 nanoflowers (NFs), MoS2 nanosheets (NSs), and molybdenum diselenide (MoSe2) NSs by monitoring the dimerization of 5-thio-2-nitrobenzoic acid (TNB) into 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB). This sulfur-vacancy-mediated reaction results in a decrease in TNB absorbance at 412 nm and the emergence of a new DTNB peak near 325 nm. The dimerization of TNB triggered by MoS2 NFs, MoS2 NSs, and MoSe2 NSs follows pseudo-first-order kinetics. The rate constants of these materials correlate positively with sulfur defect density, but their maximum absorption capability remains similar. Additionally, machine learning simulations (Open Catalyst Project, GemNet-T) show that the adsorption energy of CH₃SH (a model thiol) becomes more negative with increasing sulfur vacancies on the MoS₂ surface. The MoS2-mediated TNB dimerization system provides a simple platform to distinguish whether a thiol compound undergoes dimerization or attaches to sulfur defect sites in the presence of sulfur-defective MoS2. For example, cysteine, glutathione, and homocysteine undergo dimerization, whereas 3-sulfanylpropanoic acid, 4-aminothiophenol, and 2-sulfanylbenzoic acid primarily attach to the sulfur defect sites.
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