Substituent‐Dependent SERS Kinetics of Citrate‐Stabilized Gold Nanoparticles: Balancing Hotspot Formation and Colloidal Flocculation

Surface‐enhanced Raman scattering (SERS) is among the most sensitive vibrational techniques, but reliable quantification in liquid‐phase colloidal systems remains limited by the coupling between molecular adsorption, hotspot formation, and nanoparticle stability. Here, using citrate‐stabilized gold nanoparticles functionalized with para‐substituted benzenethiols, we show that analyte physicochemical properties determine not only the SERS intensity, but also the accessible measurement window and colloidal regime. Concentration‐dependent and time‐resolved SERS measurements, supported by UV–vis absorption spectroscopy and ζ‐potential analysis, reveal three response regimes. Halogenated thiols preserve colloidal stability and display delayed formation of SERS‐efficient configurations. Partially destabilizing thiols generate faster and stronger responses, but undergo earlier signal attenuation. Strongly destabilizing substituents produce intense but short‐lived signals governed by rapid aggregation and flocculation. The temporal response is described by a semi‐mechanistic model combining SERS‐active‐state formation with its loss through colloidal evolution. These results show that reliable quantitative SERS requires reporter selection based not only on Raman response and surface affinity, but also on the balance between hotspot generation and colloidal persistence.