The level of aggregation and aggregate morphology of metallic nanoparticles are factors that influence the SERS signal (surface-enhanced Raman scattering), affecting reproducibility and sensitivity. This study presents a systematic evaluation of the colloidal aggregation on the SERS signal by combining transmission electron microscopy and UV-vis extinction spectroscopy. It focuses on the effect of two methods of sample preparation ("external standard method-ESM" and "standard addition method-SAM") on the SERS signal using the fungicide thiabendazole (TBZ) in Ag colloid as a probe molecule. The TBZ critical concentration (concentration for which SERS reaches the maximum intensity) was 6.0 × 10^-6 mol/L for ESM and 1.5 × 10^-6 mol/L for SAM. Besides, TBZ exhibited a sigmoid-type isotherm for ESM, indicating formation of a TBZ first layer on Ag nanoparticles at lower concentrations (Ag aggregates more compact; size 2 μm). For SAM, the TBZ first layer formation was also observed at lower concentrations (Ag aggregates more branched; 2 μm). The Ag aggregation mechanisms align with reaction-limited colloidal aggregation at lower concentrations and diffusion-limited colloidal aggregation at higher concentrations. We believe these results contribute to the SERS research field despite all of the work already done over its 50-year history.

Carbendazim (MBC), a commonly used fungicide from the benzimidazole group, was applied in this study as a probe molecule to understand the influence of sample preparation on the SERS (surface-enhanced Raman scattering) signal. We applied the external standard method (ESM), preparing fresh Ag colloid samples (reduced by hydroxylamine) for each concentration and measuring with and without potassium nitrate (KNO₃) as an aggregation-inducing salt. The impact of sample dilution before or after the addition of the salt to the Ag colloid was also explored. SERS signals were correlated with Ag colloid aggregation observed via transmission electron microscopy (TEM), UV-Vis extinction, dynamic light scattering (DLS), and zeta potential, examining diffusion-limited cluster aggregation (DLCA) and reaction-limited cluster aggregation (RLCA) mechanisms. The optimal results were achieved without KNO₃, with more compact aggregates at lower concentrations and more branched ones at higher concentrations. Dilution of the Ag colloid before salt addition enabled lower detection limits than without any dilution. No SERS signal was observed when the salt was added before dilution. These findings emphasize that a consistent relationship between aggregate morphology and the SERS signal cannot be generalized across analytes. Analyte-specific properties play a crucial role in determining optimal aggregation conditions for SERS analysis.