Surfactant-free gold nanostars as a colloidal substrate for the surface-enhanced Raman spectroscopy of grouper epidermal mucus

Surface-enhanced Raman spectroscopy (SERS) is an emerging analytical method for biological analysis, leveraging the 'lightning rod' effect of metallic nanostructures to intensely amplify signals. Gold nanostars, with their numerous sharp tips, are particularly effective SERS substrates. In this work, biocompatible gold nanostars without harmful surfactants were synthesised using silver ions to control spike formations. Gold nanostars were investigated as a colloidal SERS substrate for the in-solution analysis of grouper epidermal mucus, a key indicator of fish health. The morphology of the gold nanostars was tuned by varying the concentration of silver nitrate (AgNO₃) from 0.5 to 3 mM. It was found that an increase in AgNO₃led to higher spike density but shorter, lower aspect ratio spikes. Gold nanostars synthesised with 1 mM AgNO₃produced the highest aspect ratio (3.25) and were shown to exhibit the strongest SERS enhancement. This was validated across various analytes, including rhodamine 6G (R6G), lysozyme and the grouper epidermal mucus itself. The enhancement factor of 4.72 × 10⁶and 7.62 × 10⁵were obtained with R6G and lysozyme, respectively. Ultimately, gold nanostars with long spikes were proven to be superior for in-solution SERS, achieving an enhancement factor two orders of magnitude higher than that of spherical gold nanoparticles in our previous work.