Colloidal surface-enhanced Raman spectroscopy study of hybrid grouper epidermal mucus as a non-invasive method of mucus profiling

Fish epidermal mucus is an important component in the innate immune defence against pathogens. Due to its significance in the fish physiology, detailed profiling of fish epidermal mucus will give valuable insight into fish health. However, conventional proteomics and metabolomics profiling of the biomolecules in fish epidermal mucus is time consuming. Therefore, realising the need for a rapid and minimally invasive method to profile fish epidermal mucus, this study explored a label-free colloidal surface-enhanced Raman spectroscopic (SERS) method to profile grouper epidermal mucus. The standard citrate reduction method was used to synthesise the gold nanoparticles. UV-Visible spectroscopy, dynamic light scattering, and transmission electron microscopy were used to characterise the gold nanoparticles. Acidified sodium sulphate (Na2SO4) at pH 3 was chosen as the aggregating agent due to its low affinity to form bonds with gold nanoparticles. The gold nanoparticles were preconcentrated prior to SERS analysis and 9.5 µL of the preconcentrated gold nanoparticles were mixed with 10 µL of the analyte solution followed by 0.5 µL of the aggregating agent. The SERS efficacy of the synthesised gold nanoparticles and Na2SO4 was tested with three different analyte solutions, namely, Rhodamine 6G (R6G), lysozyme and hybrid grouper (Epinephelus fuscoguttatus x Epinephelus lanceolatus) mucus. Based on the results, the optimal concentration of Na2SO4 to obtain the highest SERS enhancement for R6G and grouper mucus was 1 M, while the concentration for lysozyme was 0.1 M. This indicates that lysozyme induces a higher degree of aggregation of gold nanoparticles. There were a few overlapping peaks in the SERS spectrum of lysozyme and grouper mucus, namely, 818, 1283, 1454 and 1542 cm-1. The peaks at 818, 1283, and 1542 cm-1 may be good candidates for the detection of lysozyme in grouper epidermal mucus. Overall, the SERS protocol proposed in this work offers great potential for rapid and minimally invasive analysis of fish epidermal mucus.

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